CN101525580A - Amphiploid histidine auxotroph saccharomyces cerevisiae and constructing method thereof - Google Patents
Amphiploid histidine auxotroph saccharomyces cerevisiae and constructing method thereof Download PDFInfo
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Abstract
The invention discloses amphiploid histidine auxotroph saccharomyces cerevisiae and a constructing method thereof, belonging to the field of microorganism biological engineering. The saccharomyces cerevisiae is made from an amphiploid saccharomyces cerevisiae bacterial strain which is TSH-Sc-001(His3-/-) and contains a histidine auxotrophic mark and a g418 resistance mark. The constructing method of the amphiploid histidine auxotroph saccharomyces cerevisiae comprises the following steps: the amphiploid saccharomyces cerevisiae bacterial strain is adopted as a start bacterial strain, the start bacterial strain is transformed twice by a segment with target genes, and the amphiploid histidine auxotroph saccharomyces cerevisiae bacterial strain with two chromosomes being subjected to homologous recombination is obtained by two-time homologous recombination. The saccharomyces cerevisiae is easily identified and protected; in addition, the constructing method of the amphiploid histidine auxotroph saccharomyces cerevisiae provides a novel way and a method for the reconstruction of gene engineering of amphiploid or multiploid saccharomyces, is simple and highly efficient, and keeps the primary characteristic of the saccharomyces unchanged.
Description
Technical field
The invention belongs to microorganism biological engineering field, be specifically related to a kind of amphiploid histidine auxotroph yeast saccharomyces cerevisiae and construction process thereof.
Background technology
Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae Hansen) is traditional Alcohol Production bacterial strain, possesses the favorable industrial production traits, consumption sugar is fast, ethanol tolerance height, to toxic substance strong resistance in the hydrolysis sugar liquid; Its complete sequence is measured, and Genetic Manipulative Technology is also ripe.
The auxotroph yeast strain is playing the part of important role in genetically engineered and genetic modification field, and wherein the Histidine auxotrophy is one of auxotroph the most commonly used; In addition, Geneticin (g418) resistant gene also is a yeast selection markers commonly used.
A kind of sweet sorghum stalk solid state fermentation that is used for is produced alcoholic acid amphiploid yeast saccharomyces cerevisiae TSH-Sc-001 (deposit number is CGMCC NO.1949), has extraordinary fermentation capacity and (sees that (application number is patent " a kind of prepare the alcoholic acid method and system based on sweet broomcorn straw solid fermentation ": 200710080388.0).For to its protection with carry out further genetic engineering modifiedly, need add heredity and selection markers for it.
Existing method of yeast saccharomyces cerevisiae being carried out genetically engineered or genetic modification, all be as initial bacterial strain with haploid strains, but the yeast strain overwhelming majority who uses on the industrial production is amphiploid or polyploid, if will transform the change that can cause some characteristic behind its haploidization again.
Summary of the invention
The object of the present invention is to provide a kind of amphiploid histidine auxotroph yeast saccharomyces cerevisiae.This yeast saccharomyces cerevisiae keeps the characteristic of original Wine brewing yeast strain, is easy to identification.
Another object of the present invention provides the construction process of above-mentioned amphiploid histidine auxotroph yeast saccharomyces cerevisiae; This method provides a kind of new way for amphiploid or polyploid zymic are genetic engineering modified.
Realize that technical scheme of the present invention is as follows:
A kind of amphiploid histidine auxotroph yeast saccharomyces cerevisiae is meant that this yeast saccharomyces cerevisiae is the amphiploid Wine brewing yeast strain that contains Histidine auxotrophy mark and g418 resistance marker.
Described amphiploid Histidine auxotrophy is meant that sudden change has all taken place two chromosomal His3 genes of this amphiploid Wine brewing yeast strain, has lost the ability of synthetic Histidine, can not go up growth at minimum medium (MD); Describedly contain two karyomit(e)s that amphiploid g418 resistance marker is meant this yeast saccharomyces cerevisiae and all contain the g418 resistant gene, YPD substratum (the yeast extract 10g/L of 0.5g/L g418 can contained, peptone 20g/L, glucose 20g/L, solid medium add the agar of 20g/L) go up and grow.
Above-mentioned amphiploid histidine auxotroph yeast saccharomyces cerevisiae is meant amphiploid Wine brewing yeast strain (Saccharomyces cerevisiae) TSH-Sc-001 that contains Histidine auxotrophy and g418 resistance marker (His3-/-), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 13rd, 2008, and deposit number is: CGMCC NO.2738.This bacterial strain depositary institution address is a Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
The construction process of above-mentioned amphiploid histidine auxotroph yeast saccharomyces cerevisiae, comprise that with the amphiploid Wine brewing yeast strain be starting strain, carry out twice conversion with the fragment that has goal gene, by homologous recombination takes place twice, obtain the amphiploid histidine auxotroph Wine brewing yeast strain that homologous recombination has all taken place 2 karyomit(e)s.
Target gene fragment described in the aforesaid method be meant the goal gene two ends respectively with the dna sequence dna homology of His3 left arm or His3 right arm, g418 resistant gene and/or other genes are contained in the centre of target gene fragment.
The construction process of above-mentioned amphiploid histidine auxotroph yeast saccharomyces cerevisiae comprises the steps:
(1) according to the sequence (Genebank of S.cerevisiae His3 gene, Accession No.NC001147) and pPIC9K plasmid (Invitrogen, Cat.No.V175-20) sequences Design amplification contains the primer of homologous recombination left arm and g418 resistant gene (His3L-Kan), introduce EcoR I (GAATTC) and Sal I (GTCGAC) recognition sequence respectively at 5 ' end and 3 ' end, with the pPIC9K plasmid DNA is that template is carried out pcr amplification, the fragment His3L-Kan that must contain homologous recombination left arm and g418 resistance, purifying;
(2) with EcoR I and Sal I difference double digestion His3L-Kan fragment and pUC19 carrier, purifying enzyme is cut product, connect with the T4 ligase enzyme then, connect product with gained and transform DH5 α competent cell, the picking positive colony, extract recombinant plasmid dna, enzyme is cut evaluation, must contain the segmental recombinant plasmid pUC19-His3L-Kan of His3 homologous recombination left arm and g418 resistant gene;
(3) according to the sequence data (Genebank of S.cerevisiae His3 gene, Accession No.NC001147) the design amplification contains the primer of homologous recombination right arm (His3R), introduce Sal I (GTCGAC) and Pst I (CTGCAG) recognition site respectively at 5 ' end and 3 ' end, with the S.cerevisiae genomic dna is that template is carried out pcr amplification, get His3 homologous recombination right arm fragment His3R, purifying;
(4) with Sal I and Pst I difference double digestion His3R and plasmid pUC19-His3L-Kan, purifying enzyme is cut product, connect with the T4 ligase enzyme then, connect product with gained and transform DH5 α competent cell, the picking positive colony, extract recombinant plasmid dna, enzyme is cut evaluation, must contain the recombinant plasmid pUC19-His3L-Kan-His3R of homologous recombination left arm, g418 resistant gene and homologous recombination right arm;
(5), reclaim the fragment His3L-Kan-His3R that contains homologous recombination left arm, g418 resistant gene and homologous recombination right arm of purifying 1135bp with EcoR I and Pst I double digestion recombinant plasmid pUC19-His3L-Kan-His3R;
(6) by electroporation method the His3L-Kan-His3R fragment is transformed amphiploid yeast saccharomyces cerevisiae competent cell, be coated with the YPD substratum that contains 0.25g/L g418, the picking positive colony, extracting the laggard performing PCR of genomic dna identifies, containing the positive monoclonal bacterial strain of growing on the YPD substratum of 0.25g/L g418 through identifying, get a karyomit(e) homologous recombination has taken place, homologous recombination does not take place in a karyomit(e), is abbreviated as (His3
+/-);
(7) by electroporation method the His3L-Kan-His3R fragment is transformed (His3
+/-) competent cell, be coated with the YPD substratum that contains 0.5g/L g418, the picking positive colony extracts the laggard performing PCR of genomic dna and identifies, promptly gets the present invention and is containing the Wine brewing yeast strain (His3 that homologous recombination has all taken place two karyomit(e)s of growing on the YPD substratum of 0.5g/L g418
-/-).
Be example with amphiploid yeast saccharomyces cerevisiae (S.cerevisiae) TSH-Sc-001 below, specify the construction process of amphiploid histidine auxotroph yeast saccharomyces cerevisiae.
The construction process of above-mentioned amphiploid histidine auxotroph yeast saccharomyces cerevisiae comprises the steps:
(1) be template with the pPIC9K plasmid DNA, with His3L-1F-Kan and Kan-R is that primer carries out pcr amplification, increase the dna sequence dna shown in the SEQ ID No.9 with Kan resistant gene and His3 gene coding region 5 ' end 40bp, and add dna sequence dna the His3L '-Kan of Sal I recognition site at 3 ' end; Described primer His3L-1F-Kan is the dna sequence dna shown in the SEQ ID No.1, and described primer Kan-R is the dna sequence dna shown in the SEQ ID No.3;
(2) being template with PCR product the His3L '-Kan behind the purifying, is that primer carries out pcr amplification with His3L-2F and Kan-R, increase the dna sequence dna His3L-Kan shown in SEQ ID No.10; Purifying; Described primer His3L-2F is the dna sequence dna shown in the SEQ ID No.2;
(3) with EcoR I and Sal I difference double digestion His3L-Kan fragment and pUC19 carrier, purifying enzyme is cut product, connect with the T4 ligase enzyme then, connect product with gained and transform DH5 α competent cell, the picking positive colony, extract recombinant plasmid dna, enzyme is cut evaluation, must contain the recombinant plasmid pUC19-His3L-Kan of homologous recombination left arm and g418 resistant gene;
(4) being template with the S.cerevisiae genomic dna, is that primer carries out pcr amplification with His3R-F and His3R-R, increase the His3 homologous recombination right arm dna fragmentation His3R shown in SEQ ID No.11, purifying; Described primer His3R-F is the dna sequence dna shown in the SEQ ID No.4, and described primer His3R-R is the dna sequence dna shown in the SEQ ID No.5;
(5) with Sal I and Pst I difference double digestion His3R fragment and plasmid pUC19-His3L-Kan, purifying, connect with the T4 ligase enzyme then, connect product with gained and transform DH5 α competent cell, the picking positive colony, extract recombinant plasmid dna, enzyme is cut evaluation, must contain the recombinant plasmid pUC19-His3L-Kan-His3R of homologous recombination left arm, g418 resistant gene and homologous recombination right arm;
(6), reclaim purifying, the fragment His3L-Kan-His3R that contains His3 homologous recombination left arm, g418 resistant gene and His3 homologous recombination right arm that must be shown in SEQ ID No.12 with EcoR I and Pst I double digestion recombinant plasmid pUC19-His3L-Kan-His3R;
(7) (deposit number is: competent cell CGMCC NO.1949) by electroporation method the His3L-Kan-His3R fragment to be transformed amphiploid yeast saccharomyces cerevisiae (S.cerevisiae) TSH-Sc-001, be coated with the YPD substratum that contains 0.25g/Lg418, the picking positive colony, extracting the laggard performing PCR of genomic dna identifies, obtain the positive monoclonal bacterial strain of growing on the YPD substratum of 0.25g/L g418 containing, article one, homologous recombination has taken place in karyomit(e), article one, the Wine brewing yeast strain of homologous recombination does not take place in karyomit(e), is abbreviated as TSH-Sc-001 (His3
+/-); Described primers designed His3L is the dna sequence dna shown in the SEQ ID No.6, and described primers designed Kan-ID is the dna sequence dna shown in the SEQ ID No.7, and described primers designed His-ID is the dna sequence dna shown in the SEQ IDNo.8;
(8) by electroporation method the His3L-Kan-His3R fragment is transformed TSH-Sc-001 (His3
+/-) competent cell, be coated with the YPD substratum that contains 0.5g/L g418, the picking positive colony, extract genomic dna, carry out PCR and identify, obtain homologous recombination Wine brewing yeast strain (S.cerevisiae) TSH-Sc-001 (His3-/-) all having taken place (deposit number being: CGMCC NO.2738) containing two karyomit(e)s of positive monoclonal bacterial strain of growing on the YPD substratum of 0.5g/L g418; Described primers designed His3L is the dna sequence dna shown in the SEQ ID No.6, and described primers designed Kan-ID is the dna sequence dna shown in the SEQ ID No.7, and described primers designed His-ID is the dna sequence dna shown in the SEQ ID No.8.
The advantage that the present invention has: (1) gained yeast saccharomyces cerevisiae of the present invention has Histidine auxotrophy and g418 resistance marker, is easy to identification and protection; (2) construction process of the present invention provides a kind of new approaches and methods for amphiploid or polyploid zymic are genetic engineering modified; (3) construction process of the present invention is simple, efficient, and keeps the zymic primary characteristic constant.
Description of drawings
Fig. 1 is the schema of His3L-Kan-His3R fragment building process.
Fig. 2 is that pUC19-His3L-Kan plasmid EcoR I and Sal I double digestion are identified electrophoretogram,
M1:DL15000 wherein; M2:DL2000; 1:pUC19-His3L-Kan.
Fig. 3 is that pUC19-His3L-Kan-His3R plasmid EcoR I and Pat I double digestion are identified electrophoretogram,
1:pUC19-His3L-Kan-His3R wherein; M1:DL2000; M2:DL15000.
Fig. 4 is a His3L-Kan-His3R fragment electrophoretogram,
1:His3L-Kan-His3R wherein; M1:DL15000; M2:DL2000.
The PCR synoptic diagram that Fig. 5 identifies with primer His3-F, His-ID, Kan-ID for recombinant bacterial strain.
Fig. 6 is TSH-Sc-001, TSH-Sc-001 (His
+/-) and TSH-Sc-001 (His3-/-) PCR identify electrophoretogram, wherein
The 1:His3-F+His-ID blank; The 2:His3-F+Kan-ID blank;
The DNA of 3:TSH-Sc-001 is a template, and His3-F and His-ID are primer;
The DNA of 4:TSH-Sc-001 is a template, and His3-F and Kan-ID are primer;
5:TSH-Sc-001 (His
+/-) DNA be template, His3-F and His-ID are primer;
6:TSH-Sc-001 (His
+/-) DNA be template, His3-F and Kan-ID are primer;
7:TSH-Sc-001 (His3
-/-) DNA be template, His3-F and His-ID are primer;
8:TSH-Sc-001 (His3
-/-) DNA be template, His3-F and Kan-ID are primer;
M：DL2000。
Fig. 7 is the structural representation of plasmid pUC19-His3L-Kan-His3R.
Embodiment
The present invention is described further below in conjunction with embodiment, but the present invention is not constituted any restriction.
The structure of embodiment 1 homologous recombination fragment His3L-Kan-His3R
1.1 make up the pUC19-His3L-Kan plasmid that contains homologous recombination left arm and g418 resistant gene
1.1.1 amplification contains the His3L-Kan fragment (926bp) of homologous recombination left arm and g418 resistance
Sequence data (AccessionNo.NC001147) and pPIC9K plasmid (Invitrogen according to the S.cerevisiae His3 gene of having reported among the Genebank, Cat.No.V175-20) sequence data utilizes Primer Premier 5 software design PCR primers.
Upstream primer His3L-1F-Kan
5’-GATTGCGATCTCTTTAAAGGGTGGTCCCCTAGCGATAGAGATGAGCCATATTCAACGGG-3’(SEQ?ID?No.1)
Upstream primer His3L-2F
5’-GGAATTCATGACAGAGCAGAAAGCCCTAGTAAAGCGTATTACAAATGAAACCAAGATTCAGATTGCGATCTCTTTAAAG-3’(SEQ?ID?No.2)
Downstream primer Kan-R
5’-ACGCGTCGACTTAGAAAAACTCATCGAGCATC-3’(SEQ?ID?No.3)
1) being template with the pPIC9K plasmid, is primer amplification Kan resistant gene with His3L-1F-Kan and Kan-R, introduces the sequence of His3 gene coding region 5 ' end 40bp simultaneously, and adds Sal I recognition site at 3 ' end.With
DNA Polymerase (Takara, Cat.No.DR005A), reaction system is:
10×Pyrobest?Buffer?II 5.00μl
DNTP mixture (every kind of dNTP 2.5mM) 4.00 μ l
His3L-1F-Kan?or?His3L-2F(20μM) 1.00μl
Kan-R(20μM) 1.00μl
Pyrobest archaeal dna polymerase (5U/ μ l) 0.25 μ l
PPIC9K plasmid DNA 0.50 μ l
ddH
2O 38.25μl
Cumulative volume 50.00 μ l
PCR reaction conditions: 94 ℃ of 5min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 1min circulate 30 times; 72 ℃ of 10min.
Detect the PCR product by agarose gel electrophoresis, with the gained clip size is PCR product His3L '-Kan (SEQ ID No.9) plain agar sugar gel DNA recovery test kit (TIANGEN of 866bp, Cat.No.DP209-02) carry out purifying, operation steps and method are undertaken by product description.
2) being template with His3L '-Kan, is that primer carries out pcr amplification with His3L-2F and Kan-R, introduces the sequence of His3 gene coding region 5 ' end 93bp simultaneously, and adds EcoR I recognition site at 5 ' end, and 3 ' end adds Sal I recognition site.With
The described PCR of DNA Polymerase (Takara, Cat.No.DR005A), PCR reaction system, condition and PCR product purification method and 1) is identical.Recovery, purifying clip size are the PCR product of 926bp, must contain the His3L-Kan fragment (SEQ ID No.10) of homologous recombination left arm and g418 resistance.
1.1.2 make up the pUC19-His3L-Kan plasmid (3568bp) that contains homologous recombination left arm and g418 resistant gene
PUC19 (Takara, Cat.No.D3219) (TIANGEN is Cat.No.CB101-02) after the amplification, with the little extraction reagent kit (TIANGEN of plasmid through E.coli DH5 α for plasmid, Cat.No.DP103-02) extract, operation steps and method are undertaken by product description.With pUC19 plasmid and above-mentioned gained PCR product His3L-Kan use respectively EcoR I (Takara, Cat.No.D1040A) and Sal I (Takara Cat.No.D1080A) carries out double digestion, and it is as follows that enzyme is cut system:
A B
10×H?buffer 5.0μl 10×H?buffer 5.0μl
EcoR?I 2.0μl EcoR?I 2.0μl
Sal?I 2.0μl Sal?I 2.0μl
pUC19 20.0μl EcoRI-His3L-Kan-SalI 20.0μl
ddH
2O 21.0μl ddH
2O 21.0μl
Cumulative volume 50.0 μ l cumulative volumes 50.0 μ l
With above-mentioned system mixing, instantaneous centrifugal, 37 ℃ of enzymes are cut and are spent the night.Enzyme is cut product, and (TIANGEN Cat.No.DP209-02) carries out purifying, and operation steps and method are undertaken by product description with plain agar sugar gel DNA recovery test kit.With the endonuclease bamhi (EcoRI-pUC19-SalI and EcoRI-His3L-Kan-SalI) of purifying with the T4 dna ligase (Takara Cat.No.D2011A) connects, and the ligation system is as follows:
10×T4?DNA?ligase?buffer 2.0μl
EcoRI-pUC19-SalI 2.0μl
EcoRI-His3L-Kan-SalI 10.0μl
T4?DNA?ligase 1.0μl
ddH
2O 5.0μl
Cumulative volume 20.0 μ l
With above-mentioned system mixing, instantaneous centrifugal, 16 ℃ of connections are spent the night.
With connect product Transformed E coli DH5 α competent cell (TIANGEN, Cat.No.CB101-02), method steps is as follows:
Take out in-70 ℃ of frozen E coli DH5 α competent cells, be put in the palm of the hand and make it speed and melt, and then place on ice; Get respectively in the eppendorf pipe after 50 μ l extremely sterilize, add 5 μ l again and connect product, mixing is put ice bath 30min gently; 42 ℃ of heat-shocked 90s do not shake test tube, put into ice bath 2min rapidly; Every pipe adds the LB nutrient solution of 37 ℃ of preheatings of 950 μ l, cultivates 1h in 37 ℃ of 200rpm, makes cell recovery, and the antibiotic marker gene of expression plasmid coding.
The centrifugal 5min of 3500rpm outwells 900 μ l supernatants, with remaining 100 μ l liquid and cell precipitation mixing, coats equably on the LB plate culture medium that contains penbritin 100mg/L and kantlex 50mg/L, is inverted overnight incubation for 37 ℃.
Mono-clonal bacterium colony on the picking resistant panel is inoculated in 5ml and contains in the LB liquid nutrient medium of penbritin 100mg/L and kantlex 50mg/L, and 37 ℃, the 200rpm overnight incubation.With the little extraction reagent kit of plasmid (TIANGEN Cat.No.DP103-02) extracts plasmid, with EcoR I (Takara, Cat.No.D1040A) and Sal I (Takara, Cat.No.D1080A) double digestion is identified (Fig. 2), it is as follows that enzyme is cut system:
10×H?buffer 2.0μl
EcoR?I 1.0μl
Sal?I 1.0μl
pUC19-His3L-Kan 10.0μl
ddH
2O 6.0μl
Cumulative volume 20.0 μ l
The pUC19-His3L-Kan plasmid obtains the fragment that two sizes are respectively 915bp and 2653bp with EcoR I and Sal I double digestion, cultivates and extract plasmid with identifying that correct clone transfers in the LB liquid nutrient medium that contains penbritin 100mg/L and kantlex 50mg/L.
1.2 contain the structure of the pUC19-His3L-Kan-His3R plasmid of homologous recombination left arm, g418 resistant gene and homologous recombination right arm
1.2.1 amplification homologous recombination right arm His3R fragment (228bp)
According to the sequence data (AccessionNo.NC001147) of the S.cerevisiae His3 gene of having reported among the Genebank, utilize Primer Premier5 software design PCR primer.
Upstream primer His3R-F
5’-ACGCGTCGACGTAGGAGATCTCTCTTGCGAGATG-3’(SEQ?ID?No.4)
Downstream primer His3R-R
5’-AACTGCAGCTACATAAGAACACCTTTGGTGGAG-3’(SEQ?ID?No.5)
The purpose fragment of amplification is His3R (228bp) (SEQ ID No.11), extract test kit (TIANGEN with pastoris genomic dna, Cat.No.DP307-02) extract S.cerevisiae (CGMCC NO.1949) genomic dna, as the segmental template of amplification His3R, with His3R-F and His3R-R is the sequence of primer amplification His3 coding region 3 ' end 210bp, and adding Sal I recognition site at 5 ' end, 3 ' end adds Pst I recognition site.With
DNA Polymerase (Takara, Cat.No.DR005A), reaction system is:
10×Pyrobest?Buffer?II 5.00μl
dNTP?mix(2.5mM?each) 4.00μl
His3R-F(20μM) 1.00μl
His3R-R(20μM) 1.00μl
Pyrobest?DNA?Polymerase(5U/μl) 0.25μl
S.cerevisiae (CGMCC NO.1949) genomic dna 0.50 μ l
ddH
2O 38.25μl
Cumulative volume 50.00 μ l
PCR reaction conditions: 94 ℃ of 5min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 30s circulate 30 times; 72 ℃ of 10min.
After PCR finishes, detect the PCR product by agarose gel electrophoresis, the PCR product that detected result is correct reclaims test kit with plain agar sugar gel DNA, and (TIANGEN Cat.No.DP209-02) carries out purifying, and operation steps and method are undertaken by product description.
1.2.2 contain the structure of the pUC19-His3L-Kan-His3R plasmid (3778bp) of homologous recombination left arm, g418 resistant gene and homologous recombination right arm
1.1 make up PCR product His3R that the pUC19-His3L-Kan plasmid obtain and 1.2.1 obtain with SalI (Takara, Cat.No.D1080A) and Pst I (it is as follows that Takara, Cat.No.D1073A) double digestion, enzyme cut system:
A B
10×H?buffer 5.0μl 10×H?buffer 5.0μl
Sal?I 2.0μl Sal?I 2.0μl
Pst?I 2.0μl Pst?I 2.0μl
pUC19-His3L-Kan 20.0μl SalI-His3R-PstI 20.0μl
ddH
2O 21.0μl ddH
2O 21.0μl
Cumulative volume 50.0 μ l cumulative volumes 50.0 μ l
Endonuclease reaction condition and enzyme are cut the same 1.1.2 of product purification method.Enzyme is cut products therefrom SalI-pUC19-His3L-Kan-PstI and SalI-His3R-PstI connects with T4 dna ligase (Takara, Catalog No.D2011A), the ligation system is as follows:
10×T4?DNA?ligase?buffer 2.0μl
SalI-pUC19-His3L-Kan-Pst?I 2.0μl
SalI-His3R-PstI 10.0μl
T4?DNA?ligase 1.0μl
ddH
2O 5.0μl
Cumulative volume 20.0 μ l
The same 1.1.2 of method of ligation condition, Transformed E coli DH5 α competent cell.
Mono-clonal bacterium colony on the picking resistant panel is inoculated in 5ml and contains in the LB liquid nutrient medium of penbritin 100mg/L and kantlex 50mg/L, and 37 ℃, the 200rpm overnight incubation.With the little extraction reagent kit (TIANGEN of plasmid, Cat.No.DP103-02) extract plasmid, get pUC19-His3L-Kan-His3R plasmid (see figure 7), with EcoR I (Takara, Catalog No.D1040A) and Pst I (Takara, CatalogNo.D1073A) double digestion is identified (see figure 3), and it is as follows that enzyme is cut system:
10×H?buffer 2.0μl
EcoR?I 1.0μl
Pst?I 1.0μl
pUC19-His3L-Kan-His3R 10.0μl
ddH
2O 6.0μl
Cumulative volume 20.0 μ l
The pUC19-His3L-Kan-His3R plasmid obtains the fragment that two sizes are respectively 1135bp and 2643bp with EcoR I and Pst I double digestion.In the LB liquid nutrient medium that contains penbritin 100mg/L and kantlex 50mg/L, cultivate and extract plasmid with identifying that correct clone transfers.
1.3 contain the His3L-Kan-His3R of homologous recombination left arm, g418 resistant gene and the homologous recombination right arm segmental acquisition of practicing shooting
The pUC19-His3L-Kan-His3R plasmid with EcoR I (Takara, Cat.No.D1040A) and PstI (it is as follows that Takara, Cat.No.D1073A) double digestion, enzyme cut system:
10×H?buffer 5.0μl
EcoR?I 2.0μl
Pst?I 2.0μl
pUC19-His3L-Kan-His3R 20.0μl
ddH
2O 21.0μl
Cumulative volume 50.0 μ l
Endonuclease reaction condition and enzyme are cut the same 1.1.2 of product purification method, reclaim the His3L-Kan-His3R fragment that contains homologous recombination left arm, g418 resistant gene and homologous recombination right arm (the SEQ ID No.12) (see figure 4) of 1135bp.
The acquisition of embodiment 2 monosome exchange Wine brewing yeast strain
With the His3L-Kan-His3R fragment of 1.3 1135bp that obtain among the embodiment 1, transform S.cerevisiae TSH-Sc-001 (CGMCC NO.1949) competent cell by electroporation method.Used electroporation apparatus is Gene Pulser Xcell
TMElectroporation System (Bio-Rad company), the preparation method of S.cerevisiae competent cell is with reference to this instrument specification sheets.
For the sample that each electricity changes, prepare the small test tube of a YPD liquid nutrient medium that contains 1ml 1M sorbyl alcohol and place cooled on ice, prepare the electric revolving cup of 2mm simultaneously and place cooled on ice.
In cooled on ice, the competent cell that adds 40 μ l is in the DNA sample with His3L-Kan-His3R fragment to be transformed (100ng/5 μ l), and mixing lightly is in the about 5min of ice bath on ice.
Be ready to electroporation, carry out the conversion of brewing yeast cell by default program, the parameter that electricity transforms is C=25 μ F; PC=200ohm; V-1.5kV; 2mm cuvettes.
The DNA-competent cell is transferred to ice-cold 2mm electricity revolving cup bottom, cover lid, button shocks by electricity; Take out electric revolving cup, add the ice-cold YPD liquid nutrient medium that contains the 1M sorbyl alcohol of 1ml immediately in electric revolving cup, refund in the small test tube then.
Check that electricity changes record, the time of electric shock should cultivate 2 with the S.cerevisiae after the electricity conversion and as a child coat on the YPD plate culture medium that contains 1M sorbyl alcohol and 0.25g/L g418 in advance for about 5ms, is inverted for 30 ℃ and cultivates 3d.
Mono-clonal bacterium colony on the picking g418 resistant panel is inoculated in 5ml and contains in the YPD liquid nutrient medium of g418 (0.25g/L) 30 ℃ of 200rpm overnight incubation;
(TIANGEN Cat.No.DP307-02) carries out the extraction of S.cerevisiae genomic dna, and operation steps and method are undertaken by product description to extract test kit with pastoris genomic dna.With S.cerevisiae genomic dna initial or reorganization is template, carries out pcr amplification by following primer, judges whether homologous recombination has taken place.Upstream primer His3-F derives from one section sequence of His3 upstream region of gene promoter region; Downstream primer His-ID derives from His3 gene regions (being one section reverse complementary sequence); Downstream primer Kan-ID derives from Kan gene regions (being one section reverse complementary sequence).Therefore, homologous recombination (SEQ ID No.13) does not take place in the representative of the amplified production of primer His3-F and His-ID; The amplified production of primer His3-F and Kan-ID is then represented homologous recombination (SEQ ID No.14) has been taken place.Primer is the PCR synoptic diagram (see figure 5) of template with the genomic dna.
Upstream primer His3-F:5 '-TGCACGGTCCTGTTCCCTAGCATG-3 ' (SEQ ID No.6)
Downstream primer Kan-ID:5 '-AGACGTTTCCCGTTGAATATGGCTC-3 ' (SEQ ID No.7)
Downstream primer His-ID:5 '-GGAATGCTTGGCCAGAGCATGTATC-3 ' (SEQ ID No.8)
Initial bacterial strain TSH-Sc-001 uses this two pairs of primer amplifications respectively, and only primer His3-F and His-ID can obtain the PCR product of 681bp.Article one, the bacterial strain that reorganization, another karyomit(e) recombinates has taken place and has used this two pairs of primer amplifications respectively in karyomit(e), can obtain the PCR product of 582bp and 681bp.Article two, karyomit(e) bacterial strain that reorganization all taken place is used this two pairs of primer amplifications respectively, and only primer His3-F and Kan-ID can obtain the PCR product of 582bp.
With rTaq DNA Polymerase (Takara, Cat.No.DR100AM), reaction system is:
10×PCR?Buffer 5.0μl
dNTP?mix(2.5mM?each) 4.0μl
MgCl
2(25mM) 3.0μl
His3-F(20μM) 1.0μl
Kan-ID?or?His-ID(20μM) 1.0μl
Taq?DNA?Polymerase(5U/μl) 0.5μl
S.cerevisiae?genome?DNA 0.5μl
ddH
2O 35.0μl
Cumulative volume 50.0 μ l
PCR reaction conditions: 94 ℃ of 5min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 1min circulate 30 times; 72 ℃ of 10min.
After PCR finishes, detect PCR product clip size by agarose gel electrophoresis, as a result (see figure 6) show through electricity once transform obtain can contain the mono-clonal bacterium colony of growing on the YPD substratum of 0.25g/L g418, can obtain the PCR product (SEQ ID No.14) of 582bp with primer His3-F and Kan-ID amplification, can obtain the PCR product (SEQ ID No.13) of 681bp with primer His3-F and His-ID amplification, proof has only a karyomit(e) that homologous recombination has taken place, homologous recombination does not take place in another karyomit(e), and recombinant bacterial strain is abbreviated as TSH-Sc-001 (His3
+/-).
The acquisition of embodiment 3 diplochromosomes exchange Wine brewing yeast strain
TSH-Sc-001 (the His3 that obtains with embodiment 2
+/-) bacterial strain prepares competent cell, method for transformation is with embodiment 2.Cell after the conversion is coated on the YPD plate culture medium that contains 1M sorbyl alcohol and 0.5g/L g418, is inverted for 30 ℃ and cultivates 3d.The cultural method of mono-clonal bacterium colony, genome DNA extracting method and PCR authentication method are with embodiment 2.
The agarose gel electrophoresis result of PCR product shows (see figure 6), the mono-clonal bacterium colony of growing on the YPD substratum of 0.5g/L g418 can contained through what twice electricity conversion obtained, primer His3-F and the Kan-ID PCR product (SEQ ID No.14) that can increase and obtain 582bp only, primer His3-F and His-ID fail to increase and obtain the PCR product, prove that homologous recombination has all taken place two karyomit(e)s, obtained yeast saccharomyces cerevisiae of the present invention (S.cerevisiae) TSH-Sc-001 (His3-/-) (CGMCC NO.2738).
With TSH-Sc-001, embodiment 2 gained TSH-Sc-001 (His3
+/-) and embodiment 3 gained TSH-Sc-001 (His3-/-) (substratum consists of: no amino acid yeast nitrogen YNB13.4g/L to be inoculated into YPD, YPD-0.25g418 (g418 that contains 0.25g/L), YPD-0.5g418 (g418 that contains 0.5g/L) and MD respectively, glucose 20g/L, vitamin H 4mg/L, agar 20g/L) on the plate culture medium, is inverted for 30 ℃ and cultivates 48h.
Result's 3 bacterial strains on the YPD plate culture medium all can be grown; TSH-Sc-001 can not grow on the YPD-0.25g418 plate culture medium, TSH-Sc-001 (His
+/-) and TSH-Sc-001 (His3
-/-) can grow.TSH-Sc-001 and TSH-Sc-001 (His3 on the YPD-0.5g418 plate culture medium
+/-) can not grow TSH-Sc-001 (His3
-/-) can grow.TSH-Sc-001 and TSH-Sc-001 (His3 on the MD plate culture medium
+/-) can grow, TSH-Sc-001 (His3-/-) can not grow.
The above results explanation TSH-Sc-001 (His3
+/-) have only a karyomit(e) that reorganization has taken place, therefore can only tolerate the g418 of lower concentration (0.25g/L), because a karyomit(e) of reorganization takes place complete His3 gene is arranged simultaneously, so can grow not containing on the MD substratum of Histidine; Reorganization has all taken place in two karyomit(e)s of SH-Sc-001 (His3-/-), therefore can tolerate the g418 of high density (0.5g/L), simultaneously, can not synthesize Histidine, so can not grow not containing on the MD substratum of Histidine because two chromosomal His3 genes are all destroyed.
Fermenting experiment proves TSH-Sc-001, TSH-Sc-001 (His simultaneously
+/-) and TSH-Sc-001 (His3-/-) difference do not had aspect the fermentation producing and ethanol ability.
The present invention for it has added Histidine auxotrophy mark and g418 resistance marker, obtains purpose bacterial strain S.cerevisiae Sc001 TSH-Sc-001 (His3-/-) (CGMCC NO.2738) under the prerequisite that does not change initial bacterial strain further feature.These marks that this bacterial strain contains both can be used as a feature of bacterial strain itself, simultaneously for this bacterial strain being carried out deep heredity and genetic engineering modified providing the foundation and the available selection markers.
Sequence table
＜110〉Tsing-Hua University
＜120〉a kind of amphiploid histidine auxotroph yeast saccharomyces cerevisiae and construction process thereof
<160>14
<170>PatentIn?version?3.5
<210>1
<211>59
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉amplimer of His3 homology left arm
<400>1
gattgcgatc?tctttaaagg?gtggtcccct?agcgatagag?atgagccata?ttcaacggg 59
<210>2
<211>79
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉amplimer of His3 homology left arm
<400>2
ggaattcatg?acagagcaga?aagccctagt?aaagcgtatt?acaaatgaaa?ccaagattca?60
gattgcgatc?tctttaaag 79
<210>3
<211>32
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉amplimer of g418 resistant gene
<400>3
acgcgtcgac?ttagaaaaac?tcatcgagca?tc 32
<210>4
<211>34
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉contain the primer of homologous recombination right arm (His3R)
<400>4
acgcgtcgac?gtaggagatc?tctcttgcga?gatg 34
<210>5
<211>33
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉contain the primer of homologous recombination right arm (His3R)
<400>5
aactgcagct?acataagaac?acctttggtg?gag 33
<210>6
<211>24
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉primers designed
<400>6
tgcacggtcc?tgttccctag?catg 24
<210>7
<211>25
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉primers designed
<400>7
agacgtttcc?cgttgaatat?ggctc 25
<210>8
<211>25
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉primers designed
<400>8
ggaatgcttg?gccagagcat?gtatc 25
<210>9
<211>866
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉His3L '-Kan dna sequence dna
<400>9
gattgcgatc?tctttaaagg?gtggtcccct?agcgatagag?atgagccata?ttcaacggga 60
aacgtcttgc?tcgaggccgc?gattaaattc?caacatggat?gctgatttat?atgggtataa?120
atgggctcgc?gataatgtcg?ggcaatcagg?tgcgacaatc?tatcgattgt?atgggaagcc?180
cgatgcgcca?gagttgtttc?tgaaacatgg?caaaggtagc?gttgccaatg?atgttacaga?240
tgagatggtc?agactaaact?ggctgacgga?atttatgcct?cttccgacca?tcaagcattt?300
tatccgtact?cctgatgatg?catggttact?caccactgcg?atccccggga?aaacagcatt?360
ccaggtatta?gaagaatatc?ctgattcagg?tgaaaatatt?gttgatgcgc?tggcagtgtt?420
cctgcgccgg?ttgcattcga?ttcctgtttg?taattgtcct?tttaacagcg?atcgcgtatt?480
tcgtctcgct?caggcgcaat?cacgaatgaa?taacggtttg?gttgatgcga?gtgattttga?540
tgacgagcgt?aatggctggc?ctgttgaaca?agtctggaaa?gaaatgcata?agcttttgcc?600
attctcaccg?gattcagtcg?tcactcatgg?tgatttctca?cttgataacc?ttatttttga?660
cgaggggaaa?ttaataggtt?gtattgatgt?tggacgagtc?ggaatcgcag?accgatacca?720
ggatcttgcc?atcctatgga?actgcctcgg?tgagttttct?ccttcattac?agaaacggct?780
ttttcaaaaa?tatggtattg?ataatcctga?tatgaataaa?ttgcagtttc?atttgatgct?840
cgatgagttt?ttctaagtcg?acgcgt 866
<210>10
<211>926
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉His3L-Kan dna sequence dna
<400>10
ggaattcatg?acagagcaga?aagccctagt?aaagcgtatt?acaaatgaaa?ccaagattca 60
gattgcgatc?tctttaaagg?gtggtcccct?agcgatagag?atgagccata?ttcaacggga 120
aacgtcttgc?tcgaggccgc?gattaaattc?caacatggat?gctgatttat?atgggtataa 180
atgggctcgc?gataatgtcg?ggcaatcagg?tgcgacaatc?tatcgattgt?atgggaagcc 240
cgatgcgcca?gagttgtttc?tgaaacatgg?caaaggtagc?gttgccaatg?atgttacaga 300
tgagatggtc?agactaaact?ggctgacgga?atttatgcct?cttccgacca?tcaagcattt 360
tatccgtact?cctgatgatg?catggttact?caccactgcg?atccccggga?aaacagcatt 420
ccaggtatta?gaagaatatc?ctgattcagg?tgaaaatatt?gttgatgcgc?tggcagtgtt 480
cctgcgccgg?ttgcattcga?ttcctgtttg?taattgtcct?tttaacagcg?atcgcgtatt 540
tcgtctcgct?caggcgcaat?cacgaatgaa?taacggtttg?gttgatgcga?gtgattttga 600
tgacgagcgt?aatggctggc?ctgttgaaca?agtctggaaa?gaaatgcata?agcttttgcc 660
attctcaccg?gattcagtcg?tcactcatgg?tgatttctca?cttgataacc?ttatttttga 720
cgaggggaaa?ttaataggtt?gtattgatgt?tggacgagtc?ggaatcgcag?accgatacca 780
ggatcttgcc?atcctatgga?actgcctcgg?tgagttttct?ccttcattac?agaaacggct 840
ttttcaaaaa?tatggtattg?ataatcctga?tatgaataaa?ttgcagtttc?atttgatgct 900
cgatgagttt?ttctaagtcg?acgcgt 926
<210>11
<211>228
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉His3R dna sequence dna
<400>11
acgcgtcgac?gtaggagatc?tctcttgcga?gatgatcccg?cattttcttg?aaagctttgc 60
agaggctagc?agaattaccc?tccacgttga?ttgtctgcga?ggcaagaatg?atcatcaccg 120
tagtgagagt?gcgttcaagg?ctcttgcggt?tgccataaga?gaagccacct?cgcccaatgg 180
taccaacgat?gttccctcca?ccaaaggtgt?tcttatgtag?ctgcagtt 228
<210>12
<211>1135
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉His3L-Kan-His3R dna sequence dna
<400>12
aattcatgac?agagcagaaa?gccctagtaa?agcgtattac?aaatgaaacc?aagattcaga 60
ttgcgatctc?tttaaagggt?ggtcccctag?cgatagagat?gagccatatt?caacgggaaa 120
cgtcttgctc?gaggccgcga?ttaaattcca?acatggatgc?tgatttatat?gggtataaat 180
gggctcgcga?taatgtcggg?caatcaggtg?cgacaatcta?tcgattgtat?gggaagcccg 240
atgcgccaga?gttgtttctg?aaacatggca?aaggtagcgt?tgccaatgat?gttacagatg 300
agatggtcag?actaaactgg?ctgacggaat?ttatgcctct?tccgaccatc?aagcatttta 360
tccgtactcc?tgatgatgca?tggttactca?ccactgcgat?ccccgggaaa?acagcattcc 420
aggtattaga?agaatatcct?gattcaggtg?aaaatattgt?tgatgcgctg?gcagtgttcc 480
tgcgccggtt?gcattcgatt?cctgtttgta?attgtccttt?taacagcgat?cgcgtatttc 540
gtctcgctca?ggcgcaatca?cgaatgaata?acggtttggt?tgatgcgagt?gattttgatg 600
acgagcgtaa?tggctggcct?gttgaacaag?tctggaaaga?aatgcataag?cttttgccat 660
tctcaccgga?ttcagtcgtc?actcatggtg?atttctcact?tgataacctt?atttttgacg 720
aggggaaatt?aataggttgt?attgatgttg?gacgagtcgg?aatcgcagac?cgataccagg 780
atcttgccat?cctatggaac?tgcctcggtg?agttttctcc?ttcattacag?aaacggcttt 840
ttcaaaaata?tggtattgat?aatcctgata?tgaataaatt?gcagtttcat?ttgatgctcg 900
atgagttttt?ctaagtcgac?gtaggagatc?tctcttgcga?gatgatcccg?cattttcttg 960
aaagctttgc?agaggctagc?agaattaccc?tccacgttga?ttgtctgcga?ggcaagaatg?1020
atcatcaccg?tagtgagagt?gcgttcaagg?ctcttgcggt?tgccataaga?gaagccacct?1080
cgcccaatgg?taccaacgat?gttccctcca?ccaaaggtgt?tcttatgtag?ctgca 1135
<210>13
<211>681
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉the purpose product D NA sequence of primer His3-F and His-ID amplification
<400>13
tgcacggtcc?tgttccctag?catgtacgtg?agcgtatttc?cttttaaacc?acgacgcttt 60
gtcttcattc?aacgtttccc?attgtttttt?tctactattg?ctttgctgtg?ggaaaaactt?120
atcgaaagat?gacgactttt?tcttaattct?cgttttaaga?gcttggtgag?cgctaggagt?180
cactgccagg?tatcgtttga?acacggcatt?agtcagggaa?gtcataacac?agtcctttcc?240
cgcaattttc?tttttctatt?actcttggcc?tcctctagta?cactctatat?ttttttatgc?300
ctcggtaatg?attttcattt?ttttttttcc?acctagcgga?tgactctttt?tttttcttag?360
cgattggcat?tatcacataa?tgaattatac?attatataaa?gtaatgtgat?ttcttcgaag?420
aatatactaa?aaaatgagca?ggcaagataa?acgaaggcaa?agatgacaga?gcagaaagcc?480
ctagtaaagc?gtattacaaa?tgaaaccaag?attcagattg?cgatctcttt?aaagggtggt?540
cccctagcga?tagagcactc?gatcttccca?gaaaaagagg?cagaagcagt?agcagaacag?600
gccacacaat?cgcaagtgat?taacgtccac?acaggtatag?ggtttctgga?ccatatgata?660
catgctctgg?ccaagcattc?c 681
<210>14
<211>582
<212>DNA
<213>Artificial?Sequence
<220>
＜223〉the purpose product of primer His3-F and Kan-ID amplification
<400>14
tgcacggtcc?tgttccctag?catgtacgtg?agcgtatttc?cttttaaacc?acgacgcttt 60
gtcttcattc?aacgtttccc?attgtttttt?tctactattg?ctttgctgtg?ggaaaaactt?120
atcgaaagat?gacgactttt?tcttaattct?cgttttaaga?gcttggtgag?cgctaggagt?180
cactgccagg?tatcgtttga?acacggcatt?agtcagggaa?gtcataacac?agtcctttcc?240
cgcaattttc?tttttctatt?actcttggcc?tcctctagta?cactctatat?ttttttatgc?300
ctcggtaatg?attttcattt?ttttttttcc?acctagcgga?tgactctttt?tttttcttag?360
cgattggcat?tatcacataa?tgaattatac?attatataaa?gtaatgtgat?ttcttcgaag?420
aatatactaa?aaaatgagca?ggcaagataa?acgaaggcaa?agatgacaga?gcagaaagcc?480
ctagtaaagc?gtattacaaa?tgaaaccaag?attcagattg?cgatctcttt?aaagggtggt?540
cccctagcga?tagagatgag?ccatattcaa?cgggaaacgt?ct 582
Claims (10)
1, a kind of amphiploid histidine auxotroph yeast saccharomyces cerevisiae is characterized in that described yeast saccharomyces cerevisiae is the amphiploid Wine brewing yeast strain that contains Histidine auxotrophy mark and g418 resistance marker.
2, according to the described yeast saccharomyces cerevisiae of claim 1, it is characterized in that described Wine brewing yeast strain is meant Wine brewing yeast strain (S.cerevisiae) TSH-Sc-001 (His3-/-), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 13rd, 2008, and deposit number is: CGMCC NO.2738.
3, the construction process of the described amphiploid histidine auxotroph of claim 1 yeast saccharomyces cerevisiae, it is characterized in that with the amphiploid Wine brewing yeast strain be starting strain, carry out twice conversion with the fragment that has goal gene, by homologous recombination takes place twice, obtain the amphiploid histidine auxotroph Wine brewing yeast strain that homologous recombination has all taken place 2 karyomit(e)s.
4, according to the described construction process of claim 3, it is characterized in that two ends that its described target gene fragment is meant goal gene respectively with the dna sequence dna homology of His3 left arm or His3 right arm, g418 resistant gene and/or other genes are contained in the centre of target gene fragment.
5, the construction process of claim 1 or 3 described amphiploid histidine auxotroph yeast saccharomyces cerevisiaes comprises the steps:
(1) contains the primer of homologous recombination left arm and g418 resistant gene according to the sequences Design amplification of the sequence of S.cerevisiae His3 gene and pPIC9K plasmid, introduce EcoR I (GAATTC) and Sal I (GTCGAC) recognition sequence respectively at 5 ' end and 3 ' end, with the pPIC9K plasmid DNA is that template is carried out pcr amplification, the fragment His3L-Kan that must contain homologous recombination left arm and g418 resistance, purifying;
(2) with EcoR I and Sal I difference double digestion His3L-Kan fragment and pUC19 carrier, purifying enzyme is cut product, connect with the T4 ligase enzyme then, connect product with gained and transform DH5 α competent cell, the picking positive colony, extract recombinant plasmid dna, enzyme is cut evaluation, must contain the segmental recombinant plasmid pUC19-His3L-Kan of His3 homologous recombination left arm and g418 resistant gene;
(3) amplification contains the primer of homologous recombination right arm (His3R) according to the sequences Design of S.cerevisiae His3 gene, introduce Sal I and Pst I recognition site respectively at 5 ' end and 3 ' end, with the S.cerevisiae genomic dna is that template is carried out pcr amplification, get His3 homologous recombination right arm fragment His3R, purifying;
(4) with Sal I and Pst I difference double digestion His3R and plasmid pUC19-His3L-Kan, purifying enzyme is cut product, connect with the T4 ligase enzyme then, connect product with gained and transform DH5 α competent cell, the picking positive colony, extract recombinant plasmid dna, enzyme is cut evaluation, must contain the recombinant plasmid pUC19-His3L-Kan-His3R of homologous recombination left arm, g418 resistant gene and homologous recombination right arm;
(5), reclaim the fragment His3L-Kan-His3R that contains homologous recombination left arm, g418 resistant gene and homologous recombination right arm of purifying 1135bp with EcoR I and Pst I double digestion recombinant plasmid pUC19-His3L-Kan-His3R;
(6) by electroporation method the His3L-Kan-His3R fragment is transformed amphiploid yeast saccharomyces cerevisiae competent cell, be coated with the YPD substratum that contains 0.25g/L g418, the picking positive colony, extracting the laggard performing PCR of genomic dna identifies, containing the positive monoclonal bacterial strain of growing on the YPD substratum of 0.25g/L g418 through identifying, get a karyomit(e) homologous recombination has taken place, homologous recombination does not take place in a karyomit(e), is labeled as (His3
+/-);
(7) by electroporation method the His3L-Kan-His3R fragment is transformed amphiploid yeast saccharomyces cerevisiae (His3
+/-) competent cell, be coated with the YPD substratum that contains 0.5g/L g418, the picking positive colony extracts the laggard performing PCR of genomic dna and identifies, promptly gets the present invention and is containing the Wine brewing yeast strain (His3 that homologous recombination has all taken place two karyomit(e)s of growing on the YPD substratum of 0.5g/L g418
-/-).
6, the construction process of claim 1 or 2 described amphiploid histidine auxotroph yeast saccharomyces cerevisiaes is characterized in that comprising the steps:
(1) being template with the pPIC9K plasmid DNA, is that primer carries out pcr amplification with His3L-1F-Kan and Kan-R, increase dna sequence dna the His3L '-Kan shown in SEQ ID No.9; Described primer His3L-1F-Kan is the dna sequence dna shown in the SEQ ID No.1, and described primer Kan-R is the dna sequence dna shown in the SEQ ID No.3;
(2) being template with PCR product the His3L '-Kan behind the purifying, is that primer carries out pcr amplification with His3L-2F and Kan-R, increase the dna sequence dna His3L-Kan shown in the SEQ ID No.10; Purifying; Described primer His3L-2F is the dna sequence dna shown in the SEQ ID No.2;
(3) with EcoR I and Sal I difference double digestion His3L-Kan fragment and pUC19 carrier, purifying enzyme is cut product, connect with the T4 ligase enzyme then, connect product with gained and transform DH5 α competent cell, the picking positive colony, extract recombinant plasmid dna, enzyme is cut evaluation, must contain the recombinant plasmid pUC19-His3L-Kan of homologous recombination left arm and g418 resistant gene;
(4) being template with the S.cerevisiae genomic dna, is that primer carries out pcr amplification with His3R-F and His3R-R, contain His3 homologous recombination right arm dna fragmentation His3R, purifying shown in the SEQ ID No.11; Wherein said primer His3R-F is the dna sequence dna shown in the SEQ ID No.4, and described primer His3R-R is the dna sequence dna shown in the SEQ ID No.5;
(5) with Sal I and Pst I difference double digestion His3R fragment and plasmid pUC19-His3L-Kan, purifying, connect with the T4 ligase enzyme then, connect product with gained and transform DH5 α competent cell, the picking positive colony, extract recombinant plasmid dna, enzyme is cut evaluation, must contain the recombinant plasmid pUC19-His3L-Kan-His3R of homologous recombination left arm, g418 resistant gene and homologous recombination right arm;
(6), reclaim purifying, dna fragmentation His3L-Kan-His3R that must be shown in SEQ ID No.12 with EcoR I and Pst I double digestion recombinant plasmid pUC19-His3L-Kan-His3R;
(7) by electroporation method the His3L-Kan-His3R fragment is transformed amphiploid yeast saccharomyces cerevisiae (S.cerevisiae) TSH-Sc-001 competent cell, be coated with the YPD substratum that contains 0.25g/L g418, the picking positive colony, extracting the laggard performing PCR of genomic dna identifies, obtain the positive monoclonal bacterial strain of growing on the YPD substratum of 0.25g/L g418 containing, article one, homologous recombination has taken place in karyomit(e), and the Wine brewing yeast strain TSH-Sc-001 (His3 of homologous recombination does not take place a karyomit(e)
+/-); Wherein PCR identifies that used primer His3L is the dna sequence dna shown in the SEQ ID No.6, and described primer Kan-ID is the dna sequence dna shown in the SEQ ID No.7, and described primer His-ID is the dna sequence dna shown in the SEQ ID No.8;
(8) by electroporation method the His3L-Kan-His3R fragment is transformed TSH-Sc-001 (His3
+/-) competent cell, be coated with the YPD substratum that contains 0.5g/L g418, the picking positive colony, extract genomic dna, carrying out PCR identifies, obtain homologous recombination Wine brewing yeast strain (S.cerevisiae) TSH-Sc-001 (His3-/-) all having taken place containing two karyomit(e)s of positive monoclonal bacterial strain of growing on the YPD substratum of 0.5g/L g418, wherein PCR identifies the used same step of primer (7).
7, according to the described construction process of claim 6, it is characterized in that the primer His3L-1F-Kan described in its step (1) is the dna sequence dna shown in the SEQ ID No.1, described primer Kan-R is the dna sequence dna shown in the SEQ IDNo.3.
8,, it is characterized in that the primer His3L-2F described in its step (2) is the dna sequence dna shown in the SEQ ID No.2 according to claim 6 or 7 described construction processs.
9, according to the described construction process of claim 8, it is characterized in that the primer His3R-F described in its step (4) is the dna sequence dna shown in the SEQ ID No.4, described primer His3R-R is the dna sequence dna shown in the SEQ ID No.5.
10, according to the described construction process of claim 9, it is characterized in that PCR identifies that used primer His3L is the dna sequence dna shown in SEQ ID No.6 in its step (8), described primer Kan-ID is the dna sequence dna shown in the SEQID No.7, and described primer His-ID is the dna sequence dna shown in the SEQ ID No.8.
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| CN102399826A (en) * | 2011-10-20 | 2012-04-04 | 清华大学 | Comprehensive utilizing method of sweet sorghum stalks |
| 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 |
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| CN101033476A (en) * | 2007-01-08 | 2007-09-12 | 清华大学 | Method and system for preparing ethanol based on sweet broomcorn straw solid fermentation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102399826A (en) * | 2011-10-20 | 2012-04-04 | 清华大学 | Comprehensive utilizing method of sweet sorghum stalks |
| 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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