CN103194439A - Industrial yellow wine yeast haploid separation method - Google Patents
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Abstract
The invention discloses an industrial yellow wine yeast haploid separation method. In the method, an HO gene knockout component is formed by a Cre/loxp component and a G418 resistance maker KanMX gene, and the HO gene knockout component is successfully converted into the industrial yellow wine yeast, the HO gene of the yellow wine yeast is knocked out by homologous recombination, consequentially, a haploid strain is separated, meanwhile, a galactose culture medium induces expression of a Cre recombinant enzyme so as to cut off the kanMX gene, so that the industrial yellow wine yeast haploid without external resistance gene is obtained. The method can be used for quickly and efficiently separating the industrial yellow wine yeast haploid without external resistance gene, has the advantages of high haploid separation rate and haploid survival rate, simple technology and low production cost, and lays a material base for subsequent metabolic engineering modification and genetic research on the yellow wine yeast.
Description
Technical field
The invention belongs to biotechnology and fermentation technical field, be specifically related to the haploid efficient separation method of a kind of industrial yellow wine yeast.
Background technology
Industry yellow wine yeast bacterial strain great majority are to separate from nature or leavened prod, these wild type strains mostly are diploid even polyploid, its complicated ploidy improves to yeast, metabolic engineering and genetic research have brought many difficulties, be difficult to directly obtain the diploid mutant strain by mutagenic treatment or genetic modification, this is because above-mentioned reforming processing can only obtain dominant mutation, many recessive mutations are then missed, and because the polyploid bacterial strain lacks selection markers such as auxotroph, increased the transformation difficulty, and the yellow wine yeast that exists with the monoploid form (as the laboratory yellow wine yeast bacterial strain through transforming) gene information is simple and clear, mating ability is strong, therefore, general elder generation obtains haploid strains with diploid or polyploid bacterial strain by giving birth to the spore fractionation, at first monoploid is carried out reverting to the diploid bacterial strain by sexual mating again behind the good mutant strain of genetic engineering modified acquired character.As fully visible, haploid screening and separating is a very crucial step.
Prior art is general direct to obtain monoploid by the living spore of industrial strain, helicase enzymolysis and YPD plate isolation, and often haploid separation rate and surviving rate are lower; In addition, there are some researches show, the HO gene of yellow wine yeast can be expressed a kind of restriction endonuclease, in the living spore process of yeast, will cause monoploid to join type (a type or α type) conversion, thereby be combined with anisospore on every side and revert to diploid, therefore, the HO gene has active bacterial strain and is difficult for isolating monoploid, and industrial yellow wine yeast HO gene generally has activity, and this has just further increased its haploid separating difficulty.Given this, a kind of industrial yellow wine yeast monoploid separation method fast and efficiently of searching is very necessary.
Summary of the invention
At the above-mentioned defective that prior art exists, the applicant improves through research, provides a kind of industrial yellow wine yeast haploid separation method.The present invention can separate the industrial yellow wine yeast monoploid that acquisition does not contain external resistant gene fast and efficiently, has monoploid separation rate and surviving rate height, technology is simple, production cost is low advantage.
Technical scheme of the present invention is as follows:
The haploid separation method of a kind of industrial yellow wine yeast is to knock out by the HO gene of homologous recombination with described industrial yellow wine yeast; This separation method concrete operations step is as follows:
(1) structure of HO gene knockout assembly: the kanMX gene amplification fragment of at first utilizing the regular-PCR amplification to obtain the upstream and downstream homology arm amplified fragments of yellow wine yeast HO gene and have the G418 resistance marker, obtain the fusion fragment of above-mentioned three fragments again by the touchdown PCR amplification, utilize regular-PCR that described fusion fragment is carried out specific amplification at last and namely get yellow wine yeast HO gene knockout assembly; Described upstream homology arm amplified fragments comprises that upstream homology arm sequence reaches and the sequence (abbreviation sequence A) of kanMX gene 5 ' end complementation, and described downstream homology arm amplified fragments comprises that downstream homology arm sequence reaches the sequence (abbreviation sequence B) of holding complementation with kanMX gene 3 '; Described kanMX gene amplification fragment comprises the kanMX gene order and is positioned at these fragment two ends holds complementary sequence (being called for short sequence C and sequence D respectively) with upstream homology arm sequence 3 ' end, downstream homology arm sequence 5 ' respectively; All contain the loxp site that to be identified by the Cre recombinase between described kanMX gene order and described sequence C, the sequence D; Described sequence A, sequence B, sequence C and sequence D are 20～30bp;
(2) knocking out of yellow wine yeast HO gene: step (1) gained HO gene knockout assembly is transformed industrial yellow wine yeast bacterial strain, obtain positive transformant and evaluation by the G418 resistance screening;
(3) the haploid separation of yellow wine yeast: get and identify in the step (2) that correct positive transformant is inoculated in the YPD substratum and activates; Collect yeast slurry and it is piled up in living spore substratum, cultivate 3～5d in 26～28 ℃; Bacterium colony on the described living spore substratum of picking, get enzymolysis bacterium liquid through helicase enzymolysis broken wall treatment, spore is broken up in vibration, coats YPD again and cultivates and to be cultured to bacterium colony based on 28～30 ℃ and to grow, and picking colony is through identifying the haploid strains that obtains containing resistant gene kanMX after correct;
(4) excision of resistant gene: engineering plasmid step of converting (3) the gained haploid strains that will contain the Cre recombinase gene, get the positive colony transformant and be inoculated in 5～10 generations of semi-lactosi substratum cultured continuously, induce the Cre recombinase to express to excise resistant gene kanMX, obtain resistant gene and lose bacterial strain; This bacterial strain cultured continuously more than 10 generations, is lost above-mentioned engineering plasmid, namely get the industrial yellow wine yeast haploid strains that does not contain external resistant gene.
Its further technical scheme is:
The described touchdown PCR amplified reaction of step (1) does not have primer and participates in, its amplification condition is as follows: 95 ℃ of pre-sex change 4～5min, 98 ℃ of sex change 10～15s, 70 ℃ of annealing 15～30s, each cycle annealing temperature reduces by 1 ℃, and totally 15 circulations are extended 2.5～3min in 72 ℃, recirculation is once extended 5～10min in 72 ℃ at last again under 55 ℃ of annealing temperatures then.Preferably, the amplification condition of described touchdown PCR is as follows: 95 ℃ of pre-sex change 4min, 98 ℃ of sex change 10s, 70 ℃ of annealing 15s, each cycle annealing temperature reduces by 1 ℃, totally 15 circulations, in 72 ℃ of extension 3min, recirculation is once extended 10min in 72 ℃ at last again under 55 ℃ of annealing temperatures then.
The amplimer sequence of the described upstream of step (1) homology arm amplified fragments shown in SEQ ID NO:1 and SEQ ID NO:2,
The amplimer sequence of the described downstream of step (1) homology arm amplified fragments is shown in SEQ ID NO:3 and SEQ ID NO:4.
The amplimer sequence of the described kanMX gene amplification of step (1) fragment is shown in SEQ ID NO:7 and SEQ ID NO:8.
The specific primer sequence of the described fusion fragment of step (1) is shown in SEQ ID NO:1 and SEQ ID NO:4.
The described activation temperature of step (3) is 30 ℃, incubation time 24～36h.
The engineering plasmid of the described Cre of the containing recombinase gene of step (4) is selected from pSH47, pSH62, pSH63 or pSH65.
The described semi-lactosi substratum of step (4) contains the bleomycin (Zeocin) of 100 μ g/mL
Useful technique effect of the present invention is:
The present invention at first utilizes touchdown PCR and regular-PCR successfully to make up and obtains high specific yellow wine yeast HO gene knockout assembly (HO upstream region of gene homology arm---selection markers resistant gene kanMX---HO gene downstream homology arm) and transform industrial yellow wine yeast, knock out yellow wine yeast HO gene by homologous recombination, thereby greatly reduce the separating difficulty of yeast haploid, separate again and obtain haploid strains, simultaneously, utilize the semi-lactosi substratum to induce the expression of Cre recombinase that the kanMX gene is excised, the final industrial yellow wine yeast haploid strains that successfully obtains not contain external resistant gene.
Compare with existing industrial yellow wine yeast monoploid separation method: in the structure of HO gene knockout assembly, the present invention adopts touchdown PCR to merge each gene fragment, only need carry out a PCR and react the purpose that can reach the disposable fusion of fragment, need not to add any primer, than traditional gene knockout constructing method of component based on fusion PCR, loaded down with trivial details annealing temperature is groped process when having saved fragment fusion in twos, time saving and energy saving, thereby can obtain the highly purified HO gene knockout of high specific assembly, fusion efficiencies height by effectively enrichment purpose band; The present invention can separate the industrial yellow wine yeast monoploid that acquisition does not contain external resistant gene fast and efficiently, have monoploid separation rate and surviving rate height, technology is simple, production cost is low advantage, for follow-up yellow wine yeast metabolic engineering and genetics research have been established basic substance.
Description of drawings
Fig. 1 is HO gene upstream and downstream homology arm in the embodiment of the invention 1, the agarose gel electrophoresis figure of kanMX gene amplification fragment and HO gene knockout assembly, wherein, M:DNA molecular weight marker, 1:HOL(490bp), 2:HOR(546bp), 3:kanMX gene amplification fragment (1697bp), 4:HOL-kanMX-HOR(2632bp).
Fig. 2 is that the PCR of HO genetically deficient transformant in the embodiment of the invention 2 identifies electrophorogram, wherein, and dna molecular amount marker; A5, B6 are respectively two positive transformants; The negative contrast of N (2253bp).
Fig. 3 identifies electrophorogram for haploid strains in the embodiment of the invention 3 and the bacterium colony PCR that joins type thereof, wherein, and M:DNA molecular weight marker, a:a type monoploid (544bp), α: α type monoploid (404bp).
Fig. 4 identifies electrophorogram for the PCR that resistant gene kanMX in the embodiment of the invention 4 loses, wherein, and M:DNA molecular weight marker, the HO1/HO2 amplified fragments (1028bp) after 1:kanMX is cut.
Embodiment
Below in conjunction with accompanying drawing, and be specifically described by the present invention of embodiment.
The related experiment material of following examples is as follows:
Bacterial strain and plasmid: industrial yellow wine yeast is provided by national yellow rice wine Engineering Technical Research Centre, and pYX212-kan plasmid and pSH65 plasmid all can be bought acquisition from commercial channels.
Reagent and test kit: Prime STAR DNA Polymerase(2.5U/ μ L), Ex Taq(5U/ μ L) MiniBEST Universal Genomic DNA Extraction Kit is all available from precious biotechnology (Dalian) company limited.
Other reagent and raw material are homemade or import analytical pure product.
In embodiment 1～embodiment 4 experiment the primer sequence that uses and act on as shown in table 1.
Table 1 primer sequence table
The structure of embodiment 1 yellow wine yeast HO gene knockout assembly
The structure of yellow wine yeast HO gene knockout assembly may further comprise the steps:
(1) be template with the pYX212-kan plasmid, use primer that kan1/kan2 is carried out first round PCR, the amplification two ends have the kanMX gene in loxp site, be template with this fragment then, use primer that kan3/kan4 is carried out second and take turns PCR, the amplification two ends have the kanMX gene of HO gene upstream and downstream homology arm sequence complementary sequence respectively; The pcr amplification program: 95 ℃ of pre-sex change 4min, (72 ℃ are extended 100s for 98 ℃ of sex change 10s, 58 ℃ of annealing 15s) carries out 30 circulations, and 72 ℃ are extended 5min again.
(2) extract industrial yellow wine yeast genomic dna (concrete operation method is referring to MiniBEST Universal Genomic DNA Extraction Kit test kit working instructions), be template with this genomic dna, use primer to HO1/HO1-1 and HO2/HO2-1 amplification HO upstream region of gene homology arm HOL and downstream homology arm HOR respectively; The pcr amplification program: 95 ℃ of pre-sex change 4min, (72 ℃ are extended 30s for 98 ℃ of sex change 10s, 52 ℃ of annealing 15s) carries out 30 circulations, and 72 ℃ are extended 5min again.
The preparation of each PCR reaction system is as shown in table 2 in step (1) and the step (2):
Table 2 25 μ L PCR reaction systems
| 5×Prime?STAR?Buffer(Mg 2+plus) | 5μL |
| DNTP Mixture (each 2.5mM) | 2μL |
| Primer 1 (10 μ M) | 1μL |
| Primer 2 (10 μ M) | 1μL |
| Template DNA | 1μL |
| Prime?STAR?DNA?Polymerase(2.5U/μL) | 0.25μL |
| Sterilized water | Polishing to 25 μ L |
(3) with step (1) and step (2) gained kanMX gene amplification fragment, HOL amplified fragments and HOR amplified fragments are template, do not add any primer and carry out the touchdown PCR amplification, utilize complementary sequence between above-mentioned three fragments to connect and merge, the amplification program of touchdown PCR: 95 ℃ of pre-sex change 4min, enter the pcr amplification program then, 98 ℃ of sex change 10s, 70 ℃ of annealing 15s, each cycle annealing temperature reduces by 1 ℃, totally 15 circulations, 72 ℃ are extended 3min, recirculation is once extended 10min in 72 ℃ at last again under 55 ℃ of annealing temperatures then, and reaction finishes, must merge fragment, the preparation of touchdown PCR reaction system is as shown in table 3; Should merge fragment as template, use primer that HO1/HO2 is carried out pcr amplification, obtain HO gene knockout assembly HOL-kanMX-HOR, pcr amplification program: 95 ℃ of pre-sex change 4min, (72 ℃ are extended 160s for 94 ℃ of sex change 30s, 58 ℃ of sex change 30s) carries out 30 circulations, 72 ℃ are extended 5min again, and the preparation of PCR reaction system is as shown in table 4; The PCR product is carried out T clone and sample presentation order-checking, and checking makes up the exactness of assembly HOL-kanMX-HOR.
Table 3 25 μ L PCR reaction systems
Table 4 25 μ L PCR reaction systems
| 10×Ex?Taq?Buffer(Mg 2+Plus) | 2.5μL |
| DNTP Mixture (each 2.5mM) | 2μL |
| Primer 1 (10 μ M) | 4μL |
| Primer 2 (10 μ M) | 4μL |
| Template DNA | 1μL |
| Ex?Taq(5U/μL) | 0.25μL |
| Sterilized water | Polishing to 25 μ L |
Above HO gene upstream and downstream homology arm, the agarose gel electrophoresis result of kanMX gene amplification fragment and HO gene knockout assembly as shown in Figure 1.As shown in Figure 1, successfully increasing by regular-PCR obtains the HOL of 490bp, the HOR of 546bp, and the kanMX gene of 1697bp successfully makes up the HO gene knockout assembly HOL-kanMX-HOR that obtains 2632bp by touchdown PCR and specific amplification.
The conversion of embodiment 2 industrial yellow wine yeasts and the evaluation of transformant
Adopt electrotransformation, embodiment 1 gained HO gene knockout assembly HOL-kanMX-HOR is transformed industrial yellow wine yeast and identify, may further comprise the steps:
(1) picking industry yellow wine yeast inoculation YPD liquid nutrient medium in 30 ℃ of 200r/min incubated overnight, gets activated seed liquid;
(2) according to the inoculative proportion of volume ratio 10%, seed liquor is forwarded to the fresh YPD liquid nutrient medium of 50mL, continues to be cultured to bacterium liquid OD600 between 1.2～1.5 in 30 ℃ of 200r/min;
(3) collect thalline in the centrifugal 5min of 5000r/min, use twice of 25mL sterilized water centrifuge washing thalline;
(4) with the resuspended thalline of 8mLDTT solution, in 30 ℃ of water-bath vibration 30min, the centrifugal 5min of 5000r/min collects thalline;
(5) with the resuspended thalline of 1.2M Sorbitol Solution USP of 15mL4 ℃ of precooling, collect thalline in 5000r/min4 ℃ of centrifugal 5min;
(6) repeating step is (5) three times;
(7) with 0.5mL1.2M sorbyl alcohol resuspended thalline, get 100 μ L bacterium liquid, add 10 μ L and transform fragment, change the electric shock cup after the mixing over to, place 5min on ice after, the 1.5kv electric shock;
(8) adding the 1mLYPD substratum changes liquid with electricity and washes out, and cultivates 2h in 30 ℃ of 200r/min, coats the YPD culture medium flat plate that contains 350 μ g/mL G418, is cultured to bacterium colony in 30 ℃ and grows;
(9) picking transformant and extract genome as template uses primer that HO1/Y4 is carried out the positive transformant of pcr amplification to identify that the HO gene is knocked out, and the PCR qualification result as shown in Figure 2.
As shown in Figure 2, the negative contrast of N, be about 2.2kbp with the starting strain genome as the stripe size that template amplification goes out, and the genome amplification of A5, two transformants of A6 has gone out two bands, wherein less band is consistent with the negative control size, knock out the band that assembly obtains and be HO than big band (about 2.7kbp) after successfully transforming on another allelotrope, the living worker's biotechnology (Shanghai) of sample presentation limited-liability company is correct through sequence verification.
The separation of embodiment 3 yellow wine yeast haploid strains and join type and identify
(1) identifies among the picking embodiment 2 that correct positive transformant is inoculated in the YPD substratum, in 30 ℃ of 200r/min activation 24h, transfer in fresh YPD liquid nutrient medium continuation cultivation 24h according to the inoculum size of volume ratio 10%;
(2) collect yeast slurry in the centrifugal 8min of 4000r/min, with after the physiological saline washed twice yeast slurry being piled up in living spore substratum (1wt% Potassium ethanoate, 2wt%agar, all the other compositions are water), cultivate for 26 ℃ and give birth to spore 3～5d;
(3) picking is given birth to an amount of thalline on the spore substratum in 1mL2.5%(w/v) helicase solution in, the enzymolysis situation of observing ascus walls in 37 ℃ of water-baths vibration 50～55min(microscopies);
(4) bacterium liquid is all moved in the sterilized triangular flask that 9mL physiological saline is housed that adds granulated glass sphere, in 58 ℃ of water-baths vibration 20min;
(5) picking colony uses monoploid primer (MAT, MAT-a, MAT-α) to carry out bacterium colony PCR, identifies haploid strains and joins type, and colony PCR amplification result as shown in Figure 3.
Use primer MAT/MAT-a/MAT-α to carry out monoploid and join type when identifying, the bacterial strain that amplifies the 544bp band is a type monoploid, the bacterial strain that amplifies the 404bp band is α type monoploid, as shown in Figure 3,3 a type monoploid and 2 α type monoploid is arranged in 5 bacterial strains identifying.
The excision of embodiment 4 haploid strains resistant genes
(1) according to embodiment 2 described methods, adopt electrotransformation that plasmid pSH65 is transformed industrial yellow wine yeast, and coat contain 100 μ g/mL Zeocin(bleomycins) the YPD substratum, be cultured to bacterium colony in 30 ℃ and grow;
(2) picking colony uses primer that ble1/ble2 is carried out bacterium colony PCR, identifies the positive transformant that contains the pSH65 plasmid;
(3) positive transformant is transferred to contain 100 μ g/mL Zeocin(bleomycins) semi-lactosi substratum (2%tryptone, 1%yeast extract, 2%D-galactose, 2%agar), continuous passage 5 times induces the Cre recombinase to express to excise resistant gene kanMX;
(4) with the single bacterial colony photographic reprinting on the semi-lactosi substratum to the YPD flat board that contains 350 μ g/mL G418, select the single bacterium colony that on YPD flat board (containing G418), can not grow in semi-lactosi flat board (containing Zeocin) growth, use primer that HO1/HO2 is carried out colony PCR amplification, identify losing of resistant gene kanMX, the pcr amplification result as shown in Figure 4;
(5) inoculation that resistant gene kanMX is lost more than 10 generations, is lost the pSH65 plasmid in YPD liquid nutrient medium cultured continuously, is not finally contained the industrial yellow wine yeast haploid strains of external resistant gene.
As shown in Figure 4, the transformant of identifying has amplified the band about 1kbp, and is consistent with the expected results after the kanMX gene is lost, and the living worker's biotechnology (Shanghai) of sample presentation limited-liability company is correct through sequence verification.
In sum, the present invention has successfully made up yellow wine yeast HO gene knockout assembly, has realized knocking out of HO gene by homologous recombination, can separate the industrial yellow wine yeast haploid strains that acquisition does not contain external resistant gene fast and efficiently.
Above-described only is preferred implementation of the present invention, the invention is not restricted to above embodiment.Be appreciated that other improvement and variation that those skilled in the art directly derive or associate under the prerequisite that does not break away from spirit of the present invention and design, all should think to be included within protection scope of the present invention.
Claims (9)
1. the haploid separation method of industrial yellow wine yeast is characterized in that knocking out by the HO gene of homologous recombination with described industrial yellow wine yeast; This separation method concrete operations step is as follows:
(1) structure of HO gene knockout assembly: the kanMX gene amplification fragment of at first utilizing the regular-PCR amplification to obtain the upstream and downstream homology arm amplified fragments of yellow wine yeast HO gene and have the G418 resistance marker, obtain the fusion fragment of above-mentioned three fragments again by the touchdown PCR amplification, utilize regular-PCR that described fusion fragment is carried out specific amplification at last and namely get yellow wine yeast HO gene knockout assembly; Described upstream homology arm amplified fragments comprises that upstream homology arm sequence reaches and the sequence (abbreviation sequence A) of kanMX gene 5 ' end complementation, and described downstream homology arm amplified fragments comprises that downstream homology arm sequence reaches the sequence (abbreviation sequence B) of holding complementation with kanMX gene 3 '; Described kanMX gene amplification fragment comprises the kanMX gene order and is positioned at these fragment two ends holds complementary sequence (being called for short sequence C and sequence D respectively) with upstream homology arm sequence 3 ' end, downstream homology arm sequence 5 ' respectively; All contain the loxp site that to be identified by the Cre recombinase between described kanMX gene order and described sequence C, the sequence D; Described sequence A, sequence B, sequence C and sequence D are 20～30bp;
(2) knocking out of yellow wine yeast HO gene: step (1) gained HO gene knockout assembly is transformed industrial yellow wine yeast bacterial strain, obtain positive transformant and evaluation by the G418 resistance screening;
(3) the haploid separation of yellow wine yeast: get and identify in the step (2) that correct positive transformant is inoculated in the YPD substratum and activates; Collect yeast slurry and it is piled up in living spore substratum, cultivate 3～5d in 26～28 ℃; Bacterium colony on the described living spore substratum of picking, get enzymolysis bacterium liquid through helicase enzymolysis broken wall treatment, spore is broken up in vibration, coats YPD again and cultivates and to be cultured to bacterium colony based on 28～30 ℃ and to grow, and picking colony is through identifying the haploid strains that obtains containing resistant gene kanMX after correct;
(4) excision of resistant gene: engineering plasmid step of converting (3) the gained haploid strains that will contain the Cre recombinase gene, get the positive colony transformant and be inoculated in 5～10 generations of semi-lactosi substratum cultured continuously, induce the Cre recombinase to express to excise resistant gene kanMX, obtain resistant gene and lose bacterial strain; This bacterial strain cultured continuously more than 10 generations, is lost above-mentioned engineering plasmid, namely get the industrial yellow wine yeast haploid strains that does not contain external resistant gene.
2. according to the haploid separation method of the described industrial yellow wine yeast of claim 1, it is characterized in that: the described touchdown PCR amplified reaction of step (1) does not have primer and participates in, its amplification condition is as follows: 95 ℃ of pre-sex change 4～5min, 98 ℃ of sex change 10～15s, 70 ℃ of annealing 15～30s, each cycle annealing temperature reduces by 1 ℃, totally 15 circulations, in 72 ℃ of extension 2.5～3min, recirculation is once extended 5～10min in 72 ℃ at last again under 55 ℃ of annealing temperatures then.
3. according to the haploid separation method of the described industrial yellow wine yeast of claim 1, it is characterized in that: the amplimer sequence of the described upstream of step (1) homology arm amplified fragments is shown in SEQ ID NO:1 and SEQ ID NO:2.
4. according to the haploid separation method of the described industrial yellow wine yeast of claim 1, it is characterized in that: the amplimer sequence of the described downstream of step (1) homology arm amplified fragments is shown in SEQ ID NO:3 and SEQ ID NO:4.
5. according to the haploid separation method of the described industrial yellow wine yeast of claim 1, it is characterized in that: the amplimer sequence of the described kanMX gene amplification of step (1) fragment is shown in SEQ ID NO:7 and SEQ ID NO:8.
6. according to the haploid separation method of the described industrial yellow wine yeast of claim 1, it is characterized in that: the specific primer sequence of the described fusion fragment of step (1) is shown in SEQ ID NO:1 and SEQ ID NO:4.
7. according to the haploid separation method of the described industrial yellow wine yeast of claim 1, it is characterized in that: the described activation temperature of step (3) is 30 ℃, incubation time 24～36h.
8. according to the haploid separation method of the described industrial yellow wine yeast of claim 1, it is characterized in that: the engineering plasmid of the described Cre of the containing recombinase gene of step (4) is selected from pSH47, pSH62, pSH63 or pSH65.
9. according to the haploid separation method of the described industrial yellow wine yeast of claim 1, it is characterized in that: the described semi-lactosi substratum of step (4) contains the bleomycin of 100 μ g/mL.
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| CN109355245A (en) * | 2018-12-24 | 2019-02-19 | 江南大学 | One kind separating the haploid method of saccharomyces cerevisiae based on flow cytometry ultra-high throughput |
| CN109402162A (en) * | 2018-11-28 | 2019-03-01 | 南京工业大学 | A method of knocking out S. cervisiae HO gene |
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| CN103266104A (en) * | 2013-04-27 | 2013-08-28 | 江南大学 | Method for high-efficiency construction of saccharomyces cerevisiae HO gene knockout component |
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| CN103266104A (en) * | 2013-04-27 | 2013-08-28 | 江南大学 | Method for high-efficiency construction of saccharomyces cerevisiae HO gene knockout component |
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| CN109402162A (en) * | 2018-11-28 | 2019-03-01 | 南京工业大学 | A method of knocking out S. cervisiae HO gene |
| CN109355245A (en) * | 2018-12-24 | 2019-02-19 | 江南大学 | One kind separating the haploid method of saccharomyces cerevisiae based on flow cytometry ultra-high throughput |
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