Gene editing method for regulating rice plant type and seed grain weight
阅读说明：本技术 一种调控水稻株型和种子粒重的基因编辑方法 (Gene editing method for regulating rice plant type and seed grain weight ) 是由 不公告发明人 于 2020-03-04 设计创作，主要内容包括：本发明提供了一种调控水稻株型和种子粒重的基因编辑方法,具体地,本发明首次意外地发现,对miR156基因家族的N个成员(N≥2)进行敲除,居然可以显著改善植物性状。(The invention provides a gene editing method for regulating rice plant type and seed grain weight, and particularly, the invention unexpectedly discovers for the first time that the plant characters can be obviously improved by knocking out N members (N is more than or equal to 2) of miR156 gene family.)
1. A method of modifying a plant comprising the steps of:
(i) carrying out genetic engineering transformation on plant cells or plant tissues so as to mutate N members in a miR156 gene family, wherein N is more than or equal to 2, and the miR156 gene family comprises class I miR156 s;
(ii) regenerating the genetically engineered plant cells or plant tissues into plants, and testing the regenerated plants for traits selected from the group consisting of: plant type, thousand kernel weight, kernel type, kernel length, or combinations thereof;
(iii) and selecting plants with the required character characteristics according to the character test results.
2. The method of claim 1, wherein the desired trait characteristic is selected from the group consisting of: reducing or increasing tillering number, increasing kernel length, increasing thousand kernel weight, increasing or decreasing plant height, increasing or decreasing stem diameter, or a combination thereof.
3. The method of claim 1, wherein the class I miR156s is selected from the group consisting of: MIR156d, MIR156e, MIR156h, MIR156i, MIR156f, MIR156g, or a combination thereof.
4. The method of claim 1, wherein the miR156 gene family further comprises class II miR156 s.
5. The method of claim 4, wherein the class II miR156s is selected from the group consisting of: MIR156a, MIR156b, MIR156c, MIR156k, MIR156l, or a combination thereof.
6. The method of claim 1, wherein the mutating comprises reducing the expression or activity of N members of the miR156 gene family.
7. A genetically engineered plant tissue or plant cell comprising N members of a miR156 gene family in said plant tissue or plant cell, wherein N is greater than or equal to 2, wherein said miR156 gene family comprises class I miR156 s.
8. A method of producing genetically engineered plant tissue or plant cells comprising the steps of:
mutating N members in a miR156 gene family in plant tissues or plant cells to obtain genetically engineered plant tissues or plant cells, wherein N is more than or equal to 2, wherein the miR156 gene family comprises class I miR156 s.
9. A method of producing a genetically engineered plant comprising the steps of:
regenerating the genetically engineered plant tissue or plant cell prepared by the method of claim 8 into a plant body, thereby obtaining a genetically engineered plant.
10. A method of producing grain, comprising the steps of:
(i) planting crops, wherein N members in a miR156 gene family in the crops are mutated, wherein N is more than or equal to 2, and the miR156 gene family comprises a class I miR156 s;
(ii) the grain (grain) of the crop is harvested.
11. A method of modulating a trait in a plant comprising the steps of: reducing the expression or activity of N members in a miR156 gene family in a plant, wherein N is more than or equal to 2, and the miR156 gene family comprises a class I miR156 s.
Technical Field
The invention relates to the technical field of agriculture and biology, in particular to a gene editing method for regulating and controlling the plant type and seed grain weight of rice.
Background
Plant type and thousand kernel weight are two major determining factors of crop yield. The plant type is a comprehensive factor determining the yield of the grains and comprises the factors of tillering number, plant height, leaf length, leaf angle, spike type, stalk diameter and the like. The good plant type is beneficial to close planting, disease resistance, lodging resistance, population ventilation and improvement of photosynthesis utilization efficiency, and is a key factor for cultivating high-yield varieties. Therefore, the breeding of excellent plant types is an important subject of crop breeders.
There are 11 miR156 expression genes (MIR156 a-MIR156 i, MIR156k and MIR156l) in the rice genome, 12 miR156 precursor sequences can be generated by transcription, and the functional differentiation condition of the MIR156 family genes is not clear at present.
Therefore, there is an urgent need in the art to develop a gene editing method for regulating the plant type and seed grain weight of rice.
Disclosure of Invention
The invention aims to provide a gene editing method for regulating and controlling the plant type and seed grain weight of rice.
Another object of the present invention is to provide a method for modulating plant traits by controlling miR156 expression level or activity. More specifically, the present invention provides a method for modulating a trait of a plant, the trait comprising plant type, thousand kernel weight, kernel type, kernel length, or a combination thereof, by reducing or blocking or inhibiting the expression level of at least one gene of the miR156 family.
In a first aspect, the present invention provides a method of modifying a plant comprising the steps of:
(i) genetically engineering plant cells or plant tissues to mutate N members of the miR156 gene family, wherein N is greater than or equal to 2 (preferably N is greater than or equal to 3, more preferably N is between 4 and 10, more preferably N is between 4 and 8, and more preferably N is between 5 and 6), and the miR156 gene family comprises class I miR156 s;
(ii) regenerating the genetically engineered plant cells or plant tissues into plants, and testing the regenerated plants for traits selected from the group consisting of: plant type, thousand kernel weight, kernel type, kernel length, or combinations thereof;
(iii) and selecting plants with the required character characteristics according to the character test results.
In another preferred embodiment, when N.gtoreq.2 (preferably N.gtoreq.3, more preferably, 4 to 10, more preferably, 4 to 8, more preferably, 5 to 6), the trait is selected from the group consisting of: plant type, thousand kernel weight, kernel type, kernel length, or combinations thereof.
In another preferred example, the plant type comprises tiller number, plant height, leaf length, leaf angle, spike type and stem diameter.
In another preferred embodiment, the desired trait characteristic is selected from the group consisting of: reducing or increasing tillering number, increasing kernel length, increasing thousand kernel weight, increasing or decreasing plant height, increasing or decreasing stem diameter, or a combination thereof.
In another preferred example, the class I miR156s is selected from the group consisting of: MIR156d, MIR156e, MIR156h, MIR156i, MIR156f, MIR156g, or a combination thereof.
In another preferred example, the miR156 gene family further comprises class II miR156 s.
In another preferred example, the class II miR156s is selected from the group consisting of: MIR156a, MIR156b, MIR156c, MIR156k, MIR156l, or a combination thereof.
In another preferred example, the mutation comprises a decrease in the expression or activity of N members of the miR156 gene family.
In another preferred embodiment, the mutation comprises an insertion, deletion or substitution of one or more bases.
In another preferred embodiment, the mutation is performed by a method selected from the group consisting of: gene editing, natural variation, mutagenesis, or a combination thereof.
In another preferred example, the "decreasing" refers to decreasing the expression or activity of N members of the miR156 gene family satisfies the following condition:
the ratio of A1/A0 is less than or equal to 80 percent, preferably less than or equal to 60 percent, more preferably less than or equal to 40 percent, and most preferably 0 to 30 percent;
wherein, A1 is the expression or activity of N members in miR156 gene family; a0 is the expression or activity of N members in the same miR156 gene family in wild-type plants of the same type.
In another preferred example, said reduction refers to an expression level of a member of the miR156 family E1 in said plant which is 0-80%, preferably 0-60%, more preferably 0-40% of wild type compared to the expression level E0 of a member of the wild type miR156 family.
In another preferred example, the reduction of the expression or activity of N members of the miR156 gene family in plants is achieved by a method selected from the group consisting of: gene mutation, gene knockout, gene disruption, RNA interference techniques, criprpr techniques, or a combination thereof.
In another preferred example, the reduction of the expression or activity of N members of the miR156 gene family in plants is achieved by a method selected from the group consisting of: gene mutation, gene knockout, gene disruption, RNA interference techniques, gene editing techniques, or a combination thereof.
In another preferred example, the gene editing technology is selected from CRISPR technology, TALEN technology and ZFN technology.
In another preferred embodiment, said plant having the desired trait is selected from the group consisting of: mir156dehi, mir156deghi, mir156defghi, mir156defgh, mir156defgi, mir156dfgi, mir156efgi, mir156efgh, mir156eghi, mir156efg, mir156fgi, mir156ghi, mir156ei, mir156fg, mir156defij, or a combination thereof.
In another preferred embodiment, said plant having the desired trait is selected from the group consisting of: mir156dehi, mir156defgh, mir156defghi, or combinations thereof.
In another preferred embodiment, the plant is selected from the group consisting of: a monocot dicot, a gymnosperm, or a combination thereof.
In another preferred embodiment, the plant is selected from the group consisting of: a graminaceous plant, a leguminous plant, a cruciferous plant, or a combination thereof.
In another preferred embodiment, the plant comprises: arabidopsis, wheat, barley, oats, maize, rice, sorghum, millet, soybean, peanut, tobacco, tomato, cabbage, canola, spinach, lettuce, cucumber, garland chrysanthemum, water spinach, celery, lettuce, or combinations thereof.
In another preferred example, the genetic engineering comprises gene editing of a member of the miR156 gene family with one or more sgRNA-mediated Cas9 nuclease.
In another preferred example, the gene editing comprises gene editing of two or more genes of the miR156 gene family selected from the group consisting of: MIR156d, MIR156e, MIR156h, MIR156i, MIR156f, MIR156g, or a combination thereof.
In another preferred example, the gene editing further comprises gene editing of two or more genes of the miR156 gene family selected from the group consisting of: MIR156a, MIR156b, MIR156c, MIR156k, MIR156l, or a combination thereof.
In another preferred example, the gene editing comprises gene editing of two or more genes of the miR156 gene family selected from the group consisting of: MIR156d, MIR156e, MIR156h, MIR156i, MIR156f, MIR156g, MIR156a, MIR156b, MIR156c, MIR156k, MIR156l, or a combination thereof.
In a second aspect, the invention provides a genetically engineered plant tissue or plant cell in which N members of the miR156 gene family are mutated, wherein N is greater than or equal to 2 (preferably, N is greater than or equal to 3, more preferably, N is 4-10, more preferably, N is 4-8, more preferably, N is 5-6), wherein the miR156 gene family comprises class I miR156 s.
In another preferred example, the mutation comprises a decrease in the expression or activity of N members of the miR156 gene family.
In a third aspect, the present invention provides a method for preparing a genetically engineered plant tissue or plant cell comprising the steps of:
mutating N members of miR156 gene family in the plant tissue or the plant cell to obtain the genetically engineered plant tissue or plant cell, wherein N is more than or equal to 2 (preferably, N is more than or equal to 3, more preferably, N is 4-10, more preferably, N is 4-8, more preferably, N is 5-6), wherein the miR156 gene family comprises class I miR156 s.
In another preferred embodiment, the method comprises the following steps:
(1) introducing an expression vector for expressing a gene editing tool into the plant tissue or the plant cell, and enabling the expression vector to be integrated with a cell endogenous genome and to be expressed, wherein the gene editing tool comprises a nuclease for targeting and cutting DNA and a guide RNA for targeting and combining miR156 gene;
(2) selecting the edited plant cell or tissue.
In another preferred embodiment, the introduction method comprises agrobacterium infection, gene gun method, electric firing, polyethylene glycol transformation method, virus transformation, microinjection method.
In another preferred embodiment, the expression vector further comprises one or more selection marker genes, antibiotic genes (e.g., ampicillin or kanamycin resistance genes), herbicide resistance genes (e.g., Bar gene, Gox gene), fluorescent genes (e.g., LacZ gene), color reaction reporter genes (e.g., GUS gene).
In a fourth aspect, the present invention provides a method for preparing a genetically engineered plant, comprising the steps of:
the genetically engineered plant tissue or plant cell prepared by the method of the third aspect of the present invention is regenerated into a plant body, thereby obtaining a genetically engineered plant.
In a fifth aspect, the present invention provides a genetically engineered plant, said plant being produced by the method of the fourth aspect of the invention.
A sixth aspect of the present invention provides a method of producing grain, comprising the steps of:
(i) planting a crop in which N members of the miR156 gene family are mutated, wherein N is more than or equal to 2 (preferably, N is more than or equal to 3, more preferably, N is 4-10, more preferably, N is 4-8, more preferably, N is 5-6), wherein the miR156 gene family comprises class I miR156 s;
(ii) the grain (grain) of the crop is harvested.
In another preferred embodiment, the crop plant is selected from the group consisting of graminaceous, leguminous and cruciferous plants, more preferably rice, corn, sorghum, wheat, or soybean.
In a seventh aspect, the present invention provides a method for modulating a trait in a plant, comprising the steps of: reducing the expression or activity of N members of a miR156 gene family in a plant, wherein N is more than or equal to 2 (preferably N is more than or equal to 3, more preferably N is 4-10, more preferably N is 4-8, more preferably N is 5-6), and the miR156 gene family comprises class I miR156 s.
In another preferred embodiment, the trait is selected from the group consisting of: plant type, thousand kernel weight, kernel type, kernel length, or combinations thereof.
In another preferred embodiment, said modulating a plant trait is selected from the group consisting of: reducing or increasing tillering number, increasing kernel length, increasing thousand kernel weight, increasing or decreasing plant height, increasing or decreasing stem diameter, or a combination thereof.
In another preferred example, the method comprises the step of mutating the miR156 in the plant or adding a miR156 inhibitor.
In another preferred example, the inhibitor of miR156 is selected from the group consisting of: a small molecule compound, an antisense nucleic acid, a microRNA, a siRNA, an RNAi, a Crispr agent, or a combination thereof.
In another aspect of the invention, a method for reducing expression of miR156 is provided, comprising the step of adding an inhibitor of miR156 and allowing the inhibitor to bind to one or more genes in the miR156 family.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the MIR156 gene editing strategy. (A) Editing a single sgRNA expression vector of MIR156 gene. Arrows indicate promoters; the rectangular box after the arrow represents the sgRNA expression sequence; #, the PAM sequence used by the target site is CAG;
FIG. 2 shows that mutation of class I MIR156 gene alters rice plant type. (A) Maturity, plant types of wild type (NIP), mir156fg, mir156deghi and mir156defghi were compared. Scale, 10 cm. (B) At the grain filling stage, tillering numbers of wild type (NIP), mir156fg, mir156dehi, mir156deghi and mir156defghi were compared. (C) Maturity, plant height comparison of wild type (NIP), mir156fg, mir156dehi, mir156deghi and mir156 defghi. (D) In the mature period, the diameters of second-knot stems of a wild type (NIP), mir156fg, mir156dehi, mir156deghi and mir156defghi are compared. The second section refers to the second section from the top. (E) Maturity, phenotype comparison of wild type (XS134), mir156fg, mir156dehi and mir156 defghi. Scale, 10 cm. (F) At the grain filling stage, tillering numbers of wild type (XS134), mir156fg, mir156dehi and mir156defghi were compared. P value different from wild type less than 0.05; p value different from wild type less than 0.001. NIP, wild-type nipponbare; XS134, wild type xishui 134.
FIG. 3 shows that mutation of class I MIR156 gene increases seed length and thousand kernel weight. (A) Grain type comparisons of wild type (NIP), mir156fg and mir156 defghi. Scale, 2 cm. (B) Seed length comparison of wild type (NIP) and mir156 mutants. (C) Seed width comparisons between wild type (NIP) and mir156 mutants. (D) Wild type (NIP) and mir156 mutant seed grain thickness were compared. (E) Thousand kernel weight of wild type (NIP) and mir156 mutant seeds were compared. (F) Grain type comparisons of wild type (XS134), mir156fg and mir156 dehi. Scale, 2 cm. (G) Seed length comparisons of wild type (XS134) and mir156 mutants. (H) Seed width comparisons of wild type (XS134) and mir156 mutants. (I) Wild type (XS134) and mir156 mutant seed grain thickness were compared. (J) Thousand kernel weight of wild type (XS134) and mir156 mutant seeds were compared. P value different from wild type less than 0.001. NIP, wild-type nipponbare; XS134, wild type xishui 134.
FIG. 4 shows a plant type analysis of the type II mir156 mutants and mir156 abcdfghikl. (A) Maturity, phenotype comparison of wild type (NIP), mir156abc and mir156 abckl. Scale, 10 cm. (B) Maturity, phenotype comparison of wild type (XS134), mir156abc and mir156 abckl. Scale, 10 cm. (C) At the grain filling stage, tillering numbers of wild type (NIP) and mir156abckl were compared. (D) Tillering numbers of wild type (XS134) and mir156abckl were compared. (E) Maturity, phenotype comparison of wild type (NIP) and mir156 abcdfghikl. Scale, 10 cm. (F) At the grain filling stage, tillering numbers of wild type (NIP), mir156dehi, mir156defghi and mir156abcdfghikl were compared. P value different from wild type less than 0.001. NIP, wild-type nipponbare; XS134, wild type xishui 134.
Detailed Description
The inventor of the present invention has conducted extensive and intensive studies, and unexpectedly found for the first time that knocking out N members (N is 2 or more, preferably 3 or more, more preferably 4 to 10 or more, more preferably 4 to 8 or more, and more preferably 5 to 6) of a miR156 gene family (such as class I miR156s) can significantly improve certain traits of plants, such as reduction of tillering number, increase of kernel length, increase of thousand kernel weight, increase of plant height, increase of stem diameter, and the like. Specifically, the invention firstly utilizes a gene knockout technology (such as a CRISPR/Cas9 gene editing technology) to carry out knockout research on the rice miR156s gene, and discovers that certain required agronomic traits can be improved by knocking out members of different rice miR156s gene families, wherein mutants such as miR156dehi, miR156deghi, miR156defghi, miR156defgh, miR156defgi, miR156dfgi, miR156efgi, miR156efgh, miR156eghi, miR156efg, miR156fgi, miR156ghi, miR156ei, miR156fg and miR156defij can reduce tiller number, increase kernel length, increase thousand kernel weight, increase plant height, increase stem diameter and the like. Moreover, the applicant firstly discovers that the simultaneous knockout of the class I miR156s and the class II miR156s can also obviously improve certain traits of the plant, such as reduction of tillering number, increase of kernel length, increase of thousand kernel weight, increase of plant height, increase of stem diameter and the like. On this basis, the present inventors have completed the present invention.
As used herein, the terms "class I MIR156 s", "class II MIR156 s" refer to a subtype of the MIR156 gene family.
miR156 gene
miRNA is a non-coding RNA molecule consisting of 21-24 nucleotides. They originate from a single-stranded RNA precursor gene, are transcribed by RNA polymerase II to form a stem-loop structure, and are finally cleaved and processed to form a mature miRNA. The correct spatiotemporal accumulation of some highly conserved mirnas is essential to maintain normal plant development, such as miR 156. The target gene of miR156 is a family called SPL (SQUAMOSA promoter binding protein-like). The SPL protein family is a plant-specific family of transcription factors with highly conserved DNA binding domains, the SBP domains.
There are 11 miR156 expression genes (MIR156 a-MIR156 i, MIR156k and MIR156l) in the rice genome, and 12 miR156 precursor sequences can be generated by transcription, wherein the pre-miR156h and the pre-miR156j precursor are from the same gene, and the gene is called MIR156h in the invention.
Existing studies show that the genes of the miR156 gene family have high conservation and are crucial to the growth of plants (especially plant type, thousand kernel weight, kernel type and kernel length). The research of the invention indicates that the modification (such as knockout or down regulation) of the gene in a single miR156 gene family does not show the improvement of plant traits (such as plant type, thousand kernel weight, kernel type and kernel length).
In the present invention, MIR156 gene family members, such as class I MIR156s (including MIR156d, MIR156e, MIR156h, MIR156I, MIR156f, and MIR156g), can regulate plant type and rice grain type, and also have an effect on seed dormancy.
In the invention, the mutation of the class I MIR156 gene reduces the tillering number, increases the length and thousand seed weight of seeds, and increases the plant height and the diameter of stalks. The invention provides important gene resources and a method for improving the plant type and the yield of rice.
In the present invention, the miR156 gene family also includes class II miR156s (including miR156a, miR156b, miR156c, miR156k and miR156 l). The plant traits can also be improved by simultaneously mutating the class I MIR156 gene (i.e. class I MIR156s) and the class II MIR156 gene (i.e. class II MIR156s), for example, the tillering number can be reduced, the length and thousand kernel weight of grains can be increased, and the plant height and the diameter of stalks can be increased. In the present invention, N genes (N.gtoreq.2, preferably N.gtoreq.3, more preferably N is 4 to 10, more preferably 4 to 8, more preferably 5 to 6) of the class I MIR156 gene of any plant species may be knocked out, and representative plants include, but are not limited to: forestry plants, agricultural plants, such as gramineae, cruciferae, leguminosae, and the like, such as rice, corn, sorghum, wheat, soybean, or combinations thereof.
It is understood that different plants may contain multiple class I MIR156 genes (i.e., multiple genes from class I MIR156 genes). In the present invention, the class I MIR156 gene includes all known MIR156 genes from the plant (or species), and MIR156 genes that may be found in the future, as well as homologous genes having homology to these MIR156 genes. Wherein, the expression "having homology" means that two sequences have a homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more.
In other plants, the common name for a homologous gene to the class I MIR156 gene is the MIR156 gene, and the species latin name abbreviation may also be added to the MIR156 gene name. For example, the wheat MIR156 gene is also known as Tae-MIR 156; the maize MIR156 gene is also known as Zea-MIR 156; the soybean MIR156 gene is also called Soy-miR 156.
For example, in rice, at least 6 class I MIR156 genes are known, namely MIR156d, MIR156e, MIR156h, MIR156I, MIR156f and MIR156 g. In the present invention, two or more MIR156 genes may be combined, for example, "MIR 156 fg" means MIR156f and MIR156 g.
In a preferred embodiment, the type of rice class I MIR156 gene knockout is as follows: mir156dehi, mir156deghi, mir156defghi, mir156defgh, mir156defgi, mir156dfgi, mir156efgi, mir156efgh, mir156eghi, mir156efg, mir156fgi, mir156ghi, mir156ei, mir156fg, mir156 defij.
Genetic engineering
In the present invention, the genetic engineering refers to a technology for obtaining new genetic characteristics, or new species, or new products by modifying and utilizing nucleotides for controlling biological genetic information by means of manual intervention, including all genetic modification technologies disclosed in the art, such as methods of gene mutagenesis, transgenosis, or gene editing. Methods for gene mutagenesis include, but are not limited to, physical mutagenesis (e.g., ultraviolet mutagenesis), chemical mutagenesis (e.g., acridine dyes), biological mutagenesis (e.g., viral, phage mutagenesis), and the like. In a preferred embodiment, the genetic engineering of the invention comprises gene editing of a member of the miR156 gene family with one or more sgRNA-mediated Cas9 nuclease.
Method for improving plants
In the present invention, there is also provided a method of improving a plant, comprising the steps of:
(i) genetically engineering plant cells or plant tissues to mutate N members of a miR156 gene family, wherein N is more than or equal to 2 (preferably N is more than or equal to 3, more preferably N is 4-10, more preferably N is 4-8, more preferably N is 5-6), and the miR156 gene family comprises class I miR156 s;
(ii) regenerating the genetically engineered plant cells or plant tissues into plants, and testing the regenerated plants for traits selected from the group consisting of: plant type, thousand kernel weight, kernel type, kernel length, or combinations thereof;
(iii) and selecting plants with the required character characteristics according to the character test results.
In the present invention, the mutation comprises a decrease in the expression or activity of N members of the miR156 gene family (class I miR156 genes).
In a preferred embodiment, the "decreasing" refers to decreasing the expression or activity of N members of the miR156 gene family satisfies the following condition:
the ratio of A1/A0 is less than or equal to 80 percent, preferably less than or equal to 60 percent, more preferably less than or equal to 40 percent, and most preferably 0 to 30 percent;
wherein, A1 is the expression level or activity of N members in miR156 gene family; a0 is the expression level or activity of N members in the same miR156 gene family in wild type plants of the same type.
In a preferred embodiment, the reduction refers to an expression level E1 of the member of the miR156 gene family in the plant that is 0-80%, preferably 0-60%, more preferably 0-40% of wild type compared to the expression level E0 of the member of the wild type miR156 gene family.
In a preferred embodiment, said reducing the expression or activity of N members of the miR156 gene family in a plant is effected by a means selected from the group consisting of: gene mutation, gene knockout, gene disruption, RNA interference techniques, criprpr techniques, or a combination thereof.
In the present invention, plants with poor traits are excluded according to the results of the trait test.
In the invention, the method further comprises a step (iv) of further screening the plants with the required shape characteristics selected in the step (iii), so as to screen out plants capable of balancing the characteristics of seed dormancy, plant type, thousand kernel weight, kernel type, kernel length and the like, and the comprehensive characteristics of the plants are optimally represented.
Method for producing grain
The invention also provides a method for producing grains, which comprises the following steps:
(i) planting a crop in which N members of the miR156 gene family are mutated, wherein N is more than or equal to 2 (preferably, N is more than or equal to 3, more preferably, N is 4-10, more preferably, N is 4-8, more preferably, N is 5-6), wherein the miR156 gene family comprises class I miR156 s;
(ii) the grain (grain) of the crop is harvested.
The main advantages of the invention include:
(a) the invention discovers for the first time that the knock-out of miR156 gene family (such as I-type miR156 gene) members of different plants (such as rice) can obviously reduce the tillering number, increase the grain length, increase the thousand seed weight, increase the plant height, increase the diameter of a stem and simultaneously enhance the seed dormancy.
(b) The method tests the knockout traits of different plant (such as rice) miR156 gene families (such as I-type miR156 genes) for the first time, and picks out plants with the required trait characteristics (such as reduction of tiller number, increase of seed length, increase of thousand kernel weight, increase of plant height and increase of stem diameter).
(c) The invention discovers for the first time that mir156dehi, mir156deghi, mir156defghi, mir156defgh, mir156defgi, mir156dfgi, mir156efgi, mir156efgh, mir156eghi, mir156efg, mir156fgi, mir156ghi, mir156ei, mir156fg and mir156defij mutants can enhance seed dormancy.
(d) The invention firstly discovers that the I-type miR156s and the II-type miR156s are knocked out simultaneously, and certain traits of plants can be obviously improved, such as reduction of tillering number, increase of grain length, increase of thousand kernel weight, increase of plant height, increase of stem diameter and the like.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: a laboratory manual (New York: Cold Spring Harbor laboratory Press,1989) or a Plant Molecular Biology-laboratory manual (Plant Molecular Biology-A laboratory Manual, catalog S.Clark, Springer-verlag Berlin Heidelberg,1997), or according to the conditions suggested by the manufacturer.
Unless otherwise indicated, percentages and parts are by weight.
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