CN105037514B - Bermuda grass ' Tifway ' dehydrin protein Dehydrin-L and its encoding gene and probe - Google Patents

Abstract

The present invention relates to a kind of bermudagrass 'Tifway' dehydrin protein Dehydrin-L and its coding gene and probe, described protein is the protein of following (a) or (b): (a) by such as SEQ ID NO.4 (b) the amino acid sequence shown in SEQ ID NO.4 is substituted, deleted or added one or several amino acids and has bermudagrass 'Tifway' dehydrin protein derived from (a) protein. The present invention also provides a nucleic acid sequence encoding the above-mentioned protein, and a probe for detecting the above-mentioned nucleic acid sequence; the present invention uses genetic engineering technology to regulate the physiological response of bermudagrass 'Tifway' to drought and other adversity stress, so as to improve the drought-resistant ability of turfgrass It provides a theoretical basis for molecular breeding and has great application value.

Description

Bermudagrass 'Tifway' dehydrin protein Dehydrin-L and its coding gene and probe

technical field

The present invention relates to an important protective protein dehydrin-L and its encoding gene and probe in the response process of bermudagrass 'Tifway' to drought stress, and specifically relates to a bermudagrass 'Tifway' dehydrin protein Dehydrin-L and its encoding Genes and Probes.

Background technique

Bermudagrass (Cynodondactylon) is a very important warm-season turfgrass. It is a perennial herb of the Poaceae family. It has well-developed rhizomes and stolons. Good color and other advantages, widely used in sports grounds, playgrounds, parks and soil-fixing slope protection lawns at home and abroad. Therefore, it is one of the most valuable and widely used grass species among warm-season turfgrasses. It enjoys the title of "the master grass species" of warm-season turfgrasses, and has great social, economic and ecological values. Bermudagrass has a very strong ability to resist drought. The annual evapotranspiration is less than 400mm, which is only about a quarter of the annual water demand of crops such as corn and wheat. Some drought-resistant Bermudagrass varieties (such as 'Tifway') have annual rainfall below 250mm It can still grow well, so it is an excellent material for building water-saving lawns.

Dehydrin belongs to the LEA-Ⅱ family and is a highly heat-stable and hydrophilic LEA protein (late embryogenesis abundant proteins) in plants. It can be expressed in large quantities in the later stages of plant embryonic development and under adverse conditions, and widely exists. in the plant kingdom. It can be expressed in plants under adversity conditions such as drought, and its expression in adversity is closely related to the stress resistance of plants, and the expression level in resistant plants is higher than that in plants sensitive to adversity. This shows that it has a close relationship with the drought resistance of plants.

Bermudagrass 'Tifway' is one of the most drought tolerant varieties of Bermudagrass. Western blotting and gene expression analysis determined that dehydrin was closely related to drought tolerance in 'Tifway'. We obtained the EST sequence of a new gene by establishing the cDNA suppression subtractive library of 'Tifway' drought for 10 days, which is highly similar to the reported barley dehydrin DHN4 gene, indicating that the gene encodes a dehydrin. The results of RT-PCR proved that the expression level of this gene increased with the increase of drought degree, and the expression level in "Tifway" was significantly higher than that in drought-sensitive Bermudagrass varieties, thus showing that the dehydrin gene Played an important role in the process of Bermudagrass drought resistance.

The coding gene of dehydrin has been cloned from a variety of plants, including: Arabidopsis, rice, barley, oak, red sea olive and so on. However, the cloning, expression pattern and protein sequence of turfgrass dehydrin are still unclear. At present, there is no literature report related to the structure of bermudagrass dehydrin protein and its coding gene sequence.

Contents of the invention

The purpose of the present invention is to fill in the cloning and expression pattern analysis of Bermudagrass 'Tifway' Dehydrin-L gene and the blank of Bermudagrass 'Tifway' Dehydrin-L protein. The probe of described nucleic acid sequence; The present invention discloses the expression mode of Bermudagrass 'Tifway' Dehydrin-L protein and its nucleic acid sequence in the drought stress process of Bermudagrass 'Tifway', for the future utilization of genetic engineering technology to Dehydrin-L gene expression Regulating the spatio-temporal characteristics of bermudagrass provides a theoretical basis for improving the drought stress resistance and breeding of bermudagrass, which has great application value.

On the one hand, the present invention provides bermudagrass 'Tifway' dehydrin Dehydrin-L protein with drought stress response function and protection function, said protein is composed of amino acid sequence as shown in SEQ ID NO.4; or consists of The amino acid sequence shown in SEQ ID NO.4 is substituted, deleted or added with one or several amino acids and has the characteristics of Bermudagrass 'Tifway' Dehydrin-L protein. The expression level of this protein in cells varies greatly during different drought stress stages.

Preferably, the protein is obtained by deletion, insertion and/or substitution of 1 to 50 amino acids in the amino acid sequence shown in SEQ ID NO.4, or addition of 1 to 20 amino acids at the C-terminal and/or N-terminal sequence.

Further preferably, the protein is a sequence formed by replacing 1 to 10 amino acids in the amino acid sequence shown in SEQ ID NO.4 by amino acids with similar or similar properties.

In another aspect, the present invention provides a nucleic acid sequence encoding the above protein.

Preferably, the nucleic acid sequence is specifically: (a) the base sequence is as shown in the 1st to 543rd positions of SEQ ID NO.3; A sequence with at least 70% homology; or (c) a sequence capable of hybridizing to the nucleic acid shown in positions 1-543 of SEQ ID NO.3.

Preferably, the nucleic acid sequence is specifically the deletion, insertion and/or substitution of 1 to 90 nucleotides in the nucleic acid sequence shown in positions 1 to 543 of SEQ ID NO.3, and 5' and/or 3' A sequence formed by adding 60 or less nucleotides at the end.

In addition, the present invention also provides a probe for detecting the above-mentioned nucleic acid sequence, the probe is a nucleic acid molecule having 8 to 100 consecutive nucleotides of the above-mentioned nucleic acid sequence, and the probe can be used to detect whether there is an encoding dog in a sample. Nucleic acid molecules related to the 'Tifway' Dehydrin-L gene in tooth roots.

In the present invention, "isolated DNA" and "purified DNA" mean that the DNA or fragment has been separated from the sequences on both sides of it in the natural state, and it also means that the DNA or fragment has been accompanied by the natural state. The components of the nucleic acid are separated and have been separated from the proteins that accompany them in the cell.

In the present invention, the term "coded sequence of Bermudagrass 'Tifway' Dehydrin-L protein" refers to a nucleotide sequence encoding a polypeptide having Bermudagrass 'Tifway' Dehydrin-L protein activity, as shown in SEQ ID NO.3 The 1st to 543rd nucleotide sequence and its degenerate sequence. The degenerate sequence refers to the sequence generated after one or more codons in the 1st to 543rd nucleotides shown in SEQ ID NO.3 are replaced by degenerate codons encoding the same amino acid. Due to the degeneracy of codons, a degenerate sequence with a homology as low as about 70% to the 1st to 543rd nucleotide sequence shown in SEQ ID NO.3 can also encode the sequence shown in SEQ ID NO.4. the sequence of. The term also includes nucleotide sequences having at least 70% homology to the nucleotide sequence shown in SEQ ID NO.3.

The term also includes variants of the sequence shown in SEQ ID NO. 3 that encode the same function of native Bermudagrass 'Tifway' Dehydrin-L protein. These variations include (but are not limited to): deletions, insertions and/or substitutions, usually of 1 to 90 nucleotides, and additions of up to 60 nucleotides at the 5' and/or 3' ends.

In the present invention, the term "Bermudagrass 'Tifway' Dehydrin-L protein" refers to the polypeptide of the sequence shown in SEQ ID NO.4 having the activity of Bermudagrass 'Tifway' Dehydrin-L protein. The term also includes variants of the sequence of SEQ ID NO. 4 that have the same function as native Bermudagrass 'Tifway' Dehydrin-L protein. These variant forms include (but are not limited to): usually 1-50 amino acid deletions, insertions and/or substitutions, and addition of one or less than 20 amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of Bermudagrass 'Tifway' Dehydrin-L protein.

The variant form of Bermudagrass 'Tifway' Dehydrin-L protein of the present invention comprises: homologous sequence, conservative variant, allelic variant, natural mutant, induced mutant, can be with dog under high or low stringent condition The protein encoded by the hybridized DNA of the 'Tifway' Dehydrin-L related DNA of the tooth root, and the polypeptide or protein obtained by using the antiserum of the 'Tifway' Dehydrin-L protein of the bermudagrass.

In the present invention, "bermudagrass 'Tifway' Dehydrin-L conservative variant polypeptide" means that compared with the amino acid sequence shown in SEQ ID NO.4, at most 10 amino acids are replaced by amino acids with similar or similar properties peptide. These conservative variant polypeptides are preferably produced by substitutions according to Table 1.

Table 1

initial residue representative replacement preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn

Glu(E) Asp Asp Gly(G) Pro; Ala His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Leu

The invention also includes analogues of Bermudagrass 'Tifway' Dehydrin-L protein or polypeptide. The difference between these analogues and the related polypeptide of Bermudagrass 'Tifway' Dehydrin-L may be the difference in amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides listed above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, the expression pattern of bermudagrass 'Tifway' Dehydrin-L gene product can be analyzed by the method of real-time fluorescent quantitative PCR, that is, the presence or absence of the mRNA transcript of bermudagrass 'Tifway' Dehydrin-L gene in cells and quantity.

The detection method of the present invention for detecting whether there is a nucleotide sequence related to Bermudagrass 'Tifway' Dehydrin-L in a sample comprises using the above-mentioned probe to hybridize with the sample, and then detecting whether the probe is combined. The sample is the product of PCR amplification, wherein the PCR amplification primers correspond to the nucleotide coding sequence related to Bermudagrass 'Tifway' Dehydrin-L, and can be located on both sides or in the middle of the coding sequence. The primer length is generally 15-50 nucleotides.

In addition, according to the nucleotide sequence and amino acid sequence of Bermudagrass 'Tifway' Dehydrin-L of the present invention, bermudagrass 'Tifway' Dehydrin-L related homology can be screened on the basis of nucleic acid homology or expressed protein homology genes or homologous proteins.

In order to obtain the locus of genes related to Bermudagrass 'Tifway' Dehydrin-L, the cDNA library of Bermudagrass 'Tifway' can be screened with DNA probes, which were used under low stringency conditions to use ³² P for Bermudagrass 'Tifway' Dehydrin-L-related all or part of radioactive labeling obtained. A suitable cDNA library for screening was the library from Bermudagrass 'Tifway'. Methods for constructing cDNA libraries from cells or tissues of interest are well known in the art of molecular biology. Additionally, many such cDNA libraries are commercially available, eg, from Clontech, Stratagene, Palo Alto, Cal. This screening approach identified the nucleotide sequences of a gene family related to Bermudagrass 'Tifway' Dehydrin-L.

The bermudagrass 'Tifway' Dehydrin-L related nucleotide full-length sequence or its fragments of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

After the relevant sequences are obtained, the relevant sequences can be obtained in large quantities by the recombination method. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

In addition to recombinant production, fragments of the proteins of the present invention can also be produced by direct synthesis of peptides using solid phase techniques (Stewart et al., (1969) Solid Phase Polypeptide Synthesis, WH Freeman Co., San Francisco; Merrifield J. (1963) J. Am Chem. Soc 85:2149-2154). Protein synthesis in vitro can be performed manually or automatically. For example, peptides can be synthesized automatically using an Applied Biosystems Model 431A Peptide Synthesizer (Foster City, CA). Fragments of a protein of the invention can be chemically synthesized separately and then chemically linked to produce a full-length molecule.

Utilizing the 'Tifway' Dehydrin-L protein of Bermudagrass 'Tifway' Dehydrin-L protein of the present invention, through various conventional screening methods, substances that interact with the 'Tifway' Dehydrin-L protein of Bermudagrass 'Tifway' Dehydrin-L protein, or inhibitors and antagonists can be screened out.

Bermudagrass 'Tifway', as a common lawn grass, is widely used, and its market demand is also great. The present invention clones for the first time the coding sequence of an important response protein and a protective protein Dehydrin-L in the drought stress process of Bermudagrass 'Tifway', and analyzes the expression pattern of the Dehydrin-L gene by means of fluorescence real-time quantitative PCR, which will provide a basis for future use of genetic engineering. The technology regulates the temporal and spatial expression of Dehydrin-L gene, thus providing a theoretical basis for improving the drought resistance of turfgrass and breeding new varieties, and has great application value.

Description of drawings

Other characteristics, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

Fig. 1 is the homologous comparison (GAP) result of the nucleotide sequence of bermudagrass 'Tifway' Dehydrin-L gene and Cleistogenessongorica (Cleistogenessongorica) dehydrin gene mRNA of the present invention;

Fig. 2 is the homologous comparison (FASTA) result of the amino acid sequence of Bermudagrass 'Tifway' Dehydrin-L protein of the present invention and the long ear grass (Lophopyrumelongatum) dehydrin protein, wherein, the same amino acid is replaced by amino acid between the two sequences Single character marking.

Figure 3 is the expression level change of Bermudagrass 'Tifway' Dehydrin-L gene in the process of drought stress;

Figure 4 is the PCR verification of the positive monoclonal plaque of Bermudagrass ‘Tifway’ Dehydrin;

Figure 5 is the relative water loss rate of plants of wild type and Bermudagrass 'Tifway' Dehydrin-L transgenic Arabidopsis at different drought stress times;

Figure 6 is the drought stress phenotype observation of wild-type and Dehydrin-L transgenic Arabidopsis plants;

Fig. 7 is the plant relative conductivity value of wild type and Dehydrin-L transgenic Arabidopsis in drought stress for 4 days;

Figure 8 is a quantitative analysis of Dehydrin gene expression in wild-type and Dehydrin-L transgenic Arabidopsis plants under drought stress for 4 days.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific condition in the following examples, usually according to conventional conditions, such as molecular cloning such as Sambrook: the conditions described in the laboratory handbook (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggestion conditions of.

Embodiment 1 , the cloning of Bermudagrass 'Tifway' Dehydrin-L gene

1. Acquisition of plant material

Take bermudagrass 'Tifway' leaf tissue for RNA extraction;

2. Extraction of RNA

Use "RNA prep pure plant total RNA extraction kit" to extract total RNA (Trizol: Invitrogen), use formaldehyde denaturing gel electrophoresis to identify the integrity of RNA, and then measure the purity and concentration of RNA on a spectrophotometer (Thermo Scientific NANODROP1000Spectrophotometer) ;

3. Full-length cloning of genes

According to the EST sequence and protein function annotation results obtained from the isolation and subtraction library (SSH) established during the drought stress of Bermudagrass 'Tifway', the core fragment of Dehydrin-L gene of Bermudagrass 'Tifway' was obtained. The RACE method (SMARTer ^TM RACE cDNA Amplification Kit: Clonetech) was used for full-length cDNA cloning, which was carried out in three stages:

(1) RT-PCR to obtain the middle fragment of the gene

The extracted RNA was reverse-transcribed (Prime Script Ⅱ 1st Strand cDNA Synthesis Kit: Treasure Bioengineering (Dalian) Co., Ltd.), using the first-strand cDNA as a template, using primers Dehydrin-L F (SEQ ID NO.1) and Dehydrin- L R (SEQ ID NO.2) was used for PCR to amplify a 203bp fragment, which was recovered and connected to the pMD18-TSimple vector vector, using RV-M and M13-47 as universal primers, and using a terminator fluorescent label (Big-Dye , Perkin-Elmer, USA) method, sequenced on the ABI377 sequencer (Perkin-Elmer, USA), and the sequencing results were compared by BLAST (http://blast.ncbi.nlm.nih.gov/) on the NCBI website For the existing database (GenBank), it is known that its nucleic acid sequence and encoded protein have a high homology with the known Dehydrin genes of Cryptonium elongatum, Thinopyrum elongatum, Dactylis chinensis (Bermudagrass), and Dracula chinensis, It is initially considered to be a Dehydrin gene;

(2) 3′ RACE

Two rounds of nested PCR complete the amplification of the 3' terminal sequence.

First round: UPM+3'-GSP1 (5'-GCGAACAGTCCGTGATAACTGTCTGTCA-3')

Second round: NUP+3'-GSP2 (5'-TCGTGTAACATGATAAGATGGTCAGCCA-3')

UPM and NUP are supplied with the kit. 3'RACE obtained the 3' end sequence (228bp) of Bermudagrass 'Tifway' Dehydrin-L, recovered it, connected it to the pMD18-T Simple vector vector, used RV-M and M13-47 as universal primers, and used terminator fluorescent labeling (Big-Dye, Perkin-Elmer, USA), sequenced on the ABI377 sequencer (Perkin-Elmer, USA), and the sequencing results were carried out by BLAST (http://blast.ncbi.nlm.nih. gov/) Comparing the existing database (GenBank), it is known that its nucleic acid sequence and encoded protein have a high homology with the known Dehydrin genes of Cryptospermia amansifolia and Dactylis chinensis;

(3) 5′ RACE

Using the 5′RACE ready cDNA as a template, the 5′ terminal sequence was amplified by two rounds of nested PCR.

First round: UPM+5'-GSP1 (5'-ACCATCTTTATCATGTTACACGAACGTCG-3')

Second round: NUP+5'-GSP2 (5'-GAACGTCGTGACAGACAGTTATCACGGA-3')

UPM and NUP are supplied with the kit. 5'RACE obtained the 5' end sequence (705bp) of the bermudagrass 'Tifway' Dehydrin-L gene, recovered the connection and sequenced it with the same method as above, and spliced the sequencing results of the sequences obtained by the above three methods. The spliced sequence was submitted for BLAST analysis, and the results proved that the newly obtained Dehydrin gene from Bermudagrass 'Tifway' was indeed a dehydrin-related gene. The sequencing results were combined with NCBI's ORF Finding (http://www.ncbi.nlm.nih .gov/gorf) predicted that the start codon and stop codon of Bermudagrass 'Tifway' Dehydrin-L gene were found, and according to the obtained sequence, specific primers ORF- F(5′-ATGGAGCACCAGGGACAGTACGGC-3′), ORF-R(5′-TTAGTGCTGGCCGGGGAGCTTCTC-3′), PCR was carried out with the cDNA of Bermudagrass 'Tifway' as a template, and the 543bp of Bermudagrass 'Tifway' Dehydrin-L protein was amplified Full-length coding sequence (SEQ ID NO.3).

Example 2. Sequence Information and Homology Analysis of Bermudagrass 'Tifway' Dehydrin-L Gene

The full-length open reading frame sequence of the bermudagrass 'Tifway' Dehydrin-L gene of the present invention is 543bp, and the detailed sequence is shown in the sequence shown in SEQ ID NO.3. According to the sequence of the open reading frame, the amino acid sequence of Bermudagrass 'Tifway' Dehydrin-L protein is deduced, with a total of 180 amino acid residues, a molecular weight of 18.2kDa, and an isoelectric point (pI) of 8.78. For the detailed sequence, see SEQID NO.4 display sequence;

The open reading frame sequence of Bermudagrass 'Tifway' Dehydrin-L and the amino acid sequence of its encoded protein were compiled in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR using BLAST program Nucleotide and protein homology searches were carried out in the database, and it was found that it has 78% identity at the nucleotide level with the Dehydrin gene (accession number: FJ972827.1), as shown in Figure 1 (Query: the coding gene sequence of Bermudagrass 'Tifway' Dehydrin-L; Sbjct: the mRNA sequence of Cryptonium amansifolia Dehydrin). There are also 68% identity and 66% similarity, as shown in Figure 2 (Query: Amino acid sequence of Bermudagrass 'Tifway' Dehydrin-L protein; Sbjct: Amino acid sequence of Thiopyrum elongatum Dehydrin protein). It can be seen that the Dehydrin-L gene of Bermudagrass 'Tifway' has high homology with the Dehydrin genes of other known species in terms of both nucleic acid and protein levels.

Example 3. Differences in Expression of Bermudagrass 'Tifway' Dehydrin-L Gene at Different Stages of Drought Stress

1. Material acquisition: samples were taken from leaves of Bermudagrass 'Tifway' materials at different stages of drought stress (0 day, 5 days, 10 days, 15 days). Wrap the samples with aluminum platinum paper and put them into liquid nitrogen immediately, and then transfer them to -80°C ultra-low temperature refrigerator for storage until use;

2. Extraction of RNA: use RNA prep pure plant total RNA extraction (Trizol: Invitrogen); extract total RNA in different sample tissues of Bermudagrass ‘Tifway’;

3. Determination of the integrity, purity and concentration of RNA: use ordinary agarose gel electrophoresis (gel concentration 1.2;% 0.5×TBE electrophoresis buffer; 150v, 15min) to detect integrity, and the maximum rRNA brightness in the electrophoresis band should be The brightness of the second rRNA is 1.5 to 2.0 times, otherwise it means that the rRNA sample is degraded; for RNA with better purity, A ₂₆₀ /A ₂₈₀ and A ₂₆₀ /A ₂₃₀ are about 2.0, use a spectrophotometer to measure the OD value and calculate the RNA content;

4. Acquisition of cDNA: Using 500ng of total RNA as a template, reverse transcription was performed according to the instructions of the TaKaRa PrimeScript ^TM RT reagent Kit Perfect Real Time kit from Treasure Biotech to obtain cDNA for future use;

5. Design specific primers for real-time fluorescent quantitative PCR analysis of gene expression in various organs and tissues. According to the obtained bermudagrass 'Tifway' Dehydrin-L gene sequence, use primer design software to design for Real-time PCR Specific primers for the quantitative analysis of Dehydrin-L gene in medium, primer qDHN F (5′-CGGAGGAAGAAGGGAATC-3′), primer qDHN R (5′-AGTTCCGGAGCCGGTCA-3′), internal reference gene is ribosomal 18S gene, primer is 18S- F(5'-GTGACGGGTGACGGAGAATT-3'), 18S-R(5'-GACACTAATGCGCCCGGTAT-3');

6. Make the standard curve of the target gene and internal reference gene: use EASY Dilution (provided by the kit) to carry out gradient dilution of the standard cDNA solution, and then use the diluted cDNA solution as a template respectively, and use the specific primers of the target gene and internal reference gene Carry out Real-time PCR amplification, and draw the melting curve and standard curve after the reaction; analyze the melting curve to determine whether the melting curve of the target gene and the internal reference gene has a single peak, so as to determine whether a single PCR amplification product can be obtained by using the primer ; Determine the appropriate dilution factor of the template cDNA by a standard curve;

7. Real-time fluorescent quantitative analysis of the target gene in the sample to be tested: using the first strand of the synthesized cDNA as a template, the target gene and the internal reference gene are respectively amplified with specific primers for fluorescent quantitative analysis, and the Real-time PCR reaction is performed in BIO-RAD Chromo 4 real-time quantitative instrument, the reaction system is 20μL, the reaction adopts a three-step method, denaturation at 94°C for 20s, followed by 40 cycles: 94°C for 15s; 58°C for 15s; 72°C for 25s; after each amplification is completed , all do melting curves to check whether the amplified product is specifically produced;

8. Using the 2- ^△△Ct method for relative quantitative analysis, the results showed that the expression level of Dehydrin-L in Bermudagrass 'Tifway' increased significantly with the aggravation of drought stress (Figure 3). On the 5th day, 10th day, and 15th day of drought stress, the expression levels of the gene were 110.9, 136.8, and 313.4 times that of the control plants, respectively, indicating that the gene was significantly related to the response to drought stress and had obvious temporal and spatial differences.

Example 4 , Bermudagrass 'Tifway' Dehydrin-L Gene Transformation Model Plant Arabidopsis

(1) Construction of transformation vector

Specific primers Dehydrin_OFR-S (5′-AA GGATCC ATGGAGCACCAGGGACAGTA-3′) and Dehydrin_ORF-A (5′-A ACTAGT GTGCTGGCCGGGGAGCTT-3′) were designed from the start codon and stop codon respectively, and the full-length sequence of the gene On both sides, Bam HI and Spe I restriction sites were introduced respectively, and PCR was carried out using bermudagrass 'Tifway' cDNA as a template. The PCR product was recovered and connected to the pMD18-T Simple vector vector, and single clone plaque was picked for PCR verification. Bermudagrass 'Tifway' Dehydrin gene family has 2 members (Dehydrin-L, Dehydrin-S), and the longer fragment of about 550bp is Dehydrin-L (Figure 4, lanes 1-4), and the plasmid of the positive clone was extracted. Carry out BamHI and Spe I double digestion of the target fragment plasmid and PHB binary transformation vector, recover the digested PHB vector and Dehydrin-L fragment, and use T4 ligase to connect Dehydrin-L and PHB vector overnight in a 16°C water bath A transformation vector was constructed, and the vector was transformed into Agrobacterium GV3101.

(2) Transformation of Arabidopsis thaliana with Dehydrin-L

1. Pre-shake Agrobacterium: pick positive single clones into 25ml YEP liquid medium containing 50mg/L Kan, 50mg/L Gentamicin, and 25mg/L Rif, shake the bacteria at 200rpm at 28°C for 24h;

2. Expansion of Agrobacterium: Expand the pre-shaken Agrobacterium liquid into 400mL Kan-resistant YEP medium at a ratio of 1:100, cultivate at 28°C, 200rpm for 13-16h, and cultivate until the absorbance OD ₆₀₀ reaches 1.5- Bacteria collection between 2.0, the collection conditions are 23°C, 5000rpm, 8min;

3. Transformation of plants: (It is necessary to cut off all siliques and blooming and white florets on the plant on the day before or on the day of transformation) prepare 500mL of 1/2MS solution containing 5% sucrose, and use a small amount of MS solution to dissolve the collected siliques. Suspend the bacillus pellet, shake well, add 0.04% (v/v) Silwet L-77 and 10 μL 6-BA (mother solution is 1 mg/mL) to the remaining sucrose solution, stir well, and mix the two before transformation Mix well, soak the plant stems and inflorescences in the bacterial solution for 50 seconds, take out and drain the bacterial solution, put it in a disposable plastic bag, seal it and keep it moist. After all the plants were transformed, they were covered with a black box and incubated in the dark for 24 hours. Then take out the plants, place the plants upright, and water them for cultivation to ensure sufficient water for the plants.

(3) Screening of transgenic positive strains

The transformed plants were harvested after the siliques were fully mature, placed in a dry culture dish lined with filter paper at room temperature for one week to dry the seeds, then filtered the seeds with a 50-mesh stainless steel sieve, removed the siliques, collected the transgenic T0 generation seeds and The seeds were sown in plug trays, and the seedlings were screened for resistance with 0.05% (v/v) glyphosate to obtain transgenic plants of the T1 generation, and the screening was continued until the homozygous transgenic plants of the T3 generation were obtained.

Example 5, Physiological Research on Drought Stress of Arabidopsis Dehydrin-L Transgenic Plants

(1) Detection of water loss rate of Arabidopsis Dehydrin-L transgenic plants

Cut 0.5 g leaves of Arabidopsis wild-type and Dehydrin-L transgenic plants, place them in an aluminum box, weigh the leaves at different stages (0-12h), and calculate the water loss rate of different plants. Over time, the water loss rate of the Dehydrin-L transgenic plants was significantly lower than that of the control plants by about 5-15% ( FIG. 5 ), indicating that the Dehydrin-L gene has a better water retention effect on plants under in vitro drought conditions.

(2) Observation on drought stress phenotype of Arabidopsis Dehydrin-L transgenic plants

Arabidopsis wild-type and Dehydrin-L transgenic plants were planted in an artificial climate chamber with a temperature of 22°C, a light intensity of 6000 lux, and a photoperiod of 16 hours of light/8 hours of darkness, and were subjected to drought stress treatment for phenotypic comparison. After 4 days of drought stress, there were significant differences in the phenotypes of the wild type and Dehydrin-L transgenic Arabidopsis plants; the leaves of the wild type plants were dry and wilted, and the scapes were lodging; the leaves of the Dehydrin-L transgenic plants were less wilted, and the scapes were upright. The plant growth was also significantly greater than that of wild-type Arabidopsis (Fig. 6). It shows that Dehydrin-L transgenic plants have better drought resistance under drought conditions.

(3) Conductivity detection of Arabidopsis Dehydrin-L transgenic plants

Cut 0.2g leaves of Arabidopsis wild-type and Dehydrin-L transgenic plants under drought stress for 4 days, collect them in a 50ml centrifuge tube, add 20ml deionized water, place on a shaker for 24 hours, and measure the initial value of conductivity; Treat in a sterilizing pot (121° C.) for 15 minutes, and measure the final value of the conductivity. Calculate the relative conductivity value of the sample by using the ratio of the initial value to the final value. After 4 days of drought stress, the relative electrical conductivity of the wild-type plants was 87.7%, and that of the Dehydrin-L transgenic plants was 73.2%, significantly lower than that of the wild-type plants (Fig. 7). It shows that the degree of cell damage of Dehydrin-L transgenic plants is lower than that of wild-type plants under drought stress conditions.

(3) Differences in Dehydrin-L gene expression between wild-type and transgenic Arabidopsis plants under drought stress

Cut 0.2 g leaves of Arabidopsis wild-type and Dehydrin-L transgenic plants under drought stress for 4 days, extract RNA, prepare cDNA and perform real-time quantitative PCR analysis according to the method in Example 3 . The specific primers for quantitative analysis of Dehydrin-L gene in Real-time PCR are qDHN F (5′-CGGAGGAAGAAGGGAATC-3′), primer qDHN R (5′-TCTCCTTGATCTTGTCCAT-3′), internal reference gene is Arabidopsis actin2 gene, primer Act-F (5'-CTTGCACCAAGCAGCATGAA-3'), act-R (5'-CCGATCCAGACACTGTACTTCCTT-3'). Using the 2- ^△△Ct method for relative quantitative analysis, the results showed that the expression level of Dehydrin-L in the transgenic Arabidopsis thaliana after 4 days of drought stress was 1.52 times that of the internal reference gene actin2 and 16484 times that of the wild-type plants (Fig. 8). It indicated that Dehydrin-L was not expressed in wild-type plants.

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims (3)

1. a kind of protein that amino acid sequence by as shown in SEQ ID NO.4 forms.

2. a kind of nucleic acid for encoding protein described in claim 1.

3. nucleic acid as claimed in claim 2, it is characterized in that, the nucleic acid sequence is specially：

Base sequence is as shown in SEQ ID NO.3 the 1st~543.