CN102020707B

CN102020707B - Protein, coding genes and application thereof

Info

Publication number: CN102020707B
Application number: CN 201010528752
Authority: CN
Inventors: 麻密; 何振艳; 陈宏林
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2010-11-02
Filing date: 2010-11-02
Publication date: 2013-04-17
Anticipated expiration: 2030-11-02
Also published as: CN102020707A

Abstract

The invention discloses a protein, coding genes and an application thereof. The protein is artificially synthesized and is named as KcPCS1, and is the protein as the following (1) or (2): (1) the protein consisting of amino acid sequences expressed by a sequence 2 in a sequence list; (2) the protein which is derived from (1) by substituting and/or deleting and/or adding the amino acid sequences in the sequence 2 by one or more amino acid residues and is related to stress tolerance. The experiments show that KcPCS1 gene is subjected to yeast transformation; the transgenic yeast has excellent resistance to cadmium (Cd); and the content of Cd in the body is higher than that of the yeast cell transferring into a vector.

Description

A kind of albumen and encoding gene thereof and application

Technical field

The present invention relates to a kind of albumen and encoding gene thereof and application.

Background technology

Heavy metal generally refers to proportion greater than 4 or 5, and density is greater than 4.5g/cm ³Metal, have approximately 45 kinds, such as metals such as copper, lead, zinc, iron, cobalt, nickel, vanadium, niobium, tantalum, titanium, manganese, cadmium, mercury, tungsten, molybdenum, gold and silver.Arsenic although be non-metallic element, because it has very high toxicity, often is classified as the heavy metal ranks.

Excessive heavy metal on plants has very strong toxic action, can have influence on growing of plant.The human heavy metal Crs such as Chen Qinghua ⁶⁺Process corn seedling, found that mda (MDA) in the plant materials and the concentration of conductivity intensity and Cd are extremely significantly dependency, chlorophyll content and superoxide dismutase (SOD) then reduce along with the increase of concentration for the treatment of.The people such as Du Tianqing adopt Cd, Pb, Cr to process wheat seedling, have also found similar results, and this explanation Cd, Pb, Cr have caused serious destruction to vegetable cell, cause the saturating property of selection of plasma membrane to weaken structure deteriorate, afunction.

Occurring in nature, the living environment that plant raises day by day in order to adapt to heavy metal content has had various detoxifcations to heavy metal and has restrained oneself mechanism.These mechanism mainly comprise: (1) prevents that heavy metal from entering in the body, for example movement of rhizospheric microorganism restriction metal ion, by root cell wall and secretory product constraint, reduce the input of striding cytoplasmic membrane; (2) initiatively heavy metal is effluxed; (3) sequestering action in the plant materials; (4) separate in the plant materials and turn usefulness into; (6) effects such as self-regeneration of cell.

Present result of study shows that plant is complexing action and compartmentalization to the resistance mechanism main manifestations of heavy metal.Heavy metal ion can reduce its free ion concentration in born of the same parents after narrow spectrum binding peptide is combined in the plant materials, thereby alleviates it to the toxicity of plant.Simultaneously, plant can be further with in addition compartmentation isolation of heavy metal, as it is separated in vacuole, learn the injury that the macromolecular structure of function is subject to metal ion to avoid having important biomolecule in the cell.The heavy metal binding peptide of most study is plant complexing element (Phytochelatins-PCs) and metallothionein(MT) (Metallothionein) at present.Metallothionein(MT) is a class by the synthetic polypeptide of gene family coding, and the plant complexing element synthetic polypeptide that is rich in halfcystine of short reaction that is class of enzymes.Present research is thought: plant complexing element plays an important role in heavy metal detoxification.

Mangrove is the dominant plant that is grown in the torrid zone, river mouth, bay, subtropics, is the important primary producer of this ecosystem, and the eubiosis of safeguarding environment is had a very important role.As the unique Forest ecosystems in coastland, mangrove forest has than high-bearing capacity and tolerance the environment of bay estuary region, and especially counterweight metallic red tree table has revealed superior resistance.

The research of mangrove heavy metal resistance molecular mechanism is prerequisite and the basis of exploitation phytoremediation engineered plant.In recent years, people more and more pay attention to inquiring into from molecular level the molecular ecology mechanism of mangrove counterweight metal implement higher tolerance, in the hope of identify may be in mangrove ubiquitous efficient heavy metal binding substance and controlling gene thereof.

Summary of the invention

An object of the present invention is to provide a kind of albumen and encoding gene thereof.

The invention provides a kind of protein, synthetic, called after KcPCS1 is following 1) or 2) protein:

1) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;

2) with the aminoacid sequence of sequence 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with resistance of reverse by 1) protein of deriving.

Above-mentioned sequence 2 is comprised of 496 amino-acid residues, the replacement of described one or several amino-acid residue and/or disappearance and/or be added to replacement and/or disappearance and/or the interpolation that is no more than 10 amino-acid residues.

The encoding gene of described protein also is the scope of protection of the invention.

Above-mentioned sequence 1 is comprised of 1991 Nucleotide, the coding region be in the sequence table sequence 1 from 5 ' terminal 140-1627 position Nucleotide.

Described encoding gene is following 1), 2), 3), 4) or 5) gene:

1) dna molecular shown in the sequence 1 in the sequence table;

2) in the sequence table sequence 1 from the dna molecular shown in 5 ' the terminal 140-1627 position Nucleotide;

3) in the sequence table sequence 1 from the dna molecular shown in 5 ' the terminal 140-1658 position Nucleotide;

4) under stringent condition with 1) or 2) or 3) dna molecule hybridize that limits and the dna molecular of coding stress tolerance correlative protein;

5) with 1) or 2) or 3) dna sequence dna that limits has the dna molecular of 90% homology and coding stress tolerance correlative protein at least.

Recombinant vectors, transgenic cell line, recombinant bacterium or the expression cassette that contains described encoding gene also is the scope of protection of the invention.

Described recombinant vectors is to insert the recombinant vectors that described encoding gene obtains between the BamHI of carrier pFL61 and SalI recognition site;

Described recombinant bacterium is for importing the recombinant bacterium that Host Strains obtains with described recombinant vectors, and described Host Strains is preferably yeast (Saccharomyces cerevisiae).

The primer of described encoding gene total length or the arbitrary fragment of increasing is to also being the scope of protection of the invention, and described primer is to as follows: a primer sequence is shown in sequence in the sequence table 3, and another primer sequence is shown in sequence in the sequence table 4.

Another object of the present invention provides a kind of method of cultivating recombination yeast.

Method of the present invention is for importing the recombination yeast that host's yeast obtains with described encoding gene, and the resistance of reverse of described recombination yeast is higher than described host's yeast, and described Host Strains is preferably yeast (Saccharomyces cerevisiae).

Described resistance of reverse is anti-heavy metal and/or salt tolerant, and described heavy metal is preferably cadmium, and described heavy metal is not arsenic.

The not anti-arsenic of described recombination yeast.

Described encoding gene imports host's yeast by described recombinant vectors.

The application of described protein in the resistance of reverse of heavy metal contamination reparation and/or raising plant, or the application of described encoding gene in the resistance of reverse of heavy metal contamination reparation and/or raising plant, or the application of described recombinant vectors in the resistance of reverse of heavy metal contamination reparation and/or raising plant, or the application of described recombinant bacterium in the resistance of reverse of heavy metal contamination reparation and/or raising plant; Above-mentioned application is the scope of protection of the invention.

Described resistance of reverse is preventing from heavy metal and/or salt tolerant;

Described heavy metal is cadmium, and described heavy metal is not arsenic.

Of the present invention experimental results show that, the KcPCS1 gene has been carried out yeast conversion, detected the resistance of transformant to cadmium and arsenic, in wild-type yeast behind the expressing K cPCS1 gene, find that transgenic yeast obviously strengthens the resistance of cadmium Cd, the Cd content in its body also is higher than the yeast cell that changes empty carrier over to significantly.Because the autumn habitat of eggplant is hypersaline environment, detected the salt resistance of KcPCS1 gene yeast transformant, the result shows that the resistance of KcPCS1 gene pairs yeast salt is significantly improved, and this result shows that plant complexing element also exists certain effect to other abiotic stress outside the removing heavy metals such as salt stress etc.

Description of drawings

Fig. 1 is the agarose gel electrophoresis figure of total RNA

Fig. 2 is autumn eggplant cDNA agarose gel electrophoresis figure

Fig. 3 is conservative fragments agarose gel electrophoresis detection figure

Fig. 4 is RACE experiment agarose gel electrophoresis detection figure

Fig. 5 is that Yeast expression carrier pFL61-KcPCS1 detects electrophorogram

Fig. 6 is that yeast transformant positive colony PCR identifies

Fig. 7 is the experiment of expressing K cPCS1 gene pairs Cd resistance

Fig. 8 is the experiment of expressing K cPCS1 gene pairs As resistance

Fig. 9 is expressing K cPCS1 gene pairs Cd absorption experiment

Figure 10 is the experiment of expressing K cPCS1 gene pairs salt resistance

Embodiment

Employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

Autumn eggplant (Kadelia candel, Kc) (can available from the attached south of a city, Leizhou City cross a river management office's nursery) is picked up from the lake such as fall from the sky or outer space in the Xiamen City, Fujian Province, transplants in hot-house culture.

Intestinal bacteria: DH 5 α (being purchased from the Beijing Quanshijin Biotechnology Co., Ltd)

Yeast (Saccharomyces cerevisiae) W303 (wild-type) (can be available from Thermo Fisher Scientific company, article No. is YSC1059)

PGEM-Teasy (Promega company, ammonia benzyl mycin Amp resistance are used for the T/A clone)

PFL61 (Amp resistance, yeast shuttle expression carrier) (can be available from ATCC company, ATCC Number:77215 ^TM)

The experiment common drug is purchased from the Beijing Chemical Plant, such as NaCl, and LiCl, ethanol, LiAc;

Bacteriotrophy thing: Pepton, Trypton, Yeast Extract is purchased from OXOID company; Agar is purchased from the biological company limited of green source of students;-Leu DO Supplement is purchased from the precious biotech firm in Dalian;

Biological enzyme reagent: experiment is to the Taq enzyme, and restriction endonuclease all is purchased from the precious biotech firm in Dalian; ThermoScript II is purchased from Promag company; Marker, DL-2000 is purchased from the Beijing Quanshijin Biotechnology Co., Ltd;

The test test kit: the RACE test kit is purchased from the precious biotech firm in Dalian; The little topic test kit of common plasmid, glue reclaims test kit and is purchased from a day root biotech firm.

The acquisition of embodiment 1, KcPCS1 gene

One, the acquisition of KcPCS1

1, the total RNA of autumn eggplant extracts

A, the mortar that will extract for RNA, spoon, tweezers, reagent bottle etc. are all more than 240 ℃ of condition dry sterilization 2h; Be purchased from Invitrogen company without Rase plastics tubing and rifle head.

B, get the 0.5ml Extraction buffer (100mmolL-1 Tris-HCl, pH 8.5; 500mmolL-1EDTA.500mmolL-1 NaCl; 20%SDS; 5mmolL-1 DTT, pH=8.5; 1.5% (v/v) β, one mercaptoethanol) 65 ℃ of preheatings in the dress 1.5ml centrifuge tube;

C, get 0.2g autumn eggplant (Kadelia candel, Kc) young leaflet tablet, place liquid nitrogen, add an amount of PVP and be ground to powder, be transferred to rapidly in the extracting solution, the concuss centrifuge tube is with its mixing;

D, the centrifuge tube room temperature is kept flat 5min, 12000 turn 4 ℃ of centrifugal 10min, carefully get supernatant to new centrifuge tube;

E, Xiang Guanzhong add 0.1ml 5molL-1 NaCl, and fully mixing adds the 0.3ml trichloromethane, whirlpool concussion 2min, and abundant mixing, 4 ℃ 12000 leaves heart 10min;

F, centrifugal after, solution is divided into two-layer, carefully gets upper solution to new centrifuge tube, repeating step 5, upper solution is got in for the second time extracting, places new centrifuge tube;

G, add the 2-fourth oxyethanol of 1/4 volume in the supernatant, room temperature is placed 20min, and 4 ℃ 12000 leaves heart 10min; Get supernatant to new pipe;

The 10molL-1 LiCl of h, adding 1/4 volume, mixing ,-20 ℃ are spent the night; 4 ℃, 12000 leave heart 15min, abandon supernatant; Precipitation is washed 1-2 time with 1ml 75% (V/V) ethanol, dries; Get 30 μ l DEPC water dissolution precipitation, obtain RNA.

The Kandelia candel leaf RNA that adopts Trizol to extract is contrast.

1.2% agarose electrophoresis, result as shown in Figure 1, the Kandelia candel leaf RNA that 1 road: Trizol extracts; 2,3,4 roads: be the Kandelia candel leaf RNA of modification method extraction, as seen from the figure, 2,3,4 roads can be observed 28s clearly, 18s, 5.8s band three, and serious diffusing phenomenon has occured in the RNA that the Trizol method is extracted, and the RNA that the RNA quality that this explanation improved method extracts is extracted far above the Trizol method has preferably integrity.

Simultaneously, survey respectively the light absorption value under RNA 260nm and the 280nm, the result shows: the RNA that improved method extracts, OD ₂₆₀With OD ₂₈₀Ratio between 1.8-2.0.If OD ₂₆₀/ OD ₂₈₀Value illustrates among the RNA that extracts and contains protein contamination less than 1.8; If OD ₂₆₀/ OD ₂₈₀Value illustrates that greater than 2.2 degraded has occured the RNA that extracts.Obtain RNAOD from experiment ₂₆₀/ OD ₂₈₀The value scope has further been verified, the RNA that improved method obtains is comparatively complete, and has higher purity.

2, cDNA is synthetic

Select Promage reverse transcription test kit (Cat.No.A3800), to specifications operation.

A, get 2 μ l RNA obtained above, place ice bath;

B, add 1 μ l Oligo (dT), 15,2 μ l water (without the RNA enzyme) respectively, mixing with solution centrifugal to managing the end;

C, 70 ℃ of 5min are transferred to ice bath immediately, add 4 μ l ImProm-II ^TM5X Reaction Buffer, 2 μ l MgCl2,1 μ l dNTP Mix, 1 μ l 20U RNA enzyme inhibitors, 1 μ l ImProm-II ^TMReverseTranscriptase adds 6 μ l water (without the RNA enzyme), constant volume final volume 20 μ l at last; Mixing with solution centrifugal to managing the end;

D, 25 ℃ are hatched 5min, and 42 ℃ are extended 60min; 72 ℃ of fire extinguishing ThermoScript II 15min obtain cDNA.

1% agarose gel electrophoresis, the results are shown in shown in Figure 2,1 road, the synthetic cDNA of the Kandelia candel leaf RNA that Trizol extracts; 2,3,4 roads, the Kandelia candel leaf RNA that extracts for modification method synthesizes cDNA; As seen from the figure, improved method obtains the synthetic cDNA of RNA at 0.1-2kb, and the cDNA that the RNA that the Trizol method obtains synthesizes is all below 1kb, the RNA quality that the improved method extraction is described is higher, higher transcriptional activity is arranged, and the RNA quality that common Trizol method is extracted can not satisfy later requirement of experiment.

3, the clone of autumn eggplant KcPCS1 conservative fragments

The design pair of primers, called after:

pcs-5’：5’-AAATGGAAAGGGCCTTGGAG-3’

pcs-3’：5’-CAGCATTATATAGCCACCAATAGG-3’

Take cDNA obtained above as template, carry out pcr amplification, the results are shown in shown in Figure 3, M road, Marker DL-2000; 1 road, the PCR product can be found out to obtain the 221bp fragment.

4, the clone of autumn eggplant KcPCS1 full-length gene

Obtain full-length cDNA by the RACE means.Design a pair of special primer, called after:

pcsgsp-3’：5’-GGATTGCTGCGAGTCTTTGGATAA-3’

pcsgsp-5’：5’-TCTGATTGAAAGCCTTCCTGTTGT-3’

And a pair of anchor primer, called after:

pcs3-T：5’-CATGTGCCTCTTCTGAGAACTGT-3’

pcs5-T：5’-TGACAGTTCTCAGAAGAGGCACA-3’

1)3’RACE

A, use TaKaRa 3 '-Full RACE Core Set Ver.2.0 (Code No.D314) carry out the reverse transcription experiment;

Reaction system:

Total RNA(1ug/ul) 1μl

3’RACE Adaptor(5umol/L) 1μl

dNTP Mixture(10mmol/L each) 1μl

RNase Free dH2O 4.5μl

Reaction conditions: 70 ℃, 10min places 2min immediately on ice;

Then add following component:

Reaction conditions: 42 ℃, 60min, 70 ℃, 15min.

B, take pcsgsp-3 ' as primer, the reverse transcription cDNA of above-mentioned a is template, uses TaKaRa LA Taq

(CodeNo.DRR002A), carry out the Outer pcr amplification;

Outer PCR reaction system:

Reaction conditions:

94℃ 3min 1cycle

94℃ 30sec

60℃ 30sec 30cycles

72℃ 2min

72℃ 10min 1cycle

C, take pcs3-T as primer, reaction solution is template among the b, carries out the Inner pcr amplification; Obtain a band sequence that is about the 1400bp size, use TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 (Code No.DV805A) to cut glue and reclaim above-mentioned 3 ' RACE product, and order-checking, with the conserved sequence contrast, find that this band is to have conserved sequence to obtain.Reaction system is as follows:

Inner PCR reaction system:

Reaction conditions:

94℃ 3min 1cycle

72℃ 10min 1cycle

2)5’RACE

A, use TaKaRa 5 '-Full RACE Kit (Code No.D315) that mangrove blade Total RNA 2ug is carried out CIAP, TAP to process, and with after 5 ' RACE Adaptor is connected, cDNA is synthesized in reverse transcription;

Reaction system:

Reaction conditions: 30 ℃, 10min, 42 ℃, 60min, 70 ℃, 15min;

B, take pcsgsp-5 ' as primer, the reverse transcription cDNA of above-mentioned a is template, uses TaKaRa LA Taq (CodeNo.DRR002A), carry out the Outer pcr amplification;

Outer PCR reaction system:

Reaction conditions:

94℃ 3min 1cycle

72℃ 10min 1cycle

C, take pcs5-T as primer, reaction solution is template among the b, carries out the Inner pcr amplification;

Inner PCR reaction system:

Reaction conditions:

94℃ 3min 1cycle

72℃ 10min 1cycle

Obtain a band sequence that is about the 540bp size, use TaKaRa Agarose Gel DNAPurification Kit Ver.2.0 (Code No.DV805A) to cut glue and reclaim above-mentioned 3 ' RACE product, and order-checking, with the conserved sequence contrast, find that this band is to have conserved sequence to obtain.

3) amplification of full-length cDNA

Fragment sequence according to 3 ' RACE and 5 ' RACE acquisition designs a pair of full-length cDNA primer, called after:

PCS-F：5’-AGGCCTTGTTAACATCGGAAAAACG-3’

PCS-R：5’-GAGGCAATAGACACTAATGATA-3’

Take PCS-F, PCS-R as primer, take above-mentioned synthetic cDNA as template, use TaKaRa PrimerStar to carry out full length cDNA clone;

98℃ 3min 1cycle

72℃ 10min 1cycle

Electrophoresis PCR product, the result as shown in Figure 4, figure A be 5 ' RACE experimental result; Figure B is 3 ' RACE experimental result; Figure C is the amplification of full-length cDNA, and as seen from the figure, the fragment that figure A is cloned into is about 450bp; The fragment that figure B is cloned into is about 1500bp; Figure C obtains to be about the 1900bp fragment.

The PCR product of figure C is checked order, the result has in the sequence table sequence 1 from 5 ' terminal 65-1926 position Nucleotide for this PCR product, the unnamed gene of this PCR product is KcPCS1, the coding region of this gene be in the sequence table sequence 1 from 5 ' terminal 140-1627 position Nucleotide, 5 ' non-translational region has 139bp, and there is 364bp 3 ' terminator.The albumen called after KcPCS1 of this genes encoding, the aminoacid sequence of this albumen are sequence 2 in the sequence table, and sequence 2 is comprised of 496 amino acid, contain altogether 16 cys residues, and N end and C end respectively contain 8 cys.

Embodiment 2, transgenic yeast heavy metal resistance detect

One, the acquisition of transgenic yeast

1, the structure of Yeast expression carrier

The gene order of synthetic KcPCS1 (sequence 1) is carried out pcr amplification as template with following primer:

YPCS-BamHI:5 '-AGGGGATCCATGGCGGTGCCGGGCATATACA-3 ' (the underscore sequence is the BamHI site, sequence 3)

YPCS-SalI:5 '-GGTGTCGACTGGAAGCAATAGAGGGCGAGGAG-3 ' (the underscore sequence is the SalI site, sequence 4)

Get the above-mentioned PCR product of 1 μ g behind the BamHI/SalI double digestion, the fragment that obtains is connected with same pFL61 carrier segments through the BamHI/SalI double digestion, the connection product that obtains transforms bacillus coli DH 5 alpha, obtain transformant, transformant is extracted plasmid carry out the evaluation of BamHI/SalI double digestion, obtain the endonuclease bamhi (Fig. 5 B) of about 1840bp, carry out PCR and identify, primer is YPCS-BamHI and YPCS-SalI, obtains the purpose fragment (Fig. 5 A) of about 1840bp.Enzyme cut with PCR identify that the plasmid all obtain 1840bp purpose fragment sends to order-checking, the result for this plasmid for sequence in the sequence table 1 be inserted into the plasmid that obtains between the BamHI of pFL61 and SalI recognition site from 5 ' terminal 140-1658 position Nucleotide, with this plasmid called after pFL61-KcPCS1.

2, yeast conversion

A, yeast (Saccharomyces cerevisiae) W303 are inoculated into 5ml liquid YPD, and 30 ℃ of lower shaking culture are spent the night;

B, counting overnight culture cell density.With final 5 * 10 ⁶The cell density inoculation 100ml YPD substratum of/ml;

C, put 30 ℃, 200rpm/min shaking culture to 2 * 107/ml needs 3～5h usually;

D, the aseptic centrifuge tube of usefulness 50ml are with the centrifugal 5min of 3000 * g, harvested cell;

E, nutrient solution, suspension cell is in the 25ml sterilized water, and is centrifugal, and method is the same;

F, abandon water, cell suspension in the Lithium Acetate of the 100mmol/L of 1ml, is turned in the centrifuge tube of suspended substance to an aseptic 1.5ml;

G, centrifugal 5sec sedimentation cell, the sucking-off Lithium Acetate;

H, suspension cell wherein approximately contain the 100mmol/L Lithium Acetate of 400 μ l to final volume 500 μ l volumes;

I, 1ml single-stranded vector DNA sample is boiled 5min, in frozen water, cool off fast;

J, vibration cell suspending liquid are got 50 μ l samples and are added in the centrifuge tube of mark, and Lithium Acetate is removed in centrifugation;

K, according to following composition configuration " conversion mixed solution ":

240μl PEG(50％)

36 μ l 1.0mol/L Lithium Acetates

25 μ l single-stranded vector DNA (2.0mg/ml)

50 μ l water and pFL61-KcPCS1 (0.1～10 μ g)

L, each reaction tubes of thermal agitation are wanted 1min usually to the complete mixing of cell;

M, 30 ℃ of insulation 30min;

N, 42 ℃ of heat shock 20～25min, the centrifugal 15sec of 6000～8000rpm/min removes the conversion mixed solution;

O, suction 0.2～1.0ml sterilized water are added in each reaction tubes, and precipitation gently suspends;

P, transform mixed solution with 200 μ l of equal portions and be coated onto-the SD substratum of Leu on.

3, the screening of transformant

Cultivate 72h for 30 ℃.The picking positive colony.

Positive colony is carried out bacterium colony PCR identifies, primer YPCS-BamHI and YPCS-SalI, with the negative contrast of W303/pFL61, the results are shown in shown in Figure 6,1 road, W303/pFL61; The 2-8 road changes the different clones of KcPCS1 over to; The M road, Marker DL-2000 can find out, changes the purpose fragment that the different clones of KcPCS1 obtain about 1840bp over to.Extraction PCR obtains the plasmid of the bacterium colony of 1840bp purpose fragment and sends to order-checking, and the result is pFL61-KcPCS1 for this plasmid.The bacterium called after recombinant bacterium W303/pFL61-KcPCS1 that will contain this plasmid.

Adopting uses the same method changes empty carrier pFL61 among the yeast strain W303 over to, obtains recombinant bacterium W303/pFL61, identify with PCR, and primer YPCS-BamHI and YPCS-SalI, the result is not for containing the purpose fragment in this recombinant bacterium.Two, transgenic yeast heavy metal resistance experiment

1, transgenic yeast salt preventing from heavy metal resistance experiment

1) anti-Cd resistance experiment

The single bacterium colony of a, picking restructuring W303/pFL61-KcPCS1 in the SD-URA substratum (can be available from GENMED company, the product article No. is: GMS12117.7), 30 ℃, 200rmp culturing yeast 24hr;

The concentration of b, adjusting yeast is to OD600 value 1.0, then after diluting respectively 10 times, 100 times, 1000 times, respectively get 2 μ l and be inoculated into respectively in fermention medium 0, fermention medium 1, fermention medium 2 and the fermention medium 3 and cultivate, with recombinant bacterium W303/pFL61 and wild-type W303 in contrast;

Fermention medium 0 be the SD-URA substratum (can be available from GENMED company, the product article No. is: GMS12117.7);

Fermention medium 1 is by CdCl ₂Form CdCl with the SD-URA substratum ₂Concentration in substratum is 50 μ mol/L;

Fermention medium 2 is by CdCl ₂Form CdCl with the SD-URA substratum ₂Concentration in substratum is 75 μ mol/L;

Fermention medium 3 is by CdCl ₂Form CdCl with the SD-URA substratum ₂Concentration in substratum is 100 μ mol/L.

Observations behind c, the growth 72h.

The result as shown in Figure 7,1, stoste; 10 ^-1, 10 times of bacterium liquid dilutions; 10 ^-2, 100 times of bacterium liquid dilutions; 10 ^-3, 1000 times of bacterium liquid dilutions, as seen from the figure, the wild-type yeast cell W303 (W303/pFL61) that changes empty carrier over to shows susceptibility to Cd, when Cd reaches 50 μ mol/L, dilute 10 times the yeast bacterial plaque just the performance growth be suppressed, dilute the death of yeast bacterial plaques of 1000 times of 100 times and dilutions; When rising along with Cd concentration, thisly coerce reinforcement, when Cd concentration reached 75 μ mol/L, the bacterium liquid spot of dilution more than 10 times was almost dead, and when Cd concentration reached 100 μ mol/L, the yeast cell that changes empty carrier over to stopped growing substantially.

And expressed the wild-type yeast cell (W303/pFL61-KcPCS1) of KcPCS1 gene, although same rising along with Cd concentration, growth conditions has been subject to inhibition, but the following Cd of 100 μ mol/L all had stronger resistance, all can grow, this also sufficient proof the KcPCS1 gene pairs improve the Cd resistance and have very strong ability.

Wild-type W303 and W303/pFL61 result are without significant difference.

2) anti-As

With 1) method basic identical, unique different from fermention medium 4, fermention medium 5, fermention medium 6, fermention medium 7.With recombinant bacterium W303/pFL61 and wild-type W303 in contrast.

Fermention medium 4 is by NaAsO ₂Form NaAsO with the SD-URA substratum ₂Concentration in substratum is 0.2mmol/L;

Fermention medium 5 is by NaAsO ₂Form NaAsO with the SD-URA substratum ₂Concentration in substratum is 0.6mmol/L;

Fermention medium 6 is by Na ₃AsO ₄Form Na with the SD-URA substratum ₃AsO ₄Concentration in substratum is 0.2mmol/L;

Fermention medium 7 is by Na ₃AsO ₄Form Na with the SD-URA substratum ₃AsO ₄Concentration in substratum is 0.6mmol/L;

The result as shown in Figure 8,1, stoste; 10 ^-1, 10 times of bacterium liquid dilutions; 10 ^-2, 100 times of bacterium liquid dilutions; 10 ^-3, 1000 times of bacterium liquid dilutions, as seen from the figure, the yeast cell (W303/pFL61-KcPCS1) that changes the KcPCS1 gene over to does not all show resistance to trivalent arsenic (As III) with the yeast (recombinant bacterium W303/pFL61) that changes empty carrier over to, simultaneously also pentavalent arsenic (AsV) is not shown resistance, two primary yeasts show same result.Wild-type W303 and W303/pFL61 result are without significant difference.

2, transgenic yeast heavy metal absorption detecting

The single bacterium colony of a, picking W303/pFL61-KcPCS1 is in the SD-URA substratum, and 30 ℃, 200rmp culturing yeast 24-48h;

The concentration of b, adjusting yeast adds CdCl to OD600 value 1.0 ₂Making its final concentration is 30 μ mol/L, and 30 ℃, 200rmp follows culturing yeast;

C, respectively at 24h, 36h, 48h sampling, centrifugal collection yeast thalline is washed 3 times with clear water, 50 ℃ of oven dry yeast cell.With recombinant bacterium W303/pFL61 and wild-type W303 in contrast; Take by weighing the 0.2g dry yeast cell, add 3mL nitric acid and in microwave dissolver, clear up 15min, be settled in the 25mL volumetric flask (cadmium wavelength: 228.8nm), measure the content of Cd in the yeast cell of above-mentioned 24h, 36h, 48h sampling with the sampling Graphite Furnace Atomic Absorption spectrophotometric determination after clearing up.

The result as shown in Figure 9, recombinant bacterium W303/pFL61 is respectively 0.211,0.282 at the content of 24h, 36h, 48h Cd, the 0.289mg/g dry weight;

W303/pFL61-KcPCS1 is respectively 0.249,0.301 at the content of 24h, 36h, 48h Cd, the 0.349mg/g dry weight;

As can be seen from the figure, Cd content in the recombinant bacterium W303/pFL61 cell paste, prolongation in time and raising, but after cultivating 36h, considerable change does not just occur, the interior Cd content of yeast transformant (W303/pFL61-KcPCS1) cell paste that changes the KcPCS1 gene over to then increases with the prolongation of the time of processing always, and is higher than recombinant bacterium W303/pFL61 far away.Analyze its reason, this may be owing to change the yeast transformant of empty carrier over to, prolongation along with the Cd treatment time, Cd concentration in the body constantly increases, but after 36h, the Cd content in the body has reached the connection concentration of yeast, continues to cultivate the empty carrier yeast death has occured, therefore behind the 36h, change in the yeast conversion daughter of empty carrier Cd content over to and begin to keep certain level and obvious variation does not occur.And change the yeast transformant of KcPCS1 gene over to, then in whole culturing process, still can keep higher activity, so the Cd content in the body is raising gradually.Wild-type W303 and recombinant bacterium W303/pFL61 result are without significant difference.

3, transgenic yeast salt resistance experiment

The single bacterium colony of a, picking W303/pFL61-KcPCS1 is in the SD-URA substratum, and 30 ℃, 200rmp culturing yeast 24-48hr;

The concentration of b, adjusting yeast is to OD600 value 1.0, then after diluting respectively 10 times, 100 times, 1000 times, respectively get 2 μ l on the SD-URA substratum that does not contain NaCl and contain that (this substratum forms by NaCl and SD-URA substratum on the SD-URA substratum of 1200mM NaCl, the concentration of NaCl in substratum is 1200mM), with recombinant bacterium W303/pFL61 and wild-type W303 in contrast;

Observations behind c, the growth 72h.

The results are shown in shown in Figure 10 1, yeast stoste; 10 ^-1, 10 times of bacterium liquid dilutions; 10 ^-2, 100 times of bacterium liquid dilutions; 10 ^-3, 1000 times of bacterium liquid dilutions, as seen from the figure, do not adding on the solid medium of NaCl, W303/pFL61-KcPCS1, W303/pFL61 and wild-type W303 yeast be the energy normal growth all, when NaCl concentration reaches 1200mmol/L, W303/pFL61 and wild-type W303 yeast growth are subject to obvious inhibition, and the W303/pFL61-KcPCS1 yeast still can normal growth.

Claims

1. gene, it is characterized in that: described gene is following 1), 2) or 3) gene:

1) dna molecular shown in the sequence 1 in the sequence table;

3) in the sequence table sequence 1 from the dna molecular shown in 5 ' the terminal 140-1658 position Nucleotide.

2. the recombinant vectors that contains the described encoding gene of claim 1.

3. recombinant vectors according to claim 2 is characterized in that: the recombinant vectors that described recombinant vectors obtains for the described gene of multiple clone site insertion claim 1 at carrier pFL61.

4. the recombinant bacterium that contains the described encoding gene of claim 1.

5. recombinant bacterium according to claim 4 is characterized in that: described recombinant bacterium is for importing the recombinant bacterium that Host Strains obtain with claim 2 or 3 described recombinant vectorss, and described Host Strains is distillery yeast (Saccharomyces cerevisiae).

6. the expression cassette that contains the described encoding gene of claim 1.

7. a method of cultivating recombination yeast is the recombination yeast that gene importing host yeast claimed in claim 1 is obtained, and the resistance of reverse of described recombination yeast is higher than described host's yeast; Described resistance of reverse is anti-cadmium and/or salt tolerant; Described Host Strains is distillery yeast (Saccharomyces cerevisiae).

8. method according to claim 7 is characterized in that: described gene imports host's yeast by claim 2 or 3 described recombinant vectorss.

9. the application of the described gene of claim 1 in the resistance of reverse of heavy metal contamination reparation or raising plant; Described heavy metal is cadmium; Described resistance of reverse is anti-cadmium and/or salt tolerant.

10. claim 2 or the 3 described recombinant vectorss application in the resistance of reverse of heavy metal contamination reparation or raising plant; Described heavy metal is cadmium; Described resistance of reverse is anti-cadmium and/or salt tolerant.

11. claim 4 or the 5 described recombinant bacteriums application in the resistance of reverse of heavy metal contamination reparation or raising plant; Described heavy metal is cadmium; Described resistance of reverse is anti-cadmium and/or salt tolerant.