CN117264964A - Application of wheat TaGSKB protein and encoding gene thereof in regulation and control of plant stress tolerance - Google Patents

Abstract

The invention discloses application of wheat TaGSKB protein and a coding gene thereof in regulating and controlling plant stress tolerance. The invention specifically discloses a protein with an amino acid sequence of SEQ ID No.1 and application of a coding gene thereof in regulating and controlling plant stress resistance. According to the invention, the TaGSKB gene from wheat is introduced into a receptor plant to obtain a homozygous plant transformed with the TaGSKB gene. Experiments prove that compared with a non-transgenic receptor control, under drought stress and salt stress conditions, transgenic arabidopsis and wheat show stronger tolerance, the survival rate is obviously higher than that of the receptor control, and the growth vigor is obviously better than that of the receptor control, so that the over-expression of the TaGSKB gene can obviously improve the drought stress resistance and the salt tolerance of plants (namely, the stress resistance of the plants is improved). The gene for regulating and controlling plant stress resistance has important significance and application value for cultivating new stress-resistant varieties of plants.

Description

Application of wheat TaGSKB protein and encoding gene thereof in regulation and control of plant stress tolerance

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of wheat TaGSKB protein and a coding gene thereof in regulating and controlling plant stress tolerance.

Background

Plants inevitably suffer from various stresses during the growth and development process, such as drought, salt and alkali, high temperature, low temperature, and nutrient deficiency. Abiotic stress limits the use of cultivated land worldwide, and also seriously affects the yield and quality of crops, which is an important adversity factor for restricting agricultural development. Abiotic hypochondrium forces plants to produce various lesions: disruption of the cell membrane system, dehydration of the cells, and influence of enzyme activity, thereby causing a disturbance of the growth metabolism of the plant. Abiotic stress affects many aspects of plant physiology and causes extensive changes in cellular processes, and during long-term evolution, plants form a complex regulatory network in response to different stress signal stimuli to resist adversity stress: firstly, the plant senses an external stress signal, transmits the stress signal, and regulates and controls at the cellular and molecular level, so that the plant can generate an adaptive reaction to resist the stress, and the plant survives.

Wheat (Triticum aestivum l.) is one of the most important cereal crops in the world, being the main staple food crop for humans, however abiotic stress severely constrains the production of wheat. Improving stress resistance of wheat is an effective way for guaranteeing sustainable production of wheat and is also a potential target for crop improvement. Therefore, the development of the wheat stress resistance related genes, the research of functions and the improvement of the wheat stress resistance by utilizing molecular breeding have important economic and social significance for cultivating the wheat stress-resistant varieties and ensuring the high and stable yield of wheat.

In the process of plant resistance to adversity stress, synergism of multiple genes is required, while proteins are the final executors of cellular activities and functions, controlling and regulating many vital activities in cells. Proteins typically do not function independently in cells, they typically interact with other proteins to perform specific functions in specific times and spaces. Protein interactions are fundamental physiological interactions in cells, constituting the whole signal network within plants. Meanwhile, protein interaction is also a bridge of various important physiological processes such as replication, transcription, translation of intracellular genes, cell cycle regulation, signal transduction, immune response and the like, and many researches indicate that the protein interaction regulates the adversity stress response of plants.

Disclosure of Invention

The technical problem to be solved by the invention is how to regulate and control the stress resistance (namely stress tolerance) of plants. The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.

To solve the above technical problems, the present invention provides first an application of a protein or a substance regulating the activity and/or content of the protein, wherein the application may be any of the following:

D1 Use of a protein or a substance regulating the activity and/or content of said protein for regulating stress resistance of a plant;

d2 Use of a protein or a substance regulating the activity and/or content of said protein for the preparation of a product regulating stress resistance of a plant;

d3 Use of a protein or a substance regulating the activity and/or content of said protein for growing stress-tolerant plants;

d4 Use of a protein or a substance regulating the activity and/or content of said protein for the preparation of a product for growing stress-tolerant plants;

d5 Use of a protein or a substance regulating the activity and/or content of said protein in plant breeding;

the protein is named TaGSKB and can be any one of the following:

a1 A protein having an amino acid sequence of SEQ ID No. 1;

a2 A protein which is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence shown in SEQ ID No.1, has more than 80% of identity with the protein shown in A1) and has the same function;

a3 Fusion proteins having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of A1) or A2).

In order to facilitate purification or detection of the protein of A1), a tag protein may be attached to the amino-or carboxy-terminus of the protein consisting of the amino acid sequence shown in SEQ ID No.1 of the sequence Listing.

Such tag proteins include, but are not limited to: GST (glutathione-sulfhydryl transferase) tag protein, his6 tag protein (His-tag), MBP (maltose binding protein) tag protein, flag tag protein, SUMO tag protein, HA tag protein, myc tag protein, eGFP (enhanced green fluorescent protein), eFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

The nucleotide sequence encoding the protein TaGSKB of the present invention can be easily mutated by a person skilled in the art using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the protein TaGSKB isolated by the present invention are all derived from and are equivalent to the nucleotide sequence of the present invention as long as they encode the protein TaGSKB and have the function of the protein TaGSKB.

The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

Herein, identity refers to identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and search is performed to calculate the identity of amino acid sequences, and then the value (%) of identity can be obtained.

Herein, the 80% identity or more may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

Herein, the substance regulating the activity and/or content of the protein may be a substance regulating the expression of a gene encoding the protein TaGSKB.

In the above, the substance that regulates gene expression may be a substance that performs at least one of the following 6 regulation: 1) Regulation at the level of transcription of said gene; 2) Regulation after transcription of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) Regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); 4) Regulation of translation of the gene; 5) Regulation of mRNA degradation of the gene; 6) Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated by the gene).

The substance for regulating the expression of the genes can be specifically the biological material of any one of B1) to B3).

In the above application, the protein may be derived from wheat (Triticum aestivum l.).

The invention also provides application of the biological material related to the protein TaGSKB, wherein the application can be any one of the following:

e1 The application of the biological material related to the protein TaGSKB in regulating and controlling plant stress resistance;

e2 The application of the biological material related to the protein TaGSKB in preparing a product for regulating and controlling plant stress resistance;

e3 The application of the biological material related to the protein TaGSKB in cultivating stress-resistant plants;

e4 The application of the biological material related to the protein TaGSKB in the preparation of a product for cultivating stress-resistant plants;

e5 Use of biological material related to said protein tagsky in plant breeding;

the biomaterial may be any one of the following B1) to B7):

b1 Nucleic acid molecules encoding said protein TaGSKB;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

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

b6 A transgenic plant tissue comprising the nucleic acid molecule of B1) or a transgenic plant tissue comprising the expression cassette of B2);

b7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2).

In the above application, the nucleic acid molecule of B1) may be any of the following:

c1 A DNA molecule whose coding sequence is SEQ ID No. 2;

c2 A DNA molecule with the nucleotide sequence of SEQ ID No. 2.

The coding amino acid sequence of the DNA molecule shown in SEQ ID No.2 (TaGSKB gene for regulating plant stress resistance) is protein TaGSKB shown in SEQ ID No. 1.

The nucleotide sequence shown in SEQ ID NO.2 is the nucleotide sequence of a protein TaGSKB coding gene (CDS). The protein TaGSKB gene (TaGSKB gene) can be any nucleotide sequence capable of encoding the protein TaGSKB. In view of the degeneracy of codons and the preferences of codons of different species, one skilled in the art can use codons appropriate for expression of a particular species as desired.

B1 The nucleic acid molecules may also comprise nucleic acid molecules which have been modified by codon preference on the basis of the nucleotide sequence indicated in SEQ ID No. 2.

The nucleic acid molecules also comprise nucleic acid molecules which have more than 95% nucleotide sequence identity with the nucleotide sequence shown in SEQ ID No.2 and are derived from the same species.

The nucleic acid molecule described herein may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be an RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

Vectors described herein are well known to those of skill in the art and include, but are not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), ti plasmids, or viral vectors. Specifically, the vector pCAMBIA1302, the Peasybunt vector or the vector pWMB110 can be used.

The recombinant expression vector containing the TaGSKB gene can be constructed by using the existing plant expression vector. Such plant expression vectors include, but are not limited to, vectors such as binary Agrobacterium vectors and vectors useful for microprojectile bombardment of plants, and the like. The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to untranslated regions transcribed from the 3' end of plant genes including, but not limited to, agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase Nos genes), plant genes (e.g., soybean storage protein genes).

When the TaGSKB gene is used for constructing a recombinant plant expression vector, any one of enhanced promoters or constitutive promoters can be added before transcription initiation nucleotide of the recombinant plant expression vector, and the enhanced promoters include, but are not limited to, a cauliflower mosaic virus (CaMV) 35S promoter and a ubiquitin promoter (ubiquitin) of corn, which can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a plant expression vector, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In order to facilitate the identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, such as by adding genes encoding enzymes or luminescent compounds that produce a color change (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin markers, kanamycin markers, etc.), or anti-chemical marker genes (e.g., anti-herbicide genes), etc., which may be expressed in plants. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

The TaGSKB gene or the fragment of the gene provided by the invention is introduced into plant cells or receptor plants by using any vector capable of guiding exogenous gene to express in plants, so that a transgenic cell line with improved stress resistance and a transgenic plant can be obtained. Expression vectors carrying the TaGSKB gene can be used to transform plant cells or tissues by conventional biological methods such as Ti plasmid, ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, agrobacterium mediation, and the like, and the transformed plant tissues are cultivated into plants.

The microorganism described herein may be a yeast, bacterium, algae or fungus. Wherein the bacteria may be derived from Escherichia, erwinia, agrobacterium (Agrobacterium), flavobacterium (Flavobacterium), alcaligenes (Alcaligenes), pseudomonas, bacillus (Bacillus), etc. Specifically, the cells may be E.coli competent cells TOP10 and/or Agrobacterium tumefaciens GV3101.

The recombinant vector can be specifically Peasybunt-TaGSKB, pCAMBIA1302-TaGSKB and/or pWMB110-TaGSKB.

The recombinant vector peasyblock-TaGSKB is a recombinant vector obtained by connecting a DNA fragment with a nucleotide sequence of SEQ ID No.2 in a sequence table to a peasyblock vector by using a blunt end cloning method and keeping other sequences of the peasyblock vector unchanged.

The recombinant plasmid pCAMBIA1302-TaGSKB is a DNA molecule shown in SEQ ID No.2 inserted into the NcoI site of the vector pCAMBIA 1302.

The recombinant vector pWMB110-TaGSKB is a DNA molecule shown in SEQ ID No.2 inserted into the BamHI enzyme cutting site of the vector pWMB 110.

The recombinant microorganism can be GV3101/pCAMBIA1302-TaGSKB and/or EHA105/pWMB110-TaGSKB.

The recombinant agrobacterium GV3101/pCAMBIA1302-TaGSKB contains a TaGSKB gene with a coding sequence of SEQ ID No.2, and is a recombinant microorganism obtained by introducing the recombinant vector pCAMBIA1302-TaGSKB into agrobacterium GV 3101.

The recombinant agrobacterium EHA105/pWMB110-TaGSKB contains a TaGSKB gene with a coding sequence of SEQ ID No.2, and is a recombinant microorganism obtained by introducing the recombinant vector pWMB110-TaGSKB into the agrobacterium EHA 105.

The invention also provides a method for cultivating the stress-resistant plant, which comprises the step of improving the content and/or activity of the protein TaGSKB in the target plant to obtain the stress-resistant plant with the stress resistance higher than that of the target plant.

In the above method, the increase in the content and/or activity of the protein TaGSKB in the target plant is achieved by increasing the expression level of the gene encoding the protein TaGSKB in the target plant.

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

In the above method, the gene encoding the protein may be any of the following:

f1 A DNA molecule whose coding sequence is SEQ ID No. 2;

f2 A DNA molecule with the nucleotide sequence of SEQ ID No. 2.

Specifically, in one embodiment of the present invention, the increase in the expression level of the gene encoding the protein in the plant of interest is achieved by introducing a DNA molecule shown in SEQ ID No.2 into the plant of interest.

In one embodiment of the invention, the method of growing stress-tolerant plants comprises the steps of:

(1) Constructing a recombinant vector containing a DNA molecule shown in SEQ ID NO. 2;

(2) Transferring the recombinant vector constructed in the step (1) into a target plant (such as wheat);

(3) And screening and identifying to obtain the stress-resistant plant with stress resistance higher than that of the target plant.

In the above method, the plant may be G1) or G2):

g1 Monocotyledonous or dicotyledonous plants;

g2 Gramineous or cruciferous plants.

Herein, the plant may be a crop (crop plant).

Further, the plant described herein may specifically be arabidopsis thaliana or wheat.

The invention also provides application of the protein TaGSKB, or the biological material, or the nucleic acid molecule, or any of the methods for cultivating stress-tolerant plants in the creation of stress-tolerant plants and/or plant breeding.

The protein TaGSKB, the biological material and the nucleic acid molecule are all within the scope of the invention.

The protein TaGSKB described herein may be a wheat stress resistance related protein.

Plant breeding as described herein may be stress tolerant breeding of crops.

The regulation of plant stress resistance may be an increase in plant stress resistance or a decrease in plant stress resistance.

The expression quantity and/or activity of the TaGSKB protein or the encoding gene thereof in the target plant are improved, and the stress resistance of the plant is correspondingly improved.

Further, the stress resistance improvement is represented by:

(1) The survival rate of the target plant is improved;

(2) The chlorophyll content of the target plant is improved;

(3) The proline content of the target plant is improved;

(4) Reducing malondialdehyde content of target plant.

The stress resistance includes, but is not limited to, salt resistance, drought resistance, heat resistance, cold resistance, and/or light resistance.

In particular, the stress resistance (stress tolerance) may specifically be salt tolerance and/or drought resistance.

In the present invention, the stress-tolerant plant is understood to include not only the first generation transgenic plant obtained by transforming the target plant with the TaGSKB gene, but also the progeny thereof. The gene may be propagated in that species, or may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The stress-resistant plants include seeds, calli, whole plants and cells.

The present invention provides homozygous Arabidopsis lines (# 3 lines, #5 lines, #8 lines) and homozygous wheat lines (OE-1 lines, OE-2 lines, OE-4 lines) transformed with TaGSKB genes by introducing the TaGSKB genes, which regulate plant stress resistance, from wheat (Triticum aestivum L.), into acceptor control plants, wild-type Arabidopsis (WT, col) and wild-type wheat (Fielder), respectively. Experiments prove that under the conditions of drought stress and salt stress, the survival rate and the germination rate of transgenic arabidopsis thaliana and wheat which overexpress the TaGSKB gene are obviously higher than those of the receptor control, the growth vigor is obviously better than that of the receptor control, the over-expression of the TaGSKB gene can obviously improve the drought stress resistance and the salt tolerance of plants (namely improve the stress resistance of plants), the survival rate of the transgenic arabidopsis thaliana and the wheat which are particularly expressed by the over-expression of the TaGSKB gene is obviously improved, the germination capacity of seeds is obviously improved, the chlorophyll content and the proline content are obviously improved, the catalase activity is increased, and the malonaldehyde and H are also obviously improved ₂ O ₂ The content is obviously reduced, the relative conductivity is reduced, and each physiological index is obviously better than the receptor contrast. Therefore, under drought stress and high salt stress conditions, arabidopsis thaliana and wheat over-expressing TaGSKB genes show stronger tolerance, The TaGSKB protein and the coding gene thereof disclosed by the invention can regulate and control the stress resistance (such as drought resistance and/or salt tolerance) of plants, and the stress resistance of target plants can be obviously improved by improving the content and/or activity (such as over-expression of the TaGSKB gene) of the TaGSKB protein in the target plants.

The invention digs a new gene capable of regulating and controlling the stress resistance of plants under drought stress and salt stress conditions, and has important significance and application value for cultivating new stress-resistant varieties of plants and improving the stress resistance of the plants.

Drawings

FIG. 1 shows analysis of expression patterns of TaGSKB gene under different abiotic stresses.

FIG. 2 shows drought resistance analysis of wild-type and TaGSKB-transgenic Arabidopsis thaliana. Fig. 2 a: germination experiments of wild-type and transgenic arabidopsis under normal and PEG treatment; b in fig. 2: germination rate statistics of wild and transgenic arabidopsis under normal and PEG treatment; c in fig. 2: phenotypic analysis of wild-type and transgenic arabidopsis under normal and drought treatment; d in fig. 2: survival of wild-type and transgenic arabidopsis under normal and drought treatment. #3, #5 and #8 represent three TaGSKB-3, taGSKB-5 and TaGSKB-8 transgenic lines, respectively. * Sum indicates that the difference reached significant levels (P <0.01 and P < 0.05).

FIG. 3 shows salt tolerance analysis of wild type and TaGSKB transgenic Arabidopsis thaliana. Fig. 3 a: germination experiments of wild-type and transgenic arabidopsis thaliana under normal and NaCl treatment; b in fig. 3: germination rate statistics of wild and transgenic arabidopsis under normal and NaCl treatment; c in fig. 3: phenotypic analysis of wild-type and transgenic arabidopsis under normal and high salt treatment; fig. 3D: survival of wild-type and transgenic arabidopsis under normal and high salt treatment. #3, #5 and #8 represent three TaGSKB-3, taGSKB-5 and TaGSKB-8 transgenic lines, respectively. * Sum indicates that the difference reached significant levels (P <0.01 and P < 0.05).

FIG. 4 shows mannitol stress analysis of the receptor and TaGSKB gene-transferred wheat. Fig. 4 a: hydroponic experiments of receptor and transgenic wheat under normal and mannitol treatment; B-E in fig. 4: proline, malondialdehyde in receptor and transgenic wheat under normal and mannitol treatment、H ₂ O ₂ Content and catalase activity analysis; f in fig. 4: seedling stage phenotyping of the recipients and transgenic wheat under normal and mannitol treatment; g in fig. 4: NBT staining of recipients and transgenic wheat under normal and mannitol treatment; h in fig. 4: o of receptor and transgenic wheat under normal and mannitol treatment ^2- And (5) content analysis. OE-1, OE-2 and OE-4 represent three transgenic lines of TaGSKB-1, taGSKB-2 and TaGSKB-4, respectively. * Sum indicates that the difference reaches a significant level (p<0.01 and P<0.05)。

FIG. 5 shows drought resistance analysis of the recipients and TaGSKB gene-transferred wheat. Fig. 5 a: soil culture experiments of the receptor and transgenic wheat under normal and drought treatment; B-D in fig. 5: survival, loss and relative moisture content of the recipients and transgenic wheat under normal and drought treatment were analyzed. OE-1, OE-2 and OE-4 represent three transgenic lines of TaGSKB-1, taGSKB-2 and TaGSKB-4, respectively. * Sum indicates that the difference reached significant levels (P <0.01 and P < 0.05).

FIG. 6 shows the salt tolerance analysis of the receptor and TaGSKB gene-transferred wheat. Fig. 6 a: hydroponic experiments of recipients and transgenic wheat under normal and high salt treatment; fig. 6B: seedling stage phenotyping of recipients and transgenic wheat under normal and high salt treatment; C-D in FIG. 6: proline and malondialdehyde content analysis of normal and high salt treated recipients and transgenic wheat. OE-1, OE-2 and OE-4 represent three transgenic lines of TaGSKB-1, taGSKB-2 and TaGSKB-4, respectively. * Sum indicates that the difference reached significant levels (P <0.01 and P < 0.05).

FIG. 7 shows the salt tolerance analysis of the receptor and TaGSKB gene-transferred wheat. Fig. 7 a: soil culture experiments of recipients and transgenic wheat under normal and high salt treatment; B-D in fig. 7: survival, chlorophyll content and relative conductivity analysis of normal and high salt treated recipients and transgenic wheat. OE-1, OE-2 and OE-4 represent three transgenic lines of TaGSKB-1, taGSKB-2 and TaGSKB-4, respectively. * Sum indicates that the difference reached significant levels (P <0.01 and P < 0.05).

FIG. 8 is an expression analysis of abiotic stress related genes in recipient and TaGSKB gene-transferred wheat.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The quantitative tests in the following examples were all set up in triplicate and the results averaged. In the following examples, the percentages are by mass unless otherwise indicated.

The wheat variety "small Bai Mai" in the following examples is described in the following literature: ru JN, hou ZH, zheng L, zhao Q, wang FZ, chen J, zhou YB, chen M, ma YZ, xi YJ and Xu ZS. (2021) Genome-Wide Analysis of DEAD-box RNA Helicase Family in Wheat (Triticum aestivum) and Functional Identification of TaDEAD-box57 in Abiotic Stress responses. The "xiaobiaimai" in this document is the "small Bai Mai" of the present invention.

The wheat variety "Fielder" in the examples below is described in the following literature: cui XY, gao Y, guo J, yu TF, zheng WJ, liu YW, chen J, xu ZS, ma YZ.BES/BZR Transcription Factor TaBZR2Positively Regulates Drought Responses by Activation of TaGST1.Plant physiology.doi 10.1104/pp.19.00100.

The pEASY-Blunt Blunt end cloning vector in the following examples is a Beijing all gold Biotechnology product.

Coli competent TOP10 in the examples described below was a product of Beijing Bomaide Gene technology Co.

The agrobacterium tumefaciens GV3101 and EHA105 in the following examples are products of the allied biology company in beijing.

The wild type Arabidopsis thaliana (WT, col) in the examples below was Columbia ecological Arabidopsis thaliana (Col-0), available from SALK company.

The wheat Fielder and plant binary expression vector pWMB110 in the examples below is supplied by the national academy of agricultural sciences She Xingguo teacher laboratory. Plant eukaryotic expression vector pWMB110 vector is described in the following literature: cui XY, gao Y, guo J, yu TF, zheng WJ, liu YW, chen J, xu ZS, ma YZ.BES/BZR Transcription Factor TaBZR2Positively Regulates Drought Responses by Activation of TaGST1.Plant physiology.doi 10.1104/pp.19.00100.

The following examples were run using GraphPad Prism statistical software and the experimental results were expressed as mean ± standard deviation using ANOVA test, P < 0.05 (x) indicated significant differences and P < 0.01 (x) indicated very significant differences.

Example 1, taGSKB protein and production of the coding Gene thereof

The total RNA of wheat (small Bai Mai) which grows normally for 1 week is extracted by using a plant total RNA extraction kit of Tiangen company, and the first-strand cDNA is synthesized by using a first-strand cDNA synthesis kit of full-scale gold company. The correct TaGSKB gene is obtained by taking wheat cDNA as a template, carrying out PCR amplification by using a specific primer, and carrying out glue recovery, cloning vector connection, bacterial liquid PCR detection and sequencing comparison.

The plant stress resistance related protein gene separated and cloned from wheat variety "small Bai Mai" is named as TaGSKB gene. The coding sequence (CDS) of the TaGSKB gene is SEQ ID No.2, and the coding amino acid sequence is the protein of SEQ ID No.1, which is named as TaGSKB protein.

Example 2 real-time fluorescent quantitative PCR analysis of expression Properties of TaGSKB Gene

1. Treatment of wheat with different stresses

The small Bai Mai plants are planted in nutrient soil, and after the seedlings grow for 1 week at 22 ℃, the seedlings are subjected to different stress treatments.

1. Drought treatment: wheat seedlings were placed on absorbent filter paper to simulate drought conditions.

2. Salt treatment: wheat seedling roots were immersed in 200mM NaCl solution, simulating salt stress treatment.

3. High temperature and low temperature treatment: wheat seedlings were placed in a 42 ℃ (high temperature), 4 ℃ (low temperature) incubator for treatment.

2. Real-time fluorescent quantitative PCR

Extracting total RNA of wheat by using a total plant RNA extraction kit of Tiangen company, and synthesizing first-strand cDNA by using a first-strand cDNA synthesis kit of full-scale gold company. The cDNA treated by different stresses is properly diluted to be used as a template, wheat Actin is used as an internal reference gene, and the relative expression quantity of the TaGSKB gene under the different stresses is analyzed by a real-time fluorescent quantitative PCR technology (Relative expression level).

Amplification was performed using an ABI 7500Real Time PCR instrument, and the reaction was performed using a three-step PCR reaction program: pre-denaturation at 95℃for 15min (sufficient activation of the hot start enzyme); 95℃10s,55℃30s,72℃32s (fluorescence signal was collected), 40 cycles. Data processing after reaction was completed using 2 ^-ΔΔCt Algorithm, histogram is made with Excel software. Three biological replicates.

The primers used for detecting the action gene are as follows:

Actin-F：5’-GCCATGTACGTCGCAATTCA-3’，

Actin-R：5’-AGTCGAGAACGATACCAGTAGTACGA-3’。

the primers for detecting the expression quantity of the TaGSKB gene under different stresses are as follows:

RT-TaGSKB-F：5’-TTCAACTTCAAGCATGAACTGG-3’，

RT-TaGSKB-R：5’-AACAATAAGGTGCAAGGTTGAG-3’。

The results are shown in FIG. 1: the TaGSKB gene responds to abiotic stress treatment to varying degrees. The TaGSKB gene significantly upregulates expression after Drought (Draright), high salt (NaCl) and High temperature (High temperature) treatments, with the response to Drought being the strongest. It was shown that the TaGSKB gene may be involved in a variety of abiotic stress responses.

Example 3 Effect of TaGSKB protein on stress tolerance in Arabidopsis thaliana

1. Construction of recombinant expression vectors

1. Total RNA of the small Bai Mai leaves was extracted and reverse transcribed to obtain cDNA.

2. The cDNA in the above step 1 was used as a template, and the specific primer was used to amplify the wheat TaGSKB gene, and the PCR product was recovered. Specific amplification primers for the TaGSKB gene were as follows:

TaGSKB-F：5’-AGATAGGCGTAGGTGGATG-3’，

TaGSKB-R：5’-AAGATGGTGCAATGTTGAG-3’。

3. the PCR product amplified and recovered as described above was ligated with a Peasybunt vector (pEASY-Blunt Cloning Kit, catalog number CB101, full gold, beijing) and the ligation product transformed E.coli competent cells TOP10 and plated on solid LB medium plates containing 50. Mu.g/L kanamycin, and cultured overnight at 37 ℃. Colony PCR screening is carried out on the clone colony of the escherichia coli, positive clones are sent to a company for sequencing, the colony with correct sequencing is preserved, plasmids are extracted, and the plasmids with correct sequences are named as Peasybunt-TaGSKB.

The plasmid of the Peasyblast-TaGSKB is a recombinant vector obtained by connecting a DNA fragment with a nucleotide sequence of SEQ ID No.2 in a sequence table to a Peasyblast vector by using a blunt end cloning method and keeping other sequences of the Peasyblast vector unchanged.

4. And (3) taking the plasmid peasyblue-TaGSKB obtained in the step (3) as a template, adopting a primer pair consisting of TaGSKB-1302-F and TaGSKB-1302-R to carry out PCR amplification to obtain a PCR amplification product, and carrying out gel recovery of the PCR amplification product.

TaGSKB-1302-F：5'-GGGACTCTTGACCATGATGGAGCATCCGGCG-3'；

TaGSKB-1302-R：5'-TCAGATCTACCCATGGGCTCCCCGCATGCAC-3'。

5. The vector pCAMBIA1302 was digested with restriction enzyme NcoI, and the digested vector was recovered.

6. And (3) carrying out homologous recombination on the PCR product obtained In the step (4) and the vector obtained after the enzyme digestion In the step (5) by utilizing an In-Fusion technology. After the sequencing is correct, the recombinant plasmid pCAMBIA1302-TaGSKB is obtained.

The recombinant plasmid pCAMBIA1302-TaGSKB is a DNA molecule shown in SEQ ID No.2 inserted into the NcoI site of the vector pCAMBIA 1302.

2. Obtaining transgenic Arabidopsis thaliana

1. Recombinant plasmid pCAMBIA1302-TaGSKB is introduced into agrobacterium GV3101 to obtain recombinant agrobacterium GV3101/pCAMBIA1302-TaGSKB.

2. Transferring the bacterial solution to liquid YEP culture medium containing 50 mug/L rifampicin and 50 mug/L kanamycin, and shaking culturing at 28 ℃ and 300rpm until OD _600nm ＝1.5-3.0。

3. After the step 2 is completed, the bacterial cells are collected by centrifugation at 4000g for 10min at 4 ℃, the bacterial cells are resuspended by using an infection liquid, and the OD is adjusted ₆₀₀ The value is 0.6-0.8, and the bacterial body weight suspension (infection liquid formula: 1/2MS, 10mM MgCl) ₂ 1.0g/L MES, 5% sucrose, 100. Mu.l/L Silwet L-77; pre-configured, left at room temperature for 3 h).

4. The bacterial body weight suspension is poured into a culture dish, a flowerpot for planting Columbia ecological type Arabidopsis thaliana (Col-0) is placed down, inflorescences are completely immersed into the infection liquid for 3min, then Arabidopsis thaliana plants are horizontally placed, covered by a black plastic bag, and grown for 24h in the dark. The next day the plastic bag was opened and arabidopsis was placed upright and grown under normal light conditions. Dip-staining again after 1 week, normal growth until harvest of T ₀ Seed generation.

5. Will T ₀ Sowing seeds of the generation Arabidopsis on a solid MS culture medium flat plate containing 50mg/L kanamycin, screening Arabidopsis seedlings which can normally grow, transferring the Arabidopsis seedlings into soil for growth, and harvesting the single plant after maturation to obtain T ₁ Different strains are substituted. Screening is then continued by the same method until T is obtained ₃ And (3) replacing homozygous arabidopsis thaliana.

6. And extracting RNA from different transgenic lines obtained by screening, and performing reverse transcription to obtain cDNA. The cDNA obtained by reverse transcription is properly diluted to be used as a template of real-time fluorescence quantitative PCR, and the expression quantity of different strains is detected. From which the appropriate strain is selected for subsequent phenotypic experiments. The PCR identification primers are as follows:

RT-TaGSKB-F：5’-TTCAACTTCAAGCATGAACTGG-3’，

RT-TaGSKB-R：5’-AACAATAAGGTGCAAGGTTGAG-3’。

Three homozygous TaGSKB gene-transferred Arabidopsis lines were identified by PCR and named: #3 (i.e., taGSKB-3), #5 (i.e., taGSKB-5), #8 (i.e., taGSKB-8).

3. Stress tolerance identification of Arabidopsis thaliana

The seeds to be measured are: #3 Strain T ₃ Seed of generation plant, #5 line T ₃ Seed of generation plant, #8 line T ₃ Seeds of the generation plants and seeds of wild type Arabidopsis thaliana (Col-0).

1. Germination rate under drought stress (gemination rate): wild type and transgenic arabidopsis seeds harvested at the same time are selected, and after sterilization treatment, seeds with the same size are sown on an MS culture medium and a stress treatment culture medium added with PEG (experimental groups added with 10% and 12% of PEG are respectively arranged). After vernalization at 4℃for 3 days, the cells were transferred to a 22℃incubator with 16h light/8 h darkness and a relative humidity of 60% for growth. Germination rate was counted every 24 h. And taking the white exposure of the seeds as a germination standard, and counting that the germination rate is not changed any more. Three biological replicates.

The results are shown in fig. 2 a and B: on MS medium, there was no difference in germination rate between wild type and transgenic Arabidopsis, and all germinated on day 4. On the medium containing 10% and 12% peg, the germination rates of both wild-type and transgenic arabidopsis were slowed, but the germination rate of transgenic arabidopsis was higher than that of wild-type arabidopsis, indicating that the transgenic tagkb gene (i.e. overexpression of the tagkb gene) increased the germination rate of arabidopsis seeds under drought stress conditions.

2. Growth vigor and survival rate under drought stress treatment

When four leaves of wild type and transgenic arabidopsis were grown on MS medium, they were transferred to nutrient soil to vermiculite ratio of 1:1 (watering to saturation to ensure sufficient water), and placing in a 22 ℃ culture room with the relative humidity of 60% for growth in 16h light/8 h dark.

Drought group (drough): and (3) carrying out drought treatment on the wild type arabidopsis thaliana and the transgenic arabidopsis thaliana which normally grow for 3 weeks and have the same growth vigor, namely controlling water for two weeks, rehydrating for one week, observing the phenotype, photographing and counting the survival rate.

Normal group (Control): normal watering is continued on wild type and transgenic arabidopsis with the same growth vigor and normal growth for 3 weeks, phenotype is observed, photographing is carried out, and survival rate is counted.

Three biological replicates were set up, and 36 plants were counted for each seed tested in each replicate. The results are shown in fig. 2C and D: under normal growth conditions (beforetreatment), there was no significant difference between wild type and transgenic arabidopsis; under drought treatment (Drought treatment), transgenic arabidopsis grew significantly stronger than wild arabidopsis; statistical survival rates after rehydration of drought treated wild-type and transgenic arabidopsis thaliana were found to be significantly higher than that of wild-type arabidopsis thaliana. The expression of TaGSKB gene can obviously improve drought resistance of plants.

3. Germination rate under salt stress: wild type and transgenic Arabidopsis seeds harvested at the same time were selected, and after sterilization treatment, seeds of uniform size were sown on MS medium and stress treatment medium (MS medium) supplemented with 125mM and 150mM NaCl. After vernalization at 4℃for 3 days, the cells were transferred to a 22℃incubator with 16h light/8 h darkness and a relative humidity of 60% for growth. Germination rate was counted every 24 h. And taking the white exposure of the seeds as a germination standard, and counting that the germination rate is not changed any more. Three biological replicates.

The results are shown in fig. 3 a and B: on MS medium, there was no difference in germination rate between wild type and transgenic Arabidopsis, and all germinated on day 4. On the medium containing 125mM and 150mM NaCl, the germination rates of the wild type and the transgenic Arabidopsis thaliana are both slowed down, but the germination rate of the transgenic Arabidopsis thaliana is higher than that of the wild type Arabidopsis thaliana, which indicates that the transgenic TaGSKB gene (i.e. the overexpression of the TaGSKB gene) improves the germination rate of Arabidopsis thaliana seeds under the condition of salt stress.

4. Growth vigor and survival rate under high salt stress treatment

When four leaves of wild type and transgenic arabidopsis were grown on MS medium, they were transferred to nutrient soil to vermiculite ratio of 1:1 (watering to saturation to ensure sufficient water), and placing in a 22 ℃ culture room with the relative humidity of 60% for growth in 16h light/8 h dark.

Salt treatment group (Salt): wild type and transgenic arabidopsis thaliana which grow normally for 3 weeks and have the same growth vigor are treated with 200mM NaCl for one week until significant differences occur in the growth vigor of the wild type and transgenic arabidopsis thaliana, and the survival rate is photographed and counted.

Normal group (Control): wild-type and transgenic arabidopsis thaliana, which grew normally for 3 weeks and were identical in vigour, were continued to be watered normally, and phenotypes were observed, photographed, and Survival (survivin rate) was counted.

Three biological replicates were set up, and 36 plants were counted for each seed tested in each replicate. The results are shown in fig. 3C and D: under normal growth conditions (beforetreatment), there was no significant difference between wild type and transgenic arabidopsis; under Salt treatment (Salt treatment), the transgenic arabidopsis thaliana has a significantly stronger growth state than the wild type arabidopsis thaliana; statistical survival rates were found to be significantly higher for transgenic arabidopsis than for wild-type arabidopsis. The over-expression of the TaGSKB gene can obviously improve the salt tolerance of plants.

In summary, the TaGSKB protein and the coding gene thereof can regulate and control the stress resistance (such as drought resistance and/or salt tolerance) of the arabidopsis, and the content and/or activity (such as over-expression of the TaGSKB gene) of the TaGSKB protein in the target plant arabidopsis can obviously improve the stress resistance of the target plant arabidopsis.

Example 4 Effect of TaGSKB protein on stress tolerance of wheat

1. Construction of recombinant expression vectors

1. The plasmid (peasyblue-TaGSKB) obtained in the step 3 of the example 3 is used as a template, a primer pair consisting of TaGSKB-110-F and TaGSKB-110-R is adopted for PCR amplification, a PCR amplification product is obtained, and the PCR product is recovered by glue.

TaGSKB-110-F：5'-CGACTCTAGAGGATCCATGGAGCATCCGGCG-3'；

TaGSKB-110-R：5'-GGGTACCCGGGGATCCTTAGCTCCCCGCATG-3'。

2. The vector pWMB110 was digested with the restriction enzyme BamHI, and the digested vector was recovered.

3. And (3) carrying out homologous recombination on the PCR recovery product obtained In the step (1) and the vector subjected to enzyme digestion In the step (2) by using an In-Fusion technology. After the sequencing is correct, the recombinant plasmid pWMB110-TaGSKB is obtained.

The recombinant plasmid pWMB110-TaGSKB is a DNA molecule shown in SEQ ID No.2 inserted into the BamHI site of the vector pWMB 110.

2. Obtaining transgenic wheat

Young wheat embryos are prepared in advance, and recombinant plasmids are transformed into spring wheat field by using agrobacterium-mediated gene transformation technology. The main steps are carried out according to The references "Wang K, shi L, liang X, zhao P, wang W, liu J, chang Y, hiei Y, yanagihara C, du L, ishida Y, ye X (2022). The gene TaWOX5overcomes genotype dependency in wheat genetic transformation. Nat plants. Doi:10.1038/s41477-021-01085-8. The method comprises the following steps:

1. Recombinant plasmid pWMB110-TaGSKB is introduced into agrobacterium EHA105 to obtain recombinant agrobacterium EHA105/pWMB110-TaGSKB.

2. Transferring the bacterial solution to liquid YEP culture medium containing 50 mug/L rifampicin and 50 mug/L kanamycin, and shaking culturing at 28 ℃ and 300rpm until OD _600nm ＝1.5-3.0。

3. Wheat seed surface disinfection, taking out wheat embryo under microscope, incubating with agrobacterium infection liquid (containing acetosyringone) carrying pWMB110-TaGSKB at room temperature for 5 min, growing on WLS-AS medium at 25deg.C in dark condition for 2 days, and making scutellum upward.

4. Radicles were excised with a scalpel and transferred to WLS-Res medium for recovery. After 5 days, the tissue was transferred to callus-induced WLS-P5 medium for growth. After 2 weeks, calli were transferred to WLS-P10 medium for 3 weeks.

5. The regenerated shoots were transferred to cups containing MSF-P5 medium for elongation and rooting. After 9 days, transplanting plants with good root system development into a flowerpot for continuous growth, and obtaining transgenic wheat (T) ₀ Transgenic wheat).

6. Harvesting wheat seeds and screening homozygous lines: extracting RNA of the plant, reversely transcribing the RNA into cDNA, verifying transgenic positive plants (containing TaGSKB gene with the coding sequence of SEQ ID NO. 2) by a PCR method, and screening the transgenic positive plants into stably transformed homozygous lines which can fully grow normally in the third generation, wherein the homozygous lines are respectively named as follows: OE-1 strain (i.e., taGSKB-1 strain), OE-2 strain (i.e., taGSKB-2 strain), OE-4 strain (i.e., taGSKB-4 strain).

3. Transgenic wheat positive strain detection

For the detection of transgenic wheat, PCR and qRT-PCR analysis were used to identify transgenic wheat lines at both DNA and transcript levels. Extracting DNA by adopting a CTAB method, carrying out PCR detection, sequencing the correct strip size, and harvesting the single strain with correct sequencing into different strains.

RNA is extracted from different transgenic lines obtained by positive detection, and is reversely transcribed into cDNA. The cDNA obtained by reverse transcription is properly diluted to be used as a template of real-time fluorescence quantitative PCR, and the expression quantity of different strains is detected.

The primers used for positive detection of the TaGSKB gene were as follows:

TaGSKByxjc-F：5’-TTTAGCCCTGCCTTCATACG-3’，

TaGSKByxjc-R：5’-CCCATCTCATAAATAACGTCATGC-3’。

the primers used for detecting the action gene are as follows:

Actin-F：5’-GCCATGTACGTCGCAATTCA-3’，

Actin-R：5’-AGTCGAGAACGATACCAGTAGTACGA-3’。

the qRT-PCR primer for detecting the expression quantity of the TaGSKB positive strain is as follows:

RT-TaGSKB-F：5’-TTCAACTTCAAGCATGAACTGG-3’

RT-TaGSKB-R：5’-AACAATAAGGTGCAAGGTTGAG-3’。

4. stress tolerance identification of wheat

The seeds to be measured are: transgenic wheat TaGSKB-1 strain T ₃ Seed of generation plant, taGSKB-2 strain T ₃ Seed of generation plant, taGSKB-4 strain T ₃ Seeds of the generation plants and seeds of wild wheat (acceptor wheat).

1. Physiological index under drought stress (mannitol treatment): the recipient wheat and the transgenic wheat seeds are germinated on a culture dish, placed in an incubator at 22 ℃ (16 h illumination/8 h darkness, 60% humidity), transferred to Hoagland nutrient solution for continuous growth for 3-4 days after 3 days, subjected to 200mM and 300mM mannitol treatment, and counted for root length after 5 days. Determination of receptor and transgene 4 days after treatment For Proline (Proline), malondialdehyde (MDA), H of wheat ₂ O ₂ The determination method of the sample physiological index is operated according to the specific operation steps of the kit (Suzhou Ming Biotechnology Co., ltd.) according to the content and the Catalase (CAT) activity. Wheat root was subjected to NBT staining and O was measured ^2- The content is as follows. Three biological replicates were performed, 32 for each seed tested in each replicate.

The results are shown in FIG. 4: under normal growth conditions, there was no significant difference in growth between the receptor and transgenic wheat; proline, malondialdehyde and H of transgenic wheat ₂ O ₂ The content and the catalase activity are not obviously different from those of the acceptor wheat. Under Mannitol (Mannitol) treatment, both receptor and transgenic wheat growth was inhibited, but transgenic wheat grew stronger than the receptor wheat; the proline content and catalase activity of the transgenic wheat are higher than those of the acceptor wheat, and malondialdehyde and H of the transgenic wheat ₂ O ₂ The content is lower than that of the acceptor wheat. NBT staining of root tip of transgenic and recipient wheat, color depth and O in root tip ^2- The content is positively correlated; under the normal growth condition, the colors of the root tips of different wheat are not different; after mannitol treatment, the root tip color of the over-expressed wheat strain is obviously lighter than the receptor color, and the O of the transgenic wheat ^2- The content is lower than that of the acceptor wheat.

2. Growth under drought stress, survival rate, water loss rate and relative water content

The nutrient soil is watered to be saturated, equal amount of soil is weighed, the receptor with the same grain number and the transgenic wheat pure line are planted in a flowerpot, and the water control treatment is carried out in a 22 ℃ (16 h illumination/8 h darkness and 60% humidity) culture room. The receptor and transgenic wheat leaves were taken 12 days after the controlled water treatment to determine the water loss rate and relative water content. Drought treatment was resumed until the recipient Fielder and transgenic wheat seedlings wilted and were significantly different, and survival was counted after 2 days. Three biological replicates were performed, 60 seeds per test in each replicate.

The results are shown in FIG. 5: there was no significant difference in growth between the receptor and transgenic wheat under normal growth conditions. After drought treatment, both the growth of the receptor and the transgenic wheat is inhibited, and the growth vigor of the transgenic wheat is obviously stronger than that of wild wheat; after drought treatment, transgenic wheat has a lower Water loss rate (Water loss rate) than the recipient wheat, and a higher Relative Water Content (RWC) than the recipient wheat; after rehydration, the survival rate of transgenic wheat is significantly higher than that of recipient wheat. The expression of the TaGSKB gene can improve the drought resistance of transgenic wheat.

3. Physiological index under salt stress: the recipient Fielder and transgenic wheat seeds were germinated on petri dishes, placed in a 22℃ (16 h light/8 h dark, 60% humidity) incubator, and after 3 days the white-exposed seeds were transferred to Hoagland nutrient solution for continued growth for 3-4 days, 300mM NaCl treatment was performed, and root length was counted after 5 days. Proline and malondialdehyde content of the recipients and transgenic wheat were determined 4 days after treatment. Three biological replicates were performed, 60 seeds per test in each replicate.

The results are shown in FIG. 6: under normal growth conditions, there was no significant difference in the growth of the acceptor and transgenic wheat, and the proline and malondialdehyde content of the transgenic wheat was no significant difference from that of the acceptor wheat. After high salt treatment, the growth of the receptor and the transgenic wheat is inhibited, and the growth vigor of the transgenic wheat is obviously stronger than that of the receptor wheat; the transgenic wheat has a proline higher than the acceptor wheat and a malondialdehyde content lower than the acceptor wheat.

4. Growth vigor, survival rate, chlorophyll content and conductivity under high salt stress treatment

The nutrient soil is watered to saturation, equal amount of soil is weighed, acceptor wheat (Fielder) with the same grain number and transgenic wheat pure line are planted in a flowerpot, and the plant grows in a culture room at 22 ℃ (16 h illumination/8 h darkness and 60% humidity). Wheat grown normally for 10 days was subjected to high salt treatment with 250mM NaCl, and chlorophyll content and relative conductivity were measured after salt treatment for 7 days. Until there was a significant difference between the recipient and transgenic wheat, photographs were taken and survival was counted. Three biological replicates were performed, 60 seeds per test in each replicate.

The results are shown in FIG. 7: under normal growth conditions, there was no significant difference in the growth of the recipient and transgenic wheat, and the chlorophyll content (Chlorophyll content) and relative conductivity (Electrolyte leakage) of the transgenic wheat were no significant difference from the recipient wheat. After high salt treatment, the growth of the receptor and the transgenic wheat is inhibited, and the growth vigor of the transgenic wheat is obviously stronger than that of the receptor wheat; the chlorophyll content of the transgenic wheat is higher than that of the acceptor wheat, and the relative conductivity is lower than that of the acceptor wheat; the survival rate of transgenic wheat is higher than that of acceptor wheat. The expression of TaGSKB gene can improve the salt tolerance of transgenic plants.

In conclusion, the TaGSKB protein and the coding gene thereof can regulate and control the stress resistance (such as drought resistance and/or salt tolerance) of wheat, and the content and/or activity (such as over-expression of the TaGSKB gene) of the TaGSKB protein in the target plant wheat can obviously improve the stress resistance of the target plant wheat.

5. Molecular mechanism of transgenic wheat with improved stress tolerance

We have found genes associated with abiotic stress and tested the expression of these stress-related genes in transgenic wheat and acceptor wheat. NPF2.3 gene participates in the transportation of nitrate under salt stress, so that the capability of plants for adapting to the salt stress is improved; the Lea7 gene participates in the synthesis of cell penetrating substances, so that the resistance of plants to stress can be improved; the APX1 gene regulates the level of peroxide oxidase, can remove peroxide accumulated excessively by stress, and reduces oxidative stress caused by excessive peroxide; the RD29A gene is a key Marker gene corresponding to drought stress; by detecting the expression of abiotic stress-related genes, it is possible to help understand the stress response mechanism of plants.

The results are shown in FIG. 8: the expression of TaNPF2.3, taLea7, taAPX1 and TaRD29A genes is detected in transgenic wheat and acceptor wheat, and the results show that the expression of the genes is improved to different degrees in the wheat transformed with the TaGSKB gene, and the TaGSKB gene is used for improving the drought resistance and the salt tolerance of plants by directly or indirectly regulating the expression of abiotic stress related genes.

Therefore, the experimental results of wheat over-expressing the TaGSKB gene or Arabidopsis thaliana over-expressing the TaGSKB gene show that the TaGSKB protein and the coding gene thereof can regulate and control the stress resistance (such as drought resistance and/or salt tolerance) of plants, and the stress resistance of the target plants can be obviously improved by improving the content and/or activity (such as over-expressing the TaGSKB gene) of the TaGSKB protein in the target plants.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

SEQUENCE LISTING

<110> institute of crop science at national academy of agricultural sciences

<120> wheat TaGSKB protein and application of encoding gene thereof in regulation and control of plant stress tolerance

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 402

<212> PRT

<213> wheat (Triticum aestivum L.)

<400> 1

Met Glu His Pro Ala Pro Ala Pro Glu Pro Met Leu Leu Asp Glu Gln

1 5 10 15

Pro Pro Thr Ala Val Ala Cys Glu Lys Lys Gln Gln Asp Gly Glu Ala

20 25 30

Pro Tyr Ala Glu Gly Asn Asp Ala Met Thr Gly His Ile Ile Ser Thr

35 40 45

Thr Ile Gly Gly Lys Asn Gly Glu Pro Lys Gln Thr Ile Ser Tyr Met

50 55 60

Ala Glu Arg Val Val Gly Thr Gly Ser Phe Gly Ile Val Phe Gln Ala

65 70 75 80

Lys Cys Leu Glu Thr Gly Glu Thr Val Ala Ile Lys Lys Val Leu Gln

85 90 95

Asp Arg Arg Tyr Lys Asn Arg Glu Leu Gln Leu Met Arg Ser Met Ile

100 105 110

His Ser Asn Val Val Ser Leu Lys His Cys Phe Phe Ser Thr Thr Ser

115 120 125

Arg Asp Glu Leu Phe Leu Asn Leu Val Met Glu Tyr Val Pro Glu Thr

130 135 140

Leu Tyr Arg Val Leu Lys His Tyr Ser Asn Ala Lys Gln Gly Met Pro

145 150 155 160

Leu Ile Tyr Val Lys Leu Tyr Thr Tyr Gln Leu Phe Arg Gly Leu Ala

165 170 175

Tyr Ile His Thr Val Pro Gly Val Cys His Arg Asp Val Lys Pro Gln

180 185 190

Asn Val Leu Val Asp Pro Leu Thr His Gln Val Lys Ile Cys Asp Phe

195 200 205

Gly Ser Ala Lys Val Leu Val Ala Gly Glu Pro Asn Ile Ser Tyr Ile

210 215 220

Cys Ser Arg Tyr Tyr Arg Ala Pro Glu Leu Ile Phe Gly Ala Thr Glu

225 230 235 240

Tyr Thr Thr Ser Ile Asp Ile Trp Ser Ala Gly Cys Val Leu Ala Glu

245 250 255

Leu Leu Leu Gly Gln Pro Leu Phe Pro Gly Glu Ser Ala Val Asp Gln

260 265 270

Leu Val Glu Ile Ile Lys Val Leu Gly Thr Pro Thr Arg Glu Glu Ile

275 280 285

Arg Cys Met Asn Pro Asn Tyr Thr Glu Phe Arg Phe Pro Gln Ile Lys

290 295 300

Ala His Pro Trp His Lys Val Phe His Lys Lys Met Pro Pro Glu Ala

305 310 315 320

Ile Asp Leu Ala Ser Arg Leu Leu Gln Tyr Ser Pro Ser Leu Arg Cys

325 330 335

Thr Ala Leu Asp Ala Cys Ala His Pro Phe Phe Asp Glu Leu Trp Glu

340 345 350

Pro Asn Ala Arg Leu Pro Asn Gly Arg Pro Phe Pro Pro Leu Phe Asn

355 360 365

Phe Lys His Glu Leu Ala Asn Ala Ser Gln Asp Leu Ile Asn Arg Leu

370 375 380

Val Pro Glu His Val Arg Arg Gln Ala Gly Leu Ala Phe Val His Ala

385 390 395 400

Gly Ser

<210> 2

<211> 1209

<212> DNA

<213> wheat (Triticum aestivum L.)

<400> 2

atggagcatc cggcgccggc gccggagccg atgctgctcg acgagcagcc ccccaccgca 60

gtcgcctgcg agaagaagca gcaggatggc gaggcgccgt atgcggaggg gaacgacgcc 120

atgaccggtc acatcatctc caccaccatc ggcggcaaga acggcgagcc caagcagacg 180

attagctaca tggcggagcg cgttgtgggc actggttcgt ttggcatcgt ctttcaggct 240

aaatgcctgg agaccgggga gacagtggcc attaagaagg tactgcagga ccgacggtac 300

aagaatcgtg agctgcagct tatgcgttcg atgatccatt ccaatgttgt ctccctcaag 360

cactgcttct tctcaaccac aagtagagat gagctgtttc tgaaccttgt catggagtat 420

gtcccggaga cactctaccg cgtgcttaag cactacagta atgccaaaca ggggatgcca 480

cttatctacg tcaagcttta cacctatcag ctattcaggg ggctggcgta cattcatact 540

gttccaggag tctgtcacag ggatgtgaag ccacaaaatg ttttggttga tcctttaaca 600

catcaagtta agatctgcga ctttggaagc gcgaaagttc tggtggcggg tgagcccaat 660

atatcataca tatgctcacg ctactaccgt gctccggagc ttatatttgg tgcgactgaa 720

tatacaacat cgatagatat atggtcagct ggttgtgttc ttgcagaact gctccttggt 780

cagccattat ttccaggcga gagtgcagtc gatcaacttg tagagataat caaggttctt 840

ggaacgccaa ctcgggagga aatacgttgt atgaacccga attatacgga gtttaggttt 900

ccacagataa aagctcatcc ttggcacaag gttttccaca agaaaatgcc tcctgaagcc 960

atagatcttg cttcacgtct tcttcaatat tcaccaagcc tccgttgcac tgctcttgac 1020

gcatgtgcgc atcctttctt tgatgagcta tgggagccta acgcgcgcct gccaaatgga 1080

cgcccgttcc cacctctgtt caacttcaag catgaactgg ccaatgcttc acaagacctc 1140

atcaacaggc ttgtgcctga acatgttcgc cgacaagctg gtcttgcttt cgtgcatgcg 1200

gggagctaa 1209

Claims (10)

1. Use of a protein or a substance regulating the activity and/or content of said protein, characterized in that said use is any of the following:

d1 Use of a protein or a substance regulating the activity and/or content of said protein for regulating stress resistance of a plant;

d2 Use of a protein or a substance regulating the activity and/or content of said protein for the preparation of a product regulating stress resistance of a plant;

d3 Use of a protein or a substance regulating the activity and/or content of said protein for growing stress-tolerant plants;

d4 Use of a protein or a substance regulating the activity and/or content of said protein for the preparation of a product for growing stress-tolerant plants;

d5 Use of a protein or a substance regulating the activity and/or content of said protein in plant breeding;

the protein is any one of the following:

a1 A protein having an amino acid sequence of SEQ ID No. 1;

a2 A protein which is obtained by substituting and/or deleting and/or adding an amino acid residue in the amino acid sequence shown in SEQ ID No.1, has more than 80% of identity with the protein shown in A1) and has the same function;

A3 Fusion proteins having the same function obtained by ligating a tag to the N-terminal and/or C-terminal of A1) or A2).

2. The use according to claim 1, wherein the protein is derived from wheat.

3. Use of a biological material related to a protein as claimed in claim 1 or 2, characterized in that the use is any of the following:

e1 Use of a biological material related to the protein of claim 1 or 2 for regulating stress resistance of plants;

e2 Use of a biological material related to the protein of claim 1 or 2 for the preparation of a product for regulating stress resistance of a plant;

e3 Use of biological material related to the protein of claim 1 or 2 for breeding stress-tolerant plants;

e4 Use of a biological material related to the protein of claim 1 or 2 for the preparation of a product for breeding stress-tolerant plants;

e5 Use of biological material related to the protein of claim 1 or 2 in plant breeding;

the biomaterial is any one of the following B1) to B7):

b1 A nucleic acid molecule encoding a protein as claimed in claim 1 or 2;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

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

b5 A transgenic plant cell line comprising the nucleic acid molecule of B1) or a transgenic plant cell line comprising the expression cassette of B2);

b6 A transgenic plant tissue comprising the nucleic acid molecule of B1) or a transgenic plant tissue comprising the expression cassette of B2);

b7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2).

4. The use according to claim 3, wherein the nucleic acid molecule of B1) is any one of the following:

c1 A DNA molecule whose coding sequence is SEQ ID No. 2;

c2 A DNA molecule with the nucleotide sequence of SEQ ID No. 2.

5. A method of growing a stress-tolerant plant, comprising increasing the amount and/or activity of a protein according to claim 1 or 2 in a plant of interest to obtain a stress-tolerant plant having a stress-tolerance higher than that of the plant of interest.

6. The method according to claim 5, wherein the increase in the content and/or activity of the protein according to claim 1 or 2 in the plant of interest is achieved by increasing the expression level of a gene encoding the protein in the plant of interest.

7. The method according to claim 6, wherein the increase in the expression level of the gene encoding the protein in the target plant is achieved by introducing the gene encoding the protein in claim 1 or 2 into the target plant.

8. The method of claim 7, wherein the protein-encoding gene is any one of the following:

f1 A DNA molecule whose coding sequence is SEQ ID No. 2;

f2 A DNA molecule with the nucleotide sequence of SEQ ID No. 2.

9. The method of any one of claims 5-8, wherein the plant is G1) or G2):

g1 Monocotyledonous or dicotyledonous plants;

g2 Gramineous or cruciferous plants.

10. Use of a protein as defined in claim 1 or 2, or a biological material as defined in claim 3, or a nucleic acid molecule as defined in claim 4, or a method as defined in any one of claims 5 to 9, for creating stress-tolerant plants and/or plant breeding.