阅读说明：本技术 来源于新疆野苹果的与植物抗水分胁迫相关蛋白及编码基因的应用 (Protein derived from Malus sieversii and related to water stress resistance of plants and application of coding gene ) 是由 李天红 王彦涛 彭翔 申晓帅 李舰 黄学旺 朱圣娇 于 2020-06-03 设计创作，主要内容包括：本发明公开了来源于新疆野苹果的与植物抗水分胁迫相关蛋白及编码基因的应用。该蛋白质的名称为MsSCL26蛋白,其可为氨基酸序列是序列表中SEQ ID No.1的蛋白质。实验证明,与未转MsSCL26基因的植株相比,MsSCL26过表达转基因植株具有显著的抗水分胁迫性,说明蛋白MsSCL26能够提高植物的抗逆性。(The invention discloses a protein derived from Malus sieversii and related to water stress resistance of plants and application of a coding gene. The protein is named MsSCL26 protein, and can be a protein with an amino acid sequence of SEQ ID No.1 in a sequence table. Experiments prove that compared with plants without transgenic MsSCL26 genes, MsSCL26 overexpression transgenic plants have obvious water stress resistance, and the protein MsSCL26 can improve the stress resistance of plants.)

1. Any of the following uses of the protein:

p1, the application of the protein in regulating and controlling the stress resistance of plants,

p2, the application of the protein in preparing products for improving the stress resistance of plants,

p3, the application of the protein in cultivating stress-resistant plants,

p4, the application of the protein in preparing plant stress resistance products,

p5, use of the protein in plant breeding;

the protein is the protein of A1), A2) or A3) as follows:

A1) the amino acid sequence is protein of SEQ ID No.1 in a sequence table;

A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown by SEQ ID No.1 in the sequence table, is derived from A1) or has the same function with the protein shown by A1), has more than 90 percent of identity and is related to plant stress resistance or water stress resistance;

A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

2. Use according to claim 1, characterized in that: the stress resistance is against moisture stress and/or osmotic stress and/or salt stress and/or drought stress.

3. Use according to claim 1 or 2, characterized in that: the plant is any one of the following plants:

C1) (ii) a monocotyledonous plant which is,

C2) a woody plant, a plant which is a plant of the species,

C3) a dicotyledonous plant, a plant selected from the group consisting of dicotyledonous plants,

C4) a plant of the order Rosales,

C5) a plant of the family Rosaceae,

C6) a plant of the genus Malus,

C7) an apple.

4. Use of a biological material related to a protein according to claim 1, wherein the biological material is selected from the group consisting of:

q1, the application of the biological material in regulating and controlling the stress resistance of plants,

q2, the application of the biological material in the preparation of products for improving the stress resistance of plants,

q3, the application of the biological material in cultivating stress-resistant plants,

q4, the application of the biological material in the preparation of plant stress resistance products,

q5, use of the biomaterial in plant breeding;

the biomaterial is any one of the following B1) to B9):

B1) a nucleic acid molecule encoding the protein of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B1);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);

B8) a nucleic acid molecule that reduces the expression of the protein of claim 1;

B9) an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line comprising the nucleic acid molecule according to B8).

5. Use according to claim 4, characterized in that: B1) the nucleic acid molecule is a coding gene of the protein shown in the following b1) b2) or b 3):

b1) the coding sequence is cDNA molecule or DNA molecule of 1-1542 site nucleotide of SEQ ID No.2 in the sequence table;

b2) the nucleotide is cDNA molecule or DNA molecule of SEQ ID No.2 in the sequence table,

b3) a cDNA or DNA molecule which hybridizes with the cDNA or DNA molecule defined in b2) and encodes a protein having the same function.

6. Use according to claim 4 or 5, characterized in that: the stress resistance is water resistance stress and/or permeation resistance stress and/or salt resistance stress and/or drought resistance stress

And/or the first and/or second light sources,

C1) (ii) a monocotyledonous plant which is,

C2) a woody plant, a plant which is a plant of the species,

C3) a dicotyledonous plant, a plant selected from the group consisting of dicotyledonous plants,

C4) a plant of the order Rosales,

C5) a plant of the family Rosaceae,

C6) a plant of the genus Malus,

C7) an apple.

7. A method for cultivating stress-resistant plants, which comprises increasing the activity of the protein of claim 1 or/and the expression level of the gene coding for the protein of claim 1 in a target plant to obtain stress-resistant plants; the stress resistance of the stress-resistant plant is higher than that of the target plant.

8. Use according to claim 7, characterized in that: the improvement of the activity of the protein of claim 1 in a target plant and/or the expression level of the gene encoding the protein of claim 1 is achieved by introducing the gene encoding the protein of claim 1 into the target plant.

9. The method according to claim 7 or 8, characterized in that: the stress resistance is water resistance stress and/or permeation resistance stress and/or salt resistance stress and/or drought resistance stress,

and/or the first and/or second light sources,

the target plant is any one of the following plants:

C1) a dicotyledonous plant or a monocotyledonous plant,

C2) a woody plant, a plant which is a plant of the species,

C3) a dicotyledonous plant, a plant selected from the group consisting of dicotyledonous plants,

C4) a plant of the order Rosales,

C5) a plant of the family Rosaceae,

C6) a plant of the genus Malus,

C7) an apple.

10. A protein as claimed in claim 1 or a biomaterial as claimed in claim 4 or 5.

Technical Field

The invention relates to the technical field of biology, in particular to a water stress resistance related protein of plants from Malus sieversii and application of a coding gene thereof.

Background

Apples are the first of the four world fruits. By 2017, the total planting area of the apples in China is 3300 ten thousand mu, the total yield is 4139 ten thousand tons, which accounts for more than 50% of the cultivation area and the yield of the apples in the world and is the first in the world (Chinese apple industry development report (2017)). The intensive apple dwarf stock cultivation mode is the development direction of the modern apple industry, and in the popularization process of the apple dwarf stock close planting cultivation mode, dwarf stocks introduced from abroad generally have the problems of poor adaptability, poor stress resistance and the like. Drought stress affects the growth and development of fruit trees, and in severe cases, the trees are short and premature, and leaves fall off too early, so that the yield and quality of apples are reduced. Therefore, the molecular mechanism of apple responding to drought stress is revealed, the superior stress-resistant genes in the traditional arbor stock resources are excavated by utilizing the modern biotechnology, and the improvement and cultivation of the dwarfing stock with strong comprehensive resistance is one of the main targets of the stress-resistant breeding of the apple stock at present.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the stress resistance of plants or how to improve the drought resistance of apples or how to cultivate apple stocks with strong stress resistance.

In order to solve the technical problem, the invention firstly provides any one of the following applications of the protein:

p1, the application of the protein in regulating and controlling the stress resistance of plants,

p2, the application of the protein in preparing products for improving the stress resistance of plants,

p3, the application of the protein in cultivating stress-resistant plants,

p4, the application of the protein in preparing plant stress resistance products,

p5, use of the protein in plant breeding.

The protein is the protein of A1), A2) or A3) as follows:

A1) the amino acid sequence is protein of SEQ ID No.1 in a sequence table;

A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown by SEQ ID No.1 in the sequence table, is derived from A1) or has the same function with the protein shown by A1), has more than 90 percent of identity and is related to plant stress resistance or water stress resistance;

A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

In the protein, SEQ ID No.1 in the sequence table consists of 513 amino acid residues.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

In the above protein, the protein tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

The stress resistance in the above applications may be against moisture stress and/or osmotic stress and/or against salt stress and/or against drought stress.

In the above application, the plant is any one of the following plants:

C1) (ii) a monocotyledonous plant which is,

C2) a woody plant, a plant which is a plant of the species,

C3) a dicotyledonous plant, a plant selected from the group consisting of dicotyledonous plants,

C4) a plant of the order Rosales,

C5) a plant of the family Rosaceae,

C6) a plant of the genus Malus,

C7) an apple.

In order to solve the technical problem, the invention also provides any one of the following applications of the protein-related biological material in the above application:

q1, the application of the biological material in regulating and controlling the stress resistance of plants,

q2, the application of the biological material in the preparation of products for improving the stress resistance of plants,

q3, the application of the biological material in cultivating stress-resistant plants,

q4, the application of the biological material in the preparation of plant stress resistance products,

q5, use of the biomaterial in plant breeding;

the above biomaterial is any one of the following B1) to B9):

B1) nucleic acid molecules encoding the proteins described in the above applications;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B1);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);

B8) nucleic acid molecules which reduce the expression of said proteins in the above-mentioned applications;

B9) an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line comprising the nucleic acid molecule according to B8).

In the above biological material, the nucleic acid molecule of B1) may be a gene encoding the protein as shown in B1) B2) or B3):

b1) the coding sequence (ORF) of the coding strand is a DNA molecule of nucleotides 1 to 1542 of SEQ ID No.2 of the sequence list;

b2) the nucleotide sequence of the coding strand is a DNA molecule of SEQ ID No.2 in the sequence table;

b3) a DNA molecule which hybridizes with the DNA molecule defined in2) under stringent conditions and encodes a protein having the same function.

The above stringent conditions are hybridization and washing of the membrane 2 times 5min at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of the membrane 2 times 15min at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; alternatively, hybridization was carried out at 65 ℃ in a solution of 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS, and the membrane was washed.

In the above biological materials, the expression cassette containing a nucleic acid molecule described in B2) refers to a DNA capable of expressing the protein described in the above application in a host cell, and the DNA may include not only a promoter for initiating transcription of the gene encoding the protein but also a terminator for terminating transcription of the gene encoding the protein. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter of cauliflower mosaic virus 35S; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiology 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with jasmonic acid ester); heat shock promoters (U.S. Pat. No. 5,187)267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al (1985) EMBO J.4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (I)⁹⁸⁵) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (1991) Cell,64: 671; sanfacon et al GenesDev.,5: 141; mogen et al (1990) Plant Cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627).

The recombinant expression vector containing the protein coding gene expression cassette can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Corp.) and the like. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), marker genes for antibiotics which are expressible in plants (e.g., nptII gene which confers resistance to kanamycin and related antibiotics, bar gene which confers resistance to phosphinothricin which is a herbicide, hph gene which confers resistance to hygromycin which is an antibiotic, dhS gene which confers resistance to methatrexate, EPSPS gene which confers resistance to glyphosate), or marker genes for chemical resistance (e.g., herbicide resistance), mannose-6-phosphate isomerase gene which provides the ability to metabolize mannose, etc. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above biological material, the recombinant microorganism may be specifically yeast, bacteria, algae and fungi.

In the above application, the plant is any one of the following plants:

C1) (ii) a monocotyledonous plant which is,

C2) a woody plant, a plant which is a plant of the species,

C3) a dicotyledonous plant, a plant selected from the group consisting of dicotyledonous plants,

C4) a plant of the order Rosales,

C5) a plant of the family Rosaceae,

C6) a plant of the genus Malus,

C7) an apple.

Both the plant stress resistance enhancing product and the plant stress resistance product may be a plant stress resistance agent as described above. The plant stress tolerance agent may contain the protein or/and the biological material.

In order to solve the technical problems, the invention also provides a method for cultivating stress-resistant plants.

The method for cultivating the stress-resistant plant comprises the steps of improving the expression quantity of the protein or the coding gene thereof in a target plant to obtain the stress-resistant plant; the stress resistance of the stress-resistant plant is higher than that of the target plant.

In the above method, the increase in the expression level of the protein or the gene encoding the protein in the target plant can be achieved by introducing the gene encoding the protein into the target plant.

In the method, the coding gene of the protein can be modified as follows and then introduced into a target plant to achieve better expression effect:

1) modifying the sequence of the gene adjacent to the initiating methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;

2) linking with promoters expressed by various plants to facilitate the expression of the promoters in the plants; such promoters may include constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; for example, tissue or organ specific expression promoters, depending on the stage of development of the desired receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, desirably, dicot promoters are selected for expression in dicots and monocot promoters for expression in monocots;

3) the expression efficiency of the gene of the present invention can also be improved by linking to a suitable transcription terminator; tml from CaMV, E9 from rbcS; any available terminator which is known to function in plants may be linked to the gene of the invention;

4) enhancer sequences, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV) were introduced.

The gene encoding the protein can be introduced into Plant cells by conventional biotechnological methods using Ti plasmids, Plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. (Weissbach,1998, Method for Plant Molecular Biology VIII, academic Press, New York, pp.411-463; Geiserson and Corey,1998, Plant Molecular Biology (2nd Edition).

In the method, the stress-resistant plant can be a transgenic plant or a plant obtained by conventional breeding technologies such as hybridization and the like.

In the above methods, the transgenic plant is understood to include not only the first to second generation transgenic plants but also the progeny thereof. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

As mentioned above, the stress resistance may be against moisture stress and/or osmotic stress and/or salt stress and/or drought stress, or/and the plant of interest is any of the following:

C1) a dicotyledonous plant or a monocotyledonous plant,

C2) a woody plant, a plant which is a plant of the species,

C3) a dicotyledonous plant, a plant selected from the group consisting of dicotyledonous plants,

C4) a plant of the order Rosales,

C5) a plant of the family Rosaceae,

C6) a plant of the genus Malus,

C7) an apple.

The MsSCL26 gene from Malus sieversii is introduced into tissue culture seedling GL-3' of apple to obtain a transgenic plant over-expressing the MsSCL26 gene; compared with the untransformed apple tissue culture seedling 'GL-3' control plant, the overexpression of the MsSCL26 gene improves the tolerance of the transgenic plant 'GL-3' to adversity stress such as water stress. The protein MsSCL26 participates in regulation and control and adaptation of plants to water stress-related adversity stress, can improve apple drought resistance by utilizing the protein, and has important significance in cultivation of apple stocks with strong stress resistance.

Drawings

Figure 1 is a graph of the change in expression levels of MsSCL26 under 20% PEG 6000 treatment.

FIG. 2 shows the restriction enzyme digestion verification of pMDC83-MsSCL26 recombinant plasmid and the PCR identification of transgenic plant 'GL-3'. The left picture is enzyme digestion verification of pMDC83-MsSCL26 recombinant plasmid, lane M is Marker, lane 1 is pMDC83 empty vector without double enzyme digestion, lanes 2 and 4 are pMDC83-MsSCL26 recombinant plasmid after endonuclease Pac I/Kpn I digestion, lane 3 is pMDC83-MsSCL26 recombinant plasmid without double enzyme digestion; the right panel shows PCR identification of transgenic plants 'GL-3', lane M is Marker, and 1-8 are 8 PCR positive plants respectively.

FIG. 3 is a graph showing the expression level of SCL26 in transgenic ` GL-3 ` lines.

FIG. 4 shows the identification of resistance and the detection of physiological indexes of various strains in a culture medium.

FIG. 5 is the drought resistance identification of each strain in soil.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

The expression vectors used in the examples below, the biological material which was only used for repeating the experiments relevant to the present invention and which was not used for other purposes, were publicly available from the applicant.