CN106967724A - Afriocan agapanthus SK3Type dehydrin protein and its encoding gene and probe - Google Patents

Abstract

The present invention relates to a kind of Agapanthus praecox SK ₃ type dehydrin protein and its encoding gene and probe, said protein includes the following (a) or (b) protein: (a) composed of such as SEQ ID NO.4 A protein composed of the amino acid sequence shown; (b) the amino acid sequence shown in SEQ ID NO.4 is substituted, deleted or added with one or several amino acids and has the activity of Agapanthus SK ₃ type dehydrin protein derived from (a) of 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 invention provides a basis for improving the stress resistance of Agapanthus agapanthus and various ornamental flowers, lays a theoretical foundation for molecular assisted breeding of ornamental plants and preservation of germplasm resources, and has great application value.

Description

Agapanthus SK3 type dehydrin protein and its coding gene and probe

technical field

The invention relates to an important protective protein SK ₃ dehydrin protein in the process of responding to adversity stress of Agapanthus and its encoding gene and probe, in particular to an Agapanthus SK ₃ dehydrin protein and its encoding gene and probe.

Background technique

Dehydrin belongs to the second family of abundant proteins in the late embryonic development (LEAII), which can be expressed in large quantities and play an important role in the late stage of plant embryonic development and in plants under drought, low temperature, saline-alkali and other adversities, and has high hydrophilicity properties of stability, disorder, and oxidation resistance. A large number of plants accumulate dehydrin during embryonic development and in response to stress in response to abiotic stress environments such as drought, high temperature, severe cold, and high salinity. Many studies have confirmed that there is a positive correlation between the expression and accumulation of plant dehydrin and plant stress resistance under abiotic stress.

Agapanthus praecox, monocot perennial herbaceous flower, is native to southern Africa, also known as blue lily and African lily. Its plants are tall and straight, with beautiful leaf shape, large amount of flowers, long flowering period and high ornamental value. It is a common garden flower that is loved by people. It is mostly used in garden cultivation and cut flower production. It has the reputation of "love flower" in Europe and America.

In recent years, in the molecular research on plant stress resistance physiology, dehydrin genes have been isolated and cloned in wheat, barley, rice, corn, soybean, cotton, loquat, pear, oak and other crops and fruit trees. However, the cloning, expression pattern and protein sequence of dehydrin in ornamental plants, especially bulbous flowers, are still unclear. At present, there is no literature report related to the structure of Agapanthus dehydrin protein and its coding gene sequence.

Contents of the invention

Aiming at the defects of the prior art, the purpose of the present invention is to fill in the gaps in the cloning and expression pattern analysis of Agapanthus SK ₃ dehydrin gene and the blank of Agapanthus SK ₃ type dehydrin protein, and provide a kind of Agapanthus SK ₃ type dehydrin protein and It encodes genes and probes. The present invention discloses the physiological effects and expression patterns of Agapanthus SK ₃ gene transformed into Arabidopsis thaliana, in order to use genetic engineering technology to regulate the temporal and spatial characteristics of SK ₃ gene expression in the future, and to improve the stress resistance of Agapanthus and various ornamental flowers. The ability provides a basis, thus providing a theoretical basis for improving the stress resistance of ornamental flowers and molecular breeding, and has great application value.

In the previous study of this experiment, the cryopreservation experiment of embryogenic cells of Agapanthus was carried out, and through the comparative analysis of transcriptomics and proteomics data, it was screened that the SK ₃ type dehydrin protein was affected by the cryogenic compound stress at the transcriptional and protein levels. All have positive responses. Therefore, it is deduced that it plays an important regulatory role in protecting the cell activity of plants in the complex environment.

The purpose of the present invention is achieved through the following technical solutions:

In a first aspect, the present invention provides a kind of Agapanthus SK ₃ type dehydrin protein, including the following (a) or (b) protein:

(a) a protein consisting of the amino acid sequence shown in SEQ ID NO.4;

(b) A protein derived from (a) in which the amino acid sequence shown in SEQ ID NO.4 has been substituted, deleted or added with one or several amino acids and has the activity of Agapanthus SK ₃ type dehydrin protein.

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 the second aspect, the present invention provides a nucleic acid sequence encoding the above-mentioned Agapanthus SK ₃ type dehydrin protein.

Preferably, the nucleic acid sequence is specifically:

(a) base sequence such as SEQ ID NO.3 1st to 648th;

or (b) a sequence having at least 70% homology with the nucleic acid of SEQ ID NO.3 Nos. 1 to 648;

Or (c) a sequence capable of hybridizing to the nucleic acid of SEQ ID NO.3 1-648.

Preferably, the nucleic acid sequence is specifically the deletion, insertion and/or substitution of 1 to 90 nucleotides in the nucleic acid sequence 1 to 648 of SEQ ID NO.3, and the addition of 60 nucleotides at the 5' and/or 3' end A sequence of less than 1 nucleotides.

In the third aspect, the present invention also provides a probe for detecting the nucleic acid sequence of the above-mentioned Agapanthus SK ₃ type dehydrin protein, the probe is a nucleic acid molecule having 8 to 100 consecutive nucleotides of the above-mentioned nucleic acid sequence, and the probe The needle can be used to detect whether there is a nucleic acid molecule encoding Agapanthus SK ₃ -type dehydrin-related in the sample.

In a fourth aspect, a specific primer pair for amplifying the nucleic acid sequence of the Agapanthus SK ₃ type dehydrin protein, the primer pair is as follows:

ORF-S: 5'-ATGGCAGAGGAGAATGTGGA-3',

ORF-A: 5'-CTAATGAGCCTTCTCGGTCTC-3'.

In the fifth aspect, the present invention also provides the application of the above-mentioned Agapanthus SK ₃ type dehydrin protein coding gene, the base sequence of which is shown in the 1st to 648th positions of SEQ ID NO.3, and the application includes improving plant Resilience.

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 "Agapanthus SK ₃ type dehydrin protein coding sequence" refers to the nucleotide sequence encoding a polypeptide having the activity of Agapanthus SK ₃ type dehydrin protein, as shown in the first to the first sequence of SEQ ID NO.3. 648 acid sequence and its degenerate sequence. The degenerate sequence refers to a sequence generated after one or more codons are replaced by degenerate codons encoding the same amino acid in the 1st to 648th acids shown in SEQ ID NO.3. Due to the degeneracy of codons, a degenerate sequence with a homology as low as about 70% to the 1st to 648th acid sequence shown in SEQ ID NO.3 can also encode the sequence shown in SEQ ID NO.4. 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 can encode the same function of natural Agapanthus SK ₃ type dehydrin 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 "Agapanthus SK ₃ type dehydrin" refers to a polypeptide having the sequence shown in SEQ ID NO.4 having the activity of Agapanthus SK ₃ type dehydrin protein. The term also includes variants of SEQ ID NO. 4 that have the same function as the natural Agapanthus dehydratin SK ₃ 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 Agapanthus SK type ₃ dehydrin.

The variant form of Agapanthus SK ₃ type dehydrin of the present invention includes: homologous sequence, conservative variant, allelic variant, natural mutant, induced mutant, can be combined with Agapanthus SK under high or low stringent conditions The protein encoded by the DNA hybridized with type ₃ dehydrin-related DNA, and the polypeptide or protein obtained by using the antiserum of Agapanthus SK type ₃ dehydrin protein.

In the present invention, "Agapanthus SK ₃ type dehydrin 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 to form a polypeptide . 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 present invention also includes analogues of Agapanthus SK ₃ type dehydrin protein or polypeptide. The difference between these analogs and the Agapanthus SK ₃ type dehydrin-related polypeptide may be the difference in the 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 physiological effects of Agapanthus SK ₃ dehydrin gene in Arabidopsis can be analyzed by real-time fluorescent quantitative PCR, that is, the mRNA transcript of Agapanthus SK ₃ dehydrin gene in transgenic Arabidopsis can be analyzed presence and quantity.

The detection method of the present invention for detecting whether there is the nucleotide sequence related to Agapanthus SK ₃ type dehydrin 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 a product after PCR amplification, wherein the PCR amplification primers correspond to the nucleotide coding sequence related to the SK ₃ dehydrin of Agapanthus agapanthus, 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 Agapanthus SK ₃ type dehydrin of the present invention, it is possible to screen for homologous genes or homologous genes or homologous protein.

In order to obtain the array of genes related to Agapanthus SK ₃ dehydrin, the cDNA library of Agapanthus SK can be screened with DNA probes, which are related to Agapanthus SK ₃ dehydrin with ³² P under low stringency conditions. Fully or partially radioactively labeled. A suitable cDNA library for screening is that from Agapanthus chinensis. 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 identifies the nucleotide sequences of gene families associated with Agapanthus SK type ₃ dehydrin

The full-length sequence of SK ₃ -type dehydrin-related nucleotides of Agapanthus 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.

Using the Agapanthus SK ₃ type dehydrin of the present invention, through various conventional screening methods, substances that interact with the Agapanthus SK ₃ type dehydrin, or inhibitors and antagonists can be screened out.

Agapanthus has high ornamental value and is widely used. It is an excellent fresh-cut flower variety, and it is also the most expressive love flower besides roses. Its market demand is also increasing.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention clones the coding sequence of SK ₃ dehydrin in Agapanthus plant for the first time, and transforms it into the model plant Arabidopsis thaliana, and uses fluorescence real-time quantitative PCR to analyze the physiological function of SK ₃ dehydrin gene in Arabidopsis Effects and expression patterns provide a basis for regulating the spatiotemporal expression of SK ₃ -type dehydrin gene using genetic engineering technology in the future, improving the stress resistance of Agapanthus and various ornamental flowers, and providing a basis for molecular assisted breeding of ornamental plants and preservation of germplasm resources. The theoretical basis has been laid, thereby providing a theoretical basis for the rapid propagation of somatic embryos and the selection of 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 the SK ₃ type dehydrin gene of Agapanthus of the present invention and the nucleotide sequence of plum blossom (Prunus mume) dehydrin COR47-like gene mRNA;

Fig. 2 is the homologous comparison (FASTA) result of the amino acid sequence of Agamina SK ₃ dehydrin protein of the present invention and the dehydrin COR410 protein of Musa acuminata subsp. Malaccensis, wherein the same amino acid is between the two sequences The spaces are marked with amino acid single characters.

Fig. 3 is the phenotype observation of wild type (WT) and SK ₃ transgenic Arabidopsis plants under salt stress;

Fig. 4 is a histogram of root length of wild-type (WT) and SK ₃ transgenic Arabidopsis plants under salt stress;

Figure 5 is a histogram of weight per plant of wild-type (WT) and SK ₃ transgenic Arabidopsis plants under salt stress;

Figure 6 is the phenotype observation of wild type (WT) and SK ₃ transgenic Arabidopsis plants under osmotic temperature stress;

Figure 7 is a histogram of the rosette size of wild-type (WT) and SK ₃ transgenic Arabidopsis plants under osmotic stress;

Figure 8 is a histogram of weight per plant of wild-type (WT) and SK ₃ transgenic Arabidopsis plants under osmotic stress;

Figure 9 shows the phenotype observation of wild-type (WT) and SK ₃ transgenic Arabidopsis plants under low temperature stress;

Figure 10 is the phenotype observation of wild type (WT) and SK ₃ transgenic Arabidopsis plants under drought stress;

Figure 11 is the phenotype observation of wild-type (WT) and SK ₃ gene Arabidopsis plants under normal growth conditions;

Figure 12 is the quantitative analysis of SK ₃ gene expression in wild type (WT) and SK ₃ transgenic Arabidopsis thaliana.

detailed description

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 conditions in the following examples is usually according to conventional conditions, such as molecular cloning such as Sambrook: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention.

Cloning of embodiment 1 Agapanthus SK ₃ gene

1. Acquisition of plant material

Agapanthus praecox leaf tissue was taken for RNA extraction.

2. Extraction of RNA

Total RNA (Trizol: Invitrogen) was extracted with "RNA prep pure Plant Total RNA Extraction Kit", and the integrity of RNA was detected by 1% agarose electrophoresis, and then the purity and concentration.

3. Full-length cloning of the gene

According to the protein function annotation results of Agapanthus abaciens transcriptome sequencing (RNA-seq), the core fragment of SK ₃ gene of Agapanthus was obtained. The RACE method (SMARTerTMRACE cDNA Amplification Kit: Clonetech) was used for full-length cDNA cloning in three stages:

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

The core fragment of the gene was compared with the existing database (GenBank) by BLAST (http://blast.ncbi.nlm.nih.gov/) on the NCBI website, and its nucleic acid sequence and encoded protein were known to be similar to those of the known Ersui short The homology of the dehydrin gene of Pyrrhophyllum, Cryptomonas awnus and barley is very high, and it is preliminarily considered to be a dehydrin gene.

SK-S (SEQ ID NO. 1): ATCAAGGAAAAGCTCGGC

SK-A (SEQ ID NO. 2): TCATCGTGGCTAGCACTCT

The SK ₃ gene core fragment was amplified with the above primers to obtain a 284bp fragment. Recover and connect to the pMD18-TSimple vector carrier, and use SK-S and SK-A as sequencing primer pair, adopt the method of terminator fluorescent labeling (Big-Dye, Perkin-Elmer, USA), in ABI377 sequencer (Perkin -Elmer, USA) for sequencing.

(2) 3′ RACE

Using 3'RACE ready cDNA as a template, two rounds of nested PCR were used to amplify the 3' end sequence.

First round: UPM+3'-GSP1 (SEQ ID NO.5):

5′-CCGCCGTGGTAACCGAACAGGA-3′

The second round: NUP+3'-GSP2 (SEQ ID NO.6):

5′-CCCCGGCCACAACAAGAAGGAGG-3′

UPM and NUP are supplied with the kit. 3'RACE obtained the 3' end sequence (505bp) of the Agapanthus SK ₃ gene, recovered it, connected it to the pMD18-T Simple vector vector, and sequenced it with the same method as step (1).

(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 (SEQ ID NO.7):

5′-CGACCACGTCCTGTTCGGTTACCAC-3′

The second round: NUP+5'-GSP2 (SEQ ID NO.8):

5′-GCGGCGGTTTCTTCTTCTTTCTCGT-3′

UPM and NUP are supplied with the kit. 5' RACE obtained the 5' end sequence (444bp) of the Agapanthus SK ₃ gene, and sequenced in the same way as step (1) after recovering the connection.

The sequencing results of the sequences obtained by the above steps 1-3 were spliced, and the spliced sequences were submitted to BLAST analysis. The results proved that the newly obtained Dehydrin gene from Agapanthus was indeed a dehydrin-related gene, and the sequence splicing results were combined NCBI's ORF (Open Reading Frame) Finder (https://www.ncbi.nlm.nih.gov/orffinder/) predicted that the start codon and stop codon of Agapanthus SK ₃ gene were found, and the The ORF region of lotus SK ₃ gene. According to the obtained sequence, design specific primers from the start codon and stop codon respectively to amplify the ORF region:

ORF-S (SEQ ID NO.9): 5'-ATGGCAGAGGAGAATGTGGA-3',

ORF-A (SEQ ID NO. 10): 5'-CTAATGAGCCTTCTCGGTCTC-3'.

Using Agapanthus cDNA as a template to perform PCR, a 648bp full-length coding sequence (SEQ ID NO. 3) encoding Agapanthus SK ₃ type dehydrin protein was amplified.

Example 2 , Sequence Information and Homology Analysis of Agapanthus SK ₃ Gene

The full-length open reading frame sequence of the Agapanthus SK ₃ gene of the present invention is 648bp, 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 Agapanthus SK ₃ type dehydrin protein was deduced, with a total of 215 amino acid residues, a molecular weight of 23.7232kDa, and an isoelectric point (pI) of 4.79. For the detailed sequence, see SEQ ID NO.4 display sequence;

The open reading frame sequence of Agapanthus SK ₃ gene and the amino acid sequence of its encoded protein were stored in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR databases using BLAST program Nucleotide and protein homology searches were carried out, and it was found that it had 84% identity with Prunus mume dehydrin COR47-like gene (accession number: XM_008222942.1) at the nucleotide level, as shown in Figure 1 (Query: the coding gene sequence of Agapanthus SK ₃ ; Sbjct: the mRNA sequence of dehydrinCOR47-like gene of plum blossom); at the amino acid level, it is 56% consistent with COR410 (accession number: XP_009382058.1) , as shown in Figure 2 (Query: amino acid sequence of Agapanthus SK ₃ ; Sbjct: amino acid sequence of COR410 protein of Agapanthus chinensis).

ClustalX alignment shows that the homology of each sequence is low outside the Y/S/K conserved domain, but higher within the conserved domain, indicating that the Y/S/K functional fragment of dehydrin is highly conserved .

It can be seen that the SK ₃ gene of Agapanthus has high homology with the Dehydrin genes of other known species at both nucleic acid and protein levels.

Embodiment 3 , Agapanthus SK ₃ gene transformation model plant Arabidopsis thaliana

1. Construction of the expression vector containing the gene of interest (Agapanthus SK ₃ gene)

According to the full-length coding sequence of Agapanthus SK ₃ gene (SEQ ID NO.3), design primers to amplify the complete coding reading frame, and introduce restriction endonuclease sites on the upstream and downstream primers (depending on the vector selected). ) to construct expression vectors. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the sequence of the coding region of the Agapanthus SK ₃ gene was connected to an intermediate vector (such as pMD19-T) for sequencing, and then the sequenced correct Agapanthus The coding region sequence of the SK ₃ gene was further cloned into an expression vector (such as pHB), and transformed into Agrobacterium tumefaciens (such as GV3101) under the premise that the reading frame was correct, and the transformed Agrobacterium was identified by PCR , to ensure that the plant expression vector containing Agapanthus SK ₃ gene was successfully transformed into Agrobacterium tumefaciens.

2. Agrobacterium tumefaciens-mediated transformation of Arabidopsis

(1) Pre-shake Agrobacterium: pick positive single clones into 25ml YEP liquid medium containing 50mg/L kanamycin, 50mg/L gentamicin, 25mg/L rifampicin, shake the bacteria at 28°C and 200rpm 24h;

(2) Expansion of Agrobacterium: Expand the pre-shaken Agrobacterium liquid into 400mL of kanamycin-resistant YEP medium at a ratio of 1:100, culture at 28°C and 200rpm for 13-16 hours, and cultivate until the absorbance OD600 Achieving a harvest between 1.5-2.0, the harvesting condition is 23°C, 5000rpm, 8min;

(3) Transformed 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 add 50μL of 6BA (100mg /L) and 200 μL Silwet L-77 to form the Agrobacterium transformation buffer. Take an appropriate amount of transformation buffer solution and suspend the Agrobacterium precipitate collected in step (2). After shaking well, soak the stem and inflorescence of the plant in the bacterial solution for 1 min, then take out and drain the bacterial solution, and use an opaque black plastic Wrap the plants in bags, and culture them in the dark for 24 hours, then take out the plants, culture them normally, and infect again after one week.

3. Screening of transgenic positive lines

After the siliques of the transformed plants are all mature, the seeds are harvested, placed in a dry culture dish with filter paper at room temperature for one week, and the seeds are completely dried, and then the seeds are filtered with a 50-mesh stainless steel sieve, the siliques are removed, and the transgenic T0 generation seeds are collected and collected. The seeds were sown in plug trays, and the seedling resistance was screened with 0.05% (v/v) glyphosate to obtain T1 generation transgenic plants, and the selection was continued until T2 generation transgenic plants were obtained.

4. Expression difference of SK ₃ gene in transgenic Arabidopsis plants

Cut 0.2 g leaves of Arabidopsis wild-type and T2 generation SK ₃ transgenic plants, extract RNA, prepare cDNA and perform real-time quantitative PCR analysis. The specific primers for quantitative analysis of SK ₃ gene in Real-time PCR are:

rtSK-S (SEQ ID NO.11): 5'-AAGAGCCAAGAGGAGGTT-3',

rtSK-A (SEQ ID NO. 12): 5'-CTTCTTCTCGCCGTCTTC-3'.

The internal reference gene is the Arabidopsis UBQ5 gene, and the primers are:

UBQ5-F (SEQ ID NO.13): 5'-GACGCTTCATCTCGTCC-3',

UBQ5-R (SEQ ID NO. 14): 5'-CCACAGGTTGCGTTAG-3'.

Using SEQ ID NO.11 and SEQ ID NO.12 as primers for wild-type and transgenic Arabidopsis cDNA for RT-PCR, using 2- ^△△Ct method for relative quantitative analysis, the results show that transgenic Arabidopsis SK The expression level of ₃ was relatively high, 7.10 times that of the internal reference gene UBQ5, and SK ₃ was not expressed in the wild type (Fig. 12).

Example 4. Observation of adversity stress phenotypes of Arabidopsis SK ₃ transgenic plants

The wild-type and T2 generation SK ₃ transgenic Arabidopsis plants were grown on 1/2MS medium for 3 days, and then transferred to salt stress medium with 100mM, 150mM, 200mM sodium chloride, and 300mM, 400mM, 500mM mannitol 1/2MS high osmotic stress medium, respectively, for salt stress and osmotic stress treatment. After growing in an artificial climate chamber with a temperature of 22°C, a light intensity of 6000 lux, a photoperiod of 16 hours, and light/8 hours of darkness, photos were taken and relevant phenotypic indicators were measured and counted. At the same time, the wild type and T2 generation SK ₃ transgenic Arabidopsis were sown in the soil, and the drought and low temperature stress treatments were compared and the phenotypes were observed.

The results shown in Figures 3-5 showed that SK ₃ transgenic Arabidopsis showed better stress resistance under sodium chloride stress. Under different stress conditions, 20 transgenic and wild-type Arabidopsis plants were selected for phenotypic index determination. The experimental results showed that after stress treatment for 10 days, the root length (Fig. 4) and weight per plant (Fig. 5) of the transgenic Arabidopsis were significantly improved compared with wild-type plants under 150mM NaCl stress; Under salt stress, the survival rate of the transgenic Arabidopsis (40%) is higher than that of the wild type (10%); the results shown in Figure 6-8 show that under the mannitol stress treatment, the phenotype of the transgenic plants is also better than that of the wild type type plants. Under different stress conditions, 20 transgenic and wild-type Arabidopsis plants were selected for phenotypic index determination. The experimental results showed that after 10 days of stress treatment, under 300mM mannitol stress, the transgenic Arabidopsis rosette size (Figure 7) and weight per plant (Figure 8) were significantly increased compared with wild-type plants.

For low temperature stress, after 4 weeks of normal growth in the soil, the transgenic and wild-type Arabidopsis were placed in a light intensity of 8000 lux and grown in a low-temperature incubator at 4°C for 9 days (Fig. 9). From the results in Figure 9, it can be seen that compared with wild-type Arabidopsis, SK ₃ transgenic Arabidopsis grows better and has stronger tolerance to low temperature environment.

For drought stress, the transgenic and wild-type Arabidopsis grew normally in soil for 4 weeks, then water was cut off for 7 days, 10 days, 20 days, and then rewatered (Fig. 10). From the results in Figure 10, it can be seen that compared with the normal watered control group, the wild-type plants in the stress group showed obvious stress damage, and the growth and development of the seedlings were inhibited. Compared with wild-type Arabidopsis, SK ₃ transgenic Arabidopsis had significantly improved drought resistance, and its growth status was less affected by drought stress compared with the normal irrigation control group. It indicated that the SK ₃ gene had a positive effect on enhancing the stress resistance of Arabidopsis under drought stress.

At the same time, under normal conditions (as shown in Figure 11), the growth rate of SK ₃ transgenic Arabidopsis was significantly improved compared with wild-type Arabidopsis, showing obvious early flowering traits.

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. In the case of no conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

SEQUENCE LISTING

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gcttatattt ttttcattga agtttataat taattaaaaa aatggcagag gagaatgtgg 120

aggtgagtga gagagggttg tttggtttcg tggggaagaa ggaagagaaa gaggagaaga 180

gccaagagga ggttctcgtc gccggagtcg agagcttgaa agttgaggag gcgaagaaag 240

aagaagttaa caaggagggg ctttttgata aattgcaccg atctcatagc tctagctctt 300

cgagcgacga agaggaagtg ggcgaagacg gcgagaagaa gaagaagaag aagaagaagg 360

gaataaagga gaagatcaag gaaaagctcg gccgcgacga gaaagaagaa gaaaccgccg 420

ccgccgtggt aaccgaacag gacgtggtcg tcgcggccgc ggccgccgaa gcggaagata 480

cgaccgtcgt cgtggagaag atcgaggaga ccgtcgtcgt ggagaagatc gaggaggaag 540

agaagaaagg gttcctcgat aagatcaaac agaagctccc cggccacaac aagaaggagg 600

ctgccgccgc cgccgaagtc gaggcgccgg cggcgaagga gagtgctagc cacgatgaag 660

gaggggagaa gaagggcatt ttcggaaaga tcatggaca gataccaggg tatcacaagg 720

aggacaagga gaccgagaag gctcattaga ggaggttat aattatgtgt cgctgtttat 780

tttatgtgtg tatgttttga atattaaatg tttgtgttga tcgagtgagt gctttggtta 840

ctgttttgtt tttgatttgt tagggttgtt tctttagtaa gttgagcatg caggatgtgt 900

atggagcttg cttttttgtg cgtggcaaca atcattttgt gttttaaaat gatgagagat 960

ggagtttgga gctaggttat gaatgaatgg ctggtgttga tgtatttttc catcgtggat 1020

aaatgtttta caaaaaaaaaaaaaaaaaaaaaaaaaa 1057

<210> 4

<211> 215

<212> PRT

<213> artificial sequence

<400> 4

Met Ala Glu Glu Asn Val Glu Val Ser Glu Arg Gly Leu Phe Gly Phe

1 5 10 15

Val Gly Lys Lys Glu Glu Lys Glu Glu Lys Ser Gln Glu Glu Val Leu

20 25 30

Val Ala Gly Val Glu Ser Leu Lys Val Glu Glu Ala Lys Lys Glu Glu

35 40 45

Val Asn Lys Glu Gly Leu Phe Asp Lys Leu His Arg Ser His Ser Ser

50 55 60

Ser Ser Ser Ser Ser Asp Glu Glu Glu Val Gly Glu Asp Gly Glu Lys Lys

65 70 75 80

Lys Lys Lys Lys Lys Lys Lys Gly Ile Lys Glu Lys Ile Lys Glu Lys Leu

85 90 95

Gly Arg Asp Glu Lys Glu Glu Glu Thr Ala Ala Ala Val Val Thr Glu

100 105 110

Gln Asp Val Val Val Ala Ala Ala Ala Ala Glu Ala Glu Asp Thr Thr

115 120 125

Val Val Val Glu Lys Ile Glu Glu Thr Val Val Val Glu Lys Ile Glu

130 135 140

Glu Glu Glu Lys Lys Gly Phe Leu Asp Lys Ile Lys Gln Lys Leu Pro

145 150 155 160

Gly His Asn Lys Lys Glu Ala Ala Ala Ala Ala Glu Val Glu Ala Pro

165 170 175

Ala Ala Lys Glu Ser Ala Ser His Asp Glu Gly Gly Glu Lys Lys Gly

180 185 190

Ile Phe Gly Lys Ile Met Asp Lys Ile Pro Gly Tyr His Lys Glu Asp

195 200 205

Lys Glu Thr Glu Lys Ala His

210 215

<210> 5

<211> 22

<212>DNA

<213> artificial sequence

<400> 5

ccgccgtggt aaccgaacag ga 22

<210> 6

<211> 23

<212>DNA

<213> artificial sequence

<400> 6

ccccggccac aacaagaagg agg 23

<210> 7

<211> 25

<212>DNA

<213> artificial sequence

<400> 7

cgaccacgtc ctgttcggtt accac 25

<210> 8

<211> 25

<212>DNA

<213> artificial sequence

<400> 8

gcggcggttt cttcttcttt ctcgt 25

<210> 9

<211> 20

<212>DNA

<213> artificial sequence

<400> 9

atggcagagg agaatgtgga 20

<210> 10

<211> 21

<212>DNA

<213> artificial sequence

<400> 10

ctaatgagcc ttctcggtct c 21

<210> 11

<211> 18

<212>DNA

<213> artificial sequence

<400> 11

aagagccaag aggaggtt 18

<210> 12

<211> 18

<212>DNA

<213> artificial sequence

<400> 12

cttcttctcg ccgtcttc 18

<210> 13

<211> 17

<212>DNA

<213> artificial sequence

<400> 13

gacgcttcat ctcgtcc 17

<210> 14

<211> 16

<212>DNA

<213> artificial sequence

<400> 14

ccacaggttg cgttag 16

Claims (9)

1. a kind of Afriocan agapanthus SK₃Type dehydrin protein, it is characterized in that, include the protein of following (a) or (b)：

(a) protein being made up of the amino acid sequence as shown in SEQ ID NO.4；

(b) amino acid sequence shown in SEQ ID NO.4 is by replacing, lacking or add one or several amino acid and have Afriocan agapanthus SK₃Type dehydrin protein activity as protein derived from (a).

2. Afriocan agapanthus SK as claimed in claim 1₃Type dehydrin protein, it is characterized in that, the protein is SEQ ID NO.4 Shown amino acid sequence is by the missing of 1~50 amino acid, insertion and/or replaces, or is added in C-terminal and/or N-terminal Sequence obtained from amino acid within 1~20.

3. Afriocan agapanthus SK as claimed in claim 2₃Type dehydrin protein, it is characterized in that, the protein is SEQ ID NO.4 In shown amino acid sequence 1~10 amino acid by the similar or close amino acid of property replaced formed by sequence.

4. Afriocan agapanthus SK described in one kind coding claim 1₃The nucleotide sequence of type dehydrin protein.

5. the Afriocan agapanthus SK is encoded as claimed in claim 4₃The nucleotide sequence of type dehydrin protein, it is characterized in that, the core Acid sequence is specially：

(a) base sequence is as shown in SEQ ID NO.3 the 1st~648；

Or (b) has the sequence of at least 70% homology with the nucleic acid shown in SEQ ID NO.3 the 1st~648；

Or the sequence that (c) can be hybridized with the nucleic acid shown in SEQ ID NO.3 the 1st~648.

6. the Afriocan agapanthus SK is encoded as claimed in claim 4₃The nucleotide sequence of type dehydrin protein, it is characterized in that, the core Acid sequence be specially the missing of 1~90 nucleotides in nucleotide sequence shown in SEQ ID NO.3 the 1st~561, insertion and/ Or substitution, or the sequence formed in 5 ' and/or 3 ' end additions 60 with inner nucleotide.

7. one kind is used to detect Afriocan agapanthus SK as claimed in claim 4₃The probe of type dehydrin protein nucleotide sequence, its feature exists In the probe is the nucleic acid molecules for including 8~100 continuous nucleotides of nucleotide sequence.

8. one kind amplification Afriocan agapanthus SK as claimed in claim 4₃The specific primer pair of the nucleotide sequence of type dehydrin protein, its It is characterized in that the primer pair is as follows：

ORF-S(SEQ ID NO.9)：5 '-ATGGCAGAGGAGAATGTGGA-3 ',

ORF-A(SEQ ID NO.10)：5′-CTAATGAGCCTTCTCGGTCTC-3′.

9. a kind of Afriocan agapanthus SK as claimed in claim 1₃The application of type dehydrin protein encoding gene, it is characterised in that described The base sequence of gene is as shown in SEQ ID NO.3 the 1st~648, and described application includes improving plant stress-resistance ability.