Method for improving plant traits

文档序号：1631129 发布日期：2020-01-14 浏览：22次中文

阅读说明：本技术 用于改良植物性状的方法 (Method for improving plant traits ) 是由德罗尔·莎莉汀诺姆·格林伯格阿拉瓦·沙蒂尔·科恩于 2018-03-27 设计创作，主要内容包括：本发明公开了一种用于筛选和鉴定期望的植物改良性状的方法,所述方法包括以下步骤：(a)从预定来源的采样获得遗传物质,以及(b)从所述遗传物质构建表达文库。前述方法还包括以下步骤：(c)产生用所述表达文库转化的植物,其转化效率为至少0.05％至30％,代表至少10<Sup>2</Sup>-10<Sup>10</Sup>个转基因；(d)筛选表达所述期望性状的转化植物；和(e)鉴定所述转化植物的表达所述期望性状的所述转基因。(The present invention discloses a method for screening and identifying desirable plant improvement traits, comprising the steps of: (a) obtaining genetic material from a sample of a predetermined source, and (b) constructing an expression library from the genetic material. The aforementioned method further comprises the steps of: (c) generating plants transformed with said expression library with a transformation efficiency of at least 0.05% to 30%, representing at least 10 2 ‑10 10 A transgene; (d) screening for transformed plants that express the desired trait; and (e) identifying said transgene of said transformed plant that expresses said desired trait.)

1. A method for screening and identifying a desired plant improvement trait, the method comprising the steps of:

a. obtaining genetic material from a sample of a predetermined source;

b. constructing an expression library from said genetic material;

wherein the method further comprises the steps of:

c. producing plants transformed with said expression library with a transformation efficiency of at least 0.05% to 30%, representing at least 10²-10¹⁰A transgene;

d. screening for transformed plants that express the desired trait; and

e. identifying said transgene of said transformed plant expressing said desired trait.

2. The method of claim 1, further comprising the step of editing a target gene in a desired crop plant based on genetic information obtained from the transgene.

3. The method of claim 2, wherein the editing of the target gene is performed using any genome editing system or method, comprising using a system of engineered nucleases selected from the group consisting of: meganucleases, Zinc Finger Nucleases (ZFNs), transcription activator-like effector based nucleases (TALENs), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems, and any combination thereof.

4. The method of claim 1, wherein the predetermined source comprises a plant, microorganism, fungus, or other organism or portion thereof of an environmental niche.

5. The method of claim 1, wherein the screening step comprises a measurement of the transformed plant compared to a control plant, the measurement selected from the group consisting of: turgor measurements, plant death, leaf area, fresh weight of aerial parts of the plant, leaf number, fresh weight of branches, length of main branches, flower yield, pod or fruit yield, chlorosis, leaf damage, status or performance of the plant and any combination thereof.

6. The method of claim 5, wherein the control plant is a plant of the same genus as the transgenic plant but lacking the transgene, or a plant of the same genus as the transgenic plant, lacking the transgene, and transformed with a known gene conferring an improved trait to the plant.

7. The method of claim 1, wherein step (a) further comprises the step of enriching the genetic material by growing on an enriched or selective medium.

8. The method of claim 1, wherein said step (a) further comprises the step of enhancing expression of said desired trait by culturing said genetic material on a selective medium for said desired trait.

9. The method of claim 1, wherein said step (b) comprises the step of generating a prokaryotic cDNA library or a eukaryotic cDNA library or both.

10. The method of claim 1, wherein said step (b) further comprises the step of cloning said cDNA library into at least one binary vector.

11. The method of claim 10, wherein the binary vector comprises a constitutive promoter or a stress-inducible promoter.

12. The method of claim 10, wherein the binary vector comprises a bacterial selectable marker and a plant transformation selectable marker.

13. The method of claim 10, further comprising the step of transforming said cloned binary vector into a host cell.

14. The method according to claim 10, further comprising the step of transforming the cloned binary vector into Agrobacterium tumefaciens (Agrobacterium tumefaciens).

15. The method of claim 14, further comprising the step of introducing the transformed agrobacterium tumefaciens into at least one of: whole plants, plant tissues and plant cells.

16. The method of claim 15, comprising the step of introducing the transformed agrobacterium tumefaciens by spraying the plant with an inoculum comprising the transformed agrobacterium.

17. The method of claim 1, wherein said step (d) comprises growing said transformed plant under conditions selective for said desired trait.

18. The method of claim 1, further comprising the steps of:

f. collecting T1 seeds from the transformed plant of step (d);

g. determining the seed library transformation efficiency of the T1 seed;

h. sowing said T1 seeds of step (e) under selection conditions that allow for the screening and selection of transformed plants that express said desired trait;

i. testing the selected plants of step (g) for the presence of the transgene in the plants expressing the desired trait; and

j. isolating and sequencing the transgene of the selected transformed plant of step (h) that is positively tested for the transgene.

19. The method of claim 18, further comprising the steps of:

k. collecting T2 seeds from the plant of (h) found to be positive for the presence of the transgene;

growing plants of said T2 seed under selection conditions that allow for screening and selection of transformed plants that express said desired trait compared to control plants transformed with a known gene that confers said desired trait; and

optionally, isolating and sequencing said transgene of said selected plant of step (j).

20. The method according to any one of claims 18 and 19, comprising the steps of:

a. (ii) re-cloning and sequencing said isolated transgene of step (i) and/or (l);

b. transforming the re-cloned transgene into a plant;

c. screening said transformed plants of step (b) to select transformed plants that express said desired trait;

d. isolating the transgene from the selected plant of step (c); and

e. optionally, repeating steps (a) to (d).

21. The method of claim 4, wherein the environmental niche comprises an ecological niche, a population, a habitat, a gene bank, a prokaryotic culture, a eukaryotic culture, and any combination thereof.

22. The method of claim 4, wherein the environmental niche comprises a microbiome, a microbiota, a microbial culture, a plant, a yeast, an algae, a nematode, or any other organism or combination thereof.

23. The method of claim 4, wherein the environmental niche comprises a predetermined biological factor, a non-biological factor, and combinations thereof.

24. The method of claim 1, wherein the sample comprises a soil sample, a water sample, an organic sample, and any combination thereof.

25. The method of claim 1, wherein the desired trait is selected from the group consisting of: resistance or tolerance to at least one biotic stress, resistance or tolerance to at least one abiotic stress, increased yield, increased biomass, increased food quality and value, increased grain yield, herbicide or chemical resistance or tolerance, and any combination thereof.

26. The method of claim 25, wherein the abiotic stress is selected from the group consisting of: drought, salinity, heat, cold, fertilizer absorption, fertilizer use efficiency, and any combination thereof.

27. The method of claim 25, wherein the biotic stress is selected from the group consisting of: plant diseases, pathogens, bacteria, viruses, fungi, parasites, beneficial and harmful insects, weeds and cultivated or natural plants or any combination thereof.

28. The method of claim 1, wherein said step (a) comprises the step of extracting RNA from said sample of said predetermined environmental niche.

29. The method of claim 28, wherein the RNA extraction is performed according to a standard commercial kit or according to any other protocol for extracting RNA from environmental samples.

30. The method of claim 29, wherein the protocol for extracting RNA from environmental samples comprises the steps of:

a. obtaining a soil sample;

b. mixing the soil sample with an extraction buffer comprising 500mM phosphate buffer pH8 and 5% w/v cetyl trimethylammonium bromide (CTAB) and phenol (pH 8)/chloroform/IAA in a ratio of 25:24: 1;

c. subjecting the mixture of step (b) to shaking at 37 ℃ for 15 minutes or to a bead beater for 1 minute;

d. centrifuging the mixture of step (c) at 2500g for about 10 minutes at room temperature to obtain an aqueous phase;

e. transferring the aqueous phase to a new tube;

f. adding an equal amount of isopropanol supplemented with 20mg/ml of crystal violet solution to the aqueous phase in the tube of step (e) to obtain a violet staining solution;

g. mixing the solution by inverting the tube of step (f) and then incubating the tube at room temperature for about 30 minutes;

h. centrifuging the tube of step (g) at 2500g for about 30 minutes at room temperature to obtain a purple-stained layer;

i. transferring the purple-stained layer to a new tube and centrifuging the tube at maximum speed for about 5min to obtain a pellet and a supernatant;

j. washing the pellet with 80% v/v ice-cold ethanol and re-centrifuging for 5 minutes to obtain a pellet and a supernatant;

k. removing the supernatant of step (j) and drying the precipitate; and

suspending the dried precipitate in water in a ratio of 100 μ Ι water to 2g soil of step (a).

31. A plant comprising the transgene identified by the method of any one of claims 1 to 30.

32. The plant of claim 31, wherein said plant has at least one plant improvement trait as compared to a plant of the same genus lacking said transgene.

33. A polynucleotide sequence obtainable by a method according to any one of claims 1 to 30.

34. The polynucleotide of claim 33, wherein the polynucleotide comprises a nucleotide sequence corresponding to a sequence set forth in a polynucleotide sequence selected from the group consisting of SEQ ID NOs 1-148 and any combinations thereof.

35. A polynucleotide sequence having at least 80% sequence similarity to a polynucleotide sequence according to any one of claims 33 and 34.

36. Polypeptide sequence obtainable by the method according to claim 1.

37. The polypeptide sequence of claim 36 wherein the polypeptide comprises an amino acid sequence corresponding to a sequence as set forth in a polypeptide sequence selected from the group consisting of SEQ ID NO 149-321 and any combination thereof.

38. A polypeptide sequence having at least 60% sequence similarity to a polypeptide sequence according to any one of claims 36 and 37.

39. Use of the method of claim 1 for identifying a gene conferring a plant-improving trait selected from the group consisting of: resistance or tolerance to abiotic stress, resistance or tolerance to biotic stress, increased yield, increased biomass, increased food quality and value, increased grain yield, herbicide or chemical resistance or tolerance, and any combination thereof.

40. The use of claim 39, wherein the abiotic stress is selected from the group consisting of: drought, salinity, heat, cold, fertilizer utilization, and any combination thereof.

41. The use according to claim 39, wherein the biotic stress is selected from the group consisting of: plant diseases, pathogens, bacteria, viruses, fungi, parasites, beneficial and harmful insects, weeds and cultivated or natural plants or any combination thereof.

42. A method for screening and identifying drought or salinity resistance or tolerance improvement traits in plants, comprising the steps of:

a. obtaining genetic material from a sample of low moisture or high salinity source;

b. constructing an expression library from said genetic material;

wherein the method further comprises the steps of:

c. producing plants transformed with said expression library with a transformation efficiency of at least 0.5% to 30%, representing at least 10²-10¹⁰A transgene;

d. screening for transformed plants that are resistant or tolerant to a predetermined drought or salinity condition;

e. identifying said transgene of said drought or salinity resistant or tolerant transformed plant of step (d).

43. The method of claim 42, further comprising the step of editing a target gene in a desired crop plant based on genetic information obtained from the transgene.

44. The method of claim 43, wherein the editing of the target gene is performed using any genome editing system or method, comprising using a system of engineered nucleases selected from the group consisting of: meganucleases, Zinc Finger Nucleases (ZFNs), transcription activator-like effector based nucleases (TALENs), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems, and any combination thereof.

45. The method of claim 42, wherein the predetermined source comprises a plant, microorganism, fungus, or other organism or portion thereof of an environmental niche.

46. The method of claim 42, wherein the screening step comprises a measurement of the transformed plant compared to a control plant, the measurement selected from the group consisting of: turgor measurements, plant death, leaf area, fresh weight of aerial parts of the plant, leaf number, fresh weight of branches, length of main branches, flower yield, pod or fruit yield, chlorosis, leaf damage, status or performance of the plant and any combination thereof.

47. The method of claim 46, wherein the control plant is a plant of the same genus as the transgenic plant but lacking the transgene, or a plant of the same genus as the transgenic plant, lacking the transgene, and transformed with a known gene conferring an improved trait to the plant.

48. The method of claim 42, wherein said step (b) further comprises the step of cloning said expression library into at least one binary vector.

49. The method of claim 42, further comprising the steps of:

f. collecting T1 seeds from the transformed plant of step (c);

g. sowing said T1 seeds in soil having a water content of about 100% by volume selective for transformed plants;

h. growing the plants of said T1 seeds under drought or salinity conditions and/or without irrigation until a majority of the plants die to produce transformed plants that survive said drought or salinity conditions;

i. growing the drought or salinity-surviving transformed plant to produce T2 seeds;

j. (ii) screening said drought or salinity-surviving transformed plants of step (i) for the presence of a transgene;

k. isolating and sequencing said transgene of the positively selected plant of step (j).

50. The method of claim 49, further comprising the steps of:

collecting T2 seeds from each of said positively screened drought or salinity surviving transformed plants containing the transgene of step (j);

growing T2 plants from each of said transgenic-containing T2 seeds of step (l) under predetermined drought or salinity conditions as compared to control plants of the same genus but lacking said transgene or transformed with a known gene conferring drought or salinity tolerance or drought or salinity resistance;

subjecting each of said T2 transgenic-containing plants to a drought tolerance or resistance screening measurement as compared to said control plants, said measurement selected from the group consisting of: turgor pressure measurements, plant death, leaf area, fresh weight, leaf number, fresh weight of branches, length of main branches, flower and pod yield, chlorosis and leaf damage, status or performance of the plant, and any combination thereof;

isolating the transgene from the selected drought or salinity resistance competent T2 plant of step (n);

optionally, recloning the transgene into a binary vector;

optionally, transforming said cloned binary vector into a plant and growing said transformed plant under predetermined drought or salinity conditions; and

optionally, repeating steps (l) to (q).

51. The method according to claim 50, wherein the step of growing a T2 plant comprises the steps of:

a. sowing said T2 seeds in soil having a water content of about 100% by volume selective for transformed plants; and

b. irrigating said plant when the water content in said soil reaches about 5-10%.

52. The method of claim 50, wherein the predetermined drought or salinity condition is selected from the group consisting of: low moisture, high salinity, dry soil and heat.

53. A polynucleotide sequence obtainable by the method of claim 42.

54. The polynucleotide of claim 53, wherein the polynucleotide comprises a nucleotide sequence corresponding to a sequence set forth in a polynucleotide sequence selected from the group consisting of SEQ ID No. 1 to SEQ ID No. 148 and any combination thereof.

55. A polynucleotide sequence having at least 80% sequence similarity to a polynucleotide sequence according to any one of claims 53 and 54.

56. Polypeptide sequence obtainable by the method according to claim 42.

57. The polypeptide sequence of claim 56, wherein the polypeptide sequence comprises an amino acid sequence corresponding to a sequence as set forth in a polypeptide sequence selected from the group consisting of SEQ ID NO 149-321 and any combination thereof.

58. A polypeptide sequence having at least 60% sequence similarity to a polypeptide sequence according to any one of claims 56 and 57.

59. A method for extracting RNA from a soil sample comprising the steps of:

a. obtaining a soil sample;

b. mixing the soil sample with an extraction buffer comprising 500mM phosphate buffer pH8 and 5% w/v cetyl trimethylammonium bromide (CTAB) and phenol (pH 8)/chloroform/IAA in a ratio of 25:24: 1;

c. subjecting the mixture of step (b) to shaking at 37 ℃ for 15 minutes or to a bead beater for 1 minute;

d. centrifuging the mixture of step (c) at 2500g for about 10 minutes at room temperature to obtain an aqueous phase;

e. transferring the aqueous phase to a new tube;

f. adding an equal amount of isopropanol supplemented with 20mg/ml of crystal violet solution to the aqueous phase in the tube of step (e) to obtain a violet staining solution;

g. mixing the solution by inverting the tube of step (f) and then incubating the tube at room temperature for about 30 minutes;

h. centrifuging the tube of step (g) at 2500g for about 30 minutes at room temperature to obtain a purple-stained layer;

i. transferring the purple-stained layer to a new tube and centrifuging the tube at maximum speed for about 5min to obtain a pellet and a supernatant;

j. washing the pellet with 80% v/v ice-cold ethanol and re-centrifuging for 5 minutes to obtain a pellet and a supernatant;

k. removing the supernatant of step (j) and drying the precipitate; and

suspending the dried precipitate in water in a ratio of 100 μ Ι water to 2g soil of step (a).

60. A method for screening and identifying a desired plant improvement trait, the method comprising the steps of:

a. obtaining samples of a predetermined source;

b. extracting RNA from the sample according to the method of claim 60;

c. constructing an expression library from the RNA of step (b);

wherein the method further comprises the steps of:

d. producing plants transformed with said expression library with an efficiency of at least 0.05% to 30%, representing at least 10%²-10¹⁰A transgene;

e. screening for transformed plants that express the desired trait; and

f. identifying said transgene of said transformed plant expressing said desired trait.

61. The method of claim 60, further comprising the step of editing a target gene in a desired crop plant based on genetic information obtained from the transgene.

62. The method of claim 61, wherein the editing of the target gene is performed using any genome editing system or method, comprising using a system of engineered nucleases selected from the group consisting of: meganucleases, Zinc Finger Nucleases (ZFNs), transcription activator-like effector based nucleases (TALENs), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems, and any combination thereof.

63. The method of claim 60, wherein the predetermined source comprises a plant, microorganism, fungus, or other organism or portion thereof of an environmental niche.

64. The method of claim 60, wherein the screening step comprises a measurement of the transformed plant compared to a control plant, the measurement selected from the group consisting of: turgor measurements, plant death, leaf area, fresh weight of aerial parts of the plant, leaf number, fresh weight of branches, length of main branches, flower yield, pod or fruit yield, chlorosis, leaf damage, status or performance of the plant and any combination thereof.

65. The method of claim 64, wherein the control plant is a plant of the same genus as the transgenic plant but lacking the transgene, or a plant of the same genus as the transgenic plant, lacking the transgene, and transformed with a known gene conferring an improved trait to the plant.

66. An isolated polynucleotide having at least 80% sequence similarity to a nucleotide sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:148, and any combination thereof.

67. An isolated polypeptide having at least 60% sequence similarity to an amino acid sequence selected from the group consisting of SEQ ID NO 149-321 and any combination thereof.

Technical Field

The present invention relates generally to the field of improving plant traits. More specifically, the present invention relates to improving a trait in a plant by transforming an expression library from a predetermined source into the plant and screening for the desired trait.

Background

By 2050, the world population is estimated to reach 92 billion. To fully support this population, the total food production must be increased by 60% -70%. Climate models predict that increases in the frequency and duration of warm temperatures and drought in this century will have a negative impact on agricultural productivity. For example, african maize production may be at risk of significant yield loss, as researchers predict that if plants obtain sufficient moisture, the yield will decrease by 1% every one degree day (crop-day) the crop spends at temperatures above 30 ℃. These predictions are similar to those reported for corn yield in the united states. It is further shown that under drought conditions, the african corn yield drops by 1.7% per degree day spent by crops at temperatures above 30 ℃. In 2010, russian wheat production dropped by nearly one third, largely due to summer heat waves. Similarly, wheat yields in china and india were also significantly reduced in 2010, mainly due to drought and sudden temperature rise, respectively, resulting in forced ripening. These new global challenges require more sophisticated integrated agriculture.

In addition, global warming causes many abiotic and biotic stresses to coexist, thereby affecting agricultural productivity. The occurrence of abiotic stress can alter the interaction between a plant and a pest by enhancing the host plant's susceptibility to pathogenic organisms, insects, and reducing its ability to compete with weeds. In contrast, some pests may alter a plant's response to abiotic stress factors.

Biotic stress factors are caused by pathogens, insects, pests, weeds or intraspecies resource competition. The ability of biotic stress factors to cause yield or quality loss depends on the environment and thus may vary from region to region and also from agroecological to agroecological. For example, in australia, barley leaf disease is some of the major biotic stress factors that result in significant yield and quality loss. Although some plant species are known to be resistant to various diseases, these plant species are difficult or even impossible to reproduce in conventional ways.

The challenge is to produce crops that are resistant to biotic stress factors and that are flexible and adaptable to a variety of environments and populations. There are currently two main approaches to acclimatizing crops to new environments: development of new crops by conventional breeding (long efforts since domestication) and introduction of target traits into existing crops by plant breeding (including genetic engineering). In order to maintain productivity in the presence of increased climatic changes, populations and plant varieties are continually being developed to combat "new" extreme climates and other stresses, such as diseases, pathogens, insects, pests, and the like. In addition, there is a continuing need to find new herbicide tolerance or resistance genes for new chemicals and new modes of action of herbicides.

Genetic engineering has the potential to address some of the most challenging biological and non-biological limitations faced by farmers that are not easily addressed by conventional plant breeding alone.

Advantageous results of these genetic modifications include increased food production, reliability and yield; enhancing taste and nutritional value; and to reduce losses due to various biotic and abiotic stresses (e.g., fungal and bacterial pathogens). These goals continue to motivate modern breeders and food scientists, who are seeking newer methods of genetic modification to identify, select and analyze individual organisms with genetically enhanced characteristics.

The selection of plants transformed with foreign genes and/or genes from the same species or genus that are difficult or impossible to reproduce overcomes the species barrier and makes it possible to develop powerful "super-traits" that cannot be obtained by conventional methods. However, the molecular interactions and results of the introduced transgene and the endogenous gene are unpredictable.

When transferring genes encoding certain traits (typically from one plant species to another), the desired trait will not always be expressed unless the environment interacts with the gene in the intended manner to trigger the desired response, depending on the regulatory sequence into which the gene is inserted. This means that new transgenic varieties developed under controlled climate under laboratory conditions must be tested under field conditions as in more traditional breeding methods, so that at present there is little difference in the speed at which the new varieties are released.

Knowledge obtained from basic plant research will lay the foundation for future crop improvement, but the effective mechanism for quickly and effectively converting into public welfare agriculture still needs to be developed.

U.S. Pat. No. 6030779 and U.S. Pat. No. 6368798 disclose a method for identifying clones with a specified enzymatic activity, which is achieved by: selectively isolating a target nucleic acid from a population of genomic DNA using a polynucleotide probe that identifies a nucleic acid sequence encoding an enzyme having a particular enzymatic activity; and transforming the host with the isolated target nucleic acid to produce a clone library, which is screened for a specified enzymatic activity.

U.S. patent 6972183 discloses a method for screening expression libraries to identify clones expressing enzymes with desired activity. The method involves generating an expression library comprising a plurality of recombinant cell clones from a genomic DNA sample of one or more microorganisms, and then introducing a subset of the clones and substrate into capillaries in a capillary array. Interaction of the substrate with a clone expressing an enzyme having the desired activity produces an optically detectable signal, which can then be spatially detected to identify capillaries containing the clone producing such a signal. The clones that produce the signal can then be recovered from the identified capillaries.

European patent application 1025262 and us patent application 20020150949 teach a method for identifying clones with a specified activity of interest by: (i) generating an expression library derived from nucleic acid isolated directly from the environment; (ii) exposing the library to a specific substrate or substrates of interest; and (iii) screening the exposed library using fluorescence activated cell sorting to identify clones that react with one or more substrates.

U.S. patent application 20100152051 relates to a method for identifying and/or characterizing clones from an expression library that confer a desired biological property. The method comprises the following steps: screening for the expression of at least one (poly) peptide (e.g. a tag) expressed as a fusion protein together with the cloned recombinant inserts of the expression library. The (poly) peptide may be fused to the insert at the N-terminus or C-terminus. The method further comprises the step of contacting a ligand that specifically interacts with the (poly) peptide expressed by the insert of the clone that confers the desired biological property.

All the above methods are based on screening a DNA library (produced by a microorganism or an environmental sample) for a specific sequence or biochemical activity by interaction with a predetermined probe. In addition, the screening and selection of clones having a predetermined sequence or activity is performed before transformation into plant cells, and may be expressed in plant cells (tissue culture), but not in the whole plant. Thus, by the most recently used methods, only pre-selected clones are expressed in plants, and the expression and effect of the selected sequences in plants is unpredictable. In addition, in the above method, known activities can be screened only based on a priori knowledge. Thus, these methods are limited to the range of known enzymatic activities and enzyme families as well as previously known functions.

In view of the above, there is a long-felt need for efficient methods for screening and identifying unknown sequences that confer desirable plant improvement traits.

Disclosure of Invention

It is therefore an object of the present invention to disclose a method for screening and identifying desirable plant improving traits, said method comprisingThe following steps: (a) obtaining genetic material from a sample of a predetermined source; (b) constructing an expression library from said genetic material; wherein the method further comprises the steps of: (c) producing plants transformed with said expression library with a transformation efficiency of at least 0.05% to 30%, representing at least 10²-10¹⁰A transgene; (d) screening for transformed plants that express the desired trait; and (e) identifying said transgene of said transformed plant that expresses said desired trait.

It is a further object of the present invention to disclose the method as defined above, further comprising a step of editing a target gene in a desired crop plant based on genetic information obtained from said transgene.

It is another object of the current invention to disclose the method as defined in any of the above, wherein said editing of said target gene is performed using any genome editing system or method, comprising using a system of engineered nucleases selected from the group consisting of: meganucleases, Zinc Finger Nucleases (ZFNs), transcription activator-like effector based nucleases (TALENs), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems, and any combination thereof.

It is a further object of this invention to disclose such a method as defined in any of the above, wherein said predetermined source comprises a plant, microorganism, fungus or other organism or part thereof of an environmental niche.

It is a further object of this invention to disclose such a method as defined in any of the above, wherein said screening step comprises a measurement of said transformed plant compared to a control plant, said measurement being selected from the group consisting of: turgor measurements, plant death, leaf area, fresh weight of aerial parts of the plant, leaf number, fresh weight of branches, length of main branches, flower yield, pod or fruit yield, chlorosis, leaf damage, status or performance of the plant and any combination thereof.

It is a further object of the present invention to disclose the method as defined in any of the above, wherein said control plant is a plant of the same genus as said transgenic plant but lacking said transgene, or a plant of the same genus as said transgenic plant, lacking said transgene and transformed with a known gene conferring an improved trait to said plant.

It is a further object of this invention to disclose such a method as defined in any of the above, wherein said step (a) further comprises a step of enriching said genetic material by growing on an enriched or selective medium.

It is a further object of this invention to disclose such a method as defined in any of the above, wherein said step (a) further comprises the step of enhancing expression of said desired trait by culturing said genetic material on a selective medium for said desired trait.

It is another object of the current invention to disclose the method as defined in any of the above, wherein said step (b) comprises the step of generating a prokaryotic cDNA library or a eukaryotic cDNA library or both.

It is a further object of this invention to disclose such a method as defined in any of the above, wherein said step (b) further comprises the step of cloning said cDNA library into at least one binary vector.

It is a further object of this invention to disclose such a method as defined in any of the above, wherein said binary vector comprises a constitutive promoter or a stress inducible promoter.

It is a further object of this invention to disclose such a method as defined in any of the above, wherein said binary vector comprises a bacterial selectable marker and a plant transformation selectable marker.

It is a further object of this invention to disclose such a method as defined in any of the above, further comprising the step of transforming said cloned binary vector into a host cell.

It is a further object of this invention to disclose such a method as defined in any of the above, further comprising a step of transforming said cloned binary vector into Agrobacterium tumefaciens (Agrobacterium tumefaciens).

It is a further object of this invention to disclose such a method as defined in any of the above, further comprising a step of introducing said transformed agrobacterium tumefaciens into at least one of: whole plants, plant tissues and plant cells.

It is a further object of this invention to disclose such a method as defined in any of the above, comprising the step of introducing said transformed agrobacterium tumefaciens by spraying said plant with an inoculum comprising said transformed agrobacterium.

It is a further object of this invention to disclose such a method as defined in any of the above, wherein said step (d) comprises growing said transformed plant under conditions selective for said desired trait.

It is a further object of this invention to disclose such a method as defined in any of the above, further comprising: (f) collecting T1 seeds from the transformed plant of step (d); (g) determining the seed library transformation efficiency of the T1 seed; (h) sowing said T1 seeds of step (e) under selection conditions that allow for the screening and selection of transformed plants that express said desired trait; (i) testing the selected plants of step (g) for the presence of the transgene in the plants expressing the desired trait; and (j) isolating and sequencing the transgene of the selected transformed plant of step (h) that is positively tested for the transgene.

It is a further object of this invention to disclose such a method as defined in any of the above, further comprising: (k) collecting T2 seeds from the plant of (h) found to be positive for the presence of the transgene; (l) Growing said plant of T2 seed under selection conditions that allow for the screening and selection of transformed plants that express said desired trait compared to a control plant transformed with a known gene that confers said desired trait; and (m) optionally, isolating and sequencing said transgene of said selected plant of step (j).

It is a further object of this invention to disclose such a method as defined in any of the above, comprising: (a) (ii) re-cloning and sequencing said isolated transgene of step (i) and/or (l); (b) transforming the re-cloned transgene into a plant; (c) screening said transformed plants of step (b) to select transformed plants that express said desired trait; (d) isolating the transgene from the selected plant of step (c); and (e) optionally, repeating steps (a) through (d).

It is another object of the current invention to disclose the method as defined in any of the above, wherein the environmental niche comprises an ecological niche, a population, a habitat, a gene bank, a prokaryotic culture, a eukaryotic culture and any combination thereof.

It is another object of the current invention to disclose the method as defined in any of the above, wherein the environmental niche comprises a microbiome, a microbiota, a microbial culture, a plant, a yeast, an algae, a nematode or any other organism or combination thereof.

It is another object of the current invention to disclose the method as defined in any of the above, wherein the environmental niche comprises predetermined biological factors, non-biological factors and combinations thereof.

It is another object of the current invention to disclose the method as defined in any of the above, wherein the sampling comprises a soil sample, a water sample, an organic matter sample and any combination thereof.

It is another object of the current invention to disclose the method as defined in any of the above, wherein the desired trait is selected from the group consisting of: resistance or tolerance to at least one biotic stress, resistance or tolerance to at least one abiotic stress, increased yield, increased biomass, increased food quality and value, increased grain yield, herbicide or chemical resistance or tolerance, and any combination thereof.

It is another object of the current invention to disclose the method as defined in any of the above, wherein the abiotic stress is selected from the group consisting of: drought, salinity, heat, cold, fertilizer absorption, fertilizer use efficiency, and any combination thereof.

It is a further object of this invention to disclose such a method as defined in any of the above, wherein said biotic stress is selected from the group consisting of: plant diseases, pathogens, bacteria, viruses, fungi, parasites, beneficial and harmful insects, weeds and cultivated or natural plants or any combination thereof.

It is a further object of this invention to disclose such a method as defined in any of the above, wherein said step (a) comprises a step of extracting RNA from said sample of said predetermined environmental niche.

It is a further object of this invention to disclose such a method as defined in any of the above, wherein said RNA extraction is performed according to standard commercial kits or according to any other protocol for extracting RNA from environmental samples.

It is a further object of this invention to disclose such a method as defined in any of the above, wherein said protocol for extracting RNA from environmental samples comprises the steps of: (a) obtaining a soil sample; (b) mixing the soil sample with an extraction buffer comprising 500mM phosphate buffer pH8 and 5% w/v cetyl trimethylammonium bromide (CTAB) and phenol (pH 8)/chloroform/IAA in a ratio of 25:24: 1; (c) subjecting the mixture of step (b) to shaking at 37 ℃ for 15 minutes or to a bead beater for 1 minute; (d) centrifuging the mixture of step (c) at 2500g for about 10 minutes at room temperature to obtain an aqueous phase; (e) transferring the aqueous phase to a new tube; (f) adding an equal amount of isopropanol supplemented with 20mg/ml of crystal violet solution to the aqueous phase in the tube of step (e) to obtain a violet staining solution; (g) mixing the solution by inverting the tube of step (f) and then incubating the tube at room temperature for about 30 minutes; (h) centrifuging the tube of step (g) at 2500g for about 30 minutes at room temperature to obtain a purple-stained layer; (i) transferring the purple-stained layer to a new tube and centrifuging the tube at maximum speed for about 5min to obtain a pellet and a supernatant; (j) washing the pellet with 80% v/v ice-cold ethanol and re-centrifuging for 5 minutes to obtain a pellet and a supernatant; (k) removing the supernatant of step (j) and drying the precipitate; and (l) suspending the dried precipitate in water at a ratio of 100 μ l water to 2 grams of soil of step (a).

It is another object of the invention to disclose a plant comprising said transgene identified by the method defined in any of the above.

It is a further object of the present invention to disclose the plant as defined above, wherein said plant has at least one plant-improving trait compared to a plant of the same genus lacking said transgene.

It is another object of the invention to disclose a polynucleotide sequence obtainable by the method defined in any of the above.

It is another object of the present invention to disclose the polynucleotide as defined in any of the above, wherein said polynucleotide comprises a nucleotide sequence corresponding to a sequence as set forth in a polynucleotide sequence selected from the group consisting of SEQ ID NOs 1-148 and any combinations thereof.

It is another object of the present invention to disclose polynucleotide sequences having at least 80% sequence similarity to the polynucleotide sequences defined in any of the above.

It is a further object of the present invention to disclose a polypeptide sequence obtainable by the method defined in any of the above.

It is a further object of the present invention to disclose the polypeptide sequence as defined in any of the above, wherein said polypeptide comprises an amino acid sequence corresponding to a sequence as shown in a polypeptide sequence selected from the group consisting of SEQ ID NO 149-321 and any combination thereof.

It is another object of the invention to disclose a polypeptide sequence having at least 60% sequence similarity to the polypeptide sequence defined in any of the above.

It is another object of the invention to disclose the use of the method as defined in any of the above for identifying a gene conferring a plant improving trait selected from the group consisting of: resistance or tolerance to abiotic stress, resistance or tolerance to biotic stress, increased yield, increased biomass, increased food quality and value, increased grain yield, herbicide or chemical resistance or tolerance, and any combination thereof.

It is another object of the current invention to disclose the use as defined in any of the above, wherein the abiotic stress is selected from the group consisting of: drought, salinity, heat, cold, fertilizer utilization, and any combination thereof.

It is another object of the present invention to disclose the use as defined in any of the above, wherein the biotic stress is selected from the group consisting of: plant diseases, pathogens, bacteria, viruses, fungi, parasites, beneficial and harmful insects, weeds and cultivated or natural plants or any combination thereof.

It is another object of the present invention to disclose a method for screening and identifying drought or salinity resistance or tolerance improvement traits in plants, comprising the steps of: (a) obtaining genetic material from a sample of low moisture or high salinity source; (b) constructing an expression library from said genetic material; wherein the method further comprises the steps of: (c) producing plants transformed with said expression library with a transformation efficiency of at least 0.5% to 30%, representing at least 10²-10¹⁰A transgene; (d) screening for transformed plants that are resistant or tolerant to a predetermined drought or salinity condition; and (e) identifying said transgene of said drought or salinity resistant or tolerant transformed plant of step (d).

It is a further object of this invention to disclose such a method as defined in any of the above, further comprising the step of editing a target gene in a desired crop plant based on genetic information obtained from said transgene.

It is a further object of this invention to disclose such a method as defined in any of the above, wherein said step (b) further comprises the step of cloning said expression library into at least one binary vector.

It is a further object of this invention to disclose such a method as defined in any of the above, further comprising: (f) collecting T1 seeds from the transformed plant of step (c); (g) sowing said T1 seeds in soil having a water content of about 100% by volume selective for transformed plants; (h) growing the plants of said T1 seeds under drought or salinity conditions and/or without irrigation until a majority of the plants die to produce transformed plants that survive said drought or salinity conditions; (i) growing the drought or salinity-surviving transformed plant to produce T2 seeds; (j) (ii) screening said drought or salinity-surviving transformed plants of step (i) for the presence of a transgene; and (k) isolating the transgene from the positive selection plant of step (j) and sequencing.

It is a further object of this invention to disclose such a method as defined in any of the above, further comprising: (l) Collecting T2 seeds from each of said positively screened drought or salinity surviving transformed plants containing the transgene of step (j); (m) growing T2 plants from each of said transgenic-containing T2 seeds of step (l) under predetermined drought or salinity conditions as compared to control plants of the same genus but lacking said transgene or transformed with a known gene conferring drought or salinity tolerance or drought or salinity resistance; (n) subjecting each of said transgenic T2-containing plants to a drought tolerance or resistance screening measurement as compared to said control plants, said measurement selected from the group consisting of: turgor pressure measurements, plant death, leaf area, fresh weight, leaf number, fresh weight of branches, length of main branches, flower and pod yield, chlorosis and leaf damage, status or performance of the plant, and any combination thereof; (o) isolating the transgene from the selected drought or salinity resistance competent T2 plant of step (n); (p) optionally, recloning the transgene into a binary vector; (q) optionally, transforming said cloned binary vector into a plant and growing said transformed plant under predetermined drought or salinity conditions; and (r) optionally, repeating steps (l) through (q).

It is a further object of this invention to disclose such a method as defined in any of the above, wherein said step of growing a T2 plant comprises the steps of: (a) sowing said T2 seeds in soil having a water content of about 100% by volume selective for transformed plants; and (b) irrigating said plant when the water content in said soil reaches about 5-10%.

It is a further object of this invention to disclose such a method as defined in any of the above, wherein said predetermined drought or salinity condition is selected from a group consisting of: low moisture, high salinity, dry soil and heat.

It is another object of the invention to disclose a polynucleotide sequence obtainable by the method defined in any of the above.

It is another object of the present invention to disclose the polynucleotide as defined in any of the above, wherein said polynucleotide comprises a nucleotide sequence corresponding to a sequence shown in a polynucleotide sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:148 and any combination thereof.

It is another object of the invention to disclose polynucleotide sequences having at least 80% sequence similarity to the polynucleotide sequences defined in any of the above.

It is a further object of the present invention to disclose a polypeptide sequence obtainable by the method defined in any of the above.

It is a further object of the present invention to disclose the polypeptide sequence as defined in any of the above, comprising an amino acid sequence corresponding to the sequence as shown in the polypeptide sequence selected from the group consisting of SEQ ID NO 149-321 and any combination thereof.

It is another object of the invention to disclose a polypeptide sequence having at least 60% sequence similarity to the polypeptide sequence defined in any of the above.

It is another object of the present invention to disclose a method for extracting RNA from a soil sample, the method comprising the steps of: (a) obtaining a soil sample; (b) mixing the soil sample with an extraction buffer comprising 500mM phosphate buffer pH8 and 5% w/v cetyl trimethylammonium bromide (CTAB) and phenol (pH 8)/chloroform/IAA in a ratio of 25:24: 1; (c) subjecting the mixture of step (b) to shaking at 37 ℃ for 15 minutes or to a bead beater for 1 minute; (d) centrifuging the mixture of step (c) at 2500g for about 10 minutes at room temperature to obtain an aqueous phase; (e) transferring the aqueous phase to a new tube; (f) adding an equal amount of isopropanol supplemented with 20mg/ml of crystal violet solution to the aqueous phase in the tube of step (e) to obtain a violet staining solution; (g) mixing the solution by inverting the tube of step (f) and then incubating the tube at room temperature for about 30 minutes; (h) centrifuging the tube of step (g) at 2500g for about 30 minutes at room temperature to obtain a purple-stained layer; (i) transferring the purple-stained layer to a new tube and centrifuging the tube at maximum speed for about 5min to obtain a pellet and a supernatant; (j) washing the pellet with 80% v/v ice-cold ethanol and re-centrifuging for 5 minutes to obtain a pellet and a supernatant; (k) removing the supernatant of step (j) and drying the precipitate; and (l) suspending the dried precipitate in water at a ratio of 100 μ l water to 2 grams of soil of step (a).

It is another object of the present invention to disclose a method for screening and identifying a desired plant improvement trait, said method comprising the steps of: (a) obtaining samples of a predetermined source; (b) extracting RNA from the sample according to the method of claim 60; (c) constructing an expression library from the RNA of step (b); wherein the method further comprises the steps of: (d) producing plants transformed with said expression library with an efficiency of at least 0.05% to 30%, representing at least 10%²-10¹⁰A transgene; (e) screening tableA transformed plant that expresses the desired trait; and (f) identifying said transgene of said transformed plant that expresses said desired trait.

It is another object of the present invention to disclose an isolated polynucleotide having at least 80% sequence similarity to a nucleotide sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:148 and any combination thereof.

It is another object of the present invention to disclose an isolated polypeptide having at least 60% sequence similarity to an amino acid sequence selected from the group consisting of SEQ ID NO 149-321 and any combination thereof.

Drawings

In order to understand the invention and to see how it may be carried out in practice, various embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

FIGS. 1A-D show schematic diagrams of binary vectors used to insert amplified cDNA clones between promoters (35S, CBF3, ErdlO and Kinl) and HSP terminators. FIG. 1A shows the pPA-35H vector with a constitutive CaMV 35S promoter. FIGS. 1B-D show vectors containing the stress-inducible promoter of Arabidopsis thaliana (Arabidopsis thaliana): pPA-CH with CBF3 promoter (FIG. 1B), pPA-EH with ErdlO promoter (FIG. 1C) and pPA-KH with Kinl promoter (FIG. 1D);

FIG. 2 shows photographs of Agrobacterium libraries counted for 3 different libraries on LB plates;

figure 3 shows a photograph of tobacco tissue cultures transformed with the library: 7 days post-transformation (FIG. 3A), 40 days post-transformation (FIG. 3B) and 6-8 weeks post-transformation (FIG. 3C);

FIG. 4 shows photographs illustrating glufosinate resistance of 10-day-old Arabidopsis thaliana expression library seedlings were selected. Green plants are resistant to glufosinate, whereas small yellow plants do not have the transgene and are therefore susceptible.

FIG. 5 shows photographs of T2 and T3 control experiments in a greenhouse;

FIG. 6 shows the photographic results of screening transgenic plants for resistance to drought;

FIG. 7 shows a graph illustrating the loss of turgor pressure (dark grey) of the relativity versus soil moisture content in plants expressing the gene used as a control several days after irrigation was stopped;

figure 8 shows a graph showing the normalized mortality scale of transgenic plants expressing a positive control compared to plants expressing GFP;

FIG. 9 is a graph showing the results of various drought resistance genes identified by the methods of the present invention; and

figure 10 illustrates leaf area analysis of various transgenic plant lines expressing the identified novel drought resistance conferring genes after recloning compared to positive and negative controls.

Detailed Description

The following description is provided, along with all chapters of the present invention, to enable any person skilled in the art to make and use the present invention and sets forth the best modes contemplated by the inventors of carrying out the present invention. However, since the general principles of the present invention have been defined specifically to provide means and methods for screening and identifying desirable plant improving traits, various modifications will still be apparent to those skilled in the art.

Some plant species are known to be resistant to various diseases. However, such species are often difficult or impossible to reproduce by conventional techniques and methods.

The present invention provides a method and platform for discovering and identifying genes from plants that have unique and valuable characteristics, such as disease resistance, abiotic stress resistance or tolerance, food improvement qualities (e.g., improved oil, protein content, amino acids, vitamins, etc.), and then inserting or expressing them in desired crops by gene editing or other transformation techniques.

Thus, it is within the scope of the present invention to introduce targeted traits into existing crops by plant breeding, including genetic engineering and gene (genomic) editing.

The present invention provides a novel method for screening and identifying desirable plant improvement traits. The method comprises the following steps: (a) obtaining genetic material from a sample of a predetermined environmental niche, or extracted from other sources (e.g., plants of the same or other genera); (b) constructing an expression library from said genetic material. According to a core aspect, the invention further comprises the steps of: (c) producing plants transformed with said expression library with an efficiency of at least 0.05% to 30%, representing at least 10%²-10¹⁰A transgene; (d) screening for transformed plants that express the desired trait; and (e) identifying said transformed plantExpressing said desired trait.

The present invention provides for the first time a method for screening and selecting for unknown sequences from predetermined sources (e.g., econiches and/or plants) conferring improved traits in valuable crop plants. The method of the present invention is effective and advantageous with respect to common and conventional screening methods in the following respects:

1. expression libraries are prepared from genetic material or libraries (i.e., RNA) derived from predetermined sources, such as extreme environments, plant material, and the like. Thus, only genes expressed under preselected environmental conditions are used for screening procedures in plants.

2. The entire expression library is transformed into plants with an efficiency of 0.05% -30% and represents at least 10%²-10¹⁰A unique transgene.

3. In the method of the present invention, the screening of the desired phenotype of the expression library is performed in the target organism as a plant. This is not pre-selected and reveals new unique genes of the desired phenotype expressible in plants.

In conventional methods, the first step is to select genes for a predetermined trait in the source genetic material, for example by probing a DNA library with known sequences in prokaryotic or eukaryotic cells, before expressing the preselected genes in the plant. The results of this conventional method are limited and have the following disadvantages:

1. the screening is performed in host cells/organisms that are not the target organism (usually in prokaryotic or unicellular organisms).

2. Screening is limited because it is performed using known sequences or probes or activities. It has been found that functional screening methods require detectable levels of enzyme activity, which cannot always be achieved, for example only about 40% of the enzyme activity may be detected in an E.coli-based expression system (Gabor et al, 2004). Furthermore, it is noted herein that, despite the advanced sequencing technologies available, about 35-60% of the total protein-encoding genes show a high degree of similarity to "hypothetical proteins", "predicted proteins", or "proteins of unknown function" (Culligan et al, 2014; Venter et al, 2004).

3. Only pre-selected clones were transformed into plants.

4. The expression and effect of the preselected clone in the target plant is unpredictable.

For the reasons stated above, the novel method of the present invention for screening plants transformed with an expression library for a desired phenotype is advantageous.

Herein, it is recognized that drought and salinity are considered as two abiotic stresses with important effects on plant growth and development.

With respect to drought, it is considered the most damaging environmental stress that reduces the growth and productivity of crops. Drought severely affects plant growth and development, resulting in significant reductions in growth rate and biomass accumulation. The main consequences of drought in plants are reduced rates of cell division and expansion, leaf size, stem elongation and root proliferation, interfering with stomatal amplitude and Water Use Efficiency (WUE) (Farooq et al 2009). This phenomenon involves genetic, physiological and environmental events and their complex interactions. The rate and amount of plant growth depends on these events, which are affected by water deficit. Cell growth is one of the most drought-sensitive physiological processes due to turgor pressure and reduction in available water (Taiz and Zeiger, 2006). In the absence of water, cell elongation in higher plants may be inhibited by interruption of the water flow from the xylem to the surrounding elongated cells. Impaired mitosis, reduced cell elongation and expansion lead to a reduction in plant height, leaf area and crop growth (Nonami, 1998).

Salinity is also considered to be one of the major serious abiotic factors affecting crop growth and productivity. During salt stress, all major processes (e.g. photosynthesis, protein synthesis and energy and lipid metabolism) are affected (Parida & Das, 2005). During initial exposure to salt, plants are subjected to water stress, thereby reducing leaf expansion. Osmotic effects of salt stress can be observed immediately after salt administration and are believed to persist during exposure, resulting in inhibited cell expansion and cell division and stomatal closure. During long term exposure to salt, plants experience ionic stress, which may lead to premature senescence of adult leaves, reducing the photosynthetic area available to support continued growth. In fact, excess sodium and more importantly chloride may adversely affect plant enzymes, leading to reduced energy production and other physiological changes. It is further acknowledged that ionic stress leads to premature senescence of older leaves, as well as toxic symptoms (chlorosis, necrosis) in mature leaves. Without wishing to be bound by theory, high sodium ions affect plants by disrupting protein synthesis and interfering with the activity of enzymes (Carillo et al, 2011).

The present invention provides a method for efficiently screening for novel genes conferring drought and/or salinity resistance or increased tolerance in plants and in particular in valuable crops.

The method of the present invention overcomes the above-mentioned disadvantages by using expressed genetic material (e.g. RNA or mRNA) which represents genes expressed in the selected organism, for example due to environmental conditions (e.g. drought or high salt) and generating a cDNA library representing the "macro-Expression" (Meta-Expression) or macro-transcriptome (transcriptome) status of certain biological niches or other genetic sources. The entire cDNA library is then transformed into plants and the plants are expressed and screened for the desired phenotype.

A central aspect of the present invention is the generation of expression libraries from a variety of sources (including plants) and environments. The expression library is transformed into a plant as a target organism to improve its trait or function. The plant expression libraries are then screened for desirable traits such as salt or drought resistance or tolerance, increased biomass and yield, biotic stress (disease and pathogen) resistance or tolerance, increased nutritional value, or increased fertilizer utilization.

It is recognized herein that the environment (e.g., soil) in which the plant is growing is inhabited by a microbial community, e.g., one gram of soil contains about 10⁷-10⁹Individual microbial cells (estimated number of bacterial species varying between 2000 and 830 million per gram of soil, https:// www.ncbi.nlm.nih.gov/PMC/articles/PMC2970868/), which contain sequence information of about one gigabase (gigabase), or more. Microbial communities residing in plant growing environments (e.g., soil) are very complexMiscellaneous and, despite its economic importance, still poorly understood. Such consortia of microorganisms (consortia) provide the necessary ecosystem for plant growth, including fixation of atmospheric nitrogen, nutrient cycling, disease suppression, and sequestration of iron and other metals.

It is within the scope of the present invention to use functional metagenomic and macrotranscriptomics approaches to explore new genes conferring improved traits in plants.

Reference is now made to the metagenomic methodology employed by the present invention according to some aspects. Metagenomics is the study of genetic material from environmental samples. It generally refers to environmental genomics, ecological genomics or community genomics. Although traditional microbiology and microbial genome sequencing and genomics rely on cultured clonal cultures, environmental gene sequencing clones specific genes to generate a diversity profile in natural samples. In some aspects, metagenomics utilizes the study of genomes in microbial communities to constitute the first step in studying microbiomes. Its primary purpose is to infer a taxonomic profile of the microbial community. Whole Metagenomic Sequencing (WMS) provides data on the functional profile of a microbial community. Such work indicates that by culture-based methods, most of the microbial biodiversity is missed. In fact, it is estimated that in almost every environment on earth, over 99% of microorganisms cannot be cultured in the laboratory.

Metagenomics herein also refers to macrotranscriptomics, which studies and correlates the transcriptome of a set of interacting organisms or species. Macrotranscriptomics involve sequencing the complete (macro) transcriptome of a microbial community. In some aspects, the macro transcriptomics inform genes expressed by the entire community. By using functional annotations of expressed genes, a functional profile of the community under specific conditions can be inferred, which is usually dependent on the state of the host. While metagenomics provides data on the composition of microbial communities under different conditions, macrotranscriptomics provides data on genes co-expressed under different conditions. Macrotranscriptomics involve analysis of gene expression (RNA-seq) across a whole community. In particular aspects, macrotranscriptomics describe genes expressed in a particular microbial environment. Therefore, macrotranscriptomics are studies of the function and activity of whole transcripts (RNA-seq) from environmental samples.

It should be noted that gene expression is distributed logarithmically, e.g., the first 100 genes with the highest expression may cover up to 30% of all transcripts. Even a single gene may cover a proportion of 10%. Therefore, a very high sequencing depth is required to see genes with low expression levels.

By using methods such as "shotgun" or PCR directed sequencing, a largely unbiased gene sample can be obtained from the members of the sampled population. It is recognized herein that metagenomic approaches provide a powerful tool for exploiting microbial ecology to improve plant traits, e.g., biological mechanisms useful in agriculture and improved plant traits.

As used herein, the term "about" means ± 25% of the defined amount or measure or value.

As used herein, the term "similar" means a corresponding or similar range of about ± 20%, particularly ± 15%, more particularly about ± 10%, and even more particularly about ± 5%.

As used herein, the term "average value" refers to an average value obtained by measuring a predetermined parameter in each plant of a certain plant population and calculating the average value from the number of plants in the population.

In this specification and the appended claims, an indefinite article "a" or "an" means one or more than one of the indicated item unless the context clearly dictates otherwise. Thus, for example, reference to "a plant" includes one or more/plants, reference to "a trait" includes one or more/traits, and reference to "a cell" includes mixtures, tissues, etc. of cells.

As used herein, "plant" refers to any plant at any developmental stage, including plant seeds.

The term "plant" includes whole plants or any part or derivative thereof, such as plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant callus or callus, meristematic cells, microspores, embryos, immature embryos, pollen, ovules, anthers, fruits, flowers, leaves, cotyledons, pistils, seeds, seed coats, roots, root tips and the like.

The term "plant cell" as used herein refers to the structural and physiological unit of a plant, comprising protoplasts and a cell wall. Plant cells may be in the form of isolated single cells or cultured cells, or as a higher tissue unit such as a plant tissue, plant organ, or part of a whole plant.

As used herein, the term "plant cell culture" or "tissue culture" refers to a culture of a plant unit, such as protoplasts, regenerable cells, cell cultures, cells in plant tissue, pollen tubes, ovules, embryo sacs, zygotes and embryos at different developmental stages, leaves, roots, root tips, anthers, meristematic cells, microspores, flowers, cotyledons, pistils, fruits, seeds, seed coats, or any combination thereof.

As used herein, the term "plant material" or "plant part" refers to the leaves, stems, roots, root tips, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, seed coats, cuttings, cell or tissue cultures or any other parts or products of a plant or any combination thereof.

As used herein, "plant organ" refers to a distinct and visible structured and differentiated part of a plant, such as a root, stem, leaf, flower bud, or embryo.

As used herein, "plant tissue" refers to a group of plant cells organized into structural and functional units. Including any plant tissue in a plant or in culture. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, protoplasts, meristematic cells, callus tissue, and any group of plant cells organized into structural and/or functional units. The term is used in conjunction with or in the absence of any particular type of plant tissue listed above or otherwise encompassed by the present definition and is not intended to exclude any other type of plant tissue.

As used herein, the term "trait" refers to a characteristic or phenotype, particularly to a plant improvement characteristic or phenotype. A phenotypic trait may refer to the appearance or other detectable characteristic of an individual resulting from the interaction of its genome, proteome, and/or metabolome with the environment. For example, in the context of the present invention, a plant improved trait or a desired plant improved trait relates to resistance or tolerance to at least one biotic stress, resistance or tolerance to at least one abiotic stress, increased yield or biomass, increased grain yield, increased fertilizer uptake and use efficiency and any combination thereof.

A trait may be inherited in a dominant or recessive manner or in a partially or incompletely dominant manner. A trait may be monogenic (i.e., determined by a single locus) or polygenic (i.e., determined by more than one locus), or may result from the interaction of one or more genes with the environment. The dominant trait results in the full phenotypic manifestation at either the heterozygous or homozygous state. Usually, recessive traits are only expressed when present in a homozygous state.

Within the scope of the present invention, the term "phenotype" is understood to mean a distinguishable characteristic of a genetically controlled trait.

As used herein, the phrase "phenotypic trait" refers to the appearance or other detectable characteristic of an individual resulting from the interaction of its genome, proteome, and/or metabolome with the environment.

Within the scope of the present invention, "stress" may be defined as any external factor having a negative effect on plant growth, function and/or reproduction.

The term "abiotic stress" is generally defined herein as the negative impact of non-living factors on plants in a particular environment. Non-living variables must affect the environment beyond the normal range of variation, adversely affecting the performance or physiology of a plant or plant population in a significant manner. Non-limiting examples of abiotic or biotic stress factors or sources or environmental factors can include factors such as sunlight, wind, temperature (cold, hot), salinity, excessive watering (flooding), drought, and the like, as well as factors such as fertilizer absorption and fertilizer use efficiency, and any combination thereof. Abiotic stress resistance or tolerance can enhance the growth and productivity of plants, particularly crops. Abiotic stress factors have been shown to be the most harmful and may have synergistic effects when present together with abiotic stress factors.

The term "drought" refers hereinafter to a physical phenomenon generally caused by long periods of sub-average precipitation or irrigation. For example, insufficient or low moisture (in soil or air), water supply shortages, dry soil, moisture conditions, high salinity, heat, and any combination thereof. Drying conditions may arise for different reasons. It can have a major impact on ecosystem and agriculture, such as reduced yield and crop damage.

Many organisms are physiologically and genetically adapted for drought resistance.

"biotic stress" is defined herein as stress that occurs as a result of damage to plants by other living organisms (e.g., bacteria, viruses, fungi, whiteflies, thrips, spiders, nematodes, parasites, beneficial and harmful insects, weeds, and cultivated or native plants). The type of biotic stress imposed on a plant may depend on both geography and climate as well as the host plant and its ability to resist a particular stress.

As used herein, the phrase "resistant" refers to the ability of a plant to limit the growth and development of a particular pathogen and/or damage caused to the plant as compared to a susceptible plant under similar environmental conditions. Resistant plants may exhibit certain disease symptoms or damage under pathogen or pest stress or under abiotic stress conditions.

Further within the scope of the present invention, resistance refers to the complete immunity of a plant to a particular stress, such as an infection by a biotrophic pathogen. According to a particular embodiment of the invention, by transforming the expression library into a host plant, the transformed host acquires a resistance gene which prevents the proliferation of the pathogen and/or confers resistance to a particular abiotic stress (e.g. drought).

According to some aspects, resistance is an absolute term in which a plant is fully immune to a particular stress by itself. It should be noted that this does not mean that tolerance is not obtained under biotic or abiotic stress.

The term "tolerance" refers hereinafter to a characteristic of a plant that allows the plant to avoid, tolerate or recover from biotic or abiotic stress sources under conditions that would normally cause greater damage to other plants of the same species. These heritable characteristics affect the degree of damage done to the plant. With respect to tolerance in agricultural production, it is meant that the plant may be under stress (disease/infection/or physiological attack) but the degree of loss does not exceed the economic threshold level (the degree of loss does not hinder the economic potential of the product). According to other aspects of the invention, tolerability is a relative term. Examples of tolerance may be present in the case of plant pathogens and all abiotic stresses, especially in the case of complex traits controlled by multiple factors.

In general, "resistance" and "tolerance" are terms used to refer to a plant's ability to cope with biotic or abiotic stress.

The term "transformation" as used herein refers to a genetic alteration or modification caused by the introduction of foreign DNA into a cell. This includes integrating the foreign DNA into the host genome, and/or introducing plasmid DNA comprising the foreign DNA into the plant cell. Such transformation processes result in the uptake, incorporation and expression of exogenous genetic material (exogenous DNA, e.g., an expression library prepared from econiche samples). Plant transformation may refer to the introduction of foreign genes into plant cells, tissues or organs by direct or indirect means developed in molecular and cellular biology.

The term "environmental niche" or "ecological niche" generally refers to the behavior of a species living under specific environmental conditions. Which includes microorganisms, fungi, plants or other organisms (extreme microorganisms) residing in a given environmental location. Ecological niches describe how an organism or population responds to the distribution of resources and competitors, and in turn how those same factors are altered. The type and number of variables that make up the size of an environmental niche vary between species, and the relative importance of a particular environmental variable to a species may vary according to geographic abiotic and biotic environments.

According to other aspects, the term "environmental niche" or "ecological niche" describes the relative location of a species or population in an ecosystem. More specifically, it describes how a population responds to its rich resources and competitors and how these same factors are affected. Abiotic or physical environments are also part of the niche as they affect how and by which populations affect resources and competition. The description of the niche may include a description of the life history, habitat and location in the food chain of the organism. In the context of the present invention, an "environmental niche" or "ecological niche" may be defined in terms of biological or non-biological factors, such as high salinity, drought conditions, high temperature, cold conditions, pH or any other extreme environmental conditions.

Within the scope of the present invention, the genetic material is from a sample of a predetermined environmental niche, including from soil, water, plant biomass, microorganisms, yeast, algae, nematodes, and the like.

As used herein, the term "microbiome" or "microbiota" refers to an ecological community of commensal, and pathogenic microorganisms found in and on all multicellular organisms from plants to animals. The microbial population includes bacteria, archaea, protists, fungi, and viruses. It has been found that the microbial population is critical for immune, hormonal and metabolic homeostasis of its host. The synonymous term microbiome describes the collective genome of microorganisms present in an environmental niche or the microorganisms themselves. Microbiomes and hosts emerge during evolution as coordinated units of epigenetic and genomic characteristics, sometimes collectively referred to as symbionts (holobiots).

The term "genetic material" or "genetic library" refers hereinafter to the sum of genetic material of a population at a given time. It includes all genes and gene combinations (sum of alleles) in the population.

The term "isolated" as used hereinafter refers to the removal of a material from its original environment (e.g., from the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide that is isolated from some or all of the coexisting materials in the natural system is isolated.

Nucleic acids isolated or derived from a microorganism or any organism may preferably be inserted into a vector or plasmid. Such vectors or plasmids are preferably those which contain expression regulatory sequences including promoters, enhancers and the like suitable for expression in plants. Particularly preferred plasmids and methods for their introduction and transformation are described in detail in the schemes set forth herein.

The term "expression library" as used hereinafter refers to a collection of vectors or viruses (e.g., plant viruses used as viral vectors) or plasmids or phages comprising a representative sample of cDNA or genomic fragments constructed in such a way as to be transcribed and/or translated by a host organism (in the context of the present invention, a plant). This technique uses an expression vector to generate a library of clones, each of which transcribes one RNA and/or expresses one protein. The expression library is then screened for the desired property, and the desired clone is recovered for further analysis. One and non-limiting example is the use of expression libraries to isolate genes that can confer drought resistance or tolerance.

Within the scope of the present invention, expression libraries, typically derived from microbial genetic material, may be constructed in binary vectors (or transfer DNA (T-DNA) binary systems or shuttle vectors) capable of replication in a variety of hosts, such as e.coli and Agrobacterium tumefaciens, to produce genetically modified plants. These are artificial vectors generated from naturally occurring Ti plasmids found in agrobacterium tumefaciens. In some aspects, the expression library is transferred from agrobacterium tumefaciens to a plant.

The term "editing" or "gene editing" or "genome editing" refers hereinafter to any conventional or known genome editing system or method, including systems using engineered nucleases selected from the group consisting of: meganucleases, Zinc Finger Nucleases (ZFNs), transcription activator-like effector based nucleases (TALENs), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems, and any combination thereof. In the context of the present invention, the above-described gene editing techniques are used to edit target genes in desired crop plants based on information obtained from transgenes identified by the methods of the present invention.

The term "corresponding to a sequence" refers hereinafter to sequence homology or sequence similarity. These terms relate to two or more nucleic acid or protein sequences that are identical or have a specified percentage of amino acid residues or nucleotides that are identical when used for comparison and alignment of maximum correspondence, as determined using an available sequence comparison algorithm or by visual inspection.

According to further aspects of the invention, the term "corresponding to a nucleotide sequence" refers to variants, homologues and fragments of the indicated nucleotide sequence which have or perform the same biological function or are associated with the same phenotypic characteristics of the indicated nucleotide sequence.

Another indication that two nucleic acid sequences are substantially similar or that the sequences "correspond to nucleotide sequences" is that the two molecules hybridize to each other under stringent conditions. High stringency conditions (e.g., high hybridization temperature and low salt in hybridization buffer) allow hybridization only between highly similar nucleic acid sequences, while low stringency conditions (e.g., lower temperature and high salt conditions) allow hybridization when the sequences are less similar.

The term "similarity" or "sequence similarity" refers hereinafter to the degree of similarity between two sequences when compared. This depends on their identity and shows how well the residues are aligned. Sequence similarity refers to the best match problem (i.e., for sequence alignment). The best-fit algorithm finds the fewest number of editing operations (insertions, deletions, and substitutions) to align one sequence to another. Sequence similarity searches can identify "homologous" proteins or genes by detecting excessive similarity (i.e., statistically significant similarity reflecting common ancestry).

Within the scope of the present invention, similarity searching is an efficient and reliable strategy or tool for identifying homologues (i.e., sequences having a common evolutionary ancestor). Non-limiting examples of similarity search programs include BLAST (e.g., Altschul et al, 1997); 3.3 and 3.4 units), PSI-BLAST (e.g., Altschul et al, 1997), SSEARCH (e.g., Smith and Waterman, 1981); pearson,1991, element 3.10), FASTA (e.g., Pearson and Lipman, 1988, element 3.9), and HMMER3 (e.g., Johnson et al, 2010). Such a procedure yields accurate statistical estimates and can ensure that proteins or nucleic acid sequences with significant similarity can also have similar structures. Similarity retrieval is efficient and reliable, since sequences with significant similarity can be inferred to be homologous; i.e. having a common ancestor.

Within the scope of the present invention, similarity is understood to mean a sequence similarity of at least 60%, in particular a similarity of at least 70%, preferably of more than 80%, more preferably of more than 90%. The term "substantially similar" refers to nucleic acids that are at least 50% identical to a reference sequence when comparing the entire ORF (open reading frame), wherein the sequence similarity is preferably at least 70%, more preferably at least 80%, still more preferably at least 85%, particularly greater than about 90%, most preferably 95% or greater, particularly 98% or greater.

In some embodiments of the invention, such substantially similar sequences refer to polynucleotide or amino acid sequences that have at least about 60% similarity, preferably at least about 80% similarity, or about 90%, 95%, 96%, 97%, 98%, or 99% similarity to the indicated polynucleotide or amino acid sequence.

The present invention encompasses nucleotide sequences which have at least 60% similarity, preferably 70%, more preferably 80%, even more preferably 90% and particularly more preferably 95% similarity to a polynucleotide sequence identified by the method of the invention or to a reference sequence.

The invention also encompasses amino acid sequences that are at least 60% similar, preferably 70%, more preferably 80%, even more preferably 90% and particularly more preferably 95% similar to a polypeptide sequence identified by the methods of the invention or to a reference sequence.

As used herein, a "reference sequence" is a defined sequence that is used as a basis for sequence comparison. The reference sequence may be a subset or the entirety of the specified sequence; for example, as a fragment of a full-length cDNA or gene sequence, or the entire cDNA or gene or protein sequence.

As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage sequence identity is used to refer to proteins, it is recognized that residue positions that are not identical will often differ by conservative amino acid substitutions, wherein amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity), and therefore do not alter the functional properties of the molecule.

The term "identity" or "sequence identity" also refers hereinafter to the amount of a feature that exactly matches between two different sequences. Thus, gaps (gaps) are not calculated and the measurement is related to the shorter of the two sequences.

In other words, if the two sequences being compared to each other differ in length, the sequence identity preferably relates to the percentage of nucleotide residues in the shorter sequence that are identical to the nucleotide residues of the longer sequence. As used herein, the percent identity between two sequences is the percentage of the number of identical positions that the two sequences have, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms known in the relevant art.

Further within the scope, the terms "similarity" and "identity" also refer to local homology, identifying homologous or similar (in nucleotide and/or amino acid sequence) domains. It is well recognized that bioinformatics tools such as BLAST, sserch, FASTA and HMMER calculate local sequence alignments that identify the most similar regions between two sequences. For domains found in the context of different sequences in different proteins, the alignment should be limited to homologous domains, as domain homology provides sequence similarity captured in the score. According to some aspects, the term similarity or identity also includes sequence motifs, which are broad and have or are considered to have a biologically significant pattern of nucleotide or amino acid sequences. Proteins may have sequence motifs and/or structural motifs formed by the three-dimensional arrangement of amino acids that may not be adjacent.

According to other embodiments, protein or polynucleotide sequences having specific position or domain sequence similarity are identified by the methods of the invention. Low similarity is not meaningful when comparing residues that are not conserved, so lower overall similarity sequences with higher conservation in conserved regions are considered similar at a given range, e.g., > 60% as can be found in hmm-based search algorithms (e.g., HMMER3) (i.e., sequences that show about 37% low similarity to the closest homologous sequence but have all conserved substrate binding residues of a particular protein family).

The term "Conserved Domain Database (CDD)" refers to a collection of sequence alignments and profiles representing protein domains. It also includes domain and database (i.e. Molecular Model Database (MMDB)) known 3 dimensional protein structure alignment.

In some embodiments of the invention, such substantially identical sequences refer to polynucleotide or amino acid sequences that are at least about 60% identical, preferably at least about 80% identical, or about 90%, 95%, 96%, 97%, 98%, or 99% identical to a given polynucleotide or amino acid sequence.

Polypeptides within the scope of the invention have at least 50% identity to a protein identified by the methods of the invention; or at least 55% identity, or at least 60% identity, or at least 65% identity, or at least 70% identity, or at least 75% identity, or at least 80% identity, or at least 85% identity, or at least 90% identity to a protein identified by the methods of the invention, or to a reference sequence.

As used herein, "percent sequence identity" refers to a value determined by comparing two sequences that are optimally aligned over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentages are calculated as follows: determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

The term "substantial identity" of a polynucleotide sequence comprising at least 80% sequence identity, preferably at least 85%, more preferably at least 90%, most preferably at least 95% sequence identity as compared to a reference sequence refers to one of the alignment programs described using standard parameters. One skilled in the art will recognize that these values can be appropriately adjusted to determine the corresponding identity of the proteins encoded by the two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. For these purposes, substantial identity of amino acid sequences generally means a sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95%. Preferably, the homology alignment algorithm of Needleman et al is used for optimal alignment (1970.J. mol. biol.48: 443).

As used herein, the term "homolog" refers to a DNA or amino acid sequence that has a degree of sequence similarity in terms of a consensus amino acid or nucleotide sequence. There may be partial similarity or complete similarity (i.e., identity). For protein sequences, amino acid similarity matrices can be used, which are known in different bioinformatics programs (e.g., BLAST, FASTA, Bestfit program-Wisconsin Sequence Analysis Package, Version 8for Unix, Genetics Computer Group, University Research Park,575Science driven Madison, WI 53711, Smith Waterman). When a particular search is performed using different matrices, different results may be obtained. As is well known in the art, the degree of similarity of nucleotide sequences is based on identity matching with penalties for gaps or insertions required for optimal alignment (e.g., Altschul S.F. et al, 1990, J Mol Biol 215(3): 403-10; Altschul S.F. et al, 1997, Nucleic Acids Res.25: 3389-. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without loss of biological or activity can be found using computer programs well known in the art (e.g., DNASTAR software).

Within the scope of the present invention, the term "polymorphism" is understood to mean the presence of two or more different forms of a gene, genetic marker or genetic trait or gene product in a population, which may be obtained, for example, by alternative splicing, DNA methylation, etc.

The present invention encompasses "high throughput screening" or "HTS" techniques, which herein refers to methods for rapidly identifying genes that modulate a particular biomolecular pathway or function. It includes macrotranscriptomics and metagenomic gene expression.

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