CN104845989A - Application of Arabidopis thaliana GST gene in improvement of survivability of plants to phenol stress - Google Patents

Abstract

The invention discloses an application of Arabidopis thaliana glutathione S-transferase gene and a protein encoded by the gene in the improvement of the survivability of plants to phenol stress. The nucleotide sequence of the gene is represented by SEQ ID No.1, and the amino acid sequence of the protein encoded by the gene is represented by SEQ ID No.2. The nucleotide sequence containing the Arabidopis thaliana glutathione S-transferase gene and an exogenous promoter are connected and then are transferred to Arabidopis thaliana, so the survivability of transgenic plants to phenol stress and the degradation ability of the transgenic plants are greatly improved. The application is important for researching the stress resistance of plants and plant restoration of organic pollutants.

Description

Utilize the tolerance that Arabidopis thaliana gst gene raising plant Pyrogentisinic Acid coerces

Technical field

The present invention relates to plant genetic engineering field.Specifically, utilize Agrobacterium to be transformed in plant by arabidopsis glutathione S-transferase gene, make it high expression in plant, improve the tolerance that plant Pyrogentisinic Acid coerces.

Background technology

Phenol is widely used in raw material in dyestuff, agricultural chemicals, industry production such as medicine synthesis etc. or intermediate, and being noxious pollutant moiety main in industrial discharge waste water, is modal water pollutant, has carinogenicity, teratogenecity and mutagenicity.Therefore, in succession listed in toxic pollutant list by Environmental Protection Agency and China.Phenol in water body environment is to hydrobiont, and especially fish harm is serious.It not only can cause the fishes and shrimps generation acute poisoning in water body environment dead, and easily in hydrobiont cylinder accumulation and enrichment, affects the quality of fishery products.A large amount of contaminated water source is unprocessed enters agroecosystem by irrigation. and cause this toxic organic pollutant from water body to agrological migration, or extensively accumulate in agricultural plants and agricultural animal by water body or by agricultural soil. thus greatly have impact on the quality of food and the security of food.The organism taken place frequently in recent years reveals event, also for local environmental safety, food safety and human and livestock health problem have beaten alarm bell.

The research report of current Pyrogentisinic Acid's Pollution abatement is relatively less, and administering method mainly contains the methods such as physics, chemistry, biology.The current many places of physics, chemical process are in conceptual phase, and use cost is high, technical sophistication, also may affect the eubiosis.The microorganism of degradable phenol is utilized to carry out Pollution abatement, practicality and validity are relatively strong, but it is weak that phenol microorganism resistance to phenol impact capacity falls in majority, can only grow under lower phenol concentration, and the growth of inoculating microbe can be subject to the competitive inhibition of indigenous microorganism.In addition, inoculating microbe is introduced the pollution that can cause again another kind of form in environment, biological safety makes people worried.Therefore, utilizing the general acceptable of people, more safe and reliable biological renovation method---phytoremediation technology becomes the focus of research, in the process of heavy metal and organic pollutant, shown obvious validity.But because the hydroxylation ability of plant Pyrogentisinic Acid is more weak, the degradation capability therefore for phenol is poor.

Glutathione S-transferase (glutathione S-transferase, be called for short GST, EC2.5.1.18) be the one group of multi-functional isozyme be distributed widely in plant, animal, birds, insect and microbe, its major function is the sulfydryl coupling of the various electrophilic compound of catalysis and reduced glutathion, increase its hydrophobicity and be easy to pass through cytolemma, get rid of external after decomposing, thus reach the effect of removing toxic substances.This of glutathione S-transferase acts on phytoremediation, especially plays vital effect in the decomposition course of weedicide.That play a major role in the process of phytoremediation organic compound is phi(F) and tau(U) this two class, they by plant peculiar, and to be present in all plants with the form of gene family.Phi(F) and tau(U) transferring enzyme can fast to the detoxification of chloro-acetyl amine, chloro triazines, diphenyl ether and fragrant phenoxy acid lipid weedicide, to some herbicide-safeners as benoxacor, allyl dichloride amine and benoxacor also have same effect.In addition, this enzyme also has detoxification to tetracycline antibiotics gold toxin and anodyne Paracetamol, avoids entered environment to produce and pollutes.

Summary of the invention

The object of this invention is to provide a kind of novelty teabag of arabidopsis glutathione S-transferase gene, by this gene transferred plant, the degradation capability of tolerance that plant Pyrogentisinic Acid coerces and Pyrogentisinic Acid can be improved.

Technical scheme:

With the coding region of PCR means clone arabidopsis glutathione S-transferase gene AtGSTF2, after order-checking qualification, be connected to transformed carrier pYPX245, see Fig. 1, then transform target crop to dip in colored method.

Beneficial effect:

The present invention has cloned arabidopsis glutathione S-transferase gene, the phenol stress tolerance experimental result of Arabidopsis plant and wild-type Arabidopsis plants that this turns AtGSTF2 gene is shown: transfer-gen plant and WT lines have obvious difference on biomass (dry weight), transfer-gen plant has obvious anti-phenol ability than WT lines, and phenol residual quantity reduces in substratum, what this also showed arabidopsis glutathione S-transferase gene proceeds to the tolerance that improve Arabidopsis plant Pyrogentisinic Acid.

Accompanying drawing explanation

The structure of Fig. 1 arabidopsis glutathione S-transferase gene expression vector.

Fig. 2 RT-PCR identifies transgenic line.

After Fig. 3 arabidopsis thaliana transformation glutathione S-transferase gene, transgenic arabidopsis shows the tolerance raising that Pyrogentisinic Acid coerces.

In Fig. 4 nutrient solution, phenol remains.

Embodiment

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

Genetic manipulation conventional in following embodiment carries out (Sambook J with reference to molecular cloning document, Frets E F, Mannsdes T et al. In:Molecular Cloning. 2nd ed. Cold Spring Harbor Laboratory Press, 1989).

The clone of embodiment 1 arabidopsis glutathione S-transferring enzyme AtGSTF2 gene

The extraction agent box of plant total serum IgE is that love is pursued progress biotechnology (Hangzhou) company limited (http://axygenbio.com) product, Reverse Transcription box is Quan Shijin bio tech ltd (http://www.transgen.com.cn) product, various restriction enzyme and T ₄dNA Ligase is purchased from the precious biotechnology company limited (http://takara.com.cn) in Dalian.

Take the extracting that about 0.1g vegetable material carries out total serum IgE, concrete grammar carries out with reference to the plant sample extracting specification sheets operation of above-mentioned Axygen company, the total serum IgE of institute's extracting carries out cDNA synthesis, and concrete grammar carries out the first chain synthesis with reference to the specification sheets operation of TransGen company.With cDNA first chain of synthesis for template, under the guiding of primer GST-PF and primer GST-PR, by the sequence of Standard PCR method amplification Arabidopis thaliana GSTF2 gene.Amplification condition is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C extend 1min, coamplification 30 circulation, then 72 DEG C extend 10min.After reaction terminates, carry out 1% agarose gel electrophoresis detection to pcr amplification product, reclaim also purifying and be about the DNA fragmentation of 700bp, and this fragment is connected into pMD18-Simple carrier T, screening positive clone also checks order, the full length gene needed for acquisition.

Primer GST-PF:5'- aAGGATCCaTGGCAGGTATCAAAGTTTTC-3'(is with underscore partial sequence to be restriction enzyme BamHI recognition site and protection base);

Primer GST-PR:5'- aAGAGCTCtCACTGAACCTTCTCGGAAG-3'(is with underscore partial sequence to be restriction enzyme SacI recognition site and protection base).

Sequencing result shows, the DNA fragmentation of above-mentioned about 700bp is containing the 639bp nucleotide sequence shown in SEQ ID No 1 in ordered list, this sequence is the encoding sequence of AtGSTF2 gene, coding has the albumen of the amino acid composition of 211 shown in sequence table SEQ ID No 2, and this protein designations is AtGSTF2.

The structure of embodiment 2:AtGSTF2 gene overexpression carrier

DNA fragmentation BamHI and SacI containing Nucleotide shown in sequence table SEQ ID No 1 of the about 700bp obtained by pcr amplification in embodiment 1 carries out double digestion, reclaim after purifying with the pYPX245(Genbank:AY78049.1 containing pair 35S promoter and NOS terminator) plasmid is connected, enzyme cuts the expression of plants unit that qualification and sequencing show containing glutathione S-transferase gene, this expression unit is inserted plant expression vector pCambia1301, builds the plant expression vector pCAM:AtGSTF2 of arabidopsis glutathione S-transferring enzyme.This expression vector also comprises gus reporter gene and band intron kalamycin resistance marker gene.

The acquisition of embodiment 3 transgenic arabidopsis

The plant expression vector pCAM:AtGSTF2 of arabidopsis glutathione S-transferring enzyme embodiment 2 built is with dipping in colored method arabidopsis thaliana transformation, and concrete grammar is as follows:

1, by pCAM: AtGSTF2 with electric shocking method transform Agrobacterium tumefaciens GV3101, or EHA105, or LBA4404 bacterial strain (Biovector Co., LTD), obtain the recombinational agrobacterium containing pCAM: AtGSTF2, and coat the plate screening transformant containing kalamycin resistance.

2, be inoculated in by above-mentioned transformant in YEB liquid nutrient medium (containing 100 μ g/ml kantlex and 75 μ g/ml Rifampins), 28 DEG C of shaking culture are to OD ₆₀₀for 0.6-0.8; With the centrifugal 10min of 10,000rpm room temperature, collect thalline, suspend with the fresh sucrose solution of equal-volume 5%, and transfer in beaker after adding the Silwet-77 mixing of 0.02%.Each bacterial strain 300ml transforms, and turns 2-3 alms bowl.Immersed in penetrating fluid by thaliana flower a kind of sedge in conversion operation, take out after stirring about 10s gently, after all transforming, cover Arabidopis thaliana with preservative film, to keep moist environment, horizontal positioned 22 DEG C of lucifuges are cultivated, and remove preservative film and uprightly cultivate after 24h.After first transfonning four days, can once transform again, total cotransformation three times.Growth about after two months, is gathered in the crops seed, and is carried out transformed plant screening with 50mg/L Totomycin.

The PCR qualification of embodiment 4 transgenic arabidopsis

All plant leafs in the Arabidopis thaliana transgenic line that embodiment 3 is obtained extracting genomic dna respectively, carry out pcr amplification, the primer is: HPT-F: 5 '-TACTTCTACACAGCCATC-3 ' and HPT-R: 5 '-CGTCTGTCGAGAAGTTTC-3 ', target sequence is the partial sequence of hygromycin phosphotransferase gene, prediction object product sheet segment length is about 900bp, and the agarose gel electrophoresis result of plant part amplified production as shown in Figure 2.Containing object product is positive transgenic strain.Obtain positive T1 altogether for transgenic arabidopsis strain 12.

The phenol tolerance of embodiment 5 transgenic arabidopsis is analyzed

Conversion of plant selfing was isozygotied for 3 generations, obtains the transformant that isozygotys, results seed.Choose 3 transgenic lines, seed together with Wild type control plants is laid on MS solid medium, grows to when two panels true leaf grows and moves to MS liquid nutrient medium preculture 2 days, discarded by nutrient solution subsequently until seed germination, and add new nutrient solution and the phenol of different concns, phenol concentration is set as 0,50,75,100,125,150(mg/L), observe the tolerance of seedling under phenol is coerced.Fig. 3 shows, the biomass of wildtype Arabidopsis thaliana under phenol stress conditions (dry weight) comparatively transgenic line has obvious minimizing.When phenol concentration is 100 mg/L, the difference of two kinds of Genotypes is the most obvious.Therefore, take phenol concentration as the substratum of 100 mg/L be research object, be HPLC analyze at interval of within 24 hours, extracting nutrient solution, observe the residual quantity of phenol in nutrient solution.Use instrument for Agilent 1100 HPLC system ((Agilent company), testing conditions is: Athena C18 chromatographic column (4.6mm*150mm, 5 μm); Moving phase acetonitrile: water, 30%:70% (v/v), flow velocity 1ml/min, sample size 20 μ l, determined wavelength 213nm.Fig. 4 shows, in the substratum of plantation transgenic line, phenol residual quantity comparatively plants the less of wild-type material, illustrates that the degradation capability of transgenic line Pyrogentisinic Acid is stronger.

Attached: nucleotides sequence list of arriving involved in the present invention:

<110> Academy of Agricultural Sciences, Shanghai City

The tolerance that <120> utilizes Arabidopis thaliana gst gene raising plant Pyrogentisinic Acid to coerce

<160> 2

<170> PatentIn version 3.3

<210> SEQ ID No 1

<211> 639

<212> DNA

<213> Arabidopis thaliana ( arabidopsis thalianal.)

<400> 1

1 ATGGCAGGTA TCAAAGTTTT CGGACACCCA GCTTCCATTG CCACCAGGAG AGTCCTCATC

61 GCCCTCCACG AGAAAAACCT CGACTTTGAG CTCGTTCATG TCGAACTCAA AGACGGTGAG

121 CACAAGAAGG AGCCTTTCCT CTCCCGCAAC CCTTTTGGTC AGGTTCCAGC CTTTGAAGAT

181 GGAGACCTCA AGCTCTTCGA ATCAAGAGCG ATTACTCAGT ACATAGCTCA CCGATATGAA

241 AACCAAGGAA CCAACCTTCT CCAAACCGAC TCCAAGAACA TATCTCAGTA CGCAATCATG

301 GCCATTGGAA TGCAAGTAGA AGATCACCAG TTCGACCCAG TGGCTTCAAA GCTTGCTTTT

361 GAACAAATAT TCAAGTCCAT CTACGGCTTG ACCACAGACG AAGCCGTTGT TGCAGAAGAG

421 GAGGCTAAGT TAGCCAAGGT CCTTGATGTC TACGAGGCTA GGCTCAAGGA GTTCAAGTAT

481 TTGGCTGGTG AAACTTTCAC TTTGACTGAT CTTCACCACA TTCCCGCGAT TCAATACCTG

541 CTCGGAACTC CCACCAAGAA GCTCTTCACC GAGCGTCCAC GTGTCAATGA GTGGGTGGCT

601 GAAATCACCA AGAGGCCAGC TTCCGAGAAG GTTCAGTGA

<210> SEQ ID No 2

<211> 212

<212> PRT

<213> Arabidopis thaliana ( arabidopsis thalianal.)

<400> 2

1 MAGIKVFGHP ASIATRRVLI ALHEKNLDFE LVHVELKDGE HKKEPFLSRN PFGQVPAFED GDLKLFESRA

71 ITQYIAHRYE NQGTNLLQTD SKNISQYAIM AIGMQVEDHQ FDPVASKLAF EQIFKSIYGL TTDEAVVAEE

141 EAKLAKVLDV YEARLKEFKY LAGETFTLTD LHHIPAIQYL LGTPTKKLFT ERPRVNEWVA EITKRPASEK 211 VQ-

Claims (5)

1. the protein of arabidopsis glutathione S-transferase gene or its coding or polypeptide are improving the purposes in plant Pyrogentisinic Acid's stress tolerance and degradation capability.

2. purposes as claimed in claim 1, it is characterized in that, the sequence of described protein or polypeptide is as shown in SEQ ID No 2.

3. purposes as claimed in claim 2, is characterized in that, described arabidopsis glutathione S-transferase gene is the sequence of coding protein according to claim 2 or polypeptide.

4. purposes as claimed in claim 3, it is characterized in that, the nucleotide sequence of described arabidopsis glutathione S-transferase gene is as shown in SEQ ID No 1.

5. purposes as claimed in claim 1, is characterized in that, the tolerance that described raising plant Pyrogentisinic Acid coerces shows as: improve the biomass of plant under phenol is coerced (dry weight); The degradation capability of described raising plant Pyrogentisinic Acid shows as: reduce phenol residual quantity in substratum.