CN109081865A

CN109081865A - Moso bamboo PeVQ28 albumen and its encoding gene and application

Info

Publication number: CN109081865A
Application number: CN201811056089.8A
Authority: CN
Inventors: 项艳; 程欣然; 王玉娇; 熊瑞; 吴敏; 严涵薇
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2018-09-11
Filing date: 2018-09-11
Publication date: 2018-12-25
Anticipated expiration: 2038-09-11
Also published as: CN109081865B

Abstract

The invention discloses a kind of moso bamboo PeVQ28 albumen and its encoding gene and applications.The amino acid sequence of the albumen is as shown in SEQ ID No.1, and coding gene sequence (CDS) is as shown in SEQID No.2, and the cDNA sequence overall length of the moso bamboo PeVQ28 gene is as shown in SEQID No.3.PeVQ28 gene provided by the invention has high expression under different degrees of salt stress induction.PeVQ28 expression vector is transferred in wildtype Arabidopsis thaliana by the inflorescence dip method of mediated by agriculture bacillus, the salt-resistance of transgenosis wildtype Arabidopsis thaliana is significantly improved as the result is shown, illustrates that PeVQ28 gene can be improved the salt-resistance of transgenic arabidopsis.The vegetable material obtained using moso bamboo PeVQ28 expression vector is had certain adaptability to extraneous salt stress environment, had a good application prospect.

Description

Moso bamboo PeVQ28 albumen and its encoding gene and application

Technical field

The present invention relates to field of plant molecular biology, and in particular to a kind of moso bamboo PeVQ28 albumen and its encoding gene with Using.

Background technique

The type very abundant of China Bamboo, according to statistics about 39 belong to, more than 500 kinds, either the classification source of bamboo, growing surface Long-pending or bamboo grove accumulation and yield are all to be located at No. 1 in the world, it may be said that China is most important production bamboo wood in the world Country.Moso bamboo is also a kind of forest reserves, and area is in occupation of highly important status in south China, due to its growth speed It spends fastly and the period for reaching available standards is short, yield is also very considerable, can replace some slow raw forest reserves types, with section The development of technology, more extensively to moso bamboo use scope.Due to Mao bamboo timber speciality, the use in building gradually attracts Scenic spot, Peasants Joy operator, moulding is unique, simple and unadorned generous and low-energy environment-friendly；Bamboo besom, chair, soup ladle, toy etc. Bamboo art products are also gradually come into daily life；Content of cellulose height determines that it can be used as in papermaking former material in moso bamboo Material.Simultaneously at edible aspect, being not only bamboo shoots cannot be edible as green food materials, and the green drink of some factory process Or constituent is from extraction in the leaf of bamboo or the hydrolysis refinement from bamboo processing waste material in health care product.In addition to economic value, hair Bamboo grove also has the value of important ecology aspect, such as water conservation, soil conservation, environment purification.

VQ-motif albumen is a kind of plant specific proteins, due to gradually causing with WRKY transcription factor interaction Concern.By the research to VQ protein family in arabidopsis, VQ protein family participates in the various function controllings of plant, mainly Participate in the salt stress reaction of plant.Research discovery AtCaMBP25 (AtVQ15) is in Seed Germination of Arabidopsis Pumila and life in arabidopsis Long mesoderm growing early stage can bear tune osmotic stress, and under the conditions of NaCl and osmotic stress, transgenic line ties up to Seed Germination and growth hair Educate show it is highly sensitive.Similarly, under the conditions of discovery AtVQ9 is overexpressed plant under NaCl processing after study, either The sprouting of seed or the upgrowth situation of arabidopsis seedling have the performance being suppressed.

The growing environment of moso bamboo is more demanding to moisture, weather and soil, is suitble to be grown in moisture abundance, warmer climate And in subacidity or neutral loam environment.In recent years, with the continuous raising and the continuous deterioration of weather of Global Temperature, cause to plant Object will receive the injury of the various unfavorable conditions from environment during the growth process, and the soil salinization is world today's common concern The problem of.Salt stress is one of main limiting factor of plant growth and development.Moso bamboo be to salt stress it is sensitive, they are saline and alkaline When ground is grown, since by saline and alkaline coercion, slow growth, often blade turns yellow, is dead, falling off, and seriously affects photosynthetic Effect, sometimes even whole plant withered death.Salt stress negative-effect caused by moso bamboo is mainly manifested in: potassium, calcium, phosphorus, Nutrient Stress caused by nitrogen insufficiency of intake；Ion toxicity effect caused by the accumulation of sodium ion, chloride ion and sulfate；Soil Osmotic stress caused by middle and high concentration solute；Accumulated active oxygen causes the destruction of biomembrane, albumen and nucleic acid, i.e. oxidative stress. Equal environment-stress with high salt have significantly limited the upgrowth situation of moso bamboo, therefore, recognize plant to the reaction mechanism of salt stress, mention The salt-resistance of high plant cultivates the moso bamboo new germ plasm of a collection of salt tolerant, to the yield for improving moso bamboo, Foresty industry is promoted to stablize hair Exhibition increases national income and has great importance.

The VQ gene and its homologous gene announced at present, mostly from model plant or herbaceous plant, these plants Mostly annual plant, and the function in moso bamboo about VQ gene is not yet reported.

Summary of the invention

In order to solve the problems in the existing technology, the object of the present invention is to provide a kind of moso bamboo PeVQ28 albumen and its Encoding gene and application.

In order to achieve the object of the present invention, present invention employs following technical solutions: a kind of moso bamboo PeVQ28 egg of the invention White, the amino acid sequence of the moso bamboo PeVQ28 albumen is as shown in SEQ ID No.1.

The coding gene sequence of moso bamboo PeVQ28 albumen described in a kind of coding of the invention, it is characterised in that: described The nucleic acid sequence of moso bamboo PeVQ28 albumen is as shown in SEQ ID No.2.

Carrier containing the coding gene sequence of the invention.

The construction method of the carrier of the invention, includes the following steps:

It include the moso bamboo PeVQ28 protein coding gene sequence construct of restriction enzyme site to pEASY T1 simple by both ends It on Cloning Vector carrier, is transferred in competent escherichia coli cell TransT1, obtains T1-PeVQ28 recombinant plasmid, it is double Digestion T1-PeVQ28 recombinant plasmid and pCAMBIA1301a plasmid after connection, are transferred to competent escherichia coli cell, extract matter After grain, expression vector pCAMBIA1301a-PeVQ28 is obtained.

Engineering bacteria containing the carrier of the invention.

The primer pair for being used to clone the coding gene sequence of the invention, it is characterised in that: the primer pair includes upper Swim primer and downstream primer, the nucleotide sequence of the upstream primer as shown in SEQ ID No.4, the downstream primer Nucleotide sequence is as shown in SEQ ID No.5.

A kind of moso bamboo PeVQ28 gene of the invention, it is characterised in that: the moso bamboo PeVQ28 gene cDNA overall length Nucleic acid sequence is as shown in SEQID No.3.

Application of the moso bamboo PeVQ28 gene of the present invention in plant modification salt resistance.

Further, the gene or the coding gene sequence are transferred to by the inflorescence dip method of mediated by agriculture bacillus In plant.

The utility model has the advantages that the present invention provides the relevant PeVQ28 albumen of moso bamboo salt resistance and its encoding gene and application for the first time. PeVQ28 expression vector is transferred in wildtype Arabidopsis thaliana by the inflorescence dip method of mediated by agriculture bacillus, crosses table as the result is shown Salt-resistance up to Arabidopsis plant is remarkably reinforced, and illustrates that PeVQ28 gene can be improved the salt-resistance of transgenic arabidopsis.It utilizes The vegetable material that moso bamboo PeVQ28 expression vector obtains has certain adaptability to extraneous hypersaline environment, has fine Application prospect.

Detailed description of the invention

Fig. 1 is the amino acid sequence and structural domain division figure of the albumen of PeVQ28 coded by said gene of the present invention；

Fig. 2 is the electrophoretogram of 2 Leaves of Bamboo Phyllostachys pubescens total serum IgE of the embodiment of the present invention；M:Trans2K DNA Marker；1: Moso Bamboo Leaves Piece total serum IgE.

The plant transgene carrier pCAMBI1301a-PeVQ28 of 4 moso bamboo PeVQ28 gene of Fig. 3 embodiment of the present invention is constructed Schematic diagram；

Fig. 4 is the phenotypic map of 5 transgenic arabidopsis of the embodiment of the present invention and wildtype Arabidopsis thaliana before and after salt treatment；A: turn The phenotypic map of gene arabidopsis and wildtype Arabidopsis thaliana before and after salt treatment；B: transgenic arabidopsis and wildtype Arabidopsis thaliana are in salt Survival rate after processing 10 days；C-H: PRO, RWC before and after salt treatment of transgenic arabidopsis and wildtype Arabidopsis thaliana, SOD, The content of POD, CAT and MDA.WT: wildtype Arabidopsis thaliana；L1, L3, L10: transgenosis is overexpressed strain.

Specific embodiment

The present invention is described further with reference to the drawings and specific embodiments.It should be understood that these embodiments are merely to illustrate Purpose, rather than the limitation scope of the invention.

Reagent used in embodiment mainly includes molecular biology experiment reagent and kit etc., can pass through business Approach obtains, and specifically include: RNA extracts kit is purchased from Roche Biotechnology Co., Ltd；Reverse transcription reagent box (First- Strand cDNA Synthesis Kit) it is purchased from Promega company；SYBR Green Mix is purchased from American AB I company； Trizol is purchased from the handsome Bioisystech Co., Ltd's Ago-Gel DNA QIAquick Gel Extraction Kit in Shanghai, plasmid quickly small extraction reagent kit It is purchased from Axygen；PEASY T1simple Cloning Vector, Taq enzyme, Trans T1 competence and the purchase of relevant kit In Beijing Quan Shi King Company；Restriction enzyme Sma I and Sal I and DNAmarker are purchased from TaKaRa, and (precious bioengineering is (big Even)) Co., Ltd；Examining order is completed by Hua Da Gene science limited liability company.Provided in the embodiment of the present invention If method is conventional method without specified otherwise.

Embodiment 1

The protein sequence of one moso bamboo PeVQ28 gene coding

Protein sequence translation (SEQ is carried out to the cDNA sequence overall length of moso bamboo PeVQ28 gene using primers software ID No.1) (Fig. 1).

Embodiment 2

Moso bamboo salt resistance related gene PeVQ28 inducing expression pattern analysis

(1) Stress treatment and Total RNAs extraction

With 100mM sodium chloride solution, 150mM sodium chloride solution and 200mM sodium chloride solution pour annual hair respectively Bamboo seedling keeps other nutritional conditions constant.Take that various treated in advance with scissors (with 75% wipes of alcohol decontamination) respectively 0,1,3,6,12 and Leaves of Bamboo Phyllostachys pubescens for 24 hours, it is fitted into the dof pipe of the 2mL containing RNA protection liquid and saves backup, different time sections And sample plant is repeated 3 times.Trizol method extracts total serum IgE, and detailed step is as follows:

(1) spare Leaves of Bamboo Phyllostachys pubescens is taken out from RNA protection liquid, is firstly placed in filter paper to blot as far as possible and liquid is protected to avoid It influences, is transferred to after mortar and pours into liquid nitrogen and start grinding at once until blade becomes powder, collect powder and (mentioned in 1.5mLdorf pipe Preceding Liquid nitrogen precooler)；

(2) with pipettor draw 1mL Trizol solution be added to the dof equipped with sample pipe, quickly shake dof pipe 15s after 5min is placed at room temperature on superclean bench；

(3) 4 DEG C of centrifugation 10min, setting revolving speed are 12,000rpm.After carefully draw three-layer liquid in 800 μ L dof pipes The middle layer water phase of phase, into new dof pipe；

(4) chlorination imitates/200 μ L of isoamyl alcohol, rapid 15s concussion, and 3min is stood；

(5) 4 DEG C of centrifugation 15min, 12,000rpm, 400 μ L upper strata aqueous phases are drawn into new dof pipe, and the same volume is added Isopropanol precipitating, it is quiet to put 25min or more；

(6) repeat the centrifugation setting of " (5) ", after only stay the precipitating of dof bottom of the tube；

(7) it is precipitated, is repeated twice, revolving speed 7500rpm, centrifugation time 5min stays precipitating after the completion with 75% alcohol washes；

(8) the dof pipe uncapped is placed in superclean bench drying, room temperature 5-10min.Notice that the drying time cannot be too long, Otherwise too dry indissoluble is precipitated；

(9) the DEPC water for inhaling 30 μ L dissolves precipitating；

(10) RNA solution is obtained after mixing, and takes out 4 μ L testing results, -80 DEG C of refrigerators of balance.

(2) detection of total serum IgE and genomic DNA is gone to handle

(1) total serum IgE integrity detection

The total serum IgE for taking 3 μ L to extract mixes, electrophoresis detection, electrophoresis 5V/cm with 1 μ L loading buffer.20 minutes Afterwards, observe and take pictures in gel imaging system, if find the 28S rRNA band close from loading wells it is most bright be approximately 18S rRNA Two times, completely without miscellaneous band, then it is assumed that the RNA of extraction do not occur degrade (Fig. 2).

(2) detection of total serum IgE purity and content

Total serum IgE purity and content are detected with micro-spectrophotometer, first instrument is cleaned, then detect RNA (1 μ L). If in 1.8~2.0 ranges, it is preferable to represent RNA purity for the ratio of A260/A280, if having protein or phenol pollution in RNA, The ratio of A260/A280 will be significantly lower than 1.8, and ratio is greater than 2.0 and shows that RNA sample is degraded.

(3) removal of genomic DNA

1) following reaction solution is prepared in PCR pipe:

In micro centrifuge tube, following reagent is sequentially added:

RNase-free water is mended to 50 μ L of final volume

2) it reacts 30 minutes for 37 DEG C, the RNase-free water of 50 μ L is added；

3) 100 μ L phenol/chloroforms/isoamyl alcohol mixed liquor (ratio 25:24:1) is drawn to be added；

4) it is centrifuged (10000rpm, 10 minutes), shifts water layer to new PCR pipe, adds 10 μ L sodium acetate (3mol/L, pH= 5.2) it (is pre-chilled in advance) with 250 μ L dehydrated alcohols, -20 DEG C put 45 minutes；

5) it with the precipitating after the 70% alcohol washes centrifugation being pre-chilled in advance, is placed on superclean bench and dries up, add RNase-free water dissolution precipitating, and whether electrophoresis detection removes genomic DNA.

(3) reverse transcription

Experimental method is by Takara reverse transcription reagent box PrimeScript^TM RT Master Mix(Perfect Real Time)

Specification provides:

(1) it is loaded:

(2) sample-adding mix after, 37 DEG C child care 15 minutes, can be extended down to 30 minutes if RNA concentration is too low；

(3) 85 DEG C, 5 seconds, it is therefore an objective to inactivate reverse transcriptase, 4 DEG C of preservations；

(4) reverse transcription product cDNA is saved in -20 DEG C of refrigerators, is tested for fluorescent quantitation.

(4) design of primers

According to the cDNA sequence overall length of moso bamboo PeVQ28 gene, 5 software design fluorescent quantitation of Primer is utilized

PeVQ28 primer:

F:5 '-TGAGCCCATCAACTACACTGCC-3 '；

R:5 '-TGCTGGCGTCTGGCTTCC-3 '.

Its specificity is detected with PCR, under the premise of ensuring PCR specific amplification, can be used.With moso bamboo TIP41 gene is the positive control of fluorescent quantitation test, the primer of TIP41 gene are as follows:

F:5 '-AAAATCATTGTAGGCCATTGTCG-3 '；

R:5-ACTAAATTAAGCCAGCGGGAGTG-3 '.

Quantitative fluorescent PCR reaction system is 25 μ L, and each group is divided into 12.5 μ L of SYBR Green Mix, and template cDNA is 2 μ L, upstream primer and downstream primer (10 μm of ol/L) each 0.5 μ L finally mends deionized water to 25 μ L.PCR response parameter is as follows: 95 DEG C initial denaturation 10min；95 DEG C of 15sec, 60 DEG C of 1min, totally 40 recycle.After reaction, product is heated, is obtained The solubility curve of product.Using 2^–ΔΔCTMethod handles the signal and data of acquisition.

Embodiment 3

The clone of moso bamboo salt resistance related gene PeVQ28

According to the PeVQ28 gene C DS sequence announced on moso bamboo genome website, the primer of the PCR amplification segment is designed, Upstream primer adds Sma I restriction enzyme site, and downstream primer adds Sal I restriction enzyme site.

Primer sequence is as follows:

PeVQ28-F5′-ATACCCGGGATGGGGGAGTACCACAG-3′

PeVQ28-R5-GCCGTCGACTTAAGATGCGTACATTTCAC-3′

Using the 1st chain cDNA of Leaves of Bamboo Phyllostachys pubescens totalRNA reverse transcription as template, PCR expansion is carried out with upstream primer and downstream primer Increase, PCR reaction system is as shown in table 1:

Table 1

PCR reaction condition are as follows: initial denaturation: 98 DEG C of 10min；Denaturation: 98 DEG C of 10s；Annealing: 58 DEG C of 30s；Extend: 72 DEG C 1min, 39 circulations；Overall elongation: 72 DEG C of 10min.

After reaction, it draws PCR product and carries out 2% agarose gel electrophoresis, detect and cut in gel imaging system It after glue, is recycled using Ago-Gel DNA QIAquick Gel Extraction Kit, by recycling segment and pEASY T1 simple Cloning Vector connection is transformed into the E. coli competent TransT1 cell of Quan Shijin Bioisystech Co., Ltd；PCR and enzyme Cut detection screening positive clone；It to the tentatively judicious junction fragment of testing result, sends to Hua Da company and is sequenced, be sequenced As a result as: shown in the DNA sequence dna of SEQ ID NO:2 in sequence table, by 747bp base composition, by itself and moso bamboo gene networking The sequence alignment reported on standing, it is as a result completely the same.

Embodiment 4

The plant transgene carrier pCAMBI1301a-PeVQ28 of moso bamboo PeVQ28 gene is constructed

PeVQ28 segment and the Agrobacterium of the small fragment on pEASY T1simple Cloning Vector will be already connected to Binary vector pCAMBIA1301a carries out double digestion with SmaI and Sal I enzyme, in 37 DEG C of water-baths, 3h, and digestion 20uL system It is as shown in table 2:

Table 2

It after double digestion, is detected by 1% agarose gel electrophoresis, is recycled using Ago-Gel DNA QIAquick Gel Extraction Kit.It will Above-mentioned size segment is connected with T4DNA ligase, 25 DEG C of 2~12h of connection, and linked system is as shown in table 3:

Table 3

Carrier pCAMBIA1301a-PeVQ28 the plasmid of 5~10 μ L built is taken gently to be driven into 100 μ L EHA105 In Agrobacterium competent cell, after liquid nitrogen flash freezer 1min, 37 DEG C of water-bath 5min, 200 μ L YEP Liquid Cultures are added in ice bath 5min Base, 28 DEG C, 220 4~5h of culture；1.0×10⁴G is centrifuged 30s, abandons supernatant, and 100 μ L YEP fluid nutrient mediums are added, hang again Floating cell, is coated on the YEP solid plate containing 100 μ g/mL Kan and 50 μ g/mL Rif, 28 DEG C of culture about 24~48h；It chooses It makes even the yellowish single colonie grown on plate, is inoculated in the YEP liquid medium containing 100 μ g/mL Kan and 50 μ g/mL Rif In, shake 24~48h of bacterium；It to bacterium solution muddiness, is shown as orange-yellow, extracts plasmid；It is verified respectively with PCR and double digestion.

Embodiment 5

Plant transgene expression vector pCAMBIA1301a-PeVQ28 is transferred in wildtype Arabidopsis thaliana

1. the plantation of arabidopsis:

(1) on the super-clean bench, full wildtype Arabidopsis thaliana seed 8min is rinsed with 12% flower king's bleaching water, then to go out The distilled water repeated flushing of bacterium 7-8 times, rinses 3min every time.

(2) seed sterilized is drawn with 1mL liquid-transfering gun equably to blow and beat on 1/2MS solid medium, be put into 4 DEG C of ice It case vernalization 3 days, is then transferred into greenhouse and cultivates.

(3) after a week, the arabidopsis on plate is transplanted in the small basin equipped with vermiculite and black earth (3:1), every 4/basin. Then preservative film covers three days.Watering in growth period 3 days is primary, keeps ground moistening in flowerpot.

2. the preparation of Agrobacterium infected liquid:

The Overexpression vector Agrobacterium bacterium solution for taking 50 μ L to build, is added in 10mLYEP Liquid Culture, adds simultaneously Enter corresponding Kan and Rif, 28 DEG C, 230r/min, cultivates two days, OD₆₀₀=08-1.0.4 DEG C, 4.5 × 10³R/min, centrifugation 6min abandons supernatant.Precipitating is resuspended with conversion buffer culture medium, it is spare.

The stem of bolting is cut with scissors, breaks apical dominance, makes its mitogenetic more side shoot.To have 1/ on plant When 3 bud, a prepared bacterium solution is drawn with rubber head dropper, drop dips in bud, and cuts off existing fruit pod with scissors, reduces kind The false positive of son.It is protected from light dark culturing three days.After about 5 days, secondary infection is carried out with the method for sample.The seed finally harvested, For T0 generation.

Embodiment 6

The transgenic arabidopsis of moso bamboo PeVQ28 gene screens and identification

1. the screening of hygromycin culture medium:

It is equably laid on the MS culture medium of hygromycin, arabidopsis T0 seed such as above-mentioned steps.To 4 DEG C vernalization 3 days Afterwards, it is transferred in greenhouse and cultivates.About one week, sprouting situation is observed, it is such as normal to sprout, then it may be transgenic positive plant.

2.PCR Molecular

First according to CTAB method into extract PeVQ28 transgenic arabidopsis DNA,

(1) CTAB Extraction buffer is dissolved in water-bath (60 DEG C)；

(2) blade is placed in mortar, pours into liquid nitrogen, be ground to powdery.It is distributed into 2mLDof pipe, 1mL is added in every pipe CTAB Extraction buffer, whirlpool shake 30s, are placed in quiet 3-5min on ice, make sufficiently to crack；

(3) Dof pipe is placed in water-bath (65 DEG C), keeps the temperature 1h, keep cell completely broken；

(4) 1mL phenol: chloroform: isoamyl alcohol (25:24:1) is added into Dof pipe, and mixes well, 1.2 × 10⁴G, centrifugation 15min takes upper strata aqueous phase into new Dof pipe, isometric chloroform isoamyl alcohol is added, mixes well, and again 1.2 × 10⁴G, Centrifugation 15 minutes；

(5) supernatant of 400mL is inhaled again into new Dof pipe, and the isopropanol of 400mL is added, mixes, in -20 DEG C of ice Half an hour is stood in case, 1.2 × 10⁴G is centrifuged 15min, removes supernatant；

(6) ethyl alcohol (70%) of 1mL is added, is centrifuged 6min, abandons supernatant；

(7) step (6) are repeated；

(8) pure water for adding 50 μ L sterilized dissolves precipitating, and carries out the purity, concentration and A of DNA₂₆₀/A₂₈₀The detection of value.

Using wildtype Arabidopsis thaliana DNA as control, using the primer PeVQ28F and PeVQ28R of vector construction, and to turn The positive plant DNA of gene arabidopsis carries out PCR screening.PCR amplification program: 98 DEG C of 10min；Denaturation: 98 DEG C of 10s；Annealing: 62℃ 5s；Extend: 72 DEG C of 30s, 28 circulations；Overall elongation: 72 DEG C of 10min.After reaction, PCR product is taken, carries out 2% After agarose gel electrophoresis, detected in gel imaging system.

3. the phenotypic analysis of the transgenic arabidopsis of moso bamboo PeVQ28 gene

By transgenosis and wildtype Arabidopsis thaliana homozygote seed, plants in greenhouse, after two weeks, take pictures and observe phenotype； Salt stress processing is carried out simultaneously, during salt treatment, takes pictures and observes character mutation.And to the arabidopsis salt of different strains at Survival rate after reason counted and dry salt treatment before and after physical signs counted (Fig. 4).

Specific embodiment described herein is only an example for the spirit of the invention.The neck of technology belonging to the present invention The technical staff in domain can make various modifications or additions to the described embodiments or replace by a similar method In generation, however, it does not deviate from the spirit of the invention or beyond the scope of the appended claims.

Sequence table

<110>Agricultural University Of Anhui

<120>moso bamboo PeVQ28 albumen and its encoding gene and application

<130> 2018

<160> 5

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<210> 1

<211> 170

<212> PRT

<213>artificial sequence (PeVQ28)

<400> 1

Met Gly Glu Tyr His Arg Met Ser Pro Ser Thr Thr Leu Pro Val His

1 5 10 15

Lys Asp Ser Ser His Thr Ile Ser Lys Thr Arg Pro Lys Ile Lys Ile

20 25 30

Ile His Ile Ile Ser Pro Glu Ile Ile Lys Thr Asp Val Ala Asn Phe

35 40 45

Arg Asp Leu Val Gln Arg Leu Thr Gly Lys Pro Asp Ala Ser Thr Ala

50 55 60

Asp Val Ala Pro Ser Leu Pro Pro Val Glu Glu Glu Gln Lys Lys Glu

65 70 75 80

Ala Ile Lys Lys Arg Pro Pro Pro Pro Pro Ala Ala Glu Arg Gly Asp

85 90 95

Phe Met Val Pro Gln Glu Asn Lys Lys Lys Ile Lys Cys Glu Val Lys

100 105 110

Val Gln Glu Gly Gly Leu Gly Asp Gly Leu Asp His Asn Glu Leu Trp

115 120 125

Met Asp Leu Asn Pro Gly Gly Phe Leu Ser Phe Leu Glu Glu Asp Val

130 135 140

Phe Gln Gly Met Ala Pro Asp Phe Leu Gln Pro Leu Gly Ser Ser Arg

145 150 155 160

Met Asp Leu Val Gly Glu Met Tyr Ala Ser

165 170

<210> 2

<211> 513

<212> DNA

<213>artificial sequence (PeVQ28-CDS)

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atgggggagt accacagaat gagcccatca actacactgc ccgtgcacaa ggactcctcg 60

catactatat ccaagacgcg gcccaagata aagatcatcc acatcatttc accggagatc 120

atcaagaccg acgtcgccaa cttccgggac ctcgtgcagc ggctcactgg gaagccagac 180

gccagcacgg cggacgtggc tccgtcgctg ccgccggtag aggaggaaca gaagaaggag 240

gcgatcaaga agaggccgcc gccgccgccg gctgccgaga ggggcgattt catggtgcca 300

caagagaata agaagaagat caaatgcgag gttaaggttc aggaaggagg tttaggcgat 360

ggccttgatc acaacgagct gtggatggat ctgaatccgg gaggtttctt gagcttcttg 420

gaggaggacg tcttccaagg gatggctcct gacttcttgc agcctctcgg ctcgtcaagg 480

atggatttgg ttggtgaaat gtacgcatct taa 513

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<211> 1659

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ctatatatac ctttccattt ccctctcctc tcctcacaca ctgatctttc tagctctccc 60

cctctcttga tctctgtctg tctgcacact aatgggggag taccacagaa tgagcccatc 120

aactacactg cccgtgcaca aggactcctc gcatactata tccaagacgc ggcccaagat 180

aaagatcatc cacatcattt caccggagat catcaagacc gacgtcgcca acttccggga 240

cctcgtgcag cggctcactg ggaagccaga cgccagcacg gcggacgtgg ctccgtcgct 300

gccgccggta gaggaggaac agaagaagga ggcgatcaag aagaggccgc cgccgccgcc 360

ggctgccgag aggggcgatt tcatggtgcc acaagagaat aagaagaaga tcaaatgcga 420

ggttaaggtt caggaaggag gtttaggcga tggccttgat cacaacgagc tgtggatgga 480

tctgaatccg ggaggtttct tgagcttctt ggaggaggac gtcttccaag ggatggctcc 540

tgacttcttg cagcctctcg gctcgtcaag gatggatttg gttggtgaaa tgtacgcatc 600

ttaattgggt taagagtttt ctttgatttt attttccttt cgtgttgtga ggattttggc 660

actttggtag agaggattgg attctttttt tctcttccct ttgtctagat gaacacaatg 720

gtgttggaag ttcttgcacc cttggttgca aataatgatg tgtatgtaag gggtcactgg 780

tgggatcata gaaggaatga tcaatttggg tgcttaaatt tgttttgctc tctgttccaa 840

atgtaaataa ttgctctttg tcctgttttg aaagtaccat atcgagaaaa tcagaacagt 900

gatattttca actgaatcat ggtaagtgaa ctttaaattc tgttattttc acattgtagc 960

tgcactcttg atcatcattc tacccttgtt ttttgaagga aaattgtgct catcaatcac 1020

catcttgatg gcaattcaaa agtttttgtg tgttgtacaa gatggagctc cattattcca 1080

ttaatttggt caccaatctt acaccaagga atgtatacac ttgctctctg ctctctttga 1140

gagcaccaat ttagttttga gcccctttca gtagacctgc caaagaatgc tctgattaag 1200

tgtttgcttc agtttactaa ttgcaaagga tgtgatcaaa caggtgtcaa tgactctatg 1260

cacaactgga aattaccttg atgggaataa ctctttttta atagcattct tctatgtgaa 1320

gtttttttta gtccggatgt gtgctgccat tagcaatatg cagaggtgta agtaagctgg 1380

cagaaaacga cgcttacata ggctaatgcc ctagctcact tgtttaataa gtattatatg 1440

ttcggctatc ttaaacaaga tccaatggtt ttcttctaat atgtcctcaa tttggaagaa 1500

ccatataata gtactgacct aagatgtact acagaaaaga cagatagata gcagtgctga 1560

agggtaacat ataaattcag accaagttat tgctgaacaa atttagttga catttagttt 1620

ccattcagct ccttgtcaaa caatagccaa agactcaca 1659

<210> 4

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<213>artificial sequence (upstream primer)

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atacccggga tgggggagta ccacag 26

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<212> DNA

<213>artificial sequence (downstream primer)

<400> 5

gccgtcgact taagatgcgt acatttcac 29

Claims

1. a kind of moso bamboo PeVQ28 albumen, it is characterised in that: the amino acid sequence such as SEQ ID of the moso bamboo PeVQ28 albumen Shown in No.1.

2. a kind of coding gene sequence for encoding moso bamboo PeVQ28 albumen described in claim 1, it is characterised in that: the hair The nucleic acid sequence of bamboo PeVQ28 albumen is as shown in SEQ ID No.2.

3. the carrier containing coding gene sequence described in claim 2.

4. the construction method of carrier described in claim 3, it is characterised in that include the following steps:

5. the engineering bacteria containing carrier described in claim 3.

6. the primer pair for cloning coding gene sequence described in claim 2, it is characterised in that: the primer pair includes upstream Primer and downstream primer, the nucleotide sequence of the upstream primer is as shown in SEQ ID No.4, the core of the downstream primer Nucleotide sequence is as shown in SEQ ID No.5.

7. a kind of moso bamboo PeVQ28 gene, it is characterised in that: the nucleic acid sequence of the moso bamboo PeVQ28 gene cDNA overall length is such as Shown in SEQID No.3.

8. application of the moso bamboo PeVQ28 gene as claimed in claim 7 in plant modification salt resistance.

9. application according to claim 8, it is characterised in that: by the inflorescence dip method of mediated by agriculture bacillus by claim 7 genes or coding gene sequence as claimed in claim 2 are transferred in plant.