CN102586281B - Gene with salt resistant function, encoding protein thereof and application of encoding protein - Google Patents

Abstract

The invention relates to a gene with a salt resistant function, an encoding protein thereof and an application of the encoding protein. The gene has a DNA (Deoxyribonucleic Acid) sequence shown as SEQ ID NO:1. According to the invention, a thioredoxin gene DvTRX is firstly separated from Dunaliellaviridis, and the gene is proved to have the salt resistant function and an application value of improving the characters of crops through transforming yeast cells.

Description

Gene, its proteins encoded and application with salt tolerant function

Technical field

The present invention relates to a kind of gene, its proteins encoded and application thereof with salt tolerant function.Be particularly related to a kind of salt algae ( Dunaliella viridis) Trx (thioredoxin) gene DvTRXNucleotide sequence, and proteins encoded and improving biological salt resistance ability and the application in improvement farm crop proterties.

Background technology

World population increases day by day, and cultivated area reduces day by day, improves crop yield and becomes this important channel to contradiction of solution.Yet the soil salinization, arid, extreme temperature and nutrition are barren etc., and various environment-stress have had a strong impact on the g and D of farm crop and have reduced its output.When plant suffered environment stress, the stage can be caused plant materials ion imbalance and infiltration disorder in the early stage; The continuity of coercing or add the generation that escheat usually can be followed oxygen production and oxidative stress.Salinization is one of most important environmental stress that influences plant-growth, and it has had a strong impact on output and the productivity of farm crop.The salinification that the soil ploughed in the world 20% is serious has had a strong impact on the output of grain.Experimental results demonstrate the overexpression of the oxygen scavenging activity factor in the transgenic plant, or the mutant with high reactivity oxygen scavenging function more has stronger tolerance to environment-stress.The anti-salt mechanism that research is biological utilizes Protocols in Molecular Biology to obtain resistant gene, discloses the relation between plant and the adverse circumstance, and the supplemental crop breeding has become one of effective way that improves crop yield.

The Dunaliella salina genus ( Dunaliella) be the eucaryon unicell green alga that occurring in nature extensively exists, be distributed widely in the environment of high salinity such as salt lake, ocean, solonchak, especially be main population in the haline water territory, be easy to from physical environment, separate obtaining.The salt algae ( Dunaliella viridis) be a representative kind during Dunaliella salina belongs to.This kind claims Dunaliella salina, green Dunaliella salina, salt to give birth to green Dunaliella salina etc. again.Environment is had extremely strong adaptive faculty, is the eukaryote of known salt tolerant: can tolerate environment-stress such as high salt, heavy metal, intense UV rays radiation.Therefore, the salt algae becomes an important anti-contrary genetic resources treasure-house.

(Thioredoxin is that a class extensively is present in the multi-functional acid albumen in the organism Trx) to Trx.Sulphur oxygen also egg TRX family is the protein of small molecule amount (about 12 kD), is prevalent in the nearly all biont from prokaryotic organism to the higher eucaryote.Trx has the various biological function, has important effect to keeping redox state stable in the organism.Its function overwhelming majority is the disulfide linkage that depends in the Trx reduction target protein.All Trxs all have a conservative redox active site amino acid motif, Cys-Gly-Pro-Cys (CGPC), the active centre has two to have the also cysteine residues of activity of oxygen, 2 Cys residues can form disulphide by a transferase 12 electronics and 2 proton reversible ground, for a series of physiological responses provide redox couple.Except the disulfide linkage of reduction target protein, it also participates in adjusting to enzyme as the H+ donor, and transcription factor is regulated and control and played a role in the oxidation adverse circumstance.

Trx has functions such as regulate the cell growth, suppress apoptosis, regulatory gene is transcribed.Trx is also relevant with plant stress tolerance, as involved in plant drought resisting, the heat-resisting and anti-oxidant process of coercing, regulates the expression of adversity gene etc.At present, also there is not the document Trx of reporting for work to have the function of salt tolerant.

Summary of the invention

One of purpose of the present invention is to provide a kind of gene with salt tolerant function, this gene be from the salt algae ( Dunaliella viridis) in Trx (thioredoxin) gene separated, be called DvTRX

Two of purpose of the present invention is to provide the proteins encoded of this gene.

Three of purpose of the present invention is to provide a kind of carrier, and this carrier contains gene of the present invention.

Four of purpose of the present invention is to provide a kind of host cell that contains gene of the present invention.

Five of the object of the invention provides the application that utilizes gene to improve biological salt tolerance aspect, it is characterized in that containing DvTRXThe responsive yeast cell of the salt of gene can obtain certain salt resistance ability.

For achieving the above object, the present invention adopts following technical scheme:

A kind of gene with salt tolerant function is characterized in that this gene is the dna sequence dna shown in the SEQ ID NO:1.

The proteins encoded of above-mentioned gene is characterized in that this albumen is the aminoacid sequence shown in the SEQ ID NO 2.

A kind of recombinant expression vector, this recombinant vectors contains above-mentioned gene.

A kind of host cell, this host cell contains above-mentioned recombinant expression vector.

A kind of above-mentioned application of the gene with salt tolerant function in improving the organism salt resistance ability.

A kind of above-mentioned application of the gene with salt tolerant function in improvement farm crop proterties.

The present invention has been cloned into a thioredoxin gene by the method for environment stress from the salt algae, and has proved that by experiment it also has the function of salt tolerant.Grand analyze that the TRX gene helps the deep anti-salt mechanism of salt algae of understanding in the salt algae, also can provide genetic resources for biological resistant gene of salt engineering and molecular breeding simultaneously.The present invention first from the salt algae ( Dunaliella viridis) in be separated to Trx (thioredoxin) gene DvTRX, and prove that by transformed yeast cell this gene also has the using value of salt tolerant function and improvement farm crop proterties.

Description of drawings

Fig. 1 is of the present invention DvTRXHomology analysis figure between the species of gene coded protein.Square frame is labeled as DvTRXGene has redox active site amino acid motif, Cys-Gly-Pro-Cys (CGPC).

Fig. 2 is of the present invention DvTRXThe albumen evolutionary analysis figure of genes encoding shows that this proteins encoded belongs to Trx family.

Fig. 3 is fusion GST label of the present invention DvTRXGene coded protein SDS-polyacrylamide gel electrophoresis figure.M: albumen marker; The full bacterium of 1:pGEX4T-1; The 2:pGEX4T-1 supernatant; The 3:pGEX4T-1 precipitation; The full bacterium of 4:pGEX4T-1-TRX; The 5:pGEX4T-1-TRX supernatant; The 6:pGEX4T-1-TRX precipitation; 7: do not induce the full bacterium of pGEX4T-1-TRX; 8: do not induce the pGEX4T-1-TRX supernatant; 9: do not induce the pGEX4T-1-TRX precipitation.Above red arrow is depicted as abduction delivering fusion GST label DvTRXGene coded protein.

Fig. 4 is of the present invention DvTRXGene is at yeast mutants G19In functional analysis.That is: contain DvTRXThe est sequence of complete ORF is inserted among the Yeast expression carrier pAJ401, and transformed yeast salt sensitive mutant G19 shows that in the phenotype of the NaCl that contains different concns substratum carries out phenotype test.

Specific implementation method

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these examples only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted concrete experiment condition in the following example, usually according to normal condition, molecular cloning (Molecular Cloning:A Laboratory Manual, 3rd ed.) or yeast genetics method (Methods in Yeast Genetics:A Cold Spring Harbor Laboratory Course Manual, Adams A et al compiles, Cold Spring Harbor Laboratory, 1998 publish) described in condition, or the condition of advising according to manufacturer.

Embodiment one: DvTRXThe clone of full length coding region and analysis

To the salt algae D. viridisCDNA library est sequence (expressed sequence tag) that induced by different concns salt is analyzed, and by sequence alignment, has been separated to a salt algae thioredoxin gene, called after DvTRX DvTRXCDNA total length 841 bp, contain complete open reading frame (ORF), Kozak sequence (GGC ATGC) and poly (A) structure.

Embodiment two DvTRXEncoded protein structure, homology analysis

The analytical results of Vector NTI software shows: one of this genes encoding contains 161 amino acid whose albumen, and the molecular weight of albumen size is 18.02 kDa, and iso-electric point is 9.44. DvTRXEncoded protein has 5 β chains and 4 α chains. DvTRXHas higher homology with the Thioredoxin gene of other species.The albumen n end of this genes encoding also has conservative Cys-Gly-Pro-Cys (CGPC) sequence of Thioredoxin, and this is Trx redox active site amino acid motif (Fig. 1).Fig. 2 is of the present invention DvTRXThe albumen evolutionary analysis figure of genes encoding shows that this proteins encoded belongs to Trx family.

Embodiment three DvTRXThe location prediction of proteins encoded and prokaryotic expression analysis

By the WoLFPSORT software prediction, DvTRX albumen is positioned in the chloroplast(id), belongs to the X class subfamily of TRX.By the method for PCR, DvTRXThe gene ORF two ends add restriction endonuclease sites EcoRI and XhoI, after the order-checking of connection T carrier is correct, EcoRI and XhoI double digestion purpose fragment is connected on the prokaryotic expression carrier pGEX4T-1 of same double digestion, obtains recombinant plasmid pGEX4T-1-DvTRX.Recombinant plasmid transformed enters among the escherichia coli expression bacterial strain BL21, and 1mM IPTG induces ultrasonication thalline after 4 hours for 25 ℃.Fig. 3 is for merging the DvTRX Protein S DS-polyacrylamide gel electrophoresis figure of GST label.

Embodiment four DvTRXFunctional analysis in yeast mutants

(1) structure of Yeast expression carrier

At first will contain DvTRXThe est sequence of complete ORF (4977) is building up in the Yeast expression carrier: we utilize restriction endonuclease sites with 4977 sequences EcoR I and XhoI is building up among the Yeast expression carrier pAJ401.

(2) transformed yeast

Use LiAc heat shock conversion method will contain 4977 recombinant clone and be transformed into yeast salt sensitive mutant G19(Mat, ura3 his3 trp1 ade2 ena1 △:: ena4 △).

(3) phenotypic evaluation of yeast transformant

Used minimum medium is the AP substratum, and adds amino acid needed.Selection markers is the uridylic auxotrophy.Changing carrier over to is the transformant phenotype displaying substratum of pAJ401, and the semi-lactosi of interpolation 2% is as carbon source.Used phenotype is showed substratum: serve as the contrast growth conditions with the AP substratum that does not contain NaCl, the AP substratum that adds the NaCl of different concns is salt tolerant phenotype analytical growth conditions.

At first yeast transformant is cultured to OD in the AP substratum ₆₀₀=0.6, be a titre with 5 μ L, and be the serial dilution titre with 5 times of extent of dilution, show that in phenotype substratum shows the salt tolerant phenotype of yeast transformant.Under pAJ401 composing type strongly expressed promotor starts, the phenotype (Fig. 4) of yeast salt sensitive mutant G19 that 4977 successes are complementary; The result shows to have transformed and contains DvTRXThe yeast salt sensitive mutant of the est sequence of complete ORF can still have phenotype under 220mM NaCl, illustrated DvTRXGene can partly recover the function of yeast mutants, has proved DvTRXCan improve the salt resistance ability of organism effectively.

Although the invention describes concrete example, having a bit is significantly to those skilled in the art, namely can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.

＜110〉Shanghai University

＜120〉have gene, its proteins encoded and the application of salt tolerant function

<160> 2

<210> 1

<211> 841

<212> DNA

＜213〉the salt algae ( Dunaliellaviridis)

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aacacctctg gctggtggag tggcaggatc caggccccgt ctgcagcagc aagggcgtcg 180

cgctggcttg aggcgagtcc agacgcgcgt ggccaatgtg gatgaaacca attttgagca 240

ggaggtgctg caagagactg agagggccgt gctcgtagac ttttgggaca cctggtgcgg 300

accctgcaaa ctgatggatc ccctcatgca gtgggcggag caggagtatg gtaacgtttt 360

gaaggtggtt aaaatcaggc acgattcatg ccccaagtta atagagcgct acaaggtgta 420

cgggctgcca gcattcctcg tcttcaagaa cggcgagctt gttcccgggt gtaagaggga 480

gggtgccgtc aaccagaaaa tgttgacgga ctacctgaag cagcacgggg ccgtttaatg 540

agttgtgggg ggttgcaagg gggaattttt ggagagagca cgtaagcatt gcattttttt 600

ctgtcctggt gagttgagag actgcaggca ggcttatcta ctctgttcgt gagtttctgt 660

gcgtcgagtt gtggacctat ctttccggtg actgactaga atacctgcag acttgtgctg 720

ggatgggttt ctagtgtttt gtcttccttg gtccatcctt tcatttaatt ggtgcctatg 780

ccagtcctca aaatattgtg aacaatccac ctgcgaaaaa aaaaaaaaaa aaaaaaaaaa 840

a 841

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<211> 161

<212> PRT

＜213〉the salt algae ( Dunaliellaviridis)

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atgctcgccctgaacggcgtaaagccaagccccagcacgagactggctgcctcctccagg

M L A L N G V K P S P S T R L A A S S R

cctcaagcaacacctctggctggtggagtggcaggatccaggccccgtctgcagcagcaa

P Q A T P L A G G V A G S R P R L Q Q Q

gggcgtcgcgctggcttgaggcgagtccagacgcgcgtggccaatgtggatgaaaccaat

G R R A G L R R V Q T R V A N V D E T N

tttgagcaggaggtgctgcaagagactgagagggccgtgctcgtagacttttgggacacc

F E Q E V L Q E T E R A V L V D F W D T

tggtgcggaccctgcaaactgatggatcccctcatgcagtgggcggagcaggagtatggt

W C G P C K L M D P L M Q W A E Q E Y G

aacgttttgaaggtggttaaaatcaggcacgattcatgccccaagttaatagagcgctac

N V L K V V K I R H D S C P K L I E R Y

aaggtgtacgggctgccagcattcctcgtcttcaagaacggcgagcttgttcccgggtgt

K V Y G L P A F L V F K N G E L V P G C

aagagggagggtgccgtcaaccagaaaatgttgacggactacctgaagcagcacggggcc

K R E G A V N Q K M L T D Y L K Q H G A

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Claims (6)

1. the gene with salt tolerant function is characterized in that this gene is the dna sequence dna shown in the SEQ ID NO:1.

2. the proteins encoded of a gene according to claim 1 is characterized in that this albumen is the aminoacid sequence shown in the SEQ ID NO 2.

3. recombinant expression vector, this recombinant vectors contains gene according to claim 1.

4. host cell, this host cell contains recombinant expression vector according to claim 3.

5. the application of gene in improving the yeast cell salt resistance ability with salt tolerant function according to claim 1.

6. the application of gene in improvement yeast cell proterties with salt tolerant function according to claim 1.

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