CN110452917B

CN110452917B - Wild grape VyGOLS gene and application of encoding protein thereof in drought stress

Info

Publication number: CN110452917B
Application number: CN201910872930.9A
Authority: CN
Inventors: 余义和; 郭大龙; 杨盛迪; 李旭飞; 孟祥轩; 杨英军; 张国海
Original assignee: Henan University of Science and Technology
Current assignee: Henan University of Science and Technology
Priority date: 2019-09-16
Filing date: 2019-09-16
Publication date: 2022-08-02
Anticipated expiration: 2039-09-16
Also published as: CN110452917A

Abstract

The invention relates to wild grapeVyGOLSThe application of gene and its coded protein in drought stress belongs to the field of plant gene engineering technology. The invention utilizes the transgenic technology of strong promoter (cauliflower mosaic virus 35S promoter) driving principle to transform Yanshan grapeVyGOLSTransferring the gene overexpression vector into arabidopsis thaliana to obtain a transgenic arabidopsis thaliana plant; experiments prove that the over-expression is realized compared with the Arabidopsis thaliana plant of the transformation empty vectorVyGOLSThe gene causes the accumulation of anti-stress related substances in the transgenic arabidopsis thaliana and the expression of drought-resistant related genes, and the drought resistance of transgenic plants is enhanced. Therefore, Ampelopsis grossedentataVyGOLSThe gene and the recombinant expression vector thereof can be used for breeding plant drought-resistant varieties.

Description

Wild grape VyGOLS gene and application of encoding protein thereof in drought stress

Technical Field

The invention relates to wild grapeVyGOLSThe application of gene and its coded protein in drought stress belongs to the field of plant gene engineering technology.

Background

The grapes are various in variety and have important edible value and economic value. Drought (drough) has serious influence on the growth and development process and yield quality of grapes, and becomes one of the main factors for restricting the growth of grapes and improving the quality of fruits, and particularly, the grape industry is greatly threatened by the frequent occurrence of global climate change and southern Drought in China in recent years. Under the large background of the problem of water shortage in the world, the exploration of drought-resistant grape resources and the research of drought-resistant genes of grapes have important scientific values and significance for improving the drought resistance of grapes, cultivating new drought-resistant varieties, saving water and cultivating and the like.

The Drought resistance (Drought resistance) of grapes is a quantitative trait controlled by multiple genes, requires the participation of multiple genes for stress response and adaptation such as Drought and the like, and is regulated and controlled by multiple ways. In mild drought stress, osmotic adjustment is a main way for a plant body to adapt to water deficiency, and on one hand, the plant improves the transcription level of the enzyme gene related to the synthetic osmotic adjustment substance, promotes the expression of the gene and increases the accumulation of an expression product; on the other hand, the plant enhances the expression level of enzyme genes (such as SOD, CAT, GST and the like) related to oxidation resistance and detoxification. When the stress intensity exceeds the ability to osmotically regulate, the dehydration protective substances of LEA proteins and sugars accumulate, protecting the biomacromolecule and the biofilm system from damage. Thus, the drought resistance of grapes depends on their own tissue structure and physiological properties, and comes to be based on the difference at the molecular level. With the publication of grape whole genome sequence, many genes related to drought stress and their regulatory factors have been isolated and cloned, and transformed into grape or model plant and their functions analyzed and verified.

GOLS is a key rate-limiting enzyme in raffinose synthesis. At present, 7 of Arabidopsis thaliana have been identifiedGOLSGene and three possibilitiesGOLSThe gene(s) is (are),AtGOLS1induced expression by drought, high salt and high temperature stress;AtGOLS2is driedAnd high salt stress induced expression; whileAtGOLS3The gene is only expressed under the induction of low-temperature stress. Overexpression in ArabidopsisAtGOLS2The gene can lead the transgenic plant leaves to accumulate a large amount of galactinol and raffinose, and obviously improve the drought resistance of the plants and the tolerance of a strong oxidant-Methyl Viologen (MV). Overexpression in RiceAtGOLS2The drought tolerance of the transgenic rice is obviously improved, and the yield of the transgenic rice strain under the drought condition is obviously higher than that of a control. A galactinol synthase (C) from Thellungiella halophilaTsGOLS2) Introduced into Arabidopsis thaliana, the salt and osmotic stress tolerance of transgenic Arabidopsis thaliana is improved (Selvaraj, M. G., et al., (2017). Overexpression of an Arabidopsis thaliana alkaline hormone synthase genes promoter in transgenic and amplified grain in the field. Plant biotechnology j ounal, 15(11), 1465. f. 1477.). Treatment of Arabidopsis plants with light, heat or hydrogen peroxideAtGOLS1AndAtGOLS2the expression level of gene mRNA is increased. Arabidopsis thalianaAtGOLS1No accumulation of galactinol and raffinose could be detected in the leaves after the gene mutant atgols1 was subjected to high temperature stress, which indicates thatAtGOLS1The function of the gene may be responsible for the synthesis of galactinol under heat shock stress. Overexpression of the alfalfa inositolgalactoside synthase Gene in tobacco: (MfGOLS1) The cold resistance and the permeability resistance of the transgenic tobacco are improved, and the RFOs content in the transgenic plants is obviously improved.

Although, there are some species in the prior artGOLSGene reports, but in wild grapeGOLSThe gene has not been reported, and people can treat wild grapeGOLSThe specific function of the gene is also poorly understood.

Disclosure of Invention

The invention aims to provide wild grapeVyGOLSThe gene can increase the accumulation of stress-resistant related substances and the expression of drought-resistant related genes in transgenic plants, and promote the drought resistance of the transgenic plants to be enhanced.

The invention also provides wild grape VyGOLS protein which can promote stress resistance related substances to be accumulated in transgenic plants to cause the drought resistance of the transgenic plants to be enhanced.

The invention also provides a grape wine containing wild grapeVyGOLSRecombinant expression vector of gene, the vector carries wild grapeVyGOLSGenes, thus being capable of overexpressionVyGOLSThe gene can further enhance the drought resistance of the plant.

The invention also provides the wild grape-containing grapeVyGOLSA method for producing a recombinant expression vector of a gene, which can produce the vector.

The invention also provides the wild grapeVyGOLSThe application of the gene and the recombinant expression vector in plant variety breeding can obtain drought-resistant plant varieties.

In order to achieve the purpose, the invention adopts the technical scheme that:

wild grapeVyGOLSThe amino acid sequence of the gene is shown in SEQ ID NO. 2.

The invention utilizes the transgenic technology of strong promoter (cauliflower mosaic virus 35S promoter) driving principle to realize the gene transferVyGOLSTransferring the gene overexpression vector into arabidopsis thaliana to obtain a transgenic arabidopsis thaliana plant; experiments prove that the over-expression is realized compared with the Arabidopsis thaliana plant of the transformation empty vectorVyGOLSThe gene causes the accumulation of anti-stress related substances in the transgenic arabidopsis thaliana and the expression of drought-resistant related genes, and the drought resistance of transgenic plants is enhanced.

Wild grapeVyGOLSThe nucleotide sequence of the gene is shown as position 186-1196 in SEQ ID NO. 1.

The nucleotide sequence is a sequence naturally existing in the wild grape, and the codon optimization can be carried out according to the sequence, so that the obtained optimized sequence has the same effect.

The amino acid sequence of the wild grape VyGOLS protein is shown in SEQ ID NO. 2.

The wild grape VyGOLS protein is protein containing 336 amino acids, and can promote stress resistance related substances to be accumulated in a transgenic plant to enhance the drought resistance of the transgenic plant.

A recombinant expression vector comprising Ampelopsis brevipedunculataVyGOLSGene of the wild speciesGrapeVyGOLSThe nucleotide sequence of the gene is shown as position 186-1196 in SEQ ID NO. 1.

The recombinant expression vector is a plant over-expression vector and can over-express a target gene in a plant.

A method of making a recombinant expression vector comprising: designing a primer according to the sequence shown in the 186-1196 site in SEQ ID NO.1, and cloning the wild grapeVyGOLSGene, then said wild grape is culturedVyGOLSThe gene is connected to a pCAMBIA2300 plant expression vector to obtain the gene.

The wild grape is used in the inventionVyGOLSThe gene open reading frame is connected to a plant over-expression vector pCAMBIA2300 to form a recombinant expression vector pCAMBIA 2300-VyGOLS.

The wild grapeVyGOLSThe application of the gene in plant variety breeding; in particular to application in plant drought-resistant variety breeding; more specifically, the application in the breeding of arabidopsis drought-resistant varieties. The recombinant expression vector is applied to plant variety breeding; in particular to application in plant drought-resistant variety breeding; more specifically, the application in the breeding of arabidopsis drought-resistant varieties.

According to the invention, by a plant genetic engineering technology, a DNA fragment of a complete coding section of a drought-resistant related gene is separated and cloned from the vitis vinifera, the function of the gene is verified, accumulation of stress-resistant related substances in transgenic arabidopsis thaliana and expression of the drought-resistant related gene after overexpression are found, and the drought resistance of a transgenic plant is enhanced. Therefore, Ampelopsis grossedentataVyGOLSThe gene and the recombinant expression vector thereof can be used for breeding plant drought-resistant varieties.

Drawings

FIG. 1 shows wild grape of the present inventionVyGOLSGene expression profile analysis;

FIG. 2 shows wild grape of the present inventionVyGOLSIdentification of gene overexpression vectors;

FIG. 3 shows the rotor of the present inventionVyGOLSPCR identification picture of gene arabidopsis plant;

FIG. 4 shows the present inventionVyGOLSDrought resistance identification diagram of gene arabidopsis plant;

FIG. 5 shows the present inventionVyGOLSThe physiological characteristic analysis chart of the gene Arabidopsis plant;

FIG. 6 is an expression analysis diagram of drought-resistant related genes in transgenic Arabidopsis plants according to the present invention.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The equipment and reagents used in the examples and the experimental examples were commercially available except as specifically indicated.

Wild grapeVyGOLSExample 1 of genes

The wild grape of this exampleVyGOLSThe nucleotide sequence of the gene is shown as position 186-1196 in SEQ ID NO. 1.

Example 1 of Vietnamese VyGOLS protein

The amino acid sequence of the wild grape VyGOLS protein in the embodiment is shown in SEQ ID NO. 2.

Example 1 recombinant expression vector

In this example, the recombinant expression vector comprises Ampelopsis grossedentataVyGOLSGene of said vitis amurensisVyGOLSThe nucleotide sequence of the gene is shown as position 186-1196 in SEQ ID NO. 1.

Example 1 method for preparation of recombinant expression vector

The method for preparing the recombinant expression vector in this embodiment comprises: designing a primer according to the sequence shown in the 186-1196 site in SEQ ID NO.1, and cloning the wild grapeVyGOLSGene, then said wild grape is culturedVyGOLSThe gene is connected to a pCAMBIA2300 plant expression vector to obtain the gene.

Wild grapeVyGOLSExample 1 application of genes in plant variety breeding

The wild grape of this exampleVyGOLSThe gene can increase the accumulation of anti-drought related substances and the expression of drought-resistant related genes in transgenic plants and promote the enhancement of drought resistance of the transgenic plants, so that the gene can be applied to the breeding of plant drought-resistant varieties, and particularly can be applied to the breeding of arabidopsis drought-resistant varieties.

Example 1 application of recombinant expression vectors to plant variety Breeding

In this example, the recombinant expression vector comprises Ampelopsis grossedentataVyGOLSGenes, and thus can be transformed into plants to obtain drought resistant plant varieties.

Test example 1 grapeVyGOLSAnalysis of expression characteristics of genes

After the tissue culture seedlings of the Yanshan grape are subjected to subculture for 16 d, seedlings with consistent growth, robustness and performance are selected for various stress treatments.

Drought treatment: the grape seedlings were pulled out of the medium, placed on filter paper, exposed to room temperature (32 + -1) deg.C, relative humidity of 55%, and light cycle of 14 h/dark 10 h, and sampled at 0, 2, 6, 12, and 24 h.

Low-temperature treatment: culturing the tissue culture seedling under the conditions of temperature of 4 +/-1 ℃, relative humidity of 75% and light cycle of 14 h/dark 10 h, and sampling for 0, 2, 6, 12 and 24 h.

Salt stress: 20 mL of 100 mmol. L was put in a triangular flask ^-1 The NaCl solution was cultured at 25. + -. 1 ℃ under a relative humidity of 75% and a light cycle of 14 hours/dark 10 hours, and samples were taken at 0, 2, 6, 12 and 24 hours.

An equal volume of distilled water was added to the flask as a control for salt stress treatment. Normally cultured tissue culture seedlings served as controls for drought and low temperature treatments.

The method comprises the steps of growing Yanshan grapes of 8-10 a in a field, taking grape fruits in a color-changing period, and taking samples of root systems (first newborn lateral roots), stems (stem sections of 4-5 leaves below newly-unfolded leaves), leaves (4-5 leaves below the newly-unfolded leaves), inflorescences and tendrils (1 st branch of newly-unfolded branches) and the like in a full-bloom period. Total RNA from grape leaves was extracted using plus plant Total RNA extraction kit (Tiangen). PrimeScript for Normal reverse transcription ^II 1st Strand cDNA Synthesis Kit (TaKaRa) synthesized the first Strand of cDNA.

The specific operation steps are as follows: adding to a PCR tube: random 6 mers (50. mu.M) 1. mu.L, dNTP mix (10 mM each) 1. mu.L, Total RNA 2. mu.g, RNase free dH ₂ Supplementing O to 10 μ L, mixing, and centrifuging to obtain solutionTo the bottom of the PCR tube. The reaction was carried out on a PCR instrument at 65 ℃ for 5 min and quenched on ice.

According toVyGOLSDesigning real-time fluorescent quantitative PCR primer for gene sequence,

the forward primer sequence is qRT-VyGOLS-F: 5'-GGGGACTATGTGAAAGGGGTT-3' (shown in SEQ ID NO. 3);

the reverse primer sequence is qRT-VyGOLS-R: 5'-GGATTTGGTTCTCAGGAGGG-3' (shown in SEQ ID NO. 4).

To be provided withVyGAPDHThe gene is the internal reference, and the gene is the internal reference,

the forward primer sequence is qRT-VyGAPDH-F: 5'-CCCTTGTCCTCCCAACTCT-3' (shown in SEQ ID NO. 5);

the reverse primer sequence is qRT-VyGAPDH-R: 5'-CCTTCTCAGCACTGTCCCT-3' (shown in SEQ ID NO. 6).

Real-Time fluorescent quantitative PCR was performed on a Bio-Rad IQ5 Real-Time PCR Detection System (Bio-Rad Laboratories, Herc. mu. Les, CA) according to TaKaRa SYBR Premix Ex Taq II (Perfect Real Time) instructions. 25 μ L of reaction system: 1 microliter of reverse transcription template; 1 mu L of forward and reverse primers respectively; 12.5 μ L of 2X SYBR Premix Ex Taq ™ house (2X); 9 μ L of nucleic-free water. The reaction procedure is as follows: at 95 ℃ for 30 s; 40 cycles of 95 ℃ for 5 s; 57 ℃ for 30 s; 72 ℃ for 30 s. Results adopt 2 ^-ΔΔC(t) The method is used for analysis.

The results are shown in FIG. 1 and show thatVyGOLSThe gene is mainly expressed in root systems, and then has higher expression level in leaves and lower expression level in stems, flowers, fruits and tendrils; after low-temperature, drought and high-salt treatment for 2 hours,VyGOLStranscripts accumulated rapidly, peaking at 6h and then declining gradually.

Test example 2 wild grapeVyGOLSConstruction of Gene overexpression vector

For studying grapesVyGOLSThe function of the gene will compriseVyGOLSA total of 1011bp ORF fragments, including the gene coding region, were correctly inserted into the plant over-expression vector pCAMBIA 2300-GFP.

Cloned according to the previous stageVyGOLSGene ORF sequence designed to amplifyVyGOLSUpstream and downstream of Gene ORFPrimer and method for producing the sameVyGOLS-ORF-F andVyGOLS-ORF-R; according to the restriction enzyme cutting site on the pCAMBIA2300-GFP vector, in the primerVyGOLS5' end of ORF-F plus restriction enzyme siteXbaI, the specific sequence is as follows:

VyGOLS-ORF-XbaI-F：5’-GGGTCTAGAATGGCCCCAGGAGTGCCCGCAGA-3' (shown in SEQ ID NO. 7);

in the primerVyGOLS5' end of ORF-R plus restriction enzyme siteKpnI, the specific sequence is as follows:

VyGOLS-ORF-KpnI-R：5’-GGGGGTACCTCAAGCAGCAGAGGGTGCGGGAA-3' (shown in SEQ ID NO. 8).

pMD18-T-VyGOLSPlasmid as template, usingVyGOLS-ORF-XbaI-F andVyGOLS-ORF-Kpnamplifying the I-R, recovering a target band, connecting the target band to a pMD19-T cloning vector, transforming TOP10 competent cells, performing blue-white spot screening on an LB culture medium with Amp, performing PCR and plasmid restriction detection on bacterial liquid, and performing pMD19-T-VyGOLSPositive clones were sent to the company for sequencing. By usingXbaI、KpnI double restriction enzyme recombinant cloning vector pMD19-T-VyGOLSAnd recovering a linearized vector and a target fragment from a plant expression vector pCAMBIA2300-GFP, connecting and converting TOP10, screening by Kan antibiotics, selecting monoclonal shake bacteria, detecting bacteria liquid, and performing quality-improving enzyme digestion detection. The detection results are shown in FIG. 2, M: DNA molecular mass standard; lane 1:VyGOLSdouble enzyme digestion identification of the gene over-expression vector; lane 2:VyGOLSa gene overexpression vector; the results show that the successful construction of the plant expression vector pCAMBIA2300-VyGOLS. It is transformed into Agrobacterium for transfection of plants.

Test example 3 grapeVyGOLSOverexpression of genes in Arabidopsis

Streaking agrobacterium containing recombinant plant expression vector on LB plate (60 mg/L Gent, 100 mg/L Kan) and culturing at 28 deg.c for 24 hr; selecting a single clone, and culturing the single clone in 10 mL LB liquid medium (added with corresponding antibiotics) for 24 h at the temperature of 28 ℃; transferring 5 mL of the bacterial liquid to 50 mL of fresh LB liquid culture medium, and continuously culturing at 28 ℃ until the OD600 of the bacterial liquid reaches about 0.6; transferring to a centrifugal bottle or a centrifugal tube, centrifuging for 10min at the rotation speed of 4000 rpm at room temperature, removing supernatant and collecting thalli; resuspended in permeation buffer (0.5 × MS, 5% sucrose, 0.03% Silwet L-77 (GE Health)) and adjusted to OD600 to 0.8; removing the existing fruit pods on the inflorescence of Arabidopsis, completely immersing the inflorescence in the penetrating fluid for 10-30 s (or directly dripping the penetrating fluid on the inflorescence by using a liquid transfer device), immediately removing the penetrating fluid on the leaves or stems of Arabidopsis, flatly placing the plant in a tray, covering the tray with a plastic film, taking down the film after 24 h, and continuously culturing in a greenhouse; in order to improve the transformation efficiency, the cells are infected again by the same method after 7 days; and (4) carrying out normal management on the transformed Arabidopsis plants, and harvesting seeds when the fruit pods are white.

The kanamycin is preliminarily screenedVyGOLSTransgenic plants and transformed empty vector plants are further identified at the DNA level, and total DNA is extracted by adopting an improved SDS micro-extraction method. Respectively extracted by the aboveVyGOLSDNA of the transgenic plant and the transformed empty vector plant is used as a template, an upstream primer is designed on a 35S promoter, and a primer pair is formed by the upstream primer and a downstream primer specific to the gene for PCR detection. The primers are shown below:

detection primer-F: 5'-GAAGATGCCTCTGCCGACAGTG-3' (shown in SEQ ID NO. 9);

detection primer-R: 5'-AGTACTCCGTCGGCTACTGCCA-3' (shown in SEQ ID NO. 10). The reaction system (25 muL) is as follows: 10 × buffer 2.5 μ L; d 0.5 muL of NTPs; 0.3 muL of Taq enzyme; ddH ₂ O16.2 muL; primer F1.5 muL; primer R1.5 mu L; DNA 2.5. mu.L. The reaction procedure is as follows: pre-denaturation at 94 ℃ for 5 min; 35 cycles, denaturation at 94 ℃ for 30S, annealing at 58 ℃ for 30S, and extension at 72 ℃ for 1 min; extending for 10min at 72 ℃, and storing at 4 ℃. The PCR products were detected by electrophoresis on a 1% agarose gel.

The results are shown in FIG. 3, where M: DNA molecular mass standard; lane 1: by ddH ₂ Performing PCR by taking O as a template; lane 2: carrying out PCR by taking the Arabidopsis DNA of the transformation empty vector as a template; lane 3: carrying out PCR by taking pCAMBIA2300-VyGOLS plasmid DNA as a template; lane lane4-6: PCR was performed using the transgenic Arabidopsis DNA transformed with the VyGOLS gene as a template. As can be seen from the figure, the test example successfully obtained the transitionVyGOLSThe gene Arabidopsis plants are named as OE #1, OE #2 and OE #3 respectively.

Test example 4 drought resistance identification of transgenic Arabidopsis plants

VyGOLSTransgenic plants and Arabidopsis thaliana (EV expression) transformed with empty vector were grown on MS medium for 7 days, transferred to a nutrition pot, and watered normally for 20 days to grow into robust seedlings. And stopping watering the arabidopsis seedlings, namely performing drought treatment until the leaves of part of arabidopsis plants appear obvious water loss withering symptoms on the 7 th day. All plants were then rehydrated and the growth of the plants was observed after 48 hours.

The phenotypes of Arabidopsis plants before and after drought treatment and after rehydration were recorded by photographing, and the results are shown in FIG. 4. it can be seen from FIG. 4 that the transgenic plants were transformed into the empty vector Arabidopsis plantsVyGOLSThe drought resistance of gene Arabidopsis plants OE #1, OE #2 and OE #3 is obviously enhanced.

Test example 5 analysis of physiological and biochemical Properties of transgenic Arabidopsis plants

Determination of Water loss:VyGOLSafter the transgenic plants and the transformed empty vector plants grow normally for 3 weeks, about 0.2 g of rosette leaves are respectively taken for measuring the water loss rate. The collected rosette leaves were placed on dry filter paper, and the Fresh Weight (FW) of the leaves was measured every 10min until the end of the water loss measurement at 50 min. The ratio of the amount of water lost in each measurement to the fresh weight of the first measurement was taken as the water loss rate.

Determination of electrolyte leakage rate (conductivity): the leaves were placed in a centrifuge tube, the volume was adjusted to 10 mL with ultra-deionized water, and the conductivity of the solution was measured after 1 hour shaking at room temperature and recorded as C1 before boiling. The solution was then boiled in boiling water for 10min, and the conductance was measured after cooling to room temperature and recorded as C2. The ratio of C1 to C2 (C1/C2) was taken as the relative electrolyte leakage value.

The detection result is shown in fig. 5, (A) expression quantity detection of the VyGOLS gene in the transgenic arabidopsis plant; (B) counting the survival rate of the VyGOLS gene transferred Arabidopsis plants after 18d of drought treatment; (C) relative water loss rate of the transferred VyGOLS gene Arabidopsis leaves; (D) relative conductivity of VyGOLS transgenic Arabidopsis plants after drought treatment for 18 d. As can be seen from the figure, the expression level of the VyGOLS gene in the transgenic arabidopsis plant is higher, and the survival rate is obviously improved compared with that of the plant transformed with an empty vector; compared with the transformation of an empty vector plant, the water loss rate and the conductivity of the VyGOLS gene-transformed Arabidopsis plant are obviously reduced.

Test example 6 analysis of expression of drought-resistant Gene of transgenic Arabidopsis thaliana

And extracting the total RNA of the drought-treated transgenic arabidopsis leaves by using a plus plant total RNA extraction kit. PrimeScript for Normal reverse transcription ^II 1st Strand cDNA Synthesis Kit (TaKaRa) synthesized the first Strand of cDNA. The specific operation steps are as follows: adding to a PCR tube: random 6 mers (50. mu.M) 1. mu.L, dNTP mix (10 mM each) 1. mu.L, Total RNA 2. mu.g, RNase free dH2O were made up to 10. mu.L, mixed well and centrifuged instantaneously to bring the solution to the bottom of the PCR tube. The reaction was carried out on a PCR instrument at 65 ℃ for 5 min and quenched on ice. Detecting drought-resistant related gene in transgenic arabidopsis plant by taking arabidopsis AtActin as internal reference geneAtCOR15A、AtERD15、AtRD29A、AtP5CS1Expression of the gene. The designed primers are shown as follows:

qRT-AtActin-F: 5'-CGGTGGTTCTATCTTGGCATC-3' (shown in SEQ ID NO. 11);

qRT-AtActin-R: 5'-GTCTTTCGCTTCAATAACCCTA-3' (shown in SEQ ID NO. 12);

qRT-AtCOR15A-F: 5'-CAGCGGAGCCAAGCAGAGCAG-3' (shown as SEQ ID NO. 13);

qRT-AtCOR15A-R: 5'-CATCGAGGATGTTGCCGTCACC-3' (shown as SEQ ID NO. 14);

qRT-AtERD15-f: 5'-CCAGCGAAATGGGGAAACCA-3' (shown in SEQ ID NO. 15);

qRT-AtERD15-r: 5'-ACAAAGGTACAGTGGTGGC-3' (shown in SEQ ID NO. 16);

qRT-AtRD29A-f: 5'-GTTACTGATCCCACCAAAGAAGA-3' (shown as SEQ ID NO)ID NO. 17);

qRT-AtRD29A-r: 5'-GGAGACTCATCAGTCACTTCCA-3' (shown in SEQ ID NO. 18);

qRT-AtP5CS1-F: 5'-CGACGGAGACAATGGAATTGT-3' (shown in SEQ ID NO. 19);

qRT-AtP5CS1-R: 5'-GATCAGAAATGTGTAGGTAGC-3' (shown in SEQ ID NO. 20).

Real-Time fluorescent quantitative PCR was performed on a Bio-Rad IQ5 Real-Time PCR Detection System (Bio-Rad Laboratories, Herc. mu. Les, Calif.) according to the TaKaRa SYBR Premix Ex Taq II (Perfect Real Time) instructions. 25 μ L of reaction system: 1 microliter of reverse transcription template; 1 mu L of forward and reverse primers respectively; 12.5 μ L of 2 × SYBR Premix Ex Taq: (2 ×); 9 μ L of nucleic-free water. The reaction procedure is as follows: at 95 ℃ for 30 s; 40 cycles of 95 ℃ for 5 s; 57 ℃ for 30 s; 72 ℃ for 30 s. Results adopted 2 ^-ΔΔC(t) The method is used for analysis.

The detection result is shown in fig. 6, and it can be seen from fig. 6 that, under drought conditions, the expression level of genes related to drought resistance in transgenic arabidopsis plants is obviously increased compared with that of the transgenic empty vector arabidopsis, which indicates that wild grapes are used in the inventionVyGOLSThe gene can increase the accumulation of anti-stress related substances and the expression of drought-resistant related genes in transgenic plants, and promote the drought resistance of the transgenic plants to be enhanced.

<110> university of Henan science and technology

<120>Wild grapeVyGOLSApplication of gene and coded protein thereof in drought stress

<160> 20

<170> SIPOSequenceListing 1.0

<211> 1011

<212> DNA

<213> wild grape

<221> VyGOLSGene

<400> 1

gcgaaaccgc cccctttttg ttggtacccg ggaaaccggc cattacggcc ggggaggaac 60

aaaggcaaag tagggtggca tccacagtgt tgctggttta ctttcccaac cctcctcacc 120

accaactctc tccttaacat ttttcttgca ccaacttgaa atctcacccc aaataaacca 180

caacaatggc cccaggagtg cccgcagatg tgtttacagc cggcggaaag gtttccaccc 240

tcaacgcagg ctactcaaag ggggcctacg tcacattttt agctggaaac ggggactatg 300

tgaaaggggt tgttgggttg gctaagggtt tgcgcaaggt gaagagcgcg taccctcttg 360

tggttgcaat gttgccggat gtgcctgagg agcaccgtga gatcttaaag tctcagggct 420

gcataattcg tgaaattgag cccatctacc ctcctgagaa ccaaatccag tttgcaatgg 480

catactacgt catcaactat tccaaactcc gtatttggaa tttcgaggaa tacagcaaga 540

tggtgtattt ggatgctgat atccaagttt acgacaacat agaccacctt atggacgccc 600

cggacggcta cttttacgcg gtaatggact gcttctgtga gaagacatgg agtcacactc 660

cccagtactc cgtcggctac tgccagcagt gcccggacaa ggtgacttgg cccgctgaga 720

tgggttcacc tccacctttg tacttcaacg ctgggatgtt cgtctttgag cctagccgtt 780

tgacttatga aagccttctc catactctac ggatcactcc tccgaccgcc tttgccgagc 840

aagatttctt gaacatgttc ttccaacaca tgtacaagcc catccctctc gtatacaact 900

tggttctagc aatgctgtgg cgccacccgg agaacgttga gctcgaccag gtcaaggtgg 960

tgcactactg tgctgctgga tcaaagccat ggagatacac tgggaaagaa gcaaacatgg 1020

agagagagga catcaagatg ttggtagcca aatggtggga catttacaat gataagtctc 1080

tggatttcaa ggctgaggac agtgttccag agggagaagg attctctagg ccatcgatca 1140

tggcttccat gcctgagcct gcaatctcct atattcccgc accctctgct gcttgaagat 1200

tacaaatctt taggagagag tgtattgaag ctcagggtgt gatctatctc tttttctatt 1260

taataccttt tccaaaggct acttggt 1287

<211> 336

<212> PRT

<213> wild grape

<221> VyGOLS protein

<400> 2

MET Ala Pro Gly Val Pro Ala Asp Val Phe Thr Ala Gly Gly Lys

1 5 10 15

Val Ser Thr Leu Asn Ala Gly Tyr Ser Lys Gly Ala Tyr Val Thr

20 25 30

Phe Leu Ala Gly Asn Gly Asp Tyr Val Lys Gly Val Val Gly Leu

35 40 45

Ala Lys Gly Leu Arg Lys Val Lys Ser Ala Tyr Pro Leu Val Val

50 55 60

Ala MET Leu Pro Asp Val Pro Glu Glu His Arg Glu Ile Leu Lys

65 70 75

Ser Gln Gly Cys Ile Ile Arg Glu Ile Glu Pro Ile Tyr Pro Pro

80 85 90

Glu Asn Gln Ile Gln Phe Ala MET Ala Tyr Tyr Val Ile Asn Tyr

95 100 105

Ser Lys Leu Arg Ile Trp Asn Phe Glu Glu Tyr Ser Lys MET Val

110 115 120

Tyr Leu Asp Ala Asp Ile Gln Val Tyr Asp Asn Ile Asp His Leu

125 130 135

MET Asp Ala Pro Asp Gly Tyr Phe Tyr Ala Val MET Asp Cys Phe

140 145 150

Cys Glu Lys Thr Trp Ser His Thr Pro Gln Tyr Ser Val Gly Tyr

155 160 165

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

170 175 180

Ser Pro Pro Pro Leu Tyr Phe Asn Ala Gly MET Phe Val Phe Glu

185 190 195

Pro Ser Arg Leu Thr Tyr Glu Ser Leu Leu His Thr Leu Arg Ile

200 205 210

Thr Pro Pro Thr Ala Phe Ala Glu Gln Asp Phe Leu Asn MET Phe

215 220 225

Phe Gln His MET Tyr Lys Pro Ile Pro Leu Val Tyr Asn Leu Val

230 235 240

Leu Ala MET Leu Trp Arg His Pro Glu Asn Val Glu Leu Asp Gln

245 250 255

Val Lys Val Val His Tyr Cys Ala Ala Gly Ser Lys Pro Trp Arg

260 265 270

Tyr Thr Gly Lys Glu Ala Asn MET Glu Arg Glu Asp Ile Lys MET

275 280 285

Leu Val Ala Lys Trp Trp Asp Ile Tyr Asn Asp Lys Ser Leu Asp

290 295 300

Phe Lys Ala Glu Asp Ser Val Pro Glu Gly Glu Gly Phe Ser Arg

305 310 315

Pro Ser Ile MET Ala Ser MET Pro Glu Pro Ala Ile Ser Tyr Ile

320 325 330

Pro Ala Pro Ser Ala Ala

335 336

<211> 21

<212> DNA

<213> Artificial sequence

<221> qRT-VyGOLS-F

<400> 3

ggggactatg tgaaaggggt t 21

<211> 20

<212> DNA

<213> Artificial sequence

<221> qRT-VyGOLS-R

<400> 4

ggatttggtt ctcaggaggg 20

<211> 19

<212> DNA

<213> Artificial sequence

<221> qRT-VyGAPDH-F

<400> 5

cccttgtcct cccaactct 19

<211> 19

<212> DNA

<213> Artificial sequence

<221> qRT-VyGAPDH-R

<400> 6

ccttctcagc actgtccct 19

<211> 32

<212> DNA

<213> Artificial sequence

<221> VyGOLS-ORF-XbaI-F

<400> 7

gggtctagaa tggccccagg agtgcccgca ga 32

<211> 32

<212> DNA

<213> Artificial sequence

<221> VyGOLS-ORF-KpnI-R

<400> 8

gggggtacct caagcagcag agggtgcggg aa 32

<211> 22

<212> DNA

<213> Artificial sequence

<221> detection primer-F

<400> 9

gaagatgcct ctgccgacag tg 22

<211> 22

<212> DNA

<213> Artificial sequence

<221> detection primer-R

<400> 10

agtactccgt cggctactgc ca 22

<211> 21

<212> DNA

<213> Artificial sequence

<221> qRT-AtActin-F

<400> 11

cggtggttct atcttggcat c 21

<211> 22

<212> DNA

<213> Artificial sequence

<221> qRT-AtActin-R

<400> 12

gtctttcgct tcaataaccc ta 22

<211> 21

<212> DNA

<213> Artificial sequence

<221> qRT-AtCOR15A-F

<400> 13

cagcggagcc aagcagagca g 21

<211> 22

<212> DNA

<213> Artificial sequence

<221> qRT-AtCOR15A-R

<400> 14

catcgaggat gttgccgtca cc 22

<211> 20

<212> DNA

<213> Artificial sequence

<221> qRT-AtERD15-F

<400> 15

ccagcgaaat ggggaaacca 20

<211> 19

<212> DNA

<213> Artificial sequence

<221> qRT-AtERD15-R

<400> 16

acaaaggtac agtggtggc 19

<211> 23

<212> DNA

<213> Artificial sequence

<221> qRT-AtRD29A-F

<400> 17

gttactgatc ccaccaaaga aga 23

<211> 22

<212> DNA

<213> Artificial sequence

<221> qRT-AtRD29A-R

<400> 18

ggagactcat cagtcacttc ca 22

<211> 21

<212> DNA

<213> Artificial sequence

<221> qRT-AtP5CS1-F

<400> 19

cgacggagac aatggaattg t 21

<211> 21

<212> DNA

<213> Artificial sequence

<221> qRT-AtP5CS1-R

<400> 20

gatcagaaat gtgtaggtag c 21

Claims

1. Wild grapeVyGOLSThe application of the gene or the recombinant expression vector thereof in plant variety breeding is characterized in that: application of wild grape as drought-resistant variety of arabidopsisVyGOLSNucleotide of geneThe sequence is shown in the position 186-1196 in SEQ ID NO. 1.