CN106834335B - Method for realizing high-accuracy gene homologous cloning in saccharomycetes - Google Patents
Method for realizing high-accuracy gene homologous cloning in saccharomycetes Download PDFInfo
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Abstract
The invention provides a method for realizing high-accuracy gene homologous cloning in yeast, which comprises the steps of obtaining upstream and downstream sequences of a target cloned gene by adopting a polymerase chain reaction or whole gene synthesis method, connecting the upstream and downstream sequences, inserting the upstream and downstream sequences into a vector plasmid to construct a circular cloning vector, carrying out linearization treatment on the circular cloning vector to obtain linear double-stranded DNA, carrying out 5 ' → 3 ' exonuclease treatment on the linear double-stranded DNA to form linear 3 ' protruding long-viscous-end double-stranded DNA, and then transforming yeast cells. The invention realizes high-accuracy gene homologous cloning in yeast by means of special exogenous DNA structure types. The clone accuracy of the homologous gene can reach 50-95%, the clone workload of the yeast homologous gene can be greatly reduced, and the method has great application potential in aspects of yeast functional gene research, gene engineering strain construction and the like.
Description
Technical Field
The invention belongs to the field of genetic engineering, and mainly relates to a method for realizing high-accuracy gene homologous cloning in yeast.
Background
The yeast is a general name of a single-cell eukaryotic microorganism in the kingdom fungi, and has more than 1500 identified species which account for about 1 percent of the identified fungal species at present. The diameter of the yeast cell is about 2-6 mu m, the length is 5-30 mu m, and the yeast cell is longer in some cases, and the individual forms of the yeast cell are spherical, oval, elliptical, columnar, sausage-shaped and the like, and are widely distributed in the natural world. Most yeasts can be isolated in sugar-rich environments, such as some fruit surfaces, fermented grains, plant secretions, and even insect bodies. Most yeasts prefer to grow in a more acidic, moist, sugar-containing environment, survive both in aerobic and anaerobic environments, and are facultative anaerobes.
Yeasts are the earliest microorganisms applied in the human civilization history, and yeasts represented by saccharomyces cerevisiae are widely applied in the industries of food, medicine, feed and the like. Meanwhile, since yeast has many structures identical to those of animal and plant cells which are eukaryotes and is easily cultured, yeast is used as a model organism for studying eukaryotes, and is one of the most well-known organisms. Since the completion of the genome sequencing work of saccharomyces cerevisiae in 1996, saccharomyces cerevisiae has been widely adopted as a model organism, and plays a great role in genetics, functional genomics, proteomics, cell cycle, signal transduction, protein processing, genetic disease research and the like.
Homologous gene cloning is a gene cloning means commonly adopted in the research of yeast functional genes and is realized based on the principle of gene homologous recombination. Sequence fidelity is higher for homologous gene cloning compared to Polymerase Chain Reaction (PCR) gene cloning; compared with the cloning by a complete gene sequence synthesis method, the cost is lower. In addition, high throughput operation is possible, so that homologous gene cloning is still widely adopted in the research of yeast functional genes. At present, when yeast homologous gene cloning is carried out, linear double-stranded DNA is generally adopted as exogenous DNA, and two ends of the exogenous DNA can be blunt ends, short 5 'protruding sticky ends (usually 4bp protruding sticky ends) or short 3' protruding sticky ends (usually 4bp protruding sticky ends). Since usually the efficiency of non-homologous recombination in cells is much higher than that of homologous recombination, it is not easy to achieve a high accuracy of homologous gene cloning in yeast. When the homologous gene cloning is carried out by adopting common linear double-stranded DNA, the cloning accuracy of the homologous gene is usually lower than 50 percent. Taking the saccharomyces cerevisiae BY4743 strain as an example, when the exogenous DNA is in a blunt end structure and the length of the homologous sequence is 500bp, the cloning accuracy of the homologous gene is about 25 percent; when the non-homologous recombination inhibiting mutant BY4743 delta yku70 is used, the cloning accuracy is improved to about 37.5%. Therefore, when a large number of yeast genes are cloned, the workload of screening for correct clones is greatly increased along with the cloning flux, and the manpower and material resources are greatly consumed. If a new high-efficiency method can be established, the gene homologous cloning accuracy is improved to more than 80%, then 2-3 transformants are randomly selected for each cloning test to effectively obtain the target gene clone, and the screening workload is greatly reduced. The invention discloses a novel DNA structure and a high-accuracy yeast homologous gene cloning method mediated by the novel DNA structure, aiming at the problem of high-accuracy homologous gene cloning. The method can effectively improve the accuracy of yeast homologous gene cloning, and provides an efficient technical means for yeast genetics research, functional gene research and genetic engineering strain cultivation. The invention designs a key innovation point, namely a ' linear 3 ' protruding long sticky end double-stranded DNA ' structure and application thereof which are not reported
Disclosure of Invention
The invention aims to provide a technical method for realizing high-accuracy homologous gene cloning in yeast. Yeast homologous gene cloning is based on the gene homologous recombination principle, and the gene cloning is realized by taking an extracuclear independently-replicated DNA plasmid as a vector. In the homologous gene cloning process, invasion of the target DNA segment by the 3' single-stranded end of the exogenous DNA is the molecular basis for realizing homologous gene cloning. When the linear double-stranded DNA with a flat end or a short sticky end is used as the exogenous DNA, the DNA enters cells and needs to be treated by intracellular nuclease to form a long single-stranded end with a 3' end, and then single-stranded invasion occurs, so that gene homologous recombination is realized. Since the efficiency of forming a 3' -end long single-stranded end in a cell by exogenous DNA is low, the efficiency of homologous gene recombination is low, and the cloning accuracy of homologous genes is low. The invention prepares exogenous DNA carrying 3 ' end long single-chain end in vitro, namely ' linear 3 ' protruding long sticky end double-chain DNA ', solves the problem of low efficiency of forming the carrying 3 ' end long single-chain end in cells, thereby realizing high-accuracy gene homologous recombination, which is particularly expressed as high-accuracy homologous gene cloning in yeast.
The invention achieves the purpose through the following technical scheme:
the method for realizing high-accuracy gene homologous cloning in yeast is characterized in that after the upstream and downstream sequences of a target cloned gene are obtained by adopting a polymerase chain reaction or whole gene synthesis method, the upstream and downstream sequences are connected in the original gene direction and inserted into a vector plasmid to construct a circular cloning vector, the circular cloning vector is subjected to linearization treatment to obtain linear double-stranded DNA, and the linear double-stranded DNA is treated by 5 ' → 3 ' exonuclease to form linear 3 ' protruding long sticky end double-stranded DNA and then is transformed into yeast cells.
Furthermore, both ends of the linear 3' protruding long sticky end double-stranded DNA respectively carry 1 homologous sequence which is the same as the upstream or downstream sequence of the target clone gene, and the insertion directions are both forward insertion.
The length of the homologous sequence is 100-1000bp, and the length of the 3' -end single-stranded sticky end after nuclease treatment is 100-1000 nt.
The preparation method of the linear 3' protruding long sticky end double-stranded DNA comprises the following steps: the linear double-stranded DNA with the upstream or downstream homologous sequence of the target gene with the length of 100-1000bp at both ends is formed after 5 '→ 3' exonuclease digestion treatment, the enzyme dosage is 50-5000U/nmol of the linear double-stranded DNA carrier, the treatment temperature is 25-60 ℃, and the treatment time is 1min-1 h. The 5 '→ 3' exonuclease is T5 exonuclease, T7 exonuclease or lambda exonuclease, but may be any nuclease having 5 '→ 3' exonuclease activity.
The extension directions of two 3 ' protruding sticky ends of the linear 3 ' protruding long sticky end double-stranded DNA point to the coding region of a target gene, namely the two 3 ' protruding sticky ends point to a target clone gene segment in a mutual opposite way.
The method for transforming the yeast cell by the linear 3' protruding long-sticky-end double-stranded DNA is an electric shock mediated method, a polyethylene glycol mediated method, a lithium chloride mediated method, a lithium acetate mediated method or a protoplast mediated method, and can also be any other method capable of mediating the exogenous DNA to enter the yeast cell.
According to the invention, the linear 3' protruding long sticky end double-stranded DNA enters yeast cells and then mediates homologous gene cloning with high accuracy, and finally the homologous gene cloning accuracy (namely the percentage of correct cloning in yeast transformants) is realized, and the accuracy reaches 50-95%.
The invention relates to a high-accuracy yeast homologous gene cloning method taking linear 3' protruding long sticky end double-stranded DNA as a core innovation point, which specifically comprises the following steps:
firstly, cloning upstream and downstream sequences of a target clone gene: obtained by adopting a polymerase chain reaction or a whole gene synthesis method. The cloning sequence is an upstream sequence and a downstream sequence of a target cloning gene, the upstream sequence is called as a sequence A, the downstream sequence is called as a sequence B for convenience of presentation, and the length of the upstream sequence and the length of the downstream sequence are both 100-1000 bp.
Secondly, constructing a circular cloning vector: the sequence A and the sequence B are connected according to the original gene direction (namely, inserted in a forward direction) and are inserted into a vector plasmid to obtain a circular cloning vector, and meanwhile, a single enzyme cutting site is designed between the sequence A and the sequence B, so that the subsequent linearization treatment is facilitated.
Thirdly, linearization of the circular cloning vector: and (3) carrying out linearization treatment on the circular cloning vector by using single enzyme digestion restriction enzyme between the sequences A and B, carrying out enzyme digestion overnight according to the instruction, ensuring thorough enzyme digestion, and recovering the enzyme digestion product by using a recovery kit. The target fragment can be recovered after gel electrophoresis, and the linearized plasmid can also be recovered by an ethanol or isopropanol precipitation method and a kit recovery method.
Fourthly, exonuclease treatment is carried out to form linear 3' protruding long sticky end double-stranded DNA: and (3) quantitatively measuring the enzyme digestion product recovered in the third step, diluting the enzyme digestion product into an enzyme digestion reaction buffer solution according to the concentration of 100 ng/. mu.L, and then adding 5 '→ 3' exonuclease into an ice bath, wherein the using amount of the enzyme is 50-5000U/nmol of linear double-stranded DNA carrier, the processing temperature is 25-60 ℃, and the processing time is 1min-1 h. The processed product is the linear 3' protruding long sticky end double-stranded DNA. The 5 '→ 3' exonuclease may be any one of T5 exonuclease, T7 exonuclease and lambda exonuclease, but is not limited to these, and other nucleases having 5 '→ 3' exonuclease activity are suitable.
Step five, "linear 3' protruding long sticky end double-stranded DNA" transforms yeast cells: according to the physiological characteristics of different yeasts, a proper transformation method is selected, and the linear 3' protruding long-sticky-end double-stranded DNA is transferred into the yeast cells. The yeast transformation method may be any one of colony transformation, polyethylene glycol mediated transformation, protoplast transformation, lithium chloride mediated transformation, lithium acetate mediated transformation, electric shock transformation, and the like, but is not limited to these methods, and other methods that can introduce foreign DNA into yeast are suitable.
Sixthly, screening and identifying transformants: and (3) selecting a pseudotransformant, carrying out amplification culture, extracting plasmids and converting escherichia coli, and finally selecting an escherichia coli transformant for PCR identification, restriction enzyme digestion identification and DNA sequencing identification to obtain a correct homologous gene clone transformant. The homologous cloning accuracy (i.e., the percentage of correct clones in yeast transformants) can reach 50-95%.
Advantageous effects
The technical method is simple and convenient, is easy to operate, is easy to purchase matched reagents, can realize high-accuracy homologous gene cloning in the saccharomycetes, and has obvious significance in the aspects of improving the working efficiency of yeast homologous gene cloning, reducing labor and material resource expenditure and the like.
Drawings
FIG. 1 is a schematic diagram of the structure of exogenous DNA ("linear 3' overhanging long-sticky-end double-stranded DNA").
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples. The present invention is applicable to known common yeasts such as but not limited to Saccharomyces cerevisiae (Saccharomyces cerevisiae), Pichia pastoris (Pichia pastoris), Candida spp. For convenience of description of the embodiments, the present invention will be described in detail with reference to the Saccharomyces cerevisiae gim 2.198.198 strain as an example. Saccharomyces cerevisiae gim 2.198.198, classification name: saccharomyces cerevisiae, latin scientific name: saccharomyces cerevisiae, accession number: gim 2.198.198, knock-out strain, positive for G418 resistance, uracil auxotroph, deposited in units of: guangdong province microbial strain preservation center. The homologous cloning vector adopts a saccharomyces cerevisiae low-copy plasmid YCplac33 (the full length is 5603bp, the vector carries ura3 gene and can recover uracil auxotrophy), and is presented by the Botryowa university Huang Shi. The target cloning gene is an essential gene tor2 of saccharomyces cerevisiae, two homologous fragments (sequence A and sequence B) are respectively a region near an upstream promoter and a downstream terminator region of a tor2 gene, and an insertion site on YCplac33 is a HindIII enzyme cutting site. The restriction site of the junction between the sequence A and the sequence B is designed as Not I restriction site. The sequence A and the sequence B are obtained by PCR amplification cloning, and the sequence A, the sequence B and YCplac33 adopt a Norrespect multisegment seamless cloning kit on the connection of HindIII enzyme cutting site and NotI enzyme cutting site "
Multi S One Step Cloning Kit ". The new vector carrying the sequence A and the sequence B is named YCplac33-AB, and the linear 3' protruding long sticky end double-stranded DNA formed by the NotI enzyme digestion linearization of YCplac33-AB and the T5 exonuclease treatment is named YCplac 33-AB-T5. The saccharomyces cerevisiae transformation method is a polyethylene glycol (PEG) mediated transformation method. With SC-ura-The solid plate is the screening medium. Transformant identification was performed by PCR amplification, restriction digestion and DNA sequencing.
Specific examples are set forth below:
example 1
Step 1: the upstream (sequence A1) and the downstream (sequence B1) of the tor2 gene are cloned by 1000bp homologous sequences respectively.
Cloning by adopting a Polymerase Chain Reaction (PCR) method, wherein the used DNA Polymerase is high fidelity DNA Polymerase (Phusion DNA Polymerase) produced by NEB, an amplification template is Saccharomyces cerevisiae gim 2.198.198 genome DNA, primers are oligonucleotides which are designed and synthesized according to a Saccharomyces cerevisiae S228C sequence, PPF1, PPR1, PTF1 and PTR1, and the sequence information is as follows:
PPF1:5’-GACCATGATTACGCCAAGCTTGC AAGAAAGCGGAGGGAGAAGA-3’
PPR1:5’-GAAGATGCGGCCGCTGTTAC TTTTCATATGGGGAAAGTAA-3’
PTF1:5’-GTAACAGCGGCCGCATCTTC TTTTAATGTATTGAAAATCA-3’
PTR1:5’-GACCTGCAGGCATGCAAGCTTGG CTCTTCCGCAATTCCAGCAG-3’
the primers PPF1 and PPR1 are used for cloning a 1kb region at the upstream of the tor2, PTF1 and PTR1 are used for cloning a 1kb region at the downstream of the tor2, and the PCR reaction system is as follows:
5 XHF reaction buffer (Mg)2+Concentration 7.5mM) 10. mu.L of ddH2O31.5. mu.L, dNTP mix (10 mM each) 1.0. mu.L, forward primer (10. mu.M) 2.5. mu.L, reverse primer (10. mu.M) 2.5. mu.L, template DNA (500 ng/. mu.L) 2.0. mu.L, PhusionDNA polymerase (DNA polymerase, 2U/. mu.L) 0.5. mu.L.
The PCR amplification conditions were: (1) pre-denaturation 98 ℃ for 1min, (2) denaturation 98 ℃ for 10sec, (3) annealing at 55 ℃ for 20sec, (4) extension at 72 ℃ for 45sec, (5) final extension at 72 ℃ for 7min, and (6) reaction completion at 4 ℃ for holding.
The above step (2)) Circulating for 35 times to (4), obtaining PCR products respectively containing the upstream (sequence A) and the downstream (sequence B) of the tor2 gene after the reaction is finished, and mixing the PCR products
AxyPrepTMThe PCR clean Kit was purified to obtain the target fragment, adjusted to a concentration of 20 ng/. mu.L, and stored at-20 ℃ until use.
Step 2: construction of the circular cloning vector YCplac33-AB-1 carrying the sequence A1 and the sequence B1.
Plasmid YCplac33 was digested overnight with the restriction enzyme Hind III and
AxyPrepTMand purifying the enzyme-digested product YCplac33 by using a PCRCleanup Kit, adjusting the concentration to 50 ng/. mu.L, and freezing and storing at-20 ℃ for later use. Using Novozan multi-fragment seamless cloning kit "
The ligation of the sequences A and B to the cleavage product of YCplac33 was performed by the MultiS One Step Cloning Kit "and the orientation of the sequences A and B was identical to the orientation of the original gene genome (i.e., the forward insertion). The reaction system is as follows:
5 Xreaction buffer 4.0. mu.L, ddH2O 11.0.0. mu.L, YCplac33 enzyme cleavage product (about 50 ng/. mu.L) 1.0. mu.L, sequence A1 (about 20 ng/. mu.L) 1.0. mu.L, sequence B1 (about 20 ng/. mu.L) 1.0. mu.L, recombinase
MultiS 2.0μL。
Mixing the above components uniformly, reacting at 37 ℃ for 30min, cooling in ice water bath for 5min immediately after the reaction is finished, taking 10 microliter of the connecting product, uniformly mixing with 100 microliter of Escherichia coli DH-5 α chemically-competent cells, carrying out ice bath treatment for 30min, carrying out heat shock culture in 42 ℃ water bath for 90sec, then adding 900 microliter of liquid LB culture medium (yeast extract 5g/L, tryptone 10g/L and sodium chloride 10g/L), carrying out shake culture at 37 ℃ for 45min (180 r/min), taking 200 microliter of liquid LB culture medium (ampicillin concentration is 75 microgram/mL), carrying out inversion culture at 37 ℃ for overnight, finally picking out single clone to carry out verification to obtain the correct clone YCplac33-AB-1, and freezing and storing at-80 ℃.
And step 3: preparation of "Linear 3' overhanging Long-sticky-end double-stranded DNA" -YCplac 33-AB-T5-1.
A plasmid for the correct clone YCplac33-AB-1 was extracted and digested with the restriction enzyme NotI at 1U/. mu.g enzyme at 37 ℃ overnight. And (3) recovering the enzyme digestion product by using a PCR clean recovery kit same as the step 1, and treating the enzyme digestion product by using T5 exonuclease to obtain linear 3 'protruding long-sticky end double-stranded DNA' -YCplac 33-AB-T5-1: the enzyme reaction buffer solution is matched with 10 times buffer solution, the enzyme dosage is 1.15U/mu g YCplac33-AB-1 enzyme digestion product (equivalent to 5000U/nmol YCplac33-AB-1 enzyme digestion product), the treatment temperature is 60 ℃, the treatment time is 24 hours, and the length of the obtained 3' end single-chain sticky end is 100 bp. The treated product is numbered YCplac33-AB-T5-1 and can be directly used for subsequent yeast transformation.
And 4, step 4: and (3) transforming the saccharomyces cerevisiae.
(1) Yeast competence preparation: an inoculating loop is used for dipping gim 2.198.198 strains frozen at minus 80 ℃, the strains are coated on a YPDA culture medium and cultured for 48h at 28 ℃, and then 1 single clone is picked up and cultured in 5.0mL of liquid YPDA culture medium at 30 ℃ for 180 r/min overnight with shaking. Then 0.5mL of overnight-cultured strain was aspirated and inoculated into 25.0mL of fresh YPDA liquid medium, and shaking was continued at 30 ℃ for 4 hours at 180 rpm. Then, the cells were collected by centrifugation at 2000 Xg, and resuspended in 25.0mL of sterile ultrapure water pre-cooled in an ice bath (resistance: 18.2 M.OMEGA.) by gentle shaking. And repeating the centrifugation at 2000 Xg to collect thalli, resuspending the cells for 1 time by 25.0mL of sterile lithium acetate solution with the concentration of 0.1M, subpackaging the cell suspension into 24 sterile centrifuge tubes, centrifugally collecting the thalli at 2000 Xg and placing the thalli on ice for later use, thus obtaining the saccharomyces cerevisiae competent cells.
(2) Transformation of yeast competent cells with exogenous DNA:
adding polyethylene glycol (PEG3350) solution with mass percent concentration of 50% into the saccharomyces cerevisiae competent cells in sequence, and adding 240 mu L of the solution; 1mol/L lithium acetate solution, 36 mu L; salmon sperm DNA (2mg/mL, heated at 100 ℃ for 10 m)in, then used after cooling in ice bath for 2 min), 20 μ L; exogenous DNA YCplac33-AB-T5-1, 14. mu.L; ddH2O, 50.0. mu.L. Mixing, incubating at 30 deg.C for 30min, adding 30 μ L of analytically pure dimethyl sulfoxide, mixing, incubating at 42 deg.C for 13min, centrifuging at 2000 Xg for 30sec, collecting cells, and spreading on selective culture medium SC-ura-Above (no uracil SC medium), and inverted culture at 30 ℃ for 2d to obtain regenerated clones.
Screening Medium SC-ura-The preparation method of (1L): 6.7g of non-amino nitrogen source (containing ammonium sulfate), 0.02g of adenine sulfate, 0.15g of valine, 0.03g of isoleucine, 0.02g of arginine, 0.02g of histidine, 0.1g of leucine, 0.03g of lysine, 0.02g of methionine, 0.05g of phenylalanine, 0.02g of threonine, 0.02g of tryptophan, 0.03g of tyrosine, 0.04g of aspartic acid, 0.04g of serine, 0.01g of glutamic acid, 20.0g of glucose and Fe (NH)4)2(SO4)23 mug of agar and 20.0g of agar. Sterilizing at 121 deg.C for 30min, adding glucose, serine, glutamic acid and Fe (NH)4)2(SO4)2Each separately filter sterilized.
And 5: and (4) identifying a transformant.
(1) Colony PCR identification: randomly selecting 100 colonies for PCR identification, wherein polymerase is EX Taq, primers are M13+ and M13-universal primers, and the sequences are as follows:
m13+: 5'-AGGGTTTTCCCAGTCACG-3' and M13-: 5'-GAGCGGATAACAATTTCACAC-3'.
The PCR reaction system is as follows:
10 XEX Taq reaction buffer (Mg)2+Concentration 7.5mM) 5. mu.L, ddH2O26.5. mu.L, dNTP mix (10 mM each) 1.0. mu.L, M13+ primer (10. mu.M) 2.5. mu.L, M13-primer (10. mu.M) 2.5. mu.L, yeast colony suspension 2.0. mu.L, EX Taq (DNA polymerase, 5U/. mu.L) 0.5. mu.L.
The PCR amplification conditions were: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30sec, annealing at 50 ℃ for 30sec, elongation at 72 ℃ for 7min, and final elongation at 72 ℃ for 10min, and after the reaction is finished, maintaining at 4 ℃. And (3) performing denaturation-annealing-extension circulation for 25 times, and performing PCR product detection by agarose gel electrophoresis at the end of the reaction.
(2) And (3) positive transformant identification: the positive clones identified by PCR in (1) were inoculated into 5mL of liquid SC-ura-In the medium, shake culture was performed at 28 ℃ and 180 rpm overnight. And extracting plasmid DNA by using a yeast plasmid extraction kit, and converting the plasmid DNA into escherichia coli. Coli transformants were further subjected to genome sequencing validation.
Step 6: obtaining homologous clone transformant, and counting homologous gene clone accuracy.
The sequencing result in the step 5 shows that the Escherichia coli clone carrying the gene tor2 is the correct clone. The ratio of the number of correct clones to 94 is the cloning accuracy. The cloning accuracy in this example was 50%.
Example 2
Step 1: the upstream (sequence A2) and the downstream (sequence B2) of the tor2 gene are cloned by 100bp homologous sequences respectively.
The cloning procedure was as in example 1, with the primers PPF2, PPR1, PTF1 and PTR2, and the sequence information as follows:
PPF2:5’-GACCATGATTACGCCAAGCTTGC AAAGGGAAAA TATACCGGGT-3’
PPR1:5’-GAAGATGCGGCCGCTGTTAC TTTTCATATGGGGAAAGTAA-3’
PTF1:5’-GTAACAGCGGCCGCATCTTC TTTTAATGTATTGAAAATCA-3’
PTR2:5’-GACCTGCAGGCATGCAAGCTTGG GTAACGTCACGCTCGGAACT-3’
the primers PPF2 and PPR1 are used for cloning a 100bp region at the upstream of the primer 2, PTF1 and PTR2 are used for cloning a 100bp region at the downstream of the primer 2, and the corresponding components of the PCR reaction system are used in the same amount as that of the example 1 except that the primer PPF2 replaces PPF1, and the PPR2 replaces PPR 1. The PCR amplification conditions were the same as in example 1 except that the extension time was changed to 20sec, and the PCR product purification method was the same as in example 1.
Step 2: construction of the circular cloning vector YCplac33-AB-2 carrying the sequence A2 and the sequence B2.
HindIII digestion and product purification of plasmid YCplac33 were performed as described in example 1. The ligation reaction of the products of the restriction enzyme of the sequence A2, the sequence B2 and YCplac33 was performed as in example 1, and finally the correct clone YCplac33-AB-2 was obtained and frozen at-80 ℃.
And step 3: preparation of "Linear 3' overhanging Long-sticky-end double-stranded DNA" -YCplac 33-AB-T5-2.
The plasmid for the correct clone YCplac33-AB-2 was extracted and NotI was performed, the cleavage method was the same as in example 1. And (3) recovering the enzyme digestion product by using a PCR clean recovery kit same as the step 1, and treating the enzyme digestion product by using T5 exonuclease to obtain linear 3 'protruding long-sticky end double-stranded DNA' -YCplac 33-AB-T5-2: the enzyme reaction buffer solution is matched with 10 times buffer solution, the enzyme dosage is 0.01U/mu g YCplac33-AB-2 enzyme digestion product (equivalent to 50U/nmol YCplac33-AB-2 enzyme digestion product), the processing temperature is 25 ℃, the processing time is 1min, and the length of the obtained 3' end single-chain sticky end is 100 bp. The treated product is numbered YCplac33-AB-T5-2 and can be directly used for subsequent yeast transformation.
And 4, step 4: and (3) transforming the saccharomyces cerevisiae.
(1) Yeast competence preparation: the same as in example 1.
(2) Transformation of yeast competent cells with exogenous DNA: the procedure was as in example 1 except that the foreign DNA was replaced with YCplac 33-AB-T5-2.
And 5: and (4) identifying a transformant.
(1) Colony PCR identification: the identification method was the same as in example 1.
(2) And (3) positive transformant identification: the same as in example 1.
Step 6: obtaining homologous clone transformant, and counting homologous gene clone accuracy.
The sequencing result in the step 5 shows that the Escherichia coli clone carrying the gene tor2 is the correct clone. The ratio of the number of correct clones to 94 is the cloning accuracy. The cloning accuracy in this example was 60%.
Example 3
Step 1: the upstream (sequence A3) and the downstream (sequence B3) of the tor2 gene are cloned by 500bp homologous sequences respectively.
The cloning procedure was as in example 1, with the primers PPF3, PPR1, PTF1 and PTR3, and the sequence information as follows:
PPF3:5’-GACCATGATTACGCCAAGCTTGC TATATATTTA TTACCGTCAT-3’
PPR1:5’-GAAGATGCGGCCGCTGTTAC TTTTCATATGGGGAAAGTAA-3’
PTF1:5’-GTAACAGCGGCCGCATCTTC TTTTAATGTATTGAAAATCA-3’
PTR3:5’-GACCTGCAGGCATGCAAGCTTGG CGGTAACAGGACAACAGCCA-3’
the primers PPF3 and PPR1 are used for cloning a 500bp region at the upstream of the primer 2, PTF1 and PTR3 are used for cloning a 500bp region at the downstream of the primer 2, and the corresponding components of the PCR reaction system are used in the same amount as that of the example 1 except that the primer PPF3 replaces PPF1, and the PPR3 replaces PPR 1. The PCR amplification conditions were the same as in example 1 except that the extension time was changed to 30sec, and the PCR product purification method was the same as in example 1.
Step 2: construction of the circular cloning vector YCplac33-AB-3 carrying the sequence A3 and the sequence B3.
HindIII digestion and product purification of plasmid YCplac33 were performed as described in example 1. The ligation reaction of the products of the restriction enzyme of the sequences A3, B3 and YCplac33 was performed as in example 1, and the correct clone YCplac33-AB-3 was finally obtained and frozen at-80 ℃.
And step 3: preparation of "Linear 3' overhanging Long-sticky-end double-stranded DNA" -YCplac 33-AB-T5-3.
The plasmid for the correct clone YCplac33-AB-3 was extracted and NotI was performed, the cleavage method was the same as in example 1. And (3) recovering the enzyme digestion product by using a PCR clean recovery kit same as the step 1, and treating the enzyme digestion product by using T5 exonuclease to obtain linear 3 'protruding long-sticky end double-stranded DNA' -YCplac 33-AB-T5-3: the enzyme reaction buffer solution is a matched 10 Xbuffer solution, the enzyme dosage is 0.5U/mu g YCplac33-AB-3 enzyme digestion product (equivalent to 2500U/nmol YCplac33-AB-3 enzyme digestion product), the processing temperature is 42.5 ℃, the processing time is 30min, and the length of the obtained 3' end single-chain sticky end is 1000 bp. The treated product is numbered YCplac33-AB-T5-3 and can be directly used for subsequent yeast transformation.
And 4, step 4: and (3) transforming the saccharomyces cerevisiae.
(1) Yeast competence preparation: the same as in example 1.
(2) Transformation of yeast competent cells with exogenous DNA: the procedure was as in example 1 except that the foreign DNA was replaced with YCplac 33-AB-T5-3.
And 5: and (4) identifying a transformant.
(1) Colony PCR identification: the identification method was the same as in example 1.
(2) And (3) positive transformant identification: the same as in example 1.
Step 6: obtaining homologous clone transformant, and counting homologous gene clone accuracy.
The sequencing result in the step 5 shows that the Escherichia coli clone carrying the gene tor2 is the correct clone. The ratio of the correct clone number to 100 is the clone accuracy. The cloning accuracy in this example was 87%.
Example 4
Step 1: the upstream (sequence A3) and the downstream (sequence B3) of the tor2 gene are cloned by 500bp homologous sequences respectively.
The cloning procedure was as in example 3.
Step 2: construction of the circular cloning vector YCplac33-AB-4 carrying the sequence A3 and the sequence B3.
HindIII digestion and product purification of plasmid YCplac33 were performed as described in example 1. The ligation reaction of the products of the restriction enzyme of the sequence A3, the sequence B3 and YCplac33 was performed as in example 3, and finally the correct clone YCplac33-AB-4 was obtained and frozen at-80 ℃.
And step 3: preparation of "Linear 3' overhanging Long-sticky-end double-stranded DNA" -YCplac 33-AB-T5-4.
The plasmid for the correct clone YCplac33-AB-4 was extracted and NotI was performed, the cleavage method was the same as in example 1. And (3) recovering the enzyme digestion product by using a PCR clean recovery kit same as the step 1, and treating the enzyme digestion product by using T5 exonuclease to obtain linear 3 'protruding long-sticky end double-stranded DNA' -YCplac 33-AB-T5-4: the enzyme reaction buffer solution is matched with 10 times buffer solution, the enzyme dosage is 0.8U/mu g YCplac33-AB-4 enzyme digestion product (equivalent to 400U/nmol YCplac33-AB-4 enzyme digestion product), the processing temperature is 50 ℃, the processing time is 10min, and the length of the obtained 3' end single-chain sticky end is 450 bp. The treated product is numbered YCplac33-AB-T5-4 and can be directly used for subsequent yeast transformation.
And 4, step 4: and (3) transforming the saccharomyces cerevisiae.
(1) Yeast competence preparation: the same as in example 1.
(2) Transformation of yeast competent cells with exogenous DNA: the procedure was as in example 1 except that the foreign DNA was replaced with YCplac 33-AB-T5-4.
And 5: and (4) identifying a transformant.
(1) Colony PCR identification: the identification method was the same as in example 1.
(2) And (3) positive transformant identification: the same as in example 1.
Step 6: obtaining homologous clone transformant, and counting homologous gene clone accuracy.
The sequencing result in the step 5 shows that the Escherichia coli clone carrying the gene tor2 is the correct clone. The ratio of the correct clone number to 100 is the clone accuracy. The cloning accuracy in this example was 95%.
Example 5
Step 1: the upstream (sequence A3) and the downstream (sequence B3) of the tor2 gene are cloned by 500bp homologous sequences respectively.
The cloning procedure was as in example 3.
Step 2: construction of the circular cloning vector YCplac33-AB-5 carrying the sequence A3 and the sequence B3.
HindIII digestion and product purification of plasmid YCplac33 were performed as described in example 1. The ligation reaction of the products of the restriction enzyme of the sequence A3, the sequence B3 and YCplac33 was performed as in example 3, and finally the correct clone YCplac33-AB-5 was obtained and frozen at-80 ℃.
And step 3: preparation of "Linear 3' overhanging Long-sticky-end double-stranded DNA" -YCplac 33-AB-T5-5.
The plasmid for the correct clone YCplac33-AB-4 was extracted and NotI was performed, the cleavage method was the same as in example 1. And (3) recovering the enzyme digestion product by using a PCR clean recovery kit same as the step 1, and treating the enzyme digestion product by using T5 exonuclease to obtain linear 3 'protruding long-sticky end double-stranded DNA' -YCplac 33-AB-T5-4: the enzyme reaction buffer solution is matched with 10 times buffer solution, the enzyme dosage is 0.8U/mu g YCplac33-AB-5 enzyme digestion product (equivalent to 400U/nmol YCplac33-AB-5 enzyme digestion product), the processing temperature is 55 ℃, the processing time is 5min, and the length of the obtained 3' end single-chain sticky end is 300 bp. The treated product is numbered YCplac33-AB-T5-5 and can be directly used for subsequent yeast transformation.
And 4, step 4: and (3) transforming the saccharomyces cerevisiae.
(1) Yeast competence preparation: the same as in example 1.
(2) Transformation of yeast competent cells with exogenous DNA: the procedure was as in example 1 except that the foreign DNA was replaced with YCplac 33-AB-T5-5.
And 5: and (4) identifying a transformant.
(1) Colony PCR identification: the identification method was the same as in example 1.
(2) And (3) positive transformant identification: the same as in example 1.
Step 6: obtaining homologous clone transformant, and counting homologous gene clone accuracy.
The sequencing result in the step 5 shows that the Escherichia coli clone carrying the gene tor2 is the correct clone. The ratio of the correct clone number to 100 is the clone accuracy. The cloning accuracy in this example was 95%.
Example 6
Step 1: the upstream (sequence A3) and the downstream (sequence B3) of the tor2 gene are cloned by 500bp homologous sequences respectively.
The cloning procedure was as in example 3.
Step 2: construction of the circular cloning vector YCplac33-AB-6 carrying the sequence A3 and the sequence B3.
HindIII digestion and product purification of plasmid YCplac33 were performed as described in example 1. The ligation reaction of the products of the restriction enzyme of the sequence A3, the sequence B3 and YCplac33 was performed as in example 3, and finally the correct clone YCplac33-AB-6 was obtained and frozen at-80 ℃.
And step 3: preparation of "Linear 3' overhanging Long-sticky-end double-stranded DNA" -YCplac 33-AB-T5-6.
The plasmid for the correct clone YCplac33-AB-4 was extracted and NotI was performed, the cleavage method was the same as in example 1. And (3) recovering the enzyme digestion product by using a PCR clean recovery kit same as the step 1, and treating the enzyme digestion product by using T5 exonuclease to obtain linear 3 'protruding long-sticky end double-stranded DNA' -YCplac 33-AB-T5-6: the enzyme reaction buffer solution is matched with 10 times buffer solution, the enzyme dosage is 0.4U/mu g YCplac33-AB-6 enzyme digestion product (equivalent to 400U/nmol YCplac33-AB-6 enzyme digestion product), the processing temperature is 55 ℃, the processing time is 10min, and the length of the obtained 3' end single-chain sticky end is 300 bp. The treated product is numbered YCplac33-AB-T5-6 and can be directly used for subsequent yeast transformation.
And 4, step 4: and (3) transforming the saccharomyces cerevisiae.
(1) Yeast competence preparation: the same as in example 1.
(2) Transformation of yeast competent cells with exogenous DNA: the procedure was as in example 1 except that the foreign DNA was replaced with YCplac 33-AB-T5-5.
And 5: and (4) identifying a transformant.
(1) Colony PCR identification: the identification method was the same as in example 1.
(2) And (3) positive transformant identification: the same as in example 1.
Step 6: obtaining homologous clone transformant, and counting homologous gene clone accuracy.
The sequencing result in the step 5 shows that the Escherichia coli clone carrying the gene tor2 is the correct clone. The ratio of the correct clone number to 100 is the clone accuracy. The cloning accuracy in this example was 80%.
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Claims (3)
1. The method for realizing high-accuracy gene homologous cloning in yeast is characterized in that after the upstream and downstream sequences of a target cloning gene are obtained by adopting a polymerase chain reaction or whole gene synthesis method, the upstream and downstream sequences are connected in the original gene direction and inserted into a vector plasmid to construct a circular cloning vector, the circular cloning vector is subjected to linearization treatment to obtain linear double-stranded DNA, the linear double-stranded DNA is treated by T5 exonuclease with the enzyme dosage of 400U/nmol of the linear double-stranded DNA vector at the treatment temperature of 50-55 ℃ for 5-10min, the length of a single-stranded sticky end at the 3 ' end is 300-450bp after the nuclease treatment, the yeast cell is transformed after the linear 3 ' protruding long-sticky-end double-stranded DNA is formed, two ends of the linear 3 ' protruding long-sticky-end double-stranded DNA respectively carry 1 homologous sequence which is the same as the upstream or downstream sequence of the target cloning gene, the extending directions of the two 3 'protruding sticky ends point to the coding region of the target gene, namely the two 3' protruding sticky ends point to the target clone gene segment together in opposite directions, and the length of the homologous sequence is 500 bp.
2. The method of claim 1, wherein the method for transforming yeast cells with linear 3' overhanging long-sticky-end double-stranded DNA is electroporation, polyethylene glycol-mediated method, lithium chloride-mediated method or lithium acetate-mediated method.
3. The method of claim 1, wherein the method for transforming yeast cells with the linear 3' overhanging long-sticky-end double-stranded DNA is protoplast-mediated method.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1373803A (en) * | 1999-07-09 | 2002-10-09 | 欧洲分子生物学实验室 | Method and compositions for directed clonning and subclonning using homologous recombination |
| CN102120967A (en) * | 2010-12-09 | 2011-07-13 | 江南大学 | Preparation and application of OCH1 genetic flaw type P. pastoris X-33 bacterial strain |
| CN102634534A (en) * | 2012-03-30 | 2012-08-15 | 深圳市中联生物科技开发有限公司 | Nucleic acid molecular cloning method and related kit based on homologous recombination |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1373803A (en) * | 1999-07-09 | 2002-10-09 | 欧洲分子生物学实验室 | Method and compositions for directed clonning and subclonning using homologous recombination |
| CN102120967A (en) * | 2010-12-09 | 2011-07-13 | 江南大学 | Preparation and application of OCH1 genetic flaw type P. pastoris X-33 bacterial strain |
| CN102634534A (en) * | 2012-03-30 | 2012-08-15 | 深圳市中联生物科技开发有限公司 | Nucleic acid molecular cloning method and related kit based on homologous recombination |
Non-Patent Citations (1)
| Title |
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| 体内直接克隆大片段DNA的研究进展;朱莹 等;《中国生物工程杂志》;20141231;第34卷(第4期);第95-100页 * |
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