CN110845590B

CN110845590B - Wild grape VyPPR gene and application of encoding protein thereof in drought stress

Info

Publication number: CN110845590B
Application number: CN201911066289.6A
Authority: CN
Inventors: 余义和; 郭大龙; 李旭飞; 李敏; 张贺程; 张国海
Original assignee: Henan University of Science and Technology
Current assignee: Henan University of Science and Technology
Priority date: 2019-11-04
Filing date: 2019-11-04
Publication date: 2022-03-18
Anticipated expiration: 2039-11-04
Also published as: CN110845590A

Abstract

The invention discloses a wild grape VyPPR gene and application of a protein coded by the gene in drought stress, wherein a nucleotide sequence is shown as SEQ NO.1, the total length of a coding sequence is 1874 nucleotides, an open reading frame is 1590 nucleotides, an amino acid sequence of the gene coded protein is shown as SEQ NO.2, and the gene can code a protein containing 529 amino acids. Compared with an arabidopsis plant transformed with an empty vector, the VyPPR gene is overexpressed to cause accumulation of anti-stress related substances and expression of drought-resistant related genes in transgenic arabidopsis, and the grape VyPPR gene is cloned to increase accumulation of anti-stress related substances and expression of drought-resistant related genes in the transgenic plant and promote drought resistance enhancement of the transgenic plant.

Description

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

Technical Field

The invention relates to the technical field of plant stress resistance gene identification and genetic engineering, in particular to application of wild grape VyPPR gene and coding protein thereof in drought stress.

Background

The PPR protein, also called a triangular five-membered repeat protein, is one of the largest protein families of terrestrial plants, having over 400 members in most species, and plays an important role in various stages of plant growth and development. THA8 found in maize, which contains only 4 PPR motifs, is involved in the splicing of intron type II of the transcript of the two genes ycf3 and trnA, and this protein, because it contains only a small amount of PPR motifs, does not bind to single-stranded RNA of either ycf3 or trnA, but is able to interact with the splicing factors WTF1 and RNC1, which are also involved in the splicing of the intron of trnRNAi. Fertility restorer protein RF5 in manglietia indica type rice, belongs to PLS family, has no ability of binding with target transcript atp6-orfH79, and interaction protein GRP162 can enter mitochondria along with the target transcript and is combined with the target transcript, meanwhile, RFC3 with WD40 structural domain participates in the construction of fertility restorer protein complex, and together with RF5, GRP162 and unknown protein, the complex is constructed into 400-500 kDa complex, and the complex mediates the cleavage of atp6-orfH79 transcript at nucleotide 1169 to inhibit the translation expression of male sterile protein ORFH 79. Another fertility restorer gene Rf6, which belongs to the P family PPR protein, comprises 20 PPR motifs, interacts with hexokinase HXK6, and researches show that RF6 can also form a protein complex with other proteins, the size of the protein complex is about 400-500 kDa, and RF6 does not interact with GRP162, which suggests that the protein complexes formed by RF6 and RF5 are different. The fertility restorer complex involved in RF6 also has a function of cleaving the atp6-orfH79 transcript, and cleaves at the 1238 th nucleotide of the transcript to inhibit accumulation of ORFH79 in mitochondria, thereby allowing normal development of male gametes into mature pollen with fertility.

The grapes are the second largest fruit in the world, have a long cultivation history, are various in variety, and have important edible value and economic value. In recent years, with the global climate change, drought events occur frequently around the world, and drought hazards also occur frequently in non-drought seasons or non-drought regions. Drought has serious influence on the growth and development process and yield quality of grapes, and becomes one of main factors for restricting the growth of grapes and improving the quality of fruits, and particularly, in recent years, the grape industry is greatly threatened due to global climate change and frequent occurrence of drought in the south of China. 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.

Disclosure of Invention

The invention aims to provide a cloned grape VyPPR gene which can increase the accumulation of anti-stress related substances and the expression of drought-resistant related genes in transgenic plants and promote the enhancement of the drought resistance of the transgenic plants.

In order to achieve the purpose, the invention provides the following technical scheme: the wild grape VyPPR gene and the application of the encoding protein thereof in drought stress have the nucleotide sequence shown in SEQ NO.1, the full length of the encoding sequence is 1874 nucleotides, and the open reading frame is 1590 nucleotides.

Preferably, the amino acid sequence of the protein encoded by the gene is shown in SEQ NO.2, and the gene can encode a protein containing 529 amino acids.

The method for constructing the wild grape VyPPR gene plant over-expression vector comprises the following steps:

(1) correctly inserting the ORF fragment of 1011 bp in total, which contains the VyPPR gene coding region, into a plant over-expression vector pCAMBIA 2300-GFP;

(2) according to the ORF sequence of the previously cloned VyPPR gene, adding enzyme cutting sites XbaI and KpnI at the 5' end of a primer VyPPR-ORF-F according to the enzyme cutting sites on a pCAMBIA2300-GFP vector,

GGGTCTAGAATGGCTCCTCCCCAAAATCAAC，

GGGGGTACCCTAGTACAGACTAATCAGACTC；

(3) and (2) taking the pMD18-T-VyPPR plasmid as a template, amplifying the plasmid by using VyPPR-ORF-XbaI-F and VyPPR-ORF-KpnI-R, recovering a target band, connecting the recovered target band to a pMD19-T cloning vector, and obtaining the plant expression vector pCAMBIA2300-VyPPR after connection, transformation, screening and verification.

The application of the wild grape VyPPR gene coding protein in drought stress, and the specific method for overexpression of the wild grape VyPPR gene in arabidopsis thaliana comprises the following steps:

(1) streaking agrobacterium containing recombinant plant expression vector on LB plate, and culturing in culture box;

(2) transferring the bacterial liquid into a centrifugal bottle or a centrifugal tube, centrifuging for 10min at the rotating speed of 4000 rpm, removing supernatant, collecting thalli, and resuspending in an osmotic buffer solution;

(3) soaking the pod-removed Arabidopsis thaliana flower in a penetrating fluid, removing redundant penetrating buffer solution on the Arabidopsis thaliana flower after soaking, putting the Arabidopsis thaliana flower in an incubator for continuous culture, carrying out normal management on the transformed Arabidopsis thaliana plant, and harvesting seeds when the pod is white;

(4) the VyPPR transgenic plants and wild-type plants obtained by primary screening of kanamycin are further identified at the DNA level, and total DNA is extracted by an improved SDS micro-extraction method.

Preferably, the drought resistance of transgenic arabidopsis plants is identified.

Preferably, the transgenic arabidopsis plants are analyzed for physiological and biochemical characteristics, including the determination of water loss rate, the determination of solute leakage rate and the determination of chlorophyll content.

The invention provides an application of wild grape VyPPR gene and its coded protein in drought stress, which has the following beneficial effects:

the invention transfers the overexpression vector of the VyPPR gene into arabidopsis thaliana by a transgenic technology utilizing a strong promoter (cauliflower mosaic virus 35S promoter) driving principle, thereby obtaining a transgenic arabidopsis thaliana plant. Compared with an arabidopsis plant transformed with an empty vector, the VyPPR gene is overexpressed to cause accumulation of anti-stress related substances and expression of drought-resistant related genes in the transgenic arabidopsis, and drought resistance of the transgenic plant is enhanced, so that the grape VyPPR gene is cloned, accumulation of anti-stress related substances and expression of drought-resistant related genes in the transgenic plant can be increased, and drought resistance enhancement of the transgenic plant is promoted.

Drawings

FIG. 1 shows the expression of the VyPPR gene of the present invention in different tissues of grape;

FIG. 2 shows the expression of the VyPPR gene of the present invention after low temperature treatment;

FIG. 3 is the expression of the VyPPR gene of the present invention after drought treatment;

FIG. 4 is the expression of the VyPPR gene of the present invention after salt stress;

FIG. 5 is the drought resistance identification of VyPPR transgenic Arabidopsis plants of the present invention;

FIG. 6 is a physiological characteristic analysis of VyPPR transgenic Arabidopsis plants of the present invention;

FIG. 7 shows the expression analysis of drought-resistant related genes in transgenic Arabidopsis plants of the present invention.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

Example 1

Analysis of expression characteristics of grape VyPPR Gene

After the tissue culture seedlings of the Yanshan grape are subcultured for 16 days, seedlings which are robust in growth and consistent in 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-1 NaCl solution was added to the flask, and the mixture was incubated at 25. + -. 1 ℃ under a relative humidity of 75% and a light cycle of 14 h/dark 10 h, and samples were taken at 0, 2, 6, 12, and 24 h. 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). The first Strand of cDNA was synthesized by the PrimeScriptII1st Strand cDNA Synthesis Kit (TaKaRa) by conventional reverse transcription. 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 5min and quenched on ice. The real-time fluorescent quantitative PCR primer is designed according to the gene sequence of VyPPR, the forward primer sequence is qRT-VyPPR-F (5'CCCAACCATTTTATCTACCCTCA3'), and the reverse primer sequence is qRT-VyPPR-R (5 'CCAAGACACAACATTCCTCTCAGT 3'). The VyGAPDH gene is used as an internal reference, the forward primer sequence is qRT-VyGAPDH-F (5'CCCTTGTCCTCCCAACTCT3'), and the reverse primer sequence is qRT-VyGAPDH-R (5'CCTTCTCAGCACTGTCCCT 3'). 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 mul of reverse transcription template; 1 mul of forward and reverse primers respectively; 12.5 μ l of 2 × SYBR Premix Ex Taq: (2 ×); 9. mu.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. Result adopt

The method is used for analysis.

Example 2

Construction of grape VyPPR gene overexpression vector

To investigate the function of the VyPPR gene of grapes, a total of 1011 bp ORF fragments containing the coding region of the VyPPR gene were correctly inserted into the plant over-expression vector pCAMBIA 2300-GFP.

Designing upstream and downstream primers VyPPR-ORF-F and VyPPR-ORF-R capable of amplifying the VyPPR gene ORF according to the cloned VyPPR gene ORF sequence in the early stage; according to the restriction enzyme cutting site on the pCAMBIA2300-GFP vector, the restriction enzyme cutting site XbaI is added to the 5 'end of the primer VyPPR-ORF-F, the specific sequence is GGGTCTAGAATGGCTCCTCCCCAAAATCAAC, and the restriction enzyme cutting site KpnI is added to the 5' end of the primer VyPPR-ORF-R, the specific sequence is GGGGGTACCCTAGTACAGACTAATCAGACTC.

The pMD18-T-VyPPR plasmid is used as a template, VyPPR-ORF-XbaI-F and VyPPR-ORF-KpnI-R are used for amplification, a target band is recovered and then connected to a pMD19-T cloning vector, TOP10 competent cells are transformed, blue-white spot screening is carried out on an LB culture medium with Amp, bacterial liquid PCR and plasmid restriction enzyme detection are respectively carried out, and pMD19-T-VyPPR positive cloning is sent to a company for sequencing. The recombinant cloning vector pMD19-T-VyPPR and the plant expression vector pCAMBIA2300-GFP are subjected to double enzyme digestion by XbaI and KpnI, a linearized vector and a target fragment are recovered, the linearized vector and the target fragment are connected and converted into TOP10, the TOP10 is screened by Kan antibiotics, monoclonal shake bacteria are selected, and after bacterial liquid detection, quality improvement granule enzyme digestion detection is carried out, so that the plant expression vector pCAMBIA2300-VyPPR is formed.

Example 3:

overexpression of the grape VyPPR Gene 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 of LB liquid culture medium (added with corresponding antibiotics) for 24h at the temperature of 28 ℃; transferring 5ml of the bacterial liquid to 50ml of a 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 24h, 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 VyPPR transgenic plants and wild-type plants obtained by primary screening of kanamycin are further identified at the DNA level, and total DNA is extracted by an improved SDS micro-extraction method. Respectively taking the DNA of the VyPPR transgenic plant and the DNA of the wild plant as templates, designing an upstream primer (5'-GAAGATGCCTCTGCCGACAGTG-3') on a 35S promoter, forming a primer pair with a gene-specific downstream primer (5'-GCTGAAGTGTGATCAGAATGAGAAGC-3'), and carrying out PCR detection; 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; ddH2O 16.2 μ L; 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; extension is carried out for 10min at 72 ℃, the PCR product is stored at 4 ℃, and the electrophoresis detection is carried out on 1% agarose gel.

Example 4:

drought resistance identification of transgenic arabidopsis plants

After the VyPPR transgenic plant and the wild-type plant grow on the MS medium for 7 days, they are 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. Before and after drought treatment and after rehydration, the phenotype of arabidopsis plants was recorded by photographing.

Example 5:

analysis of physiological and biochemical characteristics of transgenic Arabidopsis plants

Determination of Water loss: after the VyPPR transgenic plant and the wild type plant grow normally for 3 weeks, about 0.2g of rosette leaves are respectively taken for water loss rate measurement. 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 (EL): 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.

Determination of chlorophyll content: cutting fresh leaves of Arabidopsis thaliana of each strain into filaments or small blocks of about 0.2 cm, uniformly mixing, weighing 0.1-0.2g, putting into a 50ml centrifuge tube, adding 0.5ml of pure acetone and 10-15ml of 80% acetone into a volumetric flask or a test tube, carefully washing the leaf scraps adhered to the edge of the bottle wall into an acetone solution, covering a bottle stopper, leaching overnight in a shaking table at room temperature, taking out the volumetric flask the next day, observing that the leaf tissues are completely whitened, indicating that chlorophyll is completely leached, then fixing the volume to 25ml by using 80% acetone, centrifuging, and carrying out colorimetric determination on the wavelength of 663nm and 652 nm.

Example 6:

transgenic arabidopsis drought-resistant related gene expression analysis

And extracting the total RNA of the drought-treated transgenic arabidopsis leaves by using a plus plant total RNA extraction kit. The first Strand cDNA was synthesized by the PrimeScriptII1st Strand cDNA Synthesis Kit (TaKaRa) by reverse transcription, which comprises the following steps:

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 5min and quenched on ice. Taking arabidopsis AtActin as an internal reference gene,

a forward primer sequence qRT-AtActin-F: 5'-CGGTGGTTCTATCTTGGCATC-3',

reverse primer sequence qRT-AtActin-R: 5'-GTCTTTCGCTTCAATAACCCTA-3'. AtCOR15A gene

The forward primer sequence qRT-AtCOR15A-F: 5'-CAGCGGAGCCAAGCAGAGCAG-3',

reverse primer sequence qRT-AtCOR15A-R: 5'-CATCGAGGATGTTGCCGTCACC-3'.

AtERD15 gene

The forward primer sequence qRT-AtERD15-F: 5'-CCAGCGAAATGGGGAAACCA-3',

reverse primer sequence qRT-AtERD15-R: 5'-ACAAAGGTACAGTGGTGGC-3'.

AtRD29A gene

The forward primer sequence qRT-AtRD29A-F: 5'-GTTACTGATCCCACCAAAGAAGA-3',

reverse primer sequence qRT-AtRD29A-R: 5'-GGAGACTCATCAGTCACTTCCA-3'.

AtP5CS1 Gene

The forward primer sequence qRT-AtP5CS1-F: 5'-CGACGGAGACAATGGAATTGT-3',

the reverse primer sequence qRT-AtP5CS 1-R: 5'-GATCAGAAATGTGTAGGTAGC-3'.

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 mul of reverse transcription template; 1 mul of forward and reverse primers respectively; 12.5 μ l of 2 × SYBR Premix Ex Taq: (2 ×); 9. mu.l of nucleic-free water. The reaction procedure is as follows: 30s at 95 ℃; 40 cycles of 95 ℃ for 5 s; 57 ℃ for 30 s; 72 ℃ for 30 s. The results were analyzed by the 2-. DELTA.C (t) method.

SEQ: NO.1

AGCTAGCCAAAGCAGGGCCCATTTCAGTTAAACTTTCAGTTGTCCAGTTACAAACTCGGAGCTCAAAGCGCCGTAGCAGTTCTGAAGAACTCAAGAACATGGCTCCTCCCCAAAATCAACTCAATCTGAACAACTCTGTCCTAGCCCTCCTTGAAAGATGCATTCATCTCAATCATCTCAAGCAGCTCCAAGCCTTTCTCATCACTCTCGGCCATGCCCAGACTCATTTCTACGCCTTCAAGCTCCTCCGCTTCTGCACTCTAGCCCTCTCCAATCTCTCCTACGCTCGCTTCATCTTCGACCACGTCGAATCCCCCAATGTCTACCTCTACACTGCAATGATCACTGCTTATGCTTCTCATTCTGATCACACTTCAGCCCTTCTTTTGTACCGCAACATGGTTCGTCGCCGTCGGCCTTGGCCCAACCATTTTATCTACCCTCATGTCTTGAAGTCGTGCACCCAGGTCGTGGGGCCGGGGAGTGCGAGAATGGTGCATTGTCAGGTGCTGAGGTCGGGTTTTGAACAATACCCAGTTGTGCAAACAGCTCTTCTTGATGCCTACTTGAGGTTTTGGTCTGATGTGGAAAGTGCGCGTCTCTTGTTTGATGAAATGACTGAGAGGAATGTTGTGTCTTGGACAGCTATGATTTCTGGGTACACGAGGCTTGGACAGATTGGGAATGCTGTATTGTTGTTTGAGGAAATGCCCGAGAGGGATGTGCCGTCTTGGAACGCTTTGATTGCTGGTTACACACAGAATGGGTTGTTCATGGAGGCGTTATCACTTTTCAGGAGAATGATTGCCGTTGAGGCGGGAGCTTGGGGTCAAGGAAATAGGCCAAATCAGGTTACTGCTGTGTGCTCACTCTCAGCTTGTGGTCACACTGGTATGCTCCGGCTTGGTAAATGGATACATGGTTATGTTTACAGAAATGGGCTTGGTTTGGATTCATTTGTATCTAATGCTCTGGTGGATATGTATGGGAAATGTGGATGTTTGAAAGAGGCAAGAAGGGTTTTTGATAGGACATTGGAGAGAAGCTTGACATCATGGAATTCCATGATCAATTGTCTCGCCCTCCATGGGCAAAGTCAGAATGCAATAAGTGTGTTTGAGGAGATGATGACATGTGGAAGTGGTGTAAAACCTGATGAAGTTACATTTATTGGCTTGTTGAATGCCTGTACCCATGGGGGTTTGGTTGAAAAAGGTTGGCTTTATTTTGAGCTGATGACTCAAAATTATGGGATAGAACCTCAGATTGAGCATTATGGTTGCTTGGTAGATCTTCTTGGTCGTGCAGGTCAGTTTGAAGAAGCTATGGAGGTTGTAAGGGGAATGAGAATTGAACCTGATGAGGTTATTTGGGGCTCTTTGCTTAATGGATGTAAGATTCATGGCCACACAGATTTGGCTGAATTTTCCATTAAAAAATTGATTGATATGGATCCAAATAATGGTGGTTATGGTATAATGTTGGCAAATATATATGGGGAGCTAGGCAAGTGGGATGAGGTTCGGAAGGTTCGGAAGGTGTTGAAGGAGCAGAATGCCCACAAGACCCCTGGTTGCAGTTGGATTGAAATTGACAACCAAGTTCATCAATTCTATTCTGTTGATAAAACACATCCTAGAACGGAGGAGATATACAATACATTGGAGAGTCTGATTAGTCTGTACTAGCTTAGAGGTGCTGGGGTTTTTCCCACAATTATAAAAACGGTTTAAAAAATAAAATTTTGAGTTTACAGGAGTTCAAATTTGATTCCTAATTCTCAAACTACATGTTTCACATGCCTACAGCACTATTCTGCTTTGGAGGGTAACCCTCTCAAGTCCTTATGGAAGTGGTTGATGAGGGGCCAAGGT

SEQ: NO.2

MAPPQNQLNLNNSVLALLERCIHLNHLKQLQAFLITLGHAQTHFYAFKLLRFCTLALSNLSYARFIFDHVESPNVYLYTAMITAYASHSDHTSALLLYRNMVRRRRPWPNHFIYPHVLKSCTQVVGPGSARMVHCQVLRSGFEQYPVVQTALLDAYLRFWSDVESARLLFDEMTERNVVSWTAMISGYTRLGQIGNAVLLFEEMPERDVPSWNALIAGYTQNGLFMEALSLFRRMIAVEAGAWGQGNRPNQVTAVCSLSACGHTGMLRLGKWIHGYVYRNGLGLDSFVSNALVDMYGKCGCLKEARRVFDRTLERSLTSWNSMINCLALHGQSQNAISVFEEMMTCGSGVKPDEVTFIGLLNACTHGGLVEKGWLYFELMTQNYGIEPQIEHYGCLVDLLGRAGQFEEAMEVVRGMRIEPDEVIWGSLLNGCKIHGHTDLAEFSIKKLIDMDPNNGGYGIMLANIYGELGKWDEVRKVRKVLKEQNAHKTPGCSWIEIDNQVHQFYSVDKTHPRTEEIYNTLESLISLY

Sequence listing

<110> university of Henan science and technology

<120> wild grape VyPPR gene and application of encoding protein thereof in drought stress

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1874

<212> DNA

<213> VyPPR

<400> 1

agctagccaa agcagggccc atttcagtta aactttcagt tgtccagtta caaactcgga 60

gctcaaagcg ccgtagcagt tctgaagaac tcaagaacat ggctcctccc caaaatcaac 120

tcaatctgaa caactctgtc ctagccctcc ttgaaagatg cattcatctc aatcatctca 180

agcagctcca agcctttctc atcactctcg gccatgccca gactcatttc tacgccttca 240

agctcctccg cttctgcact ctagccctct ccaatctctc ctacgctcgc ttcatcttcg 300

accacgtcga atcccccaat gtctacctct acactgcaat gatcactgct tatgcttctc 360

attctgatca cacttcagcc cttcttttgt accgcaacat ggttcgtcgc cgtcggcctt 420

ggcccaacca ttttatctac cctcatgtct tgaagtcgtg cacccaggtc gtggggccgg 480

ggagtgcgag aatggtgcat tgtcaggtgc tgaggtcggg ttttgaacaa tacccagttg 540

tgcaaacagc tcttcttgat gcctacttga ggttttggtc tgatgtggaa agtgcgcgtc 600

tcttgtttga tgaaatgact gagaggaatg ttgtgtcttg gacagctatg atttctgggt 660

acacgaggct tggacagatt gggaatgctg tattgttgtt tgaggaaatg cccgagaggg 720

atgtgccgtc ttggaacgct ttgattgctg gttacacaca gaatgggttg ttcatggagg 780

cgttatcact tttcaggaga atgattgccg ttgaggcggg agcttggggt caaggaaata 840

ggccaaatca ggttactgct gtgtgctcac tctcagcttg tggtcacact ggtatgctcc 900

ggcttggtaa atggatacat ggttatgttt acagaaatgg gcttggtttg gattcatttg 960

tatctaatgc tctggtggat atgtatggga aatgtggatg tttgaaagag gcaagaaggg 1020

tttttgatag gacattggag agaagcttga catcatggaa ttccatgatc aattgtctcg 1080

ccctccatgg gcaaagtcag aatgcaataa gtgtgtttga ggagatgatg acatgtggaa 1140

gtggtgtaaa acctgatgaa gttacattta ttggcttgtt gaatgcctgt acccatgggg 1200

gtttggttga aaaaggttgg ctttattttg agctgatgac tcaaaattat gggatagaac 1260

ctcagattga gcattatggt tgcttggtag atcttcttgg tcgtgcaggt cagtttgaag 1320

aagctatgga ggttgtaagg ggaatgagaa ttgaacctga tgaggttatt tggggctctt 1380

tgcttaatgg atgtaagatt catggccaca cagatttggc tgaattttcc attaaaaaat 1440

tgattgatat ggatccaaat aatggtggtt atggtataat gttggcaaat atatatgggg 1500

agctaggcaa gtgggatgag gttcggaagg ttcggaaggt gttgaaggag cagaatgccc 1560

acaagacccc tggttgcagt tggattgaaa ttgacaacca agttcatcaa ttctattctg 1620

ttgataaaac acatcctaga acggaggaga tatacaatac attggagagt ctgattagtc 1680

tgtactagct tagaggtgct ggggtttttc ccacaattat aaaaacggtt taaaaaataa 1740

aattttgagt ttacaggagt tcaaatttga ttcctaattc tcaaactaca tgtttcacat 1800

gcctacagca ctattctgct ttggagggta accctctcaa gtccttatgg aagtggttga 1860

tgaggggcca aggt 1874

<210> 2

<211> 529

<212> PRT

<213> VyPPR

<400> 2

Met Ala Pro Pro Gln Asn Gln Leu Asn Leu Asn Asn Ser Val Leu Ala

1 5 10 15

Leu Leu Glu Arg Cys Ile His Leu Asn His Leu Lys Gln Leu Gln Ala

20 25 30

Phe Leu Ile Thr Leu Gly His Ala Gln Thr His Phe Tyr Ala Phe Lys

35 40 45

Leu Leu Arg Phe Cys Thr Leu Ala Leu Ser Asn Leu Ser Tyr Ala Arg

50 55 60

Phe Ile Phe Asp His Val Glu Ser Pro Asn Val Tyr Leu Tyr Thr Ala

65 70 75 80

Met Ile Thr Ala Tyr Ala Ser His Ser Asp His Thr Ser Ala Leu Leu

85 90 95

Leu Tyr Arg Asn Met Val Arg Arg Arg Arg Pro Trp Pro Asn His Phe

100 105 110

Ile Tyr Pro His Val Leu Lys Ser Cys Thr Gln Val Val Gly Pro Gly

115 120 125

Ser Ala Arg Met Val His Cys Gln Val Leu Arg Ser Gly Phe Glu Gln

130 135 140

Tyr Pro Val Val Gln Thr Ala Leu Leu Asp Ala Tyr Leu Arg Phe Trp

145 150 155 160

Ser Asp Val Glu Ser Ala Arg Leu Leu Phe Asp Glu Met Thr Glu Arg

165 170 175

Asn Val Val Ser Trp Thr Ala Met Ile Ser Gly Tyr Thr Arg Leu Gly

180 185 190

Gln Ile Gly Asn Ala Val Leu Leu Phe Glu Glu Met Pro Glu Arg Asp

195 200 205

Val Pro Ser Trp Asn Ala Leu Ile Ala Gly Tyr Thr Gln Asn Gly Leu

210 215 220

Phe Met Glu Ala Leu Ser Leu Phe Arg Arg Met Ile Ala Val Glu Ala

225 230 235 240

Gly Ala Trp Gly Gln Gly Asn Arg Pro Asn Gln Val Thr Ala Val Cys

245 250 255

Ser Leu Ser Ala Cys Gly His Thr Gly Met Leu Arg Leu Gly Lys Trp

260 265 270

Ile His Gly Tyr Val Tyr Arg Asn Gly Leu Gly Leu Asp Ser Phe Val

275 280 285

Ser Asn Ala Leu Val Asp Met Tyr Gly Lys Cys Gly Cys Leu Lys Glu

290 295 300

Ala Arg Arg Val Phe Asp Arg Thr Leu Glu Arg Ser Leu Thr Ser Trp

305 310 315 320

Asn Ser Met Ile Asn Cys Leu Ala Leu His Gly Gln Ser Gln Asn Ala

325 330 335

Ile Ser Val Phe Glu Glu Met Met Thr Cys Gly Ser Gly Val Lys Pro

340 345 350

Asp Glu Val Thr Phe Ile Gly Leu Leu Asn Ala Cys Thr His Gly Gly

355 360 365

Leu Val Glu Lys Gly Trp Leu Tyr Phe Glu Leu Met Thr Gln Asn Tyr

370 375 380

Gly Ile Glu Pro Gln Ile Glu His Tyr Gly Cys Leu Val Asp Leu Leu

385 390 395 400

Gly Arg Ala Gly Gln Phe Glu Glu Ala Met Glu Val Val Arg Gly Met

405 410 415

Arg Ile Glu Pro Asp Glu Val Ile Trp Gly Ser Leu Leu Asn Gly Cys

420 425 430

Lys Ile His Gly His Thr Asp Leu Ala Glu Phe Ser Ile Lys Lys Leu

435 440 445

Ile Asp Met Asp Pro Asn Asn Gly Gly Tyr Gly Ile Met Leu Ala Asn

450 455 460

Ile Tyr Gly Glu Leu Gly Lys Trp Asp Glu Val Arg Lys Val Arg Lys

465 470 475 480

Val Leu Lys Glu Gln Asn Ala His Lys Thr Pro Gly Cys Ser Trp Ile

485 490 495

Glu Ile Asp Asn Gln Val His Gln Phe Tyr Ser Val Asp Lys Thr His

500 505 510

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

515 520 525

Tyr

Claims

1. The application of the wild grape VyPPR gene coding protein in drought stress is characterized in that: the specific method for overexpression of the grape VyPPR gene in Arabidopsis thaliana comprises the following steps:

GGGTCTAGAATGGCTCCTCCCCAAAATCAAC，

GGGGGTACCCTAGTACAGACTAATCAGACTC；

(3) using pMD18-T-VyPPR plasmid as a template, amplifying the plasmid by using VyPPR-ORF-XbaI-F and VyPPR-ORF-KpnI-R, recovering a target band, connecting the target band to a pMD19-T cloning vector, and obtaining a plant expression vector pCAMBIA2300-VyPPR after connection, transformation, kanamycin screening and verification;

(4) streaking agrobacterium containing recombinant plant expression vector on LB plate containing kanamycin, and culturing in culture box;

(5) transferring the bacterial liquid into a centrifugal bottle or a centrifugal tube, centrifuging for 10min at the rotating speed of 4000 rpm, removing supernatant, collecting thalli, and resuspending in an osmotic buffer solution;

(6) soaking the pod-removed Arabidopsis thaliana flower in a penetrating fluid, removing redundant penetrating buffer solution on the Arabidopsis thaliana flower after soaking, putting the Arabidopsis thaliana flower in an incubator for continuous culture, carrying out normal management on the transformed Arabidopsis thaliana plant, and harvesting seeds when the pod is white;

(7) the VyPPR transgenic plants and wild-type plants obtained by primary screening of kanamycin are further identified at the DNA level, and total DNA is extracted by an improved SDS micro-extraction method.

2. The application of the wild grape VyPPR gene coding protein in drought stress according to claim 1, which is characterized in that: and (3) identifying the drought resistance of the transgenic arabidopsis plants.

3. The application of the wild grape VyPPR gene coding protein in drought stress according to claim 1, which is characterized in that: analyzing the physiological and biochemical characteristics of the transgenic arabidopsis plants, wherein the physiological and biochemical characteristic analysis comprises the determination of the water loss rate, the determination of the electrolyte leakage rate and the determination of the chlorophyll content.