CN116769000A

CN116769000A - Application of transcription factor SmMYB13 in improving drought tolerance of plants

Info

Publication number: CN116769000A
Application number: CN202310854396.5A
Authority: CN
Inventors: 吴雪霞; 周亚茹; 黄倩茹; 张爱冬; 肖凯
Original assignee: Shanghai Academy of Agricultural Sciences
Current assignee: Shanghai Academy of Agricultural Sciences
Priority date: 2023-07-12
Filing date: 2023-07-12
Publication date: 2023-09-19

Abstract

The invention provides an application of a transcription factor SmMYB13 in improving drought tolerance of plants, belonging to the technical field of vegetables and adversity stress; the amino acid sequence of the SmMYB13 protein is shown as SEQ ID NO. 1. The SmMYB13 protein can respond to salt stress and drought stress. The SmMYB13 protein overexpression can promote the accumulation of wax on the plant epidermis, reduce the permeability of the plant epidermis, reduce the sensitivity of the plant to abscisic acid, improve the content of antioxidant enzyme in the plant and reduce the content of malondialdehyde, thereby improving the drought resistance of the plant.

Description

Application of transcription factor SmMYB13 in improving drought tolerance of plants

Technical Field

The invention belongs to the technical field of vegetables and adversity stress, and particularly relates to application of a transcription factor SmMYB13 in improving drought tolerance of plants.

Background

The surfaces of the stems, leaves, fruits and other organs of the plants are covered with a cutin layer composed of epidermal cells, and the cutin layer is divided into cutin and wax. The cuticle wax is the first barrier of plant cuticle to environmental contact, and plays an important role in protecting plants from various biotic and abiotic stresses. The wax on the plant epidermis is a mixture of multiple organic matters, the hydrophobic structure of the wax can densely cover the surface layers of plant leaves, and the physiological functional state of the wax is to effectively prevent the water loss, thereby reducing the transpiration, promoting the growth of plants in arid areas and achieving the self-balance of the water in the plants. The higher the wax thickness and the compactness of the plant epidermis leaf are, the stronger the drought resistance of the plant is.

Eggplants are a common vegetable in daily life, are rich in a large amount of anthocyanin, and have high nutritive value. In the middle and later growth stages (flowering and fruit setting) of eggplants, sufficient moisture is required, and the growth and development of eggplants can be seriously affected if drought is encountered. While there have been a great deal of research showing that plant cuticle waxes can improve drought resistance of plants, at present, such research is only conducted in corn, wheat, tomato and other species, and is rare in eggplants.

Disclosure of Invention

The invention aims to provide an application of a transcription factor SmMYB13 in improving drought tolerance of plants, wherein the SmMYB13 protein is derived from eggplants, and the SmMYB13 protein can regulate and control plant epidermis wax to plant epidermis wax.

The invention provides SmMYB13 protein, and the amino acid sequence of the SmMYB13 protein is shown as SEQ ID NO. 1.

The invention also provides a SmMYB13 gene of the SmMYB13 protein coding gene, and the nucleotide sequence of the SmMYB13 gene is shown as SEQ ID NO. 2.

The invention also provides application of the SmMYB13 protein or the SmMYB13 gene in regulating and controlling drought resistance of plants.

The invention also provides application of the SmMYB13 protein and/or the SmMYB13 gene in improving drought resistance of plants.

The invention also provides application of the SmMYB13 protein and/or the SmMYB13 gene in one or more aspects shown in 1) to 3) in positive regulation and control of the scheme: 1) Promoting the accumulation of wax on the plant epidermis; 2) Reducing the permeability of the plant epidermis; 3) Reducing the sensitivity of the plant to abscisic acid; 4) Increasing the antioxidant enzyme content and/or decreasing malondialdehyde content in plants.

Preferably, the SmMYB13 protein and/or the SmMYB13 gene in the scheme are positively regulated through reagent induction and/or through the adoption of genetic engineering means to overexpress the SmMYB13 protein and/or the SmMYB13 gene in plants; the reagent comprises NaCl, PEG6000 or abscisic acid.

Preferably, the working concentration of the abscisic acid comprises 10mg/L; the working concentration of NaCl is 0.1-0.15 mol/L; the working concentration of PEG6000 included 15% by volume.

Preferably, the plant comprises eggplant or arabidopsis thaliana.

Preferably, the antioxidant enzyme comprises one or more of SOD, POD and CAT.

The invention also provides a method for improving drought resistance of plants and/or constructing drought-resistant plants, which comprises the following steps: the SmMYB13 protein and/or the SmMYB13 gene according to the scheme are over-expressed in plants.

The invention provides SmMYB13 protein, and the amino acid sequence of the SmMYB13 protein is shown as SEQ ID NO. 1. The SmMYB13 protein can respond to salt stress and drought stress. The SmMYB13 protein overexpression can promote the accumulation of wax on the plant epidermis, reduce the permeability of the plant epidermis, reduce the sensitivity of the plant to abscisic acid, improve the content of antioxidant enzyme in the plant and reduce the content of malondialdehyde, thereby improving the drought resistance of the plant. After heterologous transformation of arabidopsis thaliana with the overexpression vector of the transcription factor SmMYB13 of the SmMYB13 protein, it was found that the wax content of arabidopsis thaliana was significantly increased. And the permeability of the arabidopsis leaves is changed after the SmMYB13 is over-expressed, and the wild type arabidopsis is found to be easier to dye and higher in water loss rate than the SmMYB13 over-expressed arabidopsis through the measurement of TB dyeing and water loss rate. Under ABA treatment, the germination rate and root length of wild type arabidopsis thaliana were significantly lower than that of SmMYB13 overexpressing lines. Through 10 days of drought treatment, wild type arabidopsis was found to be more severely stressed than SmMYB13 over-expressed strains. And SOD, POD and CAT enzyme activities all showed an increasing trend after drought stress, SOD, POD and CAT in SmMYB13 over-expressed lines were significantly higher than wild type. After the expression of the genes related to the ABA signals is measured, the genes related to the ABA are obviously up-down regulated after the SmMYB13 is over-expressed, so that the sensitivity to the ABA is reduced, and the drought resistance is enhanced.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the full length PCR cloning electrophoresis of SmMYB13 gene;

FIG. 2 is a diagram showing the analysis of the structure of SmMYB13 protein using an InterProScan on-line tool;

FIG. 3 is a prediction of the tertiary structure of SmMYB13 protein;

FIG. 4 is a validation result of SmMYB13 transcriptional activation activity;

FIG. 5 is a tissue quantification of the SmMYB13 gene;

FIG. 6 shows the relative expression of SmMYB13 gene, wherein A-G respectively represent different concentrations of NaCl treatment, different concentrations of PEG6000 treatment, 0.1mol/l LNaCl solution treatment, 10mg/LABA solution spraying, 15% PEG6000 solution treatment, 4 ℃ low temperature treatment and 45 ℃ high temperature treatment;

FIG. 7 is a screen for a resistance positive strain SmMYB 13;

FIG. 8 is a DNA level identification of an Arabidopsis over-expression strain SmMYB 13;

FIG. 9 shows the expression of SmMYB13 gene in Arabidopsis thaliana over-expression strain;

FIG. 10 is the amount and composition of cuticle wax for leaves of wild-type and SmMYB13 over-expressed strains; wherein A is total wax content, B is wax component, C is wax identified in leaves of wild type and two transgenic plants; FA-fatty acids; ALK-alkanes; ALD-aldehyde; ALC-alcohol; EST-esters; KET-ketone;

FIG. 11 is a phenotype of Arabidopsis leaves of wild-type and SmMYB13 overexpressing strains after TB staining;

FIG. 12 is a statistical result of water loss rate of wild type Arabidopsis and SmMYB13 overexpressing strain Arabidopsis;

FIG. 13 shows germination percentage test; wherein A is the distribution condition of Arabidopsis seeds in a culture dish; B-F are the germination conditions of the wild type and the over-expression strain of the arabidopsis thaliana under different treatments respectively: b is control 1/2MS, C is 1/2MS+0.25 μMABA, D is 1/2MS+0.5 μMABA, E is 1/2MS+1 μMABA, F is 1/2MS+1.5 μMABA;

FIG. 14 shows root growth of wild type Arabidopsis thaliana and SmMYB13 overexpressing strain Arabidopsis thaliana under different treatments; wherein A is 1/2MS treatment, B is 1/2MS+10mu MABA treatment, C is 1/2MS+6% ABA treatment, D is root extension length statistics, and photographing statistics are carried out after 7 days;

FIG. 15 is a drought-treated wild-type Arabidopsis and SmMYB13 overexpressing strain;

FIG. 16 shows SOD, CAT, POD activity and MDA content of wild-type Arabidopsis and overexpressing lines after salt stress, wherein A is SOD activity, B is CAT activity, C is POD activity, and D is MDA content;

FIG. 17 is an expression pattern of ABA response and regulatory genes in wild type Arabidopsis and over-expressed SmMYB13 strains.

Detailed Description

The invention provides SmMYB13 protein, wherein the amino acid sequence of the SmMYB13 protein is shown as SEQ ID NO.1, and specifically comprises the following steps:

MGRTPCCEKKGLKRGPWNKIEDDLLINYIKKNGHPNWRALPKLAGLLRCGKSCRLRWTNYLRPDIKRGNFTPQEEDAIIKLHQVLGNRWSAIAAKLPGRTDNEIKNIWHTRLKKKMSDSQPQGTPKIKVETSKSNEIFDADTEPENSKDNSEISSPKTSIEIQQQPSSSTLSSSSSEDSCSNTNATSFESKDEIIWDNLLEVDDDIWSEVLWAPVDDNNLNLSLMEENYQINSSFNDNWFWDDLFTRSNELMLELPEL。

in the present invention, the SmMYB13 protein has a molecular formula of C ₁₂₉₅ H ₂₀₃₂ N ₃₆₂ O ₄₁₆ S ₉ The molecular weight is 29617.09. The isoelectric point (pI) was 5.11. The instability factor is 55.20, greater than 40, the protein is an unstable protein, the total number of negatively charged residues (Asp+Glu) is 41, and the total number of positively charged residues (Arg+Lys) is 33. The 34 th aspartic acid of SmMYB13 protein has the strongest hydrophilcity, the hydrophobicity value is-2.8, the 41 st leucine has the strongest hydrophobicity, and the hydrophobicity value is 1.433. The average hydrophobicity (GRAVY) was-0.846, less than 0, presumably hydrophilic protein, and the SmMYB13 protein was comprehensively presumed to be a hydrophilically unstable protein.

In the present invention, the phosphate site in the SmMYB13 protein is the most serine (Ser), next threonine (Thr), and least tyrosine (Tyr).

In the present invention, the SmMYB13 protein also has a MYB domain, a SANT domain (IPR 001005) and an HTH domain (IPR 017930), respectively; the SANT domain is present in the subunits of the nuclear receptor co-inhibitor and many chromatin remodeling complexes and has strong structural similarity to the DNA binding domain of MYB-related proteins. Both are tandem repeats consisting of three alpha helices arranged in a helical motif, each alpha helix containing a large number of aromatic residues. Although generally similar, there are differences that suggest that the SANT domain is functionally different from the MYB DNA binding domain. MYB-type HTH domains are one DNA-binding helix-turn-helix (HTH) domain consisting of about 55 amino acids, typically occurring in tandem repeats of eukaryotic transcription factors.

In the present invention, the SmMYB13 protein does not have a signal peptide and a transmembrane structure.

In the invention, the secondary structure of the SmMYB13 protein contains alpha-helix and random coil; the three-dimensional diagram of the tertiary structure of the SmMYB13 protein is shown in figure 3.

In the present invention, the SmMYB13 protein is localized mainly to the nucleus and mitochondria (table 1).

TABLE 1SmMYB13 protein subcellular localization prediction

The invention also provides a coding gene SmMYB13 gene of the SmMYB13 protein, wherein the nucleotide sequence of the SmMYB13 gene is shown as SEQ ID NO.2, and the coding gene SmMYB13 gene specifically comprises the following components:

atgggaagaactccttgctgtgagaagaagggattgaaaagaggtccatggaacaaaatagaagatgatttactcatcaattacattaagaaaaatggtcatcccaattggcgtgcacttccaaaacttgcaggtctattaaggtgcggaaaaagttgtaggcttcgatggactaattacttgagacctgatattaaaagaggcaattttactcctcaagaagaagatgccattatcaaattgcatcaagttcttggaaacaggtggtctgcaattgctgcaaaattaccaggaagaacagataatgaaataaaaaatatttggcacactcgtctgaagaagaaaatgagtgattctcagcctcaaggaaccccaaagataaaagtagaaacatcaaaatccaatgaaatatttgacgcagatacggagcccgagaattccaaggataattctgaaatatcgagtcctaaaacaagtattgagattcagcaacaaccaagttcttccacactatcatcatcatcgagtgaagattcatgttcaaacacaaatgcaacgagttttgagtcgaaagatgaaataatatgggataatttattggaagttgatgacgatatttggtccgaggtactatgggcaccggtcgatgacaataatcttaatttgtcgttaatggaggaaaattaccagattaattccagctttaatgataattggttttgggatgatctttttaccagatccaatgagttgatgttagaattacctgaattatga。

the invention clones and sequences SmMYB13 gene (Sme2.5_00014.1_g00027.1), and discovers that the gene is 777bp long and codes 258 amino acids.

In the present invention, the SmMYB13 gene has transcriptional activation activity.

In the invention, the SmMYB13 gene is expressed in roots, stems, leaves, pericarps, flowers and pulps, and the expression content in the pericarps and leaves is the highest.

In the present invention, the SmMYB13 is evolutionarily closest to potato StMYB 13. The SmMYB13 gene has transcriptional activation activity through transcriptional activity analysis. SmMYB13 plays a major role in pericarp and leaf. SmMYB13 expression was significantly upregulated when eggplants were subjected to drought stress or abscisic acid (ABA) treatment.

In the invention, the improvement of drought resistance of plants is preferably realized by promoting the accumulation of wax on the surfaces of plants, reducing the permeability of the surfaces of the plants, reducing the sensitivity of the plants to abscisic acid, improving the content of antioxidant enzyme in the plants and reducing the content of malondialdehyde.

In the present invention, the promotion of the accumulation of wax on the surface of a plant skin preferably comprises promotion of the accumulation of wax on the surface of a plant leaf. In the present invention, the promotion of waxy accumulation on the surface of plant leaves is preferably achieved by one or more of (1) to (3): (1) increasing the total wax content of the plant leaf surface; (2) increasing the total alkane content on the plant leaf surface; (3) The aldehyde, ester and ketone contents of the plant leaves are reduced.

In the present invention, the reduction of plant sensitivity to abscisic acid is preferably achieved by regulating ABA-related gene expression. In the present invention, the regulation of ABA-related gene expression preferably comprises: negative regulation factors for positively regulating ABA signals and/or positive regulation factors for negatively regulating ABA signals; the negative regulator of the ABA signal preferably comprises ABI1 and/or ABI2; the positive regulator of the ABA signal preferably comprises one or more of ABF4, ABI5, GTG1, GTG2 and PYR1/RCAR 11.

In the present invention, the antioxidant enzyme preferably includes one or more of SOD, POD and CAT.

In the invention, the SmMYB13 protein and/or the SmMYB13 gene in the scheme are positively regulated through reagent induction and/or through the adoption of a genetic engineering means to overexpress the SmMYB13 protein and/or the SmMYB13 gene in plants; the reagent preferably comprises NaCl, PEG6000 or abscisic acid.

In the present invention, the working concentration of the abscisic acid preferably comprises 10mg/L; the application mode of the abscisic acid is preferably spraying; the working concentration of NaCl preferably comprises 0.1-0.15 mol/L; the working concentration of PEG6000 preferably comprises a volume concentration of 15%.

The means of the genetic engineering is not particularly limited, and the method common in the art can be adopted.

In the present invention, the plant preferably includes eggplant or arabidopsis thaliana.

The invention also provides application of the SmMYB13 protein and/or the SmMYB13 gene in improving the germination rate and/or promoting the rooting of plants under abscisic acid stress treatment and/or PEG stress treatment.

In the present invention, the working concentration at the time of the abscisic acid stress treatment is preferably 0.5 to 10. Mu.M.

In the present invention, the working concentration at the time of the PEG stress treatment is preferably 6% by volume.

The invention also provides application of the SmMYB13 protein and/or the SmMYB13 gene in regulating and controlling ABA related gene expression.

In the present invention, the regulation of ABA-related gene expression is the same as that described in the above scheme, and will not be described here.

For further explanation of the present invention, the application of the transcription factor SmMYB13 provided by the present invention to improving drought tolerance of plants is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.

Example 1 bioinformatics and expression profiling of eggplant SmMYB13 Gene

1. Materials and methods

1.1 plant Material test eggplant Material 'QPCQ' was autonomously bred by Shanghai national institute of agricultural sciences facility gardening.

1.2 reagent and instrument RNA extraction, reverse transcription and fluorescence quantitative kit are purchased from Shanghai next san Jose Biotech Co., ltd; the primers used were synthesized by Beijing engine (Shanghai). The instruments and equipment required by the test mainly comprise a pipetting gun, a centrifuge, a low-temperature refrigerated centrifuge, an ultralow temperature refrigerator, a high-temperature sterilization pot, an ultralow temperature workbench, a gel imager, a PCR instrument, a fluorescence quantitative instrument and the like.

1.3 test methods

1.3.1SmMYB13 Gene cloning

(1) Extraction of RNA

<1> transferring the eggplant tissue frozen at-80 ℃ into a mortar precooled by liquid nitrogen, and grinding into powder; <2> 100g of the powder was added to a 1.5ml centrifuge tube (RNase free) containing 500. Mu.l Buffer RLS lysate, and the mixture was vortexed at a high speed to be sufficiently lysed; <3> after the above lysate was allowed to stand for 2min, it was centrifuged at 12000rmp4℃for 2min; <4> aspirate supernatant to a new RNase free tube; <5> add 1/2 volume of absolute ethanol, transfer to PlantRNA Mini Column tube, centrifuge 12000rmp for 2min at room temperature, discard filtrate; <6> to Plant RNA Mini Column tube 600. Mu.l Buffer RWA was added, 12000rmp was centrifuged at room temperature for 1min, and the filtrate was discarded; <7> to a PlantRNA Mini Column tube, 750. Mu.l Buffer RWB (specified amount of absolute ethanol was added), and the mixture was centrifuged at 12000rmp at room temperature for 1min, and the filtrate was discarded; <8> to PlantRNA Mini Column membrane center dripping 50 u l DNase I reaction solution, room temperature standing for 15min;

<9> to PlantRNA Mini Column, 350. Mu.l Buffer RWB was added, and the mixture was centrifuged at 12000rmp for 1min at room temperature, and the filtrate was discarded; <10> to PlantRNA Mini Column, 750. Mu.l Buffer RWB was added, and the mixture was centrifuged at 12000rmp for 1min at room temperature, and the filtrate was discarded; <11> place the column of PlantRNA Mini Column on a new 2ml Collection Tube, 12000rmp centrifuge at room temperature for 2min; <12> the adsorption column of PlantRNA Mini Column was placed in a new RNase free Tube, 100. Mu.l of RNase free Water was added to the center of the membrane, and the mixture was allowed to stand at room temperature for 5min, and the mixture was centrifuged at 12000rmp for 2min at room temperature to obtain RNA, and the RNA was stored at-80 ℃.

(2) Reverse transcription of CDNA

The reaction solution was prepared as shown in Table 2, and the reverse transcription reaction was performed.

TABLE 2 composition of reaction solution

Reaction conditions: 15min at 37 ℃; 5sec at 85 ℃;4 ℃;

(3) PCR amplification

PCR was performed using cDNA as a template and MYB13-F (5'-gctgtgagaagaagggattgaa-3', SEQ ID NO. 3) and MYB13-R (5'-gcccatagtacctcggaccaaa-3', SEQ ID NO. 4) as primers to amplify the MYB13 gene. The PCR reaction system is shown in Table 3, and the PCR reaction conditions are shown in Table 4.

TABLE 3PCR reaction System

TABLE 4PCR reaction conditions

Note that: the denaturation, annealing and extension processes were carried out for 35 cycles.

1.3.2 construction of SmMYB13 Gene sequence and phylogenetic Tree

Predicting the gene structure of MYB13 by using online software FGENESH (http///ww software.. Com/berry.phtml); predicting a protein domain of MYB13 using online software InterProScan (https:// ww.ebi.ac.uk/interpro/search/sequence-search); predicting the secondary structure of MYB13 protein by adopting online software PSIPRED workbench (http:/bioin. Cs. Ucl. Ac. Uk/PSIPRED /); the tertiary structure of MYB13 protein was predicted by on-line software SWISS-MDDEL (http:// swissmodel. Expasy. Org/interactive).

Transcriptional Activity of 1.3.3MYB13 Gene

(1) And (3) constructing a carrier: inserting OFR sequences of MYB13 genes at two enzyme cutting sites of Ncol and BamHI of pGBKT7 vectors to construct recombinant vectors;

(2) Transforming empty pGBKT7 and pGBKT7-MYB13 into yeasts respectively

<1>Precooling 1.5ml centrifuge tube, adding 400ng of plasmid, 5ul of salmon sperm (Yeastmarker Carrier DNA), pre-denaturation of salmon sperm before use: 95 ℃ for 20min;<2>adding 50 μl of yeast competence, mixing, and adding 500ul PEG/LiAC for mixing;<3>incubating at 30deg.C for 30min, flicking and mixing every 10 min;<4>adding 20 μl DMSO, mixing, standing in a water bath at 42deg.C for 15min, and flicking and mixing every 5min;<5>centrifuging at high speed for 15sec, removing supernatant, adding YPDA liquid culture medium, and mixing;<6>shaking at 30 ℃ for 1h at 200rmp, centrifuging to remove supernatant, coating SD-Try and SD-Try-His plates by using 1 xTE suspension, and culturing for 2-3 days;<7>picking up the monoclonal and sterilizing with ddH ₂ Diluting with O, plating on SD-Try solid culture medium containing X-alpha-gal, culturing at 30deg.C in an inverted manner, and observing growth condition;

analysis of expression specificity of 1.3.4MYB13 Gene

Taking root, young stem, old stem, flower, pulp, peel and young leaf of QPCQ' growing for 90 days, and storing the old leaf at-80 ℃; after RNA extraction and reverse transcription of cDNA, fluorescent quantitative PCR was performed using primers MYB13-F (catgttcaaacacaaatgcaacgag, SEQ ID NO. 5) and MYB13-R (aagattattgtcatcgaccggtgcc, SEQ ID NO. 6); selecting healthy and full seeds of 'QPCQ', soaking for one night, sowing in a germination box, and moving to a plug tray after cotyledons are fully extended. When the seedlings grow to two leaves and one center, 0.1mol/l LNaCl solution treatment (continuous watering of eggplant seedlings with 0.1mol/l LNaCl solution, each watering, same amount of each seedling), 10mg/LABA solution spraying (spraying on leaf surface), PEG6000 solution treatment with volume concentration of 15%, 4 ℃ low temperature treatment and 45 ℃ high temperature treatment are carried out, and seedling leaves are taken at different time points (see D-G in FIG. 6) respectively for RNA extraction.

2. Experimental results

2.1SmMYB13 bioinformatics analysis

2.1.1 analysis of SmMYB13 Gene clone and protein Domain

The SmMYB13 gene was cloned and sequenced first, and the result shows that the SmMYB13 gene has a total length of 777bp and codes 258 amino acids (figure 1). Then, the domain of SmMYB13 protein is predicted by using InterProScan online software, and the result shows that the protein has two domains (figure 2), namely a SANT domain (IPR 001005) and an HTH domain (IPR 017930); the SANT domain is present in the subunits of the nuclear receptor co-inhibitor and many chromatin remodeling complexes and has strong structural similarity to the DNA binding domain of MYB-related proteins. Both are tandem repeats consisting of three alpha helices arranged in a helical motif, each alpha helix containing a large number of aromatic residues. Although generally similar, there are differences that suggest that the SANT domain is functionally different from the MYB DNA binding domain. MYB-type HTH domains are one DNA-binding helix-turn-helix (HTH) domain consisting of about 55 amino acids, typically occurring in tandem repeats of eukaryotic transcription factors.

2.1.2 analysis of transcriptional activation Activity of SmMYB13 Gene

In order to verify whether the SmMYB13 transcription factor has transcriptional activation activity, an OFR sequence of MYB13 gene is inserted into two enzyme cutting sites of Ncol and BamHI of pGBKT7 vector, and a recombinant vector is constructed; the recombinant vector pGBKT7-MYB13 and the empty vector are respectively transformed into yeast, and can normally grow on an SD-Try culture medium, which indicates that the yeast is successfully transformed; on SD-Try-His, pGBKT7 vector cannot grow, while pGBKT7-MYB13 vector can grow normally, indicating that His reporter gene is activated to express in yeast of pGBKT7-MYB13 vector. Meanwhile, pGBKT7-MYB13 can decompose X-alpha-gal to appear blue in the presence of X-alpha-gal, indicating that SmMYB13 transcription factor has transcriptional activation activity (FIG. 4).

2.2 analysis of expression specificity of SmMYB13 Gene

RNA is extracted from the roots, stems, leaves, pericarps, flowers and pulp of eggplants, and the expression quantity of the SmMYB13 gene in different tissues is analyzed. The results indicated that the SmMYB13 gene was expressed in roots, stems, leaves, pericarps, flowers and pulp, but the expression levels were highest in the pericarps and leaves, indicating that the SmMYB13 gene may play a major role in the pericarps and leaves (fig. 5).

Abiotic stress analysis of 2.3smyb13 gene

In order to further explore the functions of SmMYB13 in different stress conditions of eggplants, PEG6000 simulated drought treatment, salt treatment, high-temperature treatment, low-temperature treatment and ABA treatment are respectively carried out on the eggplant in the seedling stage. As can be seen from A and B in FIG. 6, the expression of SmMYB13 showed a trend of increasing and decreasing with increasing NaCl treatment and PEG6000 concentration, and the expression level was highest under stress of PEG6000 concentration at 0.1mol/LNaCl concentration and 15% by volume concentration. Selecting 0.1mol/LNaCl concentration and 15% PGE concentration for different time treatments, and the results show that after 1h of treatment, the expression level of SmMYB13 is obviously up-regulated and is 4.7 times of that of 0h, and then gradually reduced; after drought stress (PEG 6000 with 15% volume concentration) treatment for 2 hours, the expression quantity of SmMYB13 is obviously up-regulated and is 5.3 times of that of 0 hour, which shows that the SmMYB13 gene responds to salt stress and drought stress. Drought can induce the generation of ABA, so that the eggplant seedlings are subjected to ABA treatment, and after 2 hours of 10mg/ml ABA treatment, the expression quantity of SmMYB13 reaches the peak and is 12.7 times of that of 0 hour. After this, high and low temperature treatment was also performed, and the result showed that SmMYB13 hardly responded to high and low temperatures (fig. 6).

3. Discussion of the invention

And obtaining the gene MYB13 which is differentially expressed in the eggplant wax synthesis process through transcriptome sequencing screening. The SmMYB13 gene full-length sequence is 777bp, which codes 258 amino acids. And the protein has transcription activation activity, which is consistent with the characteristics of transcription factors; the qRT-PCR experiment result shows that the SmMYB13 gene is expressed in all tissues, but the expression content in the pericarp and leaf is highest, so that the gene is supposed to mainly play a role in the pericarp and leaf. Waxes play an important role in protecting against abiotic stress, and SmMYB13 was found to be most significantly upregulated mainly under drought stress and ABA treatment when eggplants were subjected to abiotic stress treatment.

Example 2SmMYB13 Gene regulates wax synthesis and response to drought stress

In order to deeply study the molecular mechanism of the SmMYB13 gene under drought stress, in the embodiment, the over-expression plant of the SmMYB13 gene in arabidopsis thaliana is adopted, and the function of the gene in the drought-tolerant process is explored by combining with the measurement of related physiological indexes, so that a theoretical basis is provided for cultivating new drought-tolerant eggplant varieties.

1. Plant material

The Arabidopsis thaliana Col-0 used in this example was stored in Shanghai national institute of agricultural science.

2. Test method

2.1 transformation of Arabidopsis inflorescence by dip-dyeing

Seed sterilization: soaking in 75% ethanol for 30s, and washing with sterile water for 5 times; after that, the mixture was shaken with 15% by volume of NaClO solution for 8min and rinsed 6 times with sterile water.

Sowing and transplanting: sowing sterilized Arabidopsis seeds on 1/2MS solid culture medium, vernalizing at 4deg.C for 72 hr,

taking out and placing in a tissue culture room at 22 ℃ for culture. The seedlings grow to the four-leaf stage, are moved into the sterilized substrate, are irradiated for 16 hours and are dark for 8 hours, and are cultured at 22 ℃.

Preparation of inflorescences before dip-dyeing: when the plant grows out of the top growing flower, the inflorescence growing points are subtracted to stimulate the growth of axillary inflorescences. Before dip dyeing, the horns and the inflorescences are cut off and water is poured.

Preparation of a dip-dyeing bacteria liquid: agrobacterium containing the SmMYB13 over-expression vector is activated, inoculated into YEP (containing 50mg/L Rif and 100 mg/LKan) liquid culture medium, cultured by shaking at 28 ℃ until OD600 is 1.2-1.5, and centrifuged at 6000rpm for 10min, and the supernatant is discarded. Resuspension with osmotic buffer (10 mM MgCl ₂ 5% sucrose, 0.05% sitfwetf-77 in 1L distilled water) to an OD600 of 0.6 to 0.7.

Dip dyeing: the previously prepared arabidopsis inflorescence was immersed in 200ml of resuspended bacteria liquid for about 1min, and the bacteria liquid was allowed to further permeate into the inflorescence by shaking continuously. Taking out, culturing in darkness for 24h, and culturing with light.

Identification and homozygosity: after the genetically transformed arabidopsis thaliana is mature, seeds are harvested, and the seeds are the T1 generation seeds.

Screening of resistant plants: the seeds of the transgenic arabidopsis thaliana are sown in the plug tray, when seedlings grow out, basta (basf European company, active ingredient 15%) is added with water according to the proportion of 1:500 to be prepared, and the seeds are sprayed on the seedlings of the transgenic arabidopsis thaliana, and the seedlings with Basta resistance survive.

2.2 extraction of DNA and RNA and identification of Positive plants

Extracting DNA of leaf by CTAB method to obtain DNA and H of non-transgenic Arabidopsis plant of same variety ₂ O is a negative control, and bacterial liquid is a positive control. Primers were designed based on the unique gene sequence on the pEarley gate 103-MYB13 recombinant vector:

pEarley gate 103-MYB13-F: ctatctgtcacttcatcgaaaggac (SEQ ID NO. 7), pEarley Gate 103-MYB13-R: cttggttgttgctgaatctcaatac (SEQ ID NO. 8), the amplified product was subjected to electrophoresis on a 1% agarose gel and photographed under observation under a gel imaging system.

2.3 Water loss Rate and TB dyeing

Water loss rate: the leaves were soaked in deionized water in the dark for 24 hours, the excess water was blotted with a napkin, and weighed on a microbalance every 30 minutes.

TB staining: 0.05% Toluidine Blue (TB) solution for 2h, then rinsed 3 times with deionized water and photographed.

2.4 analysis of tolerance of Arabidopsis to osmotic stress

Seeds of Arabidopsis WT and OE strains were sown on 1/2MS solid medium with ABA (0, 0.25, 0.5 and 1.5. Mu.M). The germination of arabidopsis seeds on these media was observed and counted using a split microscope and recorded by photographing at 4 d.

The sterilized Arabidopsis seeds were grown on 1/2MS solid medium for 4d, 10. Mu. MABA and 6% PEG in 1/2MS solid medium, and vertically cultured. Root length was measured at 7d of treatment and photographed.

2.5 drought treatment of transgenic Arabidopsis thaliana

Seeds of Col-0 and SmMYB13 over-expression lines are spread on a 1/2MS solid culture medium after being sterilized, the seeds are moved into a sterilized matrix (the matrix needs to be strictly weighed, the weight of the matrix in each pot is ensured to be consistent, the moisture is consistent), the seeds are cultured in 16h light/8 h darkness at 22 ℃ and under the condition that the moisture is proper for 4 weeks, then watering is stopped for drought treatment, and phenotypes are observed and photographed after 10 days.

2.6 determination of Arabidopsis related physiological index

SOD, POD and CAT activity and MDA content were measured using a kit from sco biotechnology limited.

2.7 analysis of expression of ABA responsive genes

For expression analysis of ABA response genes, seeds from wild-type arabidopsis and SmMYB13 over-expression lines were sown on 1/2MS solution saturated filter papers. After 21 days of growth, seedlings were treated on filter papers containing 100 μABA+1/2MS solution and sampled after 1h, 3h, 6h and 12h, respectively. RNA was extracted and the amount of fluorescence was measured. The primers are shown in Table 5.

TABLE 5ABA Signal modifier Gene primers

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2.8 determination of Arabidopsis leaf wax

Taking about 20 days of Arabidopsis leaves (before bolting Arabidopsis), taking leaves at the same position by using a puncher with the diameter of 1cm, taking 5 leaves with the diameter of 1cm from each leaf, repeating the steps as a sample, immersing the leaves into 10ml of chloroform, extracting for 1h, sucking 1ml into a centrifuge tube, drying by nitrogen, and weighing. The weight of empty centrifuge tubes was subtracted to give the total waxy content. Each material was repeated 3 times.

2.9 statistical analysis

Data analysis was performed using SPSS software and plotted using origin 9.0.

3. Results and analysis

3.1 identification of SmMYB 13-overexpressed Arabidopsis thaliana

The arabidopsis seeds over-expressing SmMYB13 are sown in the plug tray, after seedlings grow out, the seedlings are sprayed with the prepared Basta solution, as shown in fig. 7, the seedlings with Basta resistance grow strong, the seedlings without Basta resistance turn yellow, and the leaves die gradually. DNA was extracted when the plants were grown to 4 leaves, and DNA identification results showed that 10 of the 16 resistant plants tested had clear bands at approximately 777bp, and the PCR products were equal in length when primers were designed in comparison (FIG. 8). RNA is extracted from the 10 plants, three plants with highest expression plants are selected (figure 9), and after the seeds of the three plants are mature, the three plants are harvested in a separated mode and respectively named as OE-1, OE-4 and OE-6, and the plants are stored for later use for propagation so as to be used for later experiments.

3.2 waxy composition of leaves of wild type Arabidopsis and SmMYB13 overexpressing lines

GC-MS analysis showed that the total wax content on the leaf surfaces of the two overexpressing lines increased by a factor of about 2 compared to the wild-type (A in FIG. 10). The total alkane content is increased by more than 6 times compared with the wild type plant, and the fatty acid content in the over-expression strain is increased by only about 2 times compared with the wild type plant. Whereas the levels of aldehydes, esters and ketones were significantly reduced in the over-expressed lines, with the most reduced ketones, the over-expressed lines were reduced by about 3-fold compared to the wild type plants, and the esters were not significantly different in the two genotype plants (B in fig. 10). Fatty acid content was significantly increased in all of C16, C17, C18, C24, C25, C39, with C18 accounting for about 45% of the fatty acid content, and C18 content in the over-expressed strain increased by more than 4-fold compared to the wild type. The increase in alkane content is mainly dependent on about 10-fold increase in the content of C19 in the over-expressed strain compared with the wild type, and about 8-fold increase in the content of C20 and C21 in the over-expressed strain compared with the wild type. The reduction in ketone was mainly due to the reduction in C22 ketone (C in fig. 10).

3.3smyb 13 alters the permeability of the arabidopsis epidermis

Generally, the alteration of the waxy component affects the permeability of the plant epidermis, and therefore, we determined the permeability of Arabidopsis leaves by TB staining. We soaked Arabidopsis leaves of the wild type and overexpressing strains in 0.05% toluidine blue solution for 2h, and found that the wild type Arabidopsis leaves were more easily stained than the Arabidopsis leaves of the overexpressing strain (FIG. 11).

The water loss rate was measured for Arabidopsis leaves of the wild type and overexpressing strains, and it was found that the water loss rate was similar for the first 1.5h and that after 1.5h the water loss rate was progressively higher for the wild type leaves than for the overexpressing strain. These results indicate that overexpression of SmMYB13 decreases the permeability of arabidopsis leaves, which in turn can increase drought tolerance in transgenic arabidopsis (fig. 12).

3.4SmMYB13 reduces the sensitivity to ABA and improves the drought resistance of plants

In order to verify the function of the SmMYB13 gene in ABA response, the germination rates of the wild type and the overexpressing line under ABA treatment were examined, and it was found that the germination rates of the wild type and the overexpressing line on 1/2MS plates were similar, the germination rate of the overexpressing line reached about 90% or more the next day of treatment, and the remaining unmalted seeds were germinated successively at a subsequent time. Although the tendency of seed germination was reduced as compared to the control with increasing ABA concentration, the germination rate of overexpression was significantly higher after ABA treatment than that of the wild-type. For example, in the case of 1/2MS+0.5. Mu. MABA treatment, the germination rate of WT was only about 50% in the next day, whereas the over-expressed germination rate was as high as about 80% (FIG. 13).

The wild type and overexpressing strains were treated with 10 μmaba and 6% peg, and it was found by measurement analysis of root length that the elongation of the roots was inhibited for both the wild type and the overexpressing strain with 10 μmaba and 6% peg compared to the control, but the root length of the overexpressing strain was significantly higher than the wild type and there were more lateral roots, indicating that after overexpression of SmMYB13, the overexpressing strain was less affected compared to the wild type (fig. 14).

In order to explore the response of the SmMYB13 over-expressed Arabidopsis plants to drought stress, after the Arabidopsis grows for one month, watering is stopped, and after 10 days of stopping watering, almost all wild type Arabidopsis leaves are withered, but few of the over-expressed Arabidopsis leaves are dried up, so that the SmMYB13 over-expressed strain is less influenced by the drought stress compared with the wild type. (FIG. 15)

3.5 Effect of drought stress on Arabidopsis leaf MDA and antioxidant enzyme Activity

As is clear from FIGS. 16A to C, the wild-type Arabidopsis thaliana and the SmMYB13 overexpressing strain were subjected to drought treatment for 10 days, and the changes in SOD, POD and CAT activities were consistent and were induced to increase. The activities of the three antioxidase are obviously increased compared with the wild type after the drought stress treatment of the OE-1, the OE-4 and the OE-6. D in fig. 16 shows that MDA content increased significantly after 10 days drought treatment of wild-type arabidopsis and SmMYB13 overexpressing lines. The content of OE-1, OE-4 and OE-6 is significantly reduced compared to wild-type Arabidopsis thaliana.

3.6 overexpression of SmMYB13 alters expression of the ABA responsive Gene

To investigate whether SmMYB13 affects ABA signal-related gene expression, we treated wild-type arabidopsis and overexpressing strains with 100 μmaba, we first examined the expression of some stress response marker genes, and as a result found that ABA gene expression in wild-type arabidopsis and overexpressing strains, over time, the wild-type arabidopsis and overexpressing strains exhibited similar expression patterns. However, after ABA treatment, smMYB13 overexpressing lines showed significantly higher transcript levels than wild-type lines, and most transcript levels peaked after 3h ABA treatment. Thus, when we selected ABA regulatory factors, the transcriptional levels were measured 3h after treatment, and it can be seen from the figure that negative regulatory factors ABI1 and ABI2 of ABA signal were up-regulated in the over-expression system, whereas ABF4, ABI5, GTG1, GTG2, PYR1/RCAR11 positive regulatory factors were down-regulated in the over-expression system, and that the down-regulation of the relevant genes may limit ABA perception by transgenic plants, reduce ABA sensitivity, and enhance drought resistance to plants (fig. 17).

4. Discussion of the invention

This example shows that overexpression of SmMYB13 can enhance germination and root growth of arabidopsis seeds under ABA treatment, and that ABA signaling pathway can induce expression of SmMYB13 gene. We determined the expression of the stress response marker genes associated with the ABA signal, namely RD29A, RD, COR47, COR15A, NCED and KIN1, which might prevent stress-induced damage, the RD22 gene inducing drought and ABA responses through MYC and MYB pathways, while NCED3 participates in and mediates the ABA responses. When wild-type and SmMYB13 over-expressed strains are treated with ABA, these genes are both up-regulated, especially early in the treatment. The overexpressing plants exhibited higher levels of transcription compared to wild-type plants. Next, we analyzed the expression of ABA signal regulators in SmMYB13 over-expression lines, ABI1 and ABI2 encoding two negative ABA signal negative regulators, up-regulated in over-expression lines, while all positive regulators, including ABF4, ABI5, plasma membrane ABA receptor GTG1/GTG2 and PYR/PYL/RCAR, ABA receptor mediated signaling cascade genes PYR1/RCAR11, PYL2/RCAR13, snrk2.2 and snrk2.3 were down-regulated. Down-regulation of receptor-associated genes limits ABA perception by overexpressing SmMYB13 arabidopsis. Drought stress tests were performed on wild type and SmMYB13 over-expressed arabidopsis thaliana, and it was found that SmMYB13 over-expressed arabidopsis thaliana was more drought tolerant than wild type. In this example, the in-plant SOD, POD and CAT enzyme activities all showed an increasing trend after drought stress, and SOD, POD and CAT in SmMYB13 over-expressed lines were all significantly higher than wild type. SOD, POD and CAT are main antioxidase, when being stressed by the outside, the antioxidase activity in the plant body is increased, and the SOD, POD and CAT are used for removing active oxygen induced by the outside stress, reducing the toxic effect of the plant and protecting the plant.

In summary, this example identified a gene related to waxy synthesis, smMYB13, in eggplant. SmMYB13 promotes accumulation of Arabidopsis epidermal wax, reduces permeability of plant epidermis and sensitivity to ABA, thereby improving drought resistance of plants. The results of the embodiment lay a foundation for further researching the molecular mechanism of eggplant wax biosynthesis and provide candidate genes for improving the drought resistance and the fruit quality of the eggplant.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

The SmMYB13 protein is characterized in that the amino acid sequence of the SmMYB13 protein is shown as SEQ ID NO. 1.
2. The SmMYB13 gene encoding the SmMYB13 protein of claim 1, wherein the nucleotide sequence of the SmMYB13 gene is shown as SEQ ID NO. 2.
3. Use of the SmMYB13 protein of claim 1 or the SmMYB13 gene of claim 2 for modulating drought resistance in a plant.
4. Use of the SmMYB13 protein of claim 1 and/or the SmMYB13 gene of claim 2 to positively regulate and control plant drought resistance.
5. Use of the SmMYB13 protein of claim 1 and/or the SmMYB13 gene of claim 2 in the upregulation of one or more of the aspects indicated in 1) to 3):

1) Promoting the accumulation of wax on the plant epidermis;

2) Reducing the permeability of the plant epidermis;

3) Reducing the sensitivity of the plant to abscisic acid;

4) Increasing the antioxidant enzyme content and/or decreasing malondialdehyde content in plants.
6. The use according to claim 4 or 5, wherein the upregulation of the SmMYB13 protein of claim 1 and/or the SmMYB13 gene of claim 2 is achieved by agent induction and/or overexpression of the SmMYB13 protein and/or the SmMYB13 gene in plants by means of genetic engineering;

the reagent comprises NaCl, PEG6000 or abscisic acid.
7. The use according to claim 6, wherein the working concentration of abscisic acid comprises 10mg/L; the working concentration of NaCl is 0.1-0.15 mol/L; the working concentration of PEG6000 included 15% by volume.
8. The use according to claim 4 or 5, wherein the plant comprises eggplant or arabidopsis thaliana.
9. The use according to claim 5, wherein the antioxidant enzyme comprises one or more of SOD, POD and CAT.
10. A method for improving drought resistance of a plant and/or constructing a drought-tolerant plant, comprising the steps of: overexpression of the SmMYB13 protein of claim 1 and/or the SmMYB13 gene of claim 2 in a plant.