CN101130781A - Method for transforming agrobactrium tumefaciens mediated zeocin resistant gene into industrial glycerol candida - Google Patents
Method for transforming agrobactrium tumefaciens mediated zeocin resistant gene into industrial glycerol candida Download PDFInfo
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- CN101130781A CN101130781A CNA2007100241455A CN200710024145A CN101130781A CN 101130781 A CN101130781 A CN 101130781A CN A2007100241455 A CNA2007100241455 A CN A2007100241455A CN 200710024145 A CN200710024145 A CN 200710024145A CN 101130781 A CN101130781 A CN 101130781A
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Abstract
The invention discloses a converting technique which makes exogenesis gene integrate to host colorant layer by converting meloidogynosis agricillin conducting medium zeocin resistance gene to industrial candida for producing glycerol in the converting technique field of the gene engineering. The invention electric-shock converts pCAM 3300-zeocin plasmid to meloidogynosis agricillin by constructing pCAM 3300-zeocin plasmid, which achieves the conversion of pCAM 3300-zeocin for glycerol-producing candida by co culturing meloidogynosis agricillin with pCAM 3300-zeocinaim plasmid and glycerol-producing candida with zeocin resistance gene as screening mark, optimizes conversion condition according to the growth characteristic of meloidogynosis agricillin, makes the maximum conversion ratio of the cell get to two transformations /104 yeast cells when the co culturing time is 24 hours and the cell ratio is 1:500-1000, and establishes the conversion method of converting meloidogynosis agricillin conducting medium zeocin resistance gene to industrial candida for producing glycerol and creates the basis of the future research.
Description
Technical field
The transforming agrobactrium tumefaciens mediated zeocin resistant gene into industrial glycerin candida to host chromosome, belongs to exogenous origin gene integrator to transform technical field in the genetically engineered.
Background technology
Conversion is one of important step in the yeast genes operative technique.Because yeast cell has unlike prokaryotic micro-organisms such as intestinal bacteria and Bacillus subtillis and foreign DNA can be taken in intracellular special physiological state (competence), so yeast conversion once was a big limiting factor of Yeast gene engineering research.The yeast conversion method that extensively adopts mainly contains protoplast transformation method, Lithium Acetate conversion method, electric shock conversion method etc. at present.These methods have obtained great success on the Study on Genetic Transformation of yeast saccharomyces cerevisiae.
Because there is huge difference in the yeast strain that industrial application yeast strain and laboratory are used at aspects such as cell wall structure, physio-biochemical characteristics, genetic background and growing environments, when transforming the industrial application yeast, need set up corresponding transformation system.But method for transformation recited above often exists when transforming some industrial yeast bacterial strains that transformation efficiency is low, the transformant poor stability, difficulty such as be difficult to repeat, some bacterial strain even can't transform.At this problem, the researchist has proposed different improving one's methods and new method for transformation.Wherein, the genetic transformation method of agrobacterium tumefaciens (Agrobacterium tumefaciens) mediation is one of effective means.
Nineteen ninety-five Bundock etc. has successfully carried out Agrobacterium tumefaciens mediated saccharomycetic genetic transformation first.Discovering further that Agrobacterium tumefaciens mediated genetic transformation method has transforms that low copy, high frequency shift, cost is low, good stability, easy to operate and good reproducibility, can transform large fragment DNA (greatly to 50Kb foreign DNA), can obviously improve the stable advantages such as transformant of transformation efficiency acquisition, has very big potential in Application Areas.
Produce glycerin candida and be present China and be used for industrial fermentation and produce one of strain excellent of glycerine, it has, and glycerine productive rate height, anti-hypertonic pressure, fermentation condition are extensive, thalli growth speed and advantage such as fermenting speed is fast, transformation efficiency is high, fermentation raw material is simple.Except glycerine, produce glycerin candida and can also produce multiple other polyvalent alcohols, as tetrahydroxybutane, D-arabitol and N.F,USP MANNITOL etc., be the yeast that a strain has very high potential using value therefore.Adopting genetic engineering means to make up the reorganization bacterium is the approach that produces the glycerin candida performance that further improves, realize this goal and at first will obtain effective carrier and method for transformation, just might carry out autotelic transformation on this basis, to reach the purpose that improves glycerine output and improve technology to producing the glycerin candida pathways metabolism.Successfully set up its genetic conversion system and can improve glycerine production, can also create conditions for the reorganization bacterium that further makes up other polyvalent alcohols of high yield by metabolic engineering, method for transformation that is adopted and carrier construction method also can be offered reference for the genetic engineering modified of other osmophilic yeasts.Therefore, the genetic conversion system of foundation product glycerin candida is significant.
The present invention adopts the agrobacterium tumefaciens conversion system to transform the product glycerin candida, has realized producing the successful foundation of the genetic conversion system of glycerin candida.
Summary of the invention
The method that the purpose of this invention is to provide a kind of transforming agrobactrium tumefaciens mediated zeocin resistant gene into industrial glycerin candida makes up the conversion that binary vector has successfully been realized Agrobacterium tumefaciens mediated product glycerin candida.And conversion condition has been carried out suitable optimization at producing the fast characteristics of glycerin candida growth.Lay a good foundation for further studying the product glycerin candida.
Technical scheme of the present invention: the method for transforming agrobactrium tumefaciens mediated zeocin resistant gene into industrial glycerin candida, be that to be template with plasmid pPICZB obtain the zeocin resistant gene by round pcr, be connected with linearization plasmid pCAM 3300 again, obtain recombinant plasmid pCAM 3300-zeocin; Plasmid pCAM 3300-zeocin transforms agrobacterium tumefaciens lba4404, cultivates altogether with the product glycerin candida again, and agrobacterium tumefaciens lba4404/pCAM 3300-zeocin plasmid transforms industrialization and produces glycerin candida;
(1) clone of zeocin gene
Specificity design primer according to the zeocin gene:
Primer R:5 '-GTCAGTCCTGCTCCTCGGCCACGAAG-3 ';
Primers F: 5 '-ATGGCCAAGTTGACCAGTGCCGTTC-3 ';
Pcr amplification zeocin gene obtains the specific band of 300bp;
PCR method is as follows: get plasmid pPICZB template 1 μ L, and 10 * PCR damping fluid, 2 μ L, each 1 μ L of primer R, F, dNTPs2 μ L, Taq archaeal dna polymerase 0.5 μ L, total reaction volume is 20 μ L; Loop parameter: 94 ℃ of pre-sex change 5min, 94 ℃ of 45s, 56 ℃ of 90s, 72 ℃ of 90s, 35 circulations, 72 ℃ are extended 10min;
By agarose gel electrophoresis analytical reaction product, target stripe appears at the application of sample swimming lane, reclaim the target segment that test kit is purified into 0.3kb with PCR segment glue, the target segment that purifying is good is connected preservation with pEGM-T-Easyvector; Cut with corresponding EcoRI enzyme, glue reclaims zeocin target gene, EcoRI linearization for enzyme restriction plasmid pCAM3300 again; Under the effect of T4 ligase enzyme, connect and obtain pCAM3300-zeocin;
(2) plasmid pCAM3300-zeocin transforms agrobacterium tumefaciens lba4404
Directly change recombinant plasmid pCAM3300-zeocin over to agrobacterium tumefaciens lba4404 by shocking by electricity, picking resistance transformant on the kalamycin resistance flat board extracts the plasmid enzyme restriction checking, and bacterium LBA4404/pCAM3300-zeocin obtains recombinating;
(3) agrobacterium tumefaciens lba4404/pCAM3300-zeocin plasmid transforms industrialization and produces glycerin candida:
To recombinate bacterium LBA4404/pCAM3300-zeocin in LB substratum (kantlex 50 μ g/mL, sharp rich mycin 50 μ g/mL, Streptomycin sulphate 30 μ g/mL), and 30 ℃, the 200r/min shaking table is cultivated 36h, centrifugal collection thalline; Resuspended with isopyknic IM substratum, cultivate 6h; Inoculation is produced glycerin candida WL2002-5 in the YPD substratum, 30 ℃, 200r/min shaking table overnight incubation; The YPD nutrient solution that produces glycerin candida was diluted in the YPD fresh culture by 1: 5, cultivated 6h; Centrifugal collection agrobacterium tumefaciens thalline and product glycerin candida clean once with physiological saline respectively, with the resuspended thalline of IM substratum, produce the glycerin candida final concentration of cells and reach 10
9Individual/mL, the agrobacterium tumefaciens final concentration of cells reaches 10
11Individual/mL, respectively get 50 μ L and add 30 ℃, the common overnight incubation of 200r/min in the 20mL IM substratum (Syringylethanone 200 μ mol/L, kantlex 50 μ g/mL, sharp rich mycin 50 μ g/mL); Getting the IM+AS solid medium flat board that 200 μ L nutrient solutions coatings is covered with glassine paper places 25 ℃ to cultivate 24h; Glassine paper is transferred to the SM substratum, and (cefotaxime 200 μ mol/L zoecin150mg/L) go up cultivation two days, screen positive yeast transformant.
Reorganization agrobacterium tumefaciens lba4404/pCAM 3300-zeocin mediated transformation is produced the optimization conversion condition of glycerin candida WL2002-5:
(1) IM solid medium induction time is optimized: the common incubation time of optimization is 24h;
(2) cultivate the optimization of agrobacterium tumefaciens cell concentration altogether: producing glycerin candida is 1 than crown gall Agrobacterium somatic cells ratio: during 500-1000, peak rate of conversion reaches 2 transformant/10
4Individual yeast cell.
Zeocin is a kind of glycoprotein microbiotic that belongs to bleomycin family, can act on most of bacteriums in vivo and (comprise: E.coli) and fungi (as yeast).In order efficiently to select positive transformant, obtain the zeocin resistant gene from plasmid pPICZB and insert plasmid pCAM 3300, obtain recombinant plasmid pCAM 3300-zeocin.
Directly change agrobacterium tumefaciens lba4404 over to by shocking by electricity.Picking resistance transformant on the kalamycin resistance flat board extracts the plasmid enzyme restriction checking.
Beneficial effect of the present invention: agrobacterium tumefaciens transformed yeast system has (not needing to prepare protoplastis) simple to operate, transformation efficiency height, is easy to obtain good characteristics such as transformant, transformant inheritance stability.Anti-height oozes that to produce glycerin candida (Candida glycerinogenes) WL2002-5 be the strain excellent [microorganism journal Vol.39 No.1 1999.2] that the scientific worker in Southern Yangtze University industrial microorganism research centre obtains through 30 years concentrate on studies.Produce glycerin candida and have advantages such as glycerine productive rate height, anti-height ooze, grows soon, fermentation raw material is simple.Except glycerine, osmophilic yeast can also produce multiple other polyvalent alcohols, as tetrahydroxybutane, D-arabitol and N.F,USP MANNITOL etc., is the yeast that a class has very high potential using value therefore.Adopting genetic engineering means to make up the reorganization bacterium is further to improve the important channel of producing the glycerin candida performance, realize this goal and at first will obtain effective carrier and method for transformation, just might carry out autotelic transformation on this basis, to reach the purpose that improves glycerine output and improve technology to producing the glycerin candida pathways metabolism.Successfully setting up its genetic conversion system can also be for further studying the product glycerin candida by metabolic engineering except that can improving glycerine production, and the reorganization bacterium that makes up other polyvalent alcohols of high yield creates conditions.Therefore, the genetic conversion system of foundation product glycerin candida is significant.
Description of drawings
The structure of Fig. 1 recombinant plasmid pCAM 3300-zeocin
Fig. 2 plasmid pCAM 3300-zeocin enzyme is cut checking Lane1 T-zeocin/EcoRI; Lane 2DNA Marker:DL20002; Lane3 zeocin/EcoRI; Lane4 pCAM 3300/EcoRI; Lane5 pCAM 3300-zeocin/EcoRI; Lane6.pCAM 3300-zeocin/Bamh I; Lane7 DNA Marker: λ DNA/HindIII.
The specific amplified of Fig. 3 positive transformant PCR checking Lane1 zoecin gene; Lane2 DNAMarker DL2000; Lane3,4,5, the amplification of 6 positive transformant zoecin gene specifics; Lane7 produces the amplification of glycerin candida zoecin gene specific; Lane8 produces glycerin candida 18SrDNA amplification.
The different induction time transformant of Fig. 4 growing state 1. inducing culture 0h; 2. inducing culture 12h; 3. inducing culture 18h; 4. inducing culture 24h; 5. inducing culture 30h; 6. inducing culture 36h.
Embodiment
With plasmid pPICZB is that template obtains the zeocin resistant gene by round pcr, is connected with linearization plasmid pCAM 3300 again, obtains recombinant plasmid pCAM 3300-zeocin;
The clone of zeocin gene
Specificity design primer according to the Zeocin gene:
Primer R:5 '-GTCAGTCCTGCTCCTCGGCCACGAAG-3 ';
Primers F: 5 '-ATGGCCAAGTTGACCAGTGCCGTTC-3 ';
Pcr amplification zeocin gene obtains the specific band of 300bp;
PCR method is as follows: get plasmid pPICZB template 1 μ L, and 10 * PCR damping fluid, 2 μ L, each 1 μ L of primer R, F, dNTPs2 μ L, Taq archaeal dna polymerase 0.5 μ L, total reaction volume is 20 μ L; Loop parameter: 94 ℃ of pre-sex change 5min, 94 ℃ of 45s, 56 ℃ of 90s, 72 ℃ of 90s, 35 circulations, 72 ℃ are extended 10min.
By agarose gel electrophoresis analytical reaction product, target stripe appears at the application of sample swimming lane, reclaim the target segment that test kit is purified into 0.3kb with PCR segment glue, the target segment that purifying is good is connected preservation with pEGM-T-Easyvector; Cut with corresponding EcoR I enzyme, glue reclaims zeocin target gene, EcoR I linearization for enzyme restriction plasmid pCAM 3300 again; Under the effect of T4 ligase enzyme, connect and obtain pCAM 3300-zeocin.
Directly change recombinant plasmid pCAM 3300-zeocin over to agrobacterium tumefaciens lba4404 by shocking by electricity, picking resistance transformant on the kalamycin resistance flat board extracts the plasmid enzyme restriction checking, and bacterium LBA4404/pCAM 3300-zeocin obtains recombinating.
Inoculation contains the agrobacterium tumefaciens lba4404 of plasmid pCAM 3300-zeocin in LB substratum (kantlex 50 μ g/mL, rifomycin 50 μ g/mL, Streptomycin sulphate 30 μ g/mL) in, 30 ℃, the 200r/min shaking table is cultivated 36h, centrifugal collection thalline, resuspended with isopyknic IM substratum, cultivate 6h; Inoculation is produced glycerin candida in the YPD substratum, 30 ℃, 200r/min shaking table overnight incubation; The YPD nutrient solution that produces glycerin candida was diluted in the YPD fresh culture by 1: 5, cultivated 6h.Centrifugal collection yeast saccharomyces cerevisiae and agrobacterium tumefaciens thalline clean once with physiological saline respectively, and with the resuspended thalline of IM substratum, the brewing yeast cell final concentration reaches 10
9Individual/mL, the agrobacterium tumefaciens final concentration of cells reaches 10
11Individual/mL, respectively get 50 μ L and add in the 20mL IM substratum (Syringylethanone 200 μ mol/L, kantlex 50 μ g/mL, sharp rich mycin 50 μ g/mL) 30 ℃, 200r/min is overnight incubation altogether.Getting the IM+AS solid medium flat board that 200 μ L nutrient solutions coatings is covered with glassine paper places 25 ℃ to cultivate 24h; Glassine paper is transferred to the SM substratum, and (cefotaxime 200 μ mol/L zoecin150mg/L) go up cultivation two days, screen positive yeast transformant.
During the product glycerin candida positive transformant of tentatively confirming connect in the YPD liquid nutrient medium 30 ℃, 200r/min cultivates 18h, extract karyomit(e) and carry out the PCR evaluation, make negative control simultaneously, owing to produce in the glycerin candida and do not contain the zeocin gene, primer according to zeocin gene and the chromosomal difference design of product glycerin candida, produce glycerin candida karyomit(e) and can't amplify the zeocin gene, and positive transformant can increase and obtains the zeocin gene, has contained the zeocin gene in this explanation transformant.The result that PCR identifies has shown that tentatively the method by Agrobacterium tumefaciens mediated conversion has realized that the zeocin gene pairs produces glycerin candida and transforms.
(1) IM solid medium induction time is optimized:
Get the IM nutrient solution of overnight incubation, suitable dilution coating IM solid medium cultivates 0,12,18,24,30,36,40h carries out screening and culturing with the parallel SM of the transferring to solid medium of glassine paper, observes the growing state of transformant on the SM flat board altogether.Simultaneously the IM nutrient solution is carried out the yeast colony counting, calculate transformation efficiency, the common incubation time of optimization is 24h.
(2) cultivate the optimization of agrobacterium tumefaciens cell concentration altogether:
Get 10
9The yeast cell 10 μ L and 10 of individual/mL
11Agrobacterium tumefaciens cell 1,10,50,100,200, the 500 μ L mixings of individual/mL are cultivated altogether, and the screening positive transformant also calculates transformation efficiency.Cultivate transformation efficiency the best altogether with 50～100 μ L mixings, converting to producing glycerin candida is 1 than crown gall Agrobacterium somatic cells ratio: during 500-1000, peak rate of conversion reaches 2 transformant/10
4Individual yeast cell.
Claims (2)
1. the method for transforming agrobactrium tumefaciens mediated zeocin resistant gene into industrial glycerin candida, it is characterized in that with plasmid pPICZB being that template obtains the zeocin resistant gene by round pcr, be connected with linearization plasmid pCAM 3300 again, obtain recombinant plasmid pCAM 3300-zeocin; Plasmid pCAM 3300-zeocin transforms agrobacterium tumefaciens lba4404, cultivates altogether with the product glycerin candida again, and agrobacterium tumefaciens lba4404/pCAM 3300-zeocin plasmid transforms industrialization and produces glycerin candida;
(1) clone of zeocin gene
Specificity design primer according to the zeocin gene:
Primer R:5 '-GTCAGTCCTGCTCCTCGGCCACGAAG-3 ';
Primers F: 5 '-ATGGCCAAGTTGACCAGTGCCGTTC-3 ';
Pcr amplification zeocin gene obtains the specific band of 300 bp;
PCR method is as follows: get plasmid pPICZB template 1 μ L, and 10 * PCR damping fluid, 2 μ L, each 1 μ L of primer R, F, dNTPs 2 μ L, Taq archaeal dna polymerase 0.5 μ L, total reaction volume is 20 μ L; Loop parameter: 94 ℃ of pre-sex change 5min, 94 ℃ of 45s, 56 ℃ of 90s, 72 ℃ of 90s, 35 circulations, 72 ℃ are extended 10min;
By agarose gel electrophoresis analytical reaction product, target stripe appears at the application of sample swimming lane, reclaim the target segment that test kit is purified into 0.3kb with PCR segment glue, the target segment that purifying is good is connected preservation with pEGM-T-Easyvector; Cut with corresponding EcoR I enzyme, glue reclaims zeocin target gene, EcoR I linearization for enzyme restriction plasmid pCAM 3300 again; Under the effect of T4 ligase enzyme, connect and obtain pCAM 3300-zeocin;
(2) plasmid pCAM 3300-zeocin transforms agrobacterium tumefaciens lba4404
Directly change recombinant plasmid pCAM 3300-zeocin over to agrobacterium tumefaciens lba4404 by shocking by electricity, picking resistance transformant on the kalamycin resistance flat board extracts the plasmid enzyme restriction checking, and bacterium LBA4404/pCAM 3300-zeocin obtains recombinating;
(3) agrobacterium tumefaciens lba4404/pCAM 3300-zeocin plasmid transforms industrialization and produces glycerin candida:
To recombinate bacterium LBA4404/pCAM 3300-zeocin in the LB substratum, and 30 ℃, 200 r/min shaking tables are cultivated 36 h, centrifugal collection thalline; Resuspended with isopyknic IM substratum, cultivate 6h; Inoculation is produced glycerin candida WL2002-5 in the YPD substratum, 30 ℃, 200r/min shaking table overnight incubation; The YPD nutrient solution that produces glycerin candida was diluted in the YPD fresh culture by 1: 5, cultivated 6h; Centrifugal collection agrobacterium tumefaciens thalline and product glycerin candida clean once with physiological saline respectively, with the resuspended thalline of IM substratum, produce the glycerin candida final concentration of cells and reach 10
9Individual/mL, the agrobacterium tumefaciens final concentration of cells reaches 10
11Individual/mL, respectively get 50 μ L and add 30 ℃ of common overnight incubation of 200r/min in the 20mL IM substratum; Getting the IM+AS solid medium flat board that 200 μ L nutrient solutions coatings is covered with glassine paper places 25 ℃ to cultivate 24h; Glassine paper is transferred on the SM substratum cultivated two days, screen positive yeast transformant.
2. method according to claim 1, the agrobacterium tumefaciens lba4404 that it is characterized in that recombinating/pCAM3300-zeocin mediated transformation is produced the optimization conversion condition of glycerin candida WL2002-5:
(1) IM solid medium induction time is optimized: the common incubation time of optimization is 24h;
(2) cultivate the optimization of agrobacterium tumefaciens cell concentration altogether: producing glycerin candida is 1 than crown gall Agrobacterium somatic cells ratio: during 500-1000, peak rate of conversion reaches 2 transformant/10
4Individual yeast cell.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286521A (en) * | 2011-07-18 | 2011-12-21 | 江南大学 | Multifunctional shuttle expression carrier and construction method thereof |
| CN103509876A (en) * | 2013-10-24 | 2014-01-15 | 上海辰山植物园 | Method for quickly screening plant multiple stress resistance genes by using yeast |
-
2007
- 2007-07-23 CN CNA2007100241455A patent/CN101130781A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286521A (en) * | 2011-07-18 | 2011-12-21 | 江南大学 | Multifunctional shuttle expression carrier and construction method thereof |
| CN103509876A (en) * | 2013-10-24 | 2014-01-15 | 上海辰山植物园 | Method for quickly screening plant multiple stress resistance genes by using yeast |
| CN103509876B (en) * | 2013-10-24 | 2019-09-03 | 上海辰山植物园 | A method of quickly screening the multiple stress resistance gene of plant using yeast |
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