CN118546985A

CN118546985A - Plant drought-tolerance related protein TaHIN-6A and coding gene and application thereof

Info

Publication number: CN118546985A
Application number: CN202410715573.6A
Authority: CN
Inventors: 王景一; 胡格; 李超男; 李龙; 景蕊莲; 毛新国
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2024-06-04
Filing date: 2024-06-04
Publication date: 2024-08-27

Abstract

The invention discloses a plant drought-enduring related protein TaHIN-6A, and a coding gene and application thereof. The invention aims to solve the problem of how to regulate and control drought resistance of plants. TaHIN1-6A is any one of the following proteins: a1 Amino acid sequence is a protein shown in sequence 2; a2 A protein which has 80% or more identity with the protein represented by A1) and has the same function as the protein represented by A1) and is obtained by substitution and/or deletion and/or addition of an amino acid residue of the protein of A1); a3 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of A1) or A2) with a protein tag. Substances which up-regulate or enhance or increase TaHIN A gene expression can be used for breeding for improving drought tolerance of wheat.

Description

Plant drought-tolerance related protein TaHIN-6A and coding gene and application thereof

Technical Field

The application relates to a plant drought-enduring related protein TaHIN-6A, and a coding gene and application thereof.

Background

As a staple food crop which is widely planted worldwide, wheat plays a significant role in ensuring global food safety. The drought caused by water deficiency seriously threatens the wheat production and directly affects the yield. Therefore, the related genes of the drought resistance of the wheat are excavated and utilized, and a foundation is laid for improving the drought resistance of the wheat and breeding new varieties of drought-resistant high-yield wheat.

Disclosure of Invention

The invention solves the technical problem of how to improve drought resistance of plants. Especially grasses.

In order to solve the above problems, the present invention provides the following applications.

Use of a protein, a substance that upregulates or enhances or increases expression of a gene encoding said protein, or a substance that upregulates or enhances or increases activity or content of said protein, for increasing drought resistance of a gramineous plant and/or for preparing a product that increases drought resistance of a gramineous plant and/or for breeding a gramineous plant;

The protein is any one of the following:

A1 Amino acid sequence is a protein shown in sequence 2;

a2 A protein which has 80% or more identity with the protein represented by A1) and has the same function as the protein represented by A1) and is obtained by substitution and/or deletion and/or addition of an amino acid residue of the protein of A1);

A3 Fusion proteins obtained by ligating the N-terminal or/and C-terminal of A1) or A2) with a protein tag.

Among the above proteins, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with a target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.

In the above proteins, the identity refers to the identity of amino acid sequences. 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, expect values are 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 identity of a pair of amino acid sequences is searched for and calculated, and then the value (%) of identity can be obtained.

In the above protein, the 80% or more identity may be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above protein, sequence 2 (SEQ ID No. 2) consists of 362 amino acid residues.

In the above application, the protein is derived from wheat. The wheat may be a wheat variety Fielder.

The protein can be named TaHIN-6A and can be derived from wheat. The wheat can be dry-selected No. 10 wheat.

In the above, the substance that up-regulates or enhances or increases gene expression may be a substance that performs at least one of the following 6 kinds of 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).

In the above application, the substance that up-regulates or enhances or increases expression of the gene encoding the protein may be any of the following:

B1 A nucleic acid molecule encoding the above protein;

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 B1) said nucleic acid molecule, or a transgenic plant cell line comprising B2) said expression cassette, or a transgenic plant cell line comprising B3) said recombinant vector;

B6 A transgenic plant tissue comprising B1) said nucleic acid molecule, or a transgenic plant tissue comprising B2) said expression cassette, or a transgenic plant tissue comprising B3) said recombinant vector;

b7 A transgenic plant organ comprising the nucleic acid molecule of B1), or a transgenic plant organ comprising the expression cassette of B2), or a transgenic plant organ comprising the recombinant vector of B3).

In the above application, the nucleic acid molecule of B1) is any one of the following:

c1 A DNA molecule having a nucleotide sequence of the coding strand of sequence 1;

C2 A nucleic acid molecule having 80% or more identity and the same function as the nucleic acid molecule shown in C1) obtained by substitution and/or deletion and/or addition of nucleotides of the nucleic acid molecule of C1).

B1 In the nucleic acid molecules, the person skilled in the art can easily mutate the nucleotide sequence encoding the protein TaHIN a according to the invention using known methods, for example directed evolution or point mutation. Those artificially modified nucleotides having 80% or more identity to the nucleotide sequence of the protein TaHIN1-6A isolated by the present invention are all nucleotide sequences derived from the present invention and are equivalent to the sequences of the present invention, as long as they encode the protein TaHIN1-6A and have the function of the protein TaHIN 1-6A.

The 80% or more identity may be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% 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, expect values are 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.

In the above biological material, the nucleic acid molecule of B1) may be a gene encoding the protein. B1 The nucleic acid molecule may specifically be a DNA molecule in which the coding sequence of the coding strand is represented by nucleotides 54 to 1139 of the sequence 1.

Herein, such vectors are well known to those skilled in the art, including but not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), ti plasmids, or viral vectors. In particular to a vector pEASY-Blunt and/or pCAMBIA-1300;

In the above biological material, the expression cassette of B2) means a DNA capable of expressing the gene in a host cell, and the DNA may include not only a promoter for promoting transcription of the gene but also a terminator for terminating transcription of the gene. 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: a constitutive promoter of cauliflower mosaic virus 35S; wound-inducible promoters from tomato, leucine aminopeptidase ("LAP", chao et al (1999) Plant Physiol 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester); tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible with a jasmonates); 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 (CN 101063139B (China patent 200710099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al (1985) EMBO J. 4:3047-3053)). They may 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 terminator (see, e.g., odell et al (I985) 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 Genes dev, 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).

In B3) above, a recombinant expression vector containing the gene expression cassette may be constructed using a plant expression vector. The plant expression vector can be a Gateway system vector or a binary agrobacterium vector, etc., such as pGWB411、pGWB412、pGWB405、pBin438、pCAMBIA1302、pCAMBIA2301、pCAMBIA1301、pCAMBIA1300、pBI121、pCAMBIA1391-Xa or pCAMBIA1391-Xb. When TaHIN-6A is used to construct recombinant expression vectors, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi) and the like may be added before the transcription initiation nucleotide thereof, and they may 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.

c1 A DNA molecule with the nucleotide sequence of the coding strand shown as 54 th to 1139 th nucleotides of the sequence 1;

The use according to any one of the preceding claims, wherein the graminaceous plant is any one of the following G1) -G2):

g1 A) a plant of the genus Triticum,

G2 Wheat.

In order to solve the problems, the invention also provides a method for improving drought resistance of the gramineous plants.

The method comprises increasing drought resistance of a gramineous plant by up-regulating or enhancing or increasing the expression of a gene encoding a protein according to claim 1 or 2 or up-regulating or enhancing or increasing the activity and/or content of a protein as described above.

In order to solve the problems, the invention also provides a method for breeding gramineous plants.

The method comprises up-regulating or enhancing or increasing the expression level of the coding gene of the protein or the activity and/or the content of the protein in the target gramineous plant to obtain the drought-resistant gramineous plant, wherein the drought resistance of the drought-resistant gramineous plant is higher than that of the target gramineous plant.

The gramineous plant may be a seedling stage gramineous plant. The seedling stage may be a 2-leaf-one-heart stage.

The drought resistance index may be survival rate.

The coding gene of the target protein can be DNA molecules described by 54 th to 1139 th nucleotides of the sequence 1.

The application increases the transcription abundance of the target protein coding gene by inserting the coding gene of the target protein through external assistance, and increases the drought resistance of the target gramineous plant. The coding gene of the foreign insertion target protein can be realized by a transgenic means.

The plant expression vector used in the transgenic means can be a Gateway system vector or a binary agrobacterium vector.

In particular, the present application uses a vector which may be a binary agrobacterium vector.

Specifically, the binary agrobacterium vectors used in the present application may be pWMB and pCAMBIA series vectors.

Specifically, the pCAMBIA series vector used in the present application may be pCAMBIA1300 vector.

In the above method, the up-regulating or enhancing or increasing the expression level of the gene encoding the protein in the target gramineous plant or the activity and/or content of the protein is to introduce the gene encoding the protein into the target gramineous plant.

The drought resistance refers to the capability of a plant to maintain normal growth and development under drought conditions through a series of physiological, biochemical and morphological structure changes. This ability enables plants to survive and reproduce in a water deficient environment. The purpose of plant breeding includes growing drought-resistant plants.

The use as claimed in any one of the preceding claims or the method as claimed in any one of the preceding claims or the use or the method as claimed in any one of the preceding claims, the graminaceous plant being any one of the following:

g1 A) a plant of the genus Triticum,

G2 Wheat.

The wheat may be a wheat variety Fielder.

Advantageous effects

The coding gene TaHIN A is introduced into wheat to obtain transgenic wheat plant over expressing TaHIN A1-6A gene. Repeated drought treatment is carried out on wheat plants transformed with TaHIN-6A genes, and compared with wild wheat, the wheat plants transformed with TaHIN-6A genes are found to have enhanced drought resistance, which is manifested by high survival rate, enhanced cell membrane stability and enhanced superoxide dismutase activity under the drought treatment. The TaHIN A protein and the coding gene thereof provided by the invention play an important role in the drought stress resisting process of plants, and can be used for improving the drought resistance of the plants.

Drawings

FIG. 1 shows the expression of TaHIN-6A gene in different stress and hormone-treated wheat.

FIG. 2 shows the expression of TaHIN-6A gene in different organs of wheat at different stages of wheat development.

FIG. 3 shows the localization of TaHIN-6A protein in tobacco leaves.

FIG. 4 shows the relative expression levels of TaHIN-6A genes in different transgenic wheat lines.

FIG. 5 is a graph showing the identification of drought resistance phenotype of TaHIN-6A transgenic wheat lines.

FIG. 6 is a comparison of cell membrane stability before and after drought treatment of TaHIN-6A transgenic wheat lines.

FIG. 7 is a comparison of superoxide dismutase content of TaHIN-6A transgenic wheat lines before and after drought treatment.

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 following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

Fielder wheat: hexaploid wheat, cultivated in the united states in 1974, was commonly used for agrobacterium-mediated transformation and gene editing receptors. Reference to the literature ：Sato K,Abe F,Mascher M,Haberer G,Gundlach H,Spannagl M,Shirasawa K,Isobe S.Chromosome-scale genome assembly of the transformation-amenable common wheat cultivar'Fielder'.DNA Res.2021Jun25;28(3):dsab008.doi:10.1093/dnares/dsab008.PMID:34254113;PMCID:PMC8320877.

Dry-selecting No. 10 wheat: national germplasm pool (http:// www.cgris.net /), numbered: ZM009279.

The pCAMBIA1300-GFP vector and pWMB110,110 vector are described in the following documents: the "reference ：Wang J,Li C,Li L,Gao L,Hu G,Zhang Y,Reynolds MP,Zhang X,Jia J,Mao X,Jing R.DIW1encoding a clade IPP2C phosphatase negatively regulates drought tolerance by de-phosphorylating TaSnRK1.1in wheat.J Integr Plant Biol.2023Aug;65(8):1918-1936.doi:10.1111/jipb.13504.Epub 2023Jun 9.PMID:37158049".pWMB110 vector" is known in the literature as pWMB110vector and the pCAMBIA1300-GFP vector "is known in the literature as pCAMBIA1300vector, which are available from crop science research at the national academy of agricultural sciences.

PEASY-Blunt vector: beijing full gold Biotechnology Co., catalog number: CB101.

Agrobacterium tumefaciens EHA105: reference ：Torisky RS,Kovacs L,Avdiushko S,Newman JD,Hunt AG,Collins GB.Development of a binary vector system for plant transformation based on the supervirulent Agrobacterium tumefaciens strain Chry5.Plant Cell Reports,(1997)17:102-108.; is publicly available from the national academy of agricultural sciences of crop science.

Cloning of the Gene of examples 1, taHIN1-6A

The sequence of TraesCS A02G117700 is obtained through http:// writes.sdau.edu.cn/website, and the protein shown in the sequence 2 of the sequence table is named as protein TaHIN-6A and consists of 362 amino acid residues. The gene sequence of protein TaHIN A was designated TaHIN1-6A gene. The full length of TaHIN1-6A gene is shown as sequence 1 (1593 bp) in the sequence table, the 1 st-53 st nucleotide from the 5' end is 5' UTR (53 bp), the 54 th-1142 nd nucleotide is open reading frame (1089 bp), the 1143 th-1593 st nucleotide is 3' UTR (451 bp). The protein whose coding sequence is as that of sequence 2 is designated as protein TaHIN A.

EXAMPLES 2, taHIN expression Pattern analysis of 1-6A Gene

1. Response of TaHIN A1-6A Gene to different stress

1. The wheat plants No. 10 which are in a leaf-one heart period and have consistent growth conditions are divided into eight groups, 15 plants in each group are respectively subjected to the following treatments (the first seven groups are respectively treated by different stress and phytohormones, and the last group is treated by a control):

(1) Drought stress: carrying out water culture by adopting PEG-6000 aqueous solution (osmotic potential is-0.5 MPa), wherein the culture condition is 20 ℃, and light and dark are alternated (12 hours of illumination/12 hours of darkness);

(2) High salt stress: carrying out water culture by adopting 250mM NaCl water solution under the culture condition of 20 ℃ and light and dark alternation (12 hours of illumination/12 hours of darkness);

(3) Low temperature stress: carrying out water culture by adopting deionized water under the culture conditions of 4 ℃ and light-dark alternation (12-hour illumination/12-hour darkness);

(4) Exogenous IAA (indoleacetic acid) treatment: water culture with deionized water (initial spraying with 0.1MIAA aqueous solution until the leaves are all wet) under 20 deg.C alternating light and dark (12 hr light/12 hr dark);

(5) Exogenous ABA (abscisic acid) treatment: hydroponic culture with deionized water (initially sprayed with 50 μm ABA aqueous solution until the leaves are fully wet) at 20deg.C with alternating light and dark (12 hr light/12 hr dark);

Leaves were collected at 0, 0.5, 1,2,3, 6, 12, 24, 48 and 72 hours of treatment, respectively, and were snap frozen with liquid nitrogen and stored at-80℃for further use.

2. The total RNA of wheat leaves collected in the step 1 is extracted by a Trizol method, a Quant REVERSE TRANSCRIPTASE reverse transcription kit is used for synthesizing a first-strand cDNA (Tiangen Biochemical technologies Co., ltd.), a Real-time quantitative PCR (Real-time quantitative PCR, qRT-PCR) method is adopted for detecting the response condition of TaHIN1-6A genes (detection primers are shown in table 2) to various adversity stresses and hormones, and a TUBLIN gene expressed in a constitutive mode is used as an internal reference (detection primers are shown in table 2). The primers for qRT-PCR are shown in Table 2.

TABLE 2qRT-PCR primers

Calculated according to the formula presented by LIVAK AND SCHMITTGEN: the relative expression amount (N),N＝2^－△△CT,△△CT＝(CT_(TaHIN1,Time _x)－CT_(TUBLIN,Time _x))－(CT_(TaHIN1,Time ₀₎－CT_(TUBLIN,Time ₀₎). of TaHIN A genes under 4 kinds of stress (drought stress, high-salt stress and low-temperature stress) and 2 kinds of hormone treatment is shown in the specification, wherein the meaning of CT value is as follows: the number of cycles that the fluorescent signal in each reaction tube undergoes when reaching a set threshold. When the PCR cycle reaches the cycle number of the CT value, the real exponential amplification period (logarithmic period) is just entered, and the minor error is not amplified at the moment, so that the reproducibility of the CT value is excellent, namely, the CT value obtained by amplification of the same template at different times or amplification in different tubes at the same time is constant. time x represents different processing time points; time 0 represents the zero point of the process.

Experiments were repeated 3 times and the results averaged and the results are shown in figure 1. In FIG. 1, A is that under the condition that PEG (polyethylene glycol) simulates drought, taHIN-6A firstly descends in leaves and then ascends slightly, then starts to descend after 2 hours, and after 24 hours of treatment, the expression level ascends again and then descends. There was little response to PEG stress in the roots. B is that under NaCl treatment, taHIN-6A reaches a peak value when the expression level in the blade is 3 hours, then descends, gradually ascends after 12 hours, and reaches a higher level after 72 hours; in roots, the expression level thereof was at a low level under salt stress, and after 24 hours, it began to rise and then declined. C is that under low temperature stress, the expression level of TaHIN-6A in the leaf gradually rises along with the extension of time, and the expression level reaches the highest level at 1 h. TaHIN1-6A in roots tended to stabilize at low temperature treatment. D is that under the treatment of indoleacetic acid (IAA), the expression level of TaHIN1-6A in roots rises along with the extension of time, reaches a first peak at 3h, then falls, reaches another peak at 48h, and reaches a peak value. And then descends. TaHIN 1A was expressed in roots similarly to leaves, reaching a maximum at 1h, but not in leaves. E is that under abscisic acid (ABA) treatment, the expression amount of TaHIN-6A in roots is higher than that in leaves, and the expression amount in the leaves shows a tendency of descending and then ascending, and reaches a peak value at 48 hours. While there was no obvious trend in the early stage of expression in roots, the first peak was reached at 48h, followed by a decrease.

The result shows that TaHIN-6A gene participates in the response to drought, high salt, low temperature stress response and hormone such as indoleacetic acid, abscisic acid and the like.

2. Expression level of TaHIN A1-6A gene in different development stages of wheat

The following materials were taken for the following periods:

The dry-selected No. 10 wheat seeds are planted in root tubes with the depth of 1.8m in 10 months of the last year, and the root systems of the wheat are cleaned and sampled in the jointing booting stage, the flowering stage and the heading stage respectively in 4-5 months of the second year. Ear picking, new leaf, sub-ear segment, two-segment, three-segment, root 0-30cm, root 30-60cm, root 60-90cm, root 90-120cm, and root 120-150cm. Flag leaves, upper small ears, middle small ears, lower ear sections, lower ear internodes, inverted two-node, inverted three-node, root 0-30cm, root 30-60cm, root 60-90cm, root 90-120cm, root 120-150cm and root 150-180cm are taken in flowering stage and heading stage.

The materials are preserved at-80 ℃ and used for tissue specific expression analysis of target genes.

Extracting total RNA of the materials, synthesizing first-strand cDNA by using a Quant REVERSE TRANSCRIPTASE reverse transcription kit, detecting the expression condition of TaHIN-6A genes in different tender tissues at different development periods by using a qRT-PCR method (TUBLIN genes are taken as internal reference genes), detecting the expression of TUBLIN genes by using a primer pair consisting of a primer F1 and a primer R1, and detecting the expression of TaHIN-6A genes by using a primer pair consisting of a primer F2 and a primer R2 (the primer sequences are shown in a table 1).

The experiment was repeated 3 times, and the relative expression amount results are shown in FIG. 2. The results showed that TaHIN A was expressed in various tissues at various times. The expression level is highest in the leaves in the seedling stage, the jointing stage, the heading stage and the flowering stage. There is also a small expression in the root of each period.

EXAMPLE 3 TaHIN1-6A subcellular localization

1. Recombinant vector construction

1. And (3) extracting mRNA of wheat No.10 in dry selection, and carrying out RT-PCR by using the mRNA as a template and adopting a primer pair consisting of L and R to obtain a PCR product 1.

L：5′-GCACTGTATGAGCAGCATGAGCT-3′；

R：5′-CAGTTCGTTCACAGAGCGT-3′；

2. And (3) taking the PCR product 1 in the step (1) as a template, and adopting a primer pair consisting of LF and LR to carry out PCR amplification.

LF:5′-CCAAATCGACTCTAGAATGGCTGATCGACGTTGCTTG-3′；

LR:5′-TGCTCACCATGGTACCATTTTGATCATTTTTTTGGGCACGTGC-3′；

In LF and LR, xbaI and KpnI cleavage sites are underlined, respectively.

2. And (5) recovering the PCR amplification product to obtain a PCR amplification product 2.

3. The pCMBIA-GFP vector was digested simultaneously with the restriction enzymes XbaI and KpnI. Recovering the cut carrier skeleton to obtain the double enzyme-cut carrier skeleton.

4. And (3) connecting the PCR amplification product 2 of the step (3) and the double-enzyme-cut vector skeleton of the step (4) by using T4 ligase to obtain a recombinant vector 35s which is TaHIN < 1 > -GFP. Sequencing results show that the structure of the recombinant vector 35s:: taHIN1-GFP is described as follows:

The recombinant vector 35s is TaHIN-GFP, wherein a small fragment between XbaI and KpnI cleavage sites of the pCMBIA-GFP vector is substituted for a DNA molecule shown as 54-1139 nucleotides from the 5' end of the sequence 1 of a sequence table, and the rest sequences are kept unchanged, so that the recombinant vector is named as the recombinant vector 35s is TaHIN-GFP. The recombinant vector 35s is TaHIN1-GFP expressing TaHIN-GFP fusion protein.

2. Transferring the target plasmid with correct base sequence comparison into agrobacterium strain

Recombinant plasmid pCMBIA1300-TaHIN1-GFP was transformed into EHA105 Agrobacterium competent, and after about two days of culture, we observed the growth of monoclonal colonies. After the PCR identification and confirmation, the correct monoclonal bacterial liquid is properly stored in a refrigerator for subsequent use. Next, we will conduct a tobacco transient expression experiment.

3. TaHIN1 positioning of 1-6A in tobacco lamina

1. Sowing tobacco seeds and cultivating seedlings: first, we uniformly broadcast the tobacco seeds on a substantially moist substrate. Over a period of about one week, the seeds germinated and seedlings developed. Subsequently, we transplanted each plantlet individually and when it grew to a stage with 4-5 leaves, these plants were ready for subsequent injection experiments.

2. Activating agrobacterium and preparing bacterial liquid: the agrobacterium preserved before is taken and streaked and activated on a plate containing double anti-Kan and Rif antibodies. The monoclonal was inoculated into LB liquid medium. Culturing until the OD value of the bacterial liquid is about 0.6-0.8, and the bacterial liquid is moderate in concentration at the moment and can be used for subsequent conversion experiments.

3. Resuspension of bacterial fluid and injection: and (3) centrifuging the agrobacterium tumefaciens bacterial liquid, re-suspending the agrobacterium tumefaciens bacterial liquid by using a heavy suspension, enabling the bacterial liquid concentration to reach an OD 600 of 0.1-0.5, culturing the agrobacterium tumefaciens bacterial liquid in dark place for 2-3 hours, and then injecting the agrobacterium tumefaciens bacterial liquid.

4. Observing and shooting positioning conditions: dark culture is carried out for 24 hours after injection, then transfer is carried out to culture under normal light for 48 hours, then tabletting is carried out, and a zeiss LSM900 inverted laser confocal microscope is used for observing TaHIN-6A protein GFP fluorescence signals.

As a result, GFP was distributed in the cell membrane, nucleus and cytoplasm, whereas TaHIN-GFP fusion protein was present only in the nucleus, indicating that TaHIN protein is a typical nuclear localization protein, as shown in FIG. 3.

Application of example 4, taHIN1-6A genes in improving drought resistance of plants

1. Obtaining transgenic wheat over-expressing TaHIN-6A Gene

1. Extracting mRNA of wheat No. 10 in dry selection, taking the mRNA as a template, and adopting a primer pair consisting of F and R to carry out RT-PCR to obtain a PCR amplification product 1 (through sequencing, the PCR amplification product 1 contains DNA molecules shown by nucleotides 1-1593 from the 5' end of the sequence 1 in a sequence table).

The sequence 1 is specifically as follows:

CCTCGGCCGGCCTCCTTGCTTCTATTCCTCCGTATAGGAAGTCCAGCAACAAGATGGCTGATCGACGTTGCTTGTCTGAGCGACAAGGCCATCATCTGCAATCAAATGGTGCAATTGATCTTGAAGTTGATGGTGGGACTACAGGTCTTCATCCAGAAATAGGAATCACAAAAGGTAACTCTCGAGCCAAATGGAGTCACCAAATGAAGCTGTACCTTATTAAACTCCTAAAAGATCACGATGTGCCTGGTTTTCGGACGCAGAATGCTTGGAGCAAGGAGGCATGGAATAATATTCTTCATCAACTCAATCAAAAGTTTGACCAGTCATTTACTCTCAACCAAGTCAAACAGAAGGAGCAAGATCTGAAGAGAGACTATCATACTGTGAAAAAGTTGTTGGATGTGAGTGGCTTTGGATGGGATAAGGACAGAAAAATGGTAGACGCACCTGATAGTGTTTGGGCAAGCTTTACTGCTCGTACGAACAGCAAGGATGCTCTCCAATGGAAAGAAAGATCATTTCCCTTTTATGAAGAATTAGCTCCACTCTATGAAGGTCGTTATGCTGAAGGGAGAACTCGCCAAGGCATGGACCATTATACTAGCAAGAGGAAGTATGCACCAGTTCCCTTGTCACAGTTGACACCGATGGCTGATCTCGATCAATCACCATCACCTACTATGCCGGTTACTGGTGAGTCGGACATGCGGTTTACTTTAGATGAAGAACTTGAGGAAACCAACTTGGATTCTCCCCCGCATCTATCTACACCTATTCAGCATGTGCAAGCCCCTCCAAGATCCACACAAATGGAGAAACATGACACTAGGCGTGGGAAAAAACAGAAGCGTAGTGCTACTGATGACTTCCATGAGAAGTATTTAAAACTGAAGAAGGAAGAAATTGATCGATTTGCTGCCATTGAGGAAAAGAAGCTAGAGGACCCCTACAGCATCAACAAGTGTATCACAACAATTGAAGGCTTGGAAGGTCTACAACTAGGAGATATGCTGATGGCATCAGATATCTTCAAATGTAAGGAGAACAGGGAAGTTTTCTTGTCCTACTCTACTAATGAACTACGGTTGGCTTGGCTTAAAAGAGAGATTGCACGTGCCCAAAAAAATGATCAAAATTAGCCATTGTGGTGGACTGGTGGTGATCCACTATGATCAGAGGTCATATTTTAGGGAAGCTTTTGGTCTGTGGGTGTCATATACTTTTGTCTAGTTAAATTGGTCATAGAACTGACCATGATGGTTTAGTATATTTTGGTAGGTATGTCGAGAGACTTTTGGTTAGGCATGCAGCAAAACTAGCTTGATCATAAGCATGAAGTCCAAGAATGGTATTACAACTTGCATCTCATATTTCAAGTACATGAATGGTAGCAGAACTTGGATAAAATATTACATATTCTGAGATCTTCGTGATACATATTGTGCCCACATTTGTTGTGCTATATAATCTCGTTTTTGATTCCCAGCTTCTCTGGAGTAATTAAAAGCATGTATATCATTGTGATAGTTGTGGTCTCCACTTGGCACATCAACAAATTGTTCTTGATCAATCTCCACAGTAGTATTCTCA

the sequence 2 is specifically as follows:

MADRRCLSERQGHHLQSNGAIDLEVDGGTTGLHPEIGITKGNSRAKWSHQMKLYLIKLLKDHDVPGFRTQNAWSKEAWNNILHQLNQKFDQSFTLNQVKQKEQDLKRDYHTVKKLLDVSGFGWDKDRKMVDAPDSVWASFTARTNSKDALQWKERSFPFYEELAPLYEGRYAEGRTRQGMDHYTSKRKYAPVPLSQLTPMADLDQSPSPTMPVTGESDMRFTLDEELEETNLDSPPHLSTPIQHVQAPPRSTQMEKHDTRRGKKQKRSATDDFHEKYLKLKKEEIDRFAAIEEKKLEDPYSINKCITTIEGLEGLQLGDMLMASDIFKCKENREVFLSYSTNELRLAWLKREIARAQKNDQN

F：5′-GCACTGTATGAGCAGCATGAGCT-3′；

R：5′-GCTGCATGCCTAACCAAAAGTCTCT-3′；

2. and (2) taking the PCR amplification product 1 obtained in the step (1) as a template, adopting a primer pair consisting of TF1 and TR1 to carry out RT-PCR amplification, and adopting high-fidelity enzyme Pfu to amplify a target gene to obtain a PCR amplification product 2.

TF1：5′-CGACTCTAGAGGATCCATGGCTGATCGACGTTGCTTG-3′；

TR1：5′-AGCTCGGTACCCGGGGATCCATTTTGATCATTTTTTTGGGCACGTGC-3′；

In TF1 and TR1, bamHI cleavage sites are underlined; the underlined front (5' side) sequence is the homology arm (the same fragment as the sequence near the insertion position of the vector pWMB band).

3. The vector pWMB was cut with the restriction enzyme BamHI, and the about 10kb vector backbone was recovered to obtain the digested vector 1.

4. The PCR amplified product 2 recovered In step 2 and the digested vector 1 (10 kb of vector bone) obtained In step 3 were subjected to homologous recombination by using a seamless ligase [ seamless ligase In-fusion was a seamless ligase In-fusion, which was purchased from TaKaRa (cat. Number: 638947) ] and the two were mixed and incubated at 37 for 30min, followed by conventional transformation to obtain a recombinant expression vector pWMB110-TaHIN1.

Sequencing results show that the recombinant expression vector pWMB-TaHIN 1 is obtained by replacing a small fragment between BamHI recognition sites of the vector pWMB by a DNA molecule shown as 54-1139 th nucleotides from the 5 'end of the sequence 1 in a sequence table, so that the DNA molecule shown as 54-1139 th nucleotides from the 5' end of the sequence 1 in the sequence table can be correctly expressed, and other nucleotides of the vector pWMB are kept unchanged, and is named as the recombinant expression vector pWMB110-TaHIN1.

5. Obtaining transgenic wheat OE16, OE18 and OE22 overexpressing TaHIN A Gene

Transferring the TaHIN-6A wheat expression vector (recombinant expression vector pWMB-TaHIN 1) into a wheat variety Fielder through agrobacterium (agrobacterium EHA 105), obtaining 10 homozygous lines through selfing, taking the 10 homozygous lines of the obtained TaHIN1-6A gene over-expressed wheat as test lines, respectively named as OE2, OE3, OE5, OE7, OE16, OE17, OE18, OE22, OE23 and OE24, detecting the relative expression quantity of TaHIN1-6A genes (taking TUBLIN genes as reference genes) in T3 generation plants (OE 2, OE3, OE5, OE7, OE16, OE17, OE18, OE22, OE23 and OE 24) of each test line by qRT-PCR, the expression of the reference gene TUBLIN was detected by using a primer set composed of F1 (5'-CGTGCTGTCTTTGTAGATCTCG-3') and R1 (5'-GACCAGTGCAGTTGTCTGAAAG-3'), the expression of TaHIN-6A gene was detected by using a primer set composed of F2 (GAGTGGCTTTGGATGGGATAA) and R2 (GCATAACGACCTTCATAGAGTGG), and as a result, as shown in FIG. 4, the receptor material Fielder had a very small amount of TaHIN-6A gene expression, and the expression of TaHIN-6A gene was detected in the above-mentioned over-expression lines, and OE16, OE18 and OE22 were selected as the strains to be tested for the following drought resistance detection.

2. Drought resistance identification of transgenic wheat

And (3) measuring plants: transgenic recipient variety wheat Fielder (negative control), T3 generation plants overexpressing lines OE16, OE18 and OE 22.

The experiment is divided into two groups, namely a drought treatment group and a control group;

The dry treatment group performs the following operations:

1. After treating the wheat seeds to be treated (seeds of wheat Fielder (negative control), over-expressed strains OE16, OE18 and OE 22) with 1%H ₂O₂ ℃ for 24h, sterile water was used for germination.

2. After germination and exposure, culturing the WT and the transgenic wheat seeds until the length of 2d coleoptile is 2cm, selecting seedlings with consistent germination and growth vigor, transplanting the seedlings to a major engineering building of the national academy of agricultural science, deeply burying the seedlings outside a plastic box with the depth of 40cm multiplied by 25cm multiplied by 11cm (length multiplied by width multiplied by height) under the ground for culture, filling nutrient soil (moist, just opening bags; brand PINDSTRUP) in each basin, adding 0.066kg of slow-release fertilizer (brand Osmocote controlled release fertilizer) and uniformly mixing. Firstly, arranging seeds in order, uniformly scattering soil for covering, and covering 0.2kg of the nutrient soil. Tap water was poured 2L per pot.

3. The test was repeated 3 times, wheat seedlings were cultivated outdoors in wheat growing season, water was replenished once every 4-5 days, the water replenishment amount was 1L/pot, when seedlings were grown to 2 leaves 1 heart (after 6 days of cultivation), a recording phenotype was photographed (recorded as before drought treatment, before Drought in FIG. 5), drought treatment was started after the recording phenotype was photographed (no additional watering was performed, other cultivation conditions were the same as above), when the WT and transgenic wheat phenotype plants of the drought treatment group exhibited significant differences (after 8 days of drought treatment, i.e., after 16 days of seed cultivation), a difference phenotype occurred), a recording phenotype was photographed, cell membrane drought stability and SOD activity were detected, and drought treatment was continued, until seedlings were drought to all dry and all withered and the root base had elasticity (after 19 days of drought treatment), rehydration was started (evenly watered for flowers, soil moisture of the cultivation box was wet, and the bottom had no excess water, green new leaves were grown after leaves were continued to be buried outdoors, and when the leaves were yellow, the number of surviving plants was counted when small Miao Bu distributed (after 7 days of rehydration), and the surviving plants were recorded (recorded in FIG. 5, phenotype, after Drought).

Statistics of drought survival rate of wheat at seedling stage:

After restoring the aqueous environment, we performed extensive statistics on individual strains and the number of surviving plants in each repeat. Subsequently, we calculate the survival rate using a specific formula: survival (%) = (number of surviving plants/number of initial plants) ×100%. From this calculation we have derived accurate survival data as shown in table 3.

Table 3 survival data

Strains of plants	WT	OE16	OE18	OE22
					Repeat 1 survival rate	23.33％	33.33％	46.67％	63.33％
Repeat 2 survival rate	30.00％	46.67％	43.33％	56.67％
					Repeat 3 survival rate	26.67％	50.00％	46.67％	66.67％

The results are shown in FIG. 5 (where WT is wheat Fielder, OE16, OE18 and OE22 are progeny plants of the wheat over-expressed strains OE16, OE18 and OE22, respectively, the upper half of FIG. 5 (Before Drought) is a pre-drought-treatment phenotype picture, the lower half of FIG. After Drought is a post-drought-treatment phenotype picture) and Table 3, where the pre-drought-treatment wild-type (wild-type, WT) and transgenic strains grow consistently, the number of surviving wild-type wheat is significantly less and the number of surviving transgenic strains is significantly greater after drought-treatment rehydration, indicating that the drought resistance of transgenic wheat is significantly enhanced over WT.

The control group was subjected to the following operations:

the control group was only different from the drought-treated group in that no drought treatment was performed, and the rest of the operations were identical to those of the drought stress group.

Identification of cell membrane stability:

Relative conductivities of WT, OE16, OE18 and OE22 offspring seedlings were determined by boiling and cell membrane stability (cell membrane stability,CMS)(Mao,X.,Zhang,H.,Tian,S.,Chang,X.and Jing,R.(2010)TaSnRK2.4,an SNF1-type serine/threonine protein kinase of wheat(Triticum aestivum L.),confers enhanced multistress tolerance in Arabidopsis.Journal of Experimental Botany.61,683-696).CMS(％)＝(1– conductivity before boiling/conductivity after boiling was assessed by 100%. The greater the CMS value, the higher the cell membrane stability, and the lower the extent of heat injury.

Three replicates were performed, 3 seedlings per line in each replicate, and the results averaged. The differences between the different strains were analyzed by SPSS (16.0) comparison. The results are shown in FIG. 6 (in FIG. 6, CK is a control group, drought is a drought treated group, WT is wheat Fielder, OE16, OE18 and OE22 are T3-homozygous plants for the wheat overexpressing strains OE16, OE18 and OE22, respectively). By detecting the cell membrane stability, the cell membrane stability of the over-expressed wheat is found to be obviously higher than that of the wild type after the drought treatment, which indicates that the cell membrane damage degree of the transgenic wheat is obviously lower than that of the wild type.

Detecting the content of superoxide dismutase (SOD):

Superoxide dismutase (Superoxide Dismutase, SOD) catalyzes the disproportionation of superoxide anions to produce H ₂O₂ and O ₂. SOD is not only superoxide anion scavenging enzyme, but also H ₂O₂ is mainly generated enzyme, and has important function in biological antioxidant system. The content of superoxide dismutase (SOD) of the sample to be measured was determined by using a content detection test box (BC 0175) provided by Beijing Soy Biotechnology Co., ltd. About 0.1g of sample (W) is weighed, ground into powder by a proofing machine, added with 1mL of extracting solution, vibrated and mixed uniformly, and centrifuged for 10min at the temperature of 5000 g and 4 ℃, and the supernatant is taken and placed on ice for testing.

Sample measurement:

Reagents in table 4 were added sequentially in 96 well plates or.

TABLE 4 Table 4

Reagent name (mu L)	Measuring tube	Control tube	Blank pipe 1	Blank pipe 2
					Sample of	20	20	-	-
Reagent one	45	45	45	45
					Reagent II	20	-	20	-
Reagent III	35	35	35	35
					Distilled water	70	90	90	110
Reagent IV	10	10	10	10

After thoroughly mixing, water bath was carried out at 37℃for 30min, the absorbance A of each tube was measured at 560 nm. Delta aassay = a assay-a control, delta a blank = A1 blank-A2 blank. If there is precipitate at the bottom, mixing, and measuring. ( The blank pipe 1 and the blank pipe 2 are respectively 1-2 pipes; with one control tube per sample )

1. Calculation of percent inhibition

Percent inhibition = (Δa blank- Δa assay) +.Δa blank x 100%

Calculated on the basis of sample mass

SOD activity (U/g mass) = [ percent inhibition ] percentage) x V inverse total/(W x V sample/(V sample total) x F

=10×Percent inhibition ≡ (1-percent inhibition) ≡w×f

The SOD activity of the overexpressed wheat before drought stress treatment was not different from that of the wild type except OE16, and after drought stress, the SOD activity was significantly higher than that of the wild type, as shown in FIG. 7 (in FIG. 7, CK is a control group, drought is a drought treated group, WT is wheat Fielder, OE16, OE18 and OE22 are T3 generation homozygous plants of the wheat overexpressing strains OE16, OE18 and OE22, respectively). The result shows that the drought resistance of wheat of which the number TaHIN A is over-expressed is increased.

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

Claims

1. Use of a protein, a substance that upregulates or enhances or increases expression of a gene encoding said protein, or a substance that upregulates or enhances or increases activity or content of said protein, for increasing drought resistance of a gramineous plant and/or for preparing a product that increases drought resistance of a gramineous plant and/or for breeding a gramineous plant;

The protein is any one of the following:

A1 Amino acid sequence is a protein shown in sequence 2;

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

3. The use according to claim 1 or 2, wherein the substance that up-regulates or enhances or increases expression of a gene encoding the protein is any of the following:

b1 A nucleic acid molecule encoding the protein of claim 1 or 2;

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

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

5. The use according to any one of claims 1 to 4, characterized in that the graminaceous plant is any one of the following G1) -G2):

g1 A) a plant of the genus Triticum,

G2 Wheat.

6. A method of improving drought resistance in a gramineous plant, comprising improving drought resistance in a gramineous plant by up-regulating or enhancing or increasing expression of a gene encoding a protein according to claim 1 or 2 or up-regulating or enhancing or increasing activity and/or content of a protein according to claim 1 or 2.

7. A method of breeding a gramineous plant, comprising up-regulating or enhancing or increasing the expression level of a gene encoding the protein of claim 1 or 2 or the activity and/or content of the protein in a gramineous plant of interest to obtain a drought-resistant gramineous plant having a drought resistance higher than that of the gramineous plant of interest.

8. The method according to claim 7, wherein the up-regulation or enhancement or increase of the expression level of the gene encoding the protein according to claim 1 or 2 or the activity and/or the content of the protein in the target plant is the introduction of the gene encoding the protein according to claim 1 or 2 into the target gramineous plant.

9. The use according to any one of claims 1 to 5, the method according to any one of claims 6 to 8, wherein the indicator of drought resistance is survival.

10. The use according to any one of claims 1 to 5, the method according to any one of claims 6 to 8 or the use or method according to claim 9, wherein the graminaceous plant is any one of the following:

g1 A) a plant of the genus Triticum,

G2 Wheat.