CN103497969B - A kind of Escherichia coli-yeast-agrobacteshuttle shuttle expression vector and application thereof - Google Patents

Abstract

The invention discloses a kind of Escherichia coli-yeast-agrobacteshuttle shuttle expression vector and application thereof.Described Escherichia coli-yeast-agrobacteshuttle shuttle expression vector is the binary vector containing cerevisiae dna replicon and yeast screening assay marker gene.The described Escherichia coli-yeast-agrobacteshuttle shuttle expression vector that is applied as is to the application in fungi or transforming plant cells DNA.Escherichia coli-yeast-agrobacteshuttle shuttle expression vector of the present invention be in the world reported first, can copy in yeast, intestinal bacteria and Agrobacterium simultaneously, breed and the plasmid of stable existence, this shuttle expression carrier directly can transform fungi or vegetable cell by agriculture bacillus mediated mode, shorten flow process and the time of genetic manipulation, easy to use, working efficiency is high, and preparation process is easy, quick, efficient.

Description

A kind of Escherichia coli-yeast-agrobacteshuttle shuttle expression vector and application thereof

Technical field

The present invention relates to gene engineering technology field, be specifically related to a kind of Escherichia coli-yeast-agrobacteshuttle shuttle expression vector and application thereof.

Background technology

Transgenic technology is that a kind of DNA fragmentation artificial separation or modified imports in organism genome, causes the expression of certain gene or does not express, making the proterties of organism that heritable change occur.In production and scientific research, plant and fungi are the transgenosis objects that two classes are extensively carried out.The DNA fragmentation transformed is generally used for genetic expression, gene knockout or gene silencing etc., and object is the function studying gene, improves the resistance against diseases of plant, volume increase, oil recovery enhancement, improves the production efficiency etc. of industrial fungi.The DNA of plant and fungi transforms many methods, and wherein Agrobacterium-medialed transformation method (Agrobacteriumtumefaciensmediatedtransformation, ATMT) is a kind of most effective method.Agriculture bacillus mediated needs utilizes binary vector (binaryvector), and binary vector contains one section of T-DNA sequence, and T-DNA, by infecting plant or fungal cell, can be inserted in Plant Genome by Agrobacterium.

The binary vector of current use has pCAMBIA series (as pCAMBIA1300), pBHt1, pSN1301, pUN1301, PSN1301 etc.These binary vectors, generally containing colibacillary amplicon dna and bacteria screening mark (as resistant genes such as Amp, Kana, TET), contain amplicon dna (as pVS1_origin, pVS1-REP etc.) and the T-DNA sequence of Agrobacterium simultaneously.Thus, these binary vectors are E. coli-agrobacterium shuttle expression carrier, can copy, breed in intestinal bacteria and Agrobacterium.But all these binary vectors all can not copy, breed in yeast cell at present.

DNA fragmentation for transforming fungi or plant is generally made up of several small segment, when carrying out DNA conversion, normally first these fragments are inserted into by the method for digestion with restriction enzyme in the binary vector in intestinal bacteria body and build recombinant vectors, then recombinant vectors DNA is extracted from intestinal bacteria, be transformed in Agrobacterium (as AGL1) body, then be transformed into plant or fungal cell.

Above-mentioned carrier construction method weak point is, very low, the consuming time length of efficiency, be unsuitable for high-throughout DNA transform and gene knockout operation.

And yeast homologous restructuring be one efficient, fast and be suitable for the carrier construction method that high-throughput DNA transforms.The DNA vector with yeast replicon and yeast screening assay mark may be used for the homologous recombination of DNA.These carriers have pYES2, pYIP5, pPICZ, pRS426 etc., its feature is containing a cerevisiae dna replicon (as 2micro2_origin), yeast screening assay marker gene (as URA3, LEU2, HIS3 etc.), and these carriers are generally also containing colibacillary amplicon dna and bacteria screening mark (as resistant genes such as Amp, Kana, TET) simultaneously.Thus, these yeast plasmids are E. coli-Yeast shuttle expression carrier, can copy, breed in yeast and Bacillus coli cells.

But all these E. coli-Yeast shuttle expression carriers all can not copy, breed in agrobatcerium cell at present, agriculture bacillus mediated DNA conversion process therefore can not be directly used in.After DNA fusion fragment (expressed sequence, gene knockout structure etc.) utilizing yeast homologous recombination method to build needs to use restriction enzyme to cut target DNA fragment from yeast plasmid, insert E. coli-agrobacterium shuttle expression carrier again, then transform for agriculture bacillus mediated DNA.This makes the inefficiency of vector construction, be unsuitable for high-throughout ATMT operates.

Summary of the invention

The invention provides a kind of Escherichia coli-yeast-agrobacteshuttle shuttle expression vector, this shuttle expression carrier can copy propagation in intestinal bacteria, Agrobacterium and yeast cell, can be used for carrying out efficient, quick, high-throughout DNA to fungi or vegetable cell and transforms.

A kind of Escherichia coli-yeast-agrobacteshuttle shuttle expression vector, it is the binary vector containing cerevisiae dna replicon and yeast screening assay marker gene.

Binary vector is a kind of E. coli-agrobacterium shuttle vectors, existing intestinal bacteria replication orgin has Agrobacterium replication origin again, cerevisiae dna replicon is recombinated in binary vector by the present invention, the replication orgin of cerevisiae dna replicon is by the replication-associated protein identification of yeast cell, thus described Escherichia coli-yeast-agrobacteshuttle shuttle expression vector obtained copy in yeast cell, breed and the ability of stable existence, transform for carrying out efficient, quick, high-throughout DNA.

As preferably, described binary vector is pCAMBIA serial carrier, pBHt1, pSN1301 or pUN1301.As preferred further, described binary vector is pCAMBIA serial carrier.As most preferably, described binary vector is pCAMBIA1300.

Amplicon dna pVS1_origin and the T-DNA sequence of colibacillary amplicon dna pBR322_origin and Agrobacterium is contained in pCAMBIA1300 plasmid, also containing Kana resistant gene, Kana resistant gene is the common screening-gene of intestinal bacteria and Agrobacterium, directly as selection markers, can need not insert resistant gene in addition.Certainly, also Kana resistant gene can be replaced with Amp, Tet, Cmr and Spec resistant gene as required.

In the present invention, described cerevisiae dna replicon can select 2micro2_origin, 25 μm, ARS1, ARS3002 or ARS3003.Particular requirement is not had to the on position of cerevisiae dna replicon in described binary vector.

Screen after transformed yeast cell for ease of this Escherichia coli-yeast-agrobacteshuttle shuttle expression vector and transform successful bacterium colony, described Escherichia coli-yeast-agrobacteshuttle shuttle expression vector is also containing yeast screening assay marker gene.As preferably, described yeast screening assay marker gene is URA3, HIS3, LEU2, LYS2, TRP1, MET15, ADE2 or ADE1.

Escherichia coli-yeast-agrobacteshuttle shuttle expression vector of the present invention also comprises H3 promotor and fluorescence protein gene, whether described fluorescence protein gene can select green fluorescence protein gene or red fluorescent protein gene, be used to indicate the goal gene that Escherichia coli-yeast-agrobacteshuttle shuttle expression vector of the present invention carries and express in recipient cell.

H3 promotor is expressed in recipient cell for driving fluorescence protein gene, and its base sequence is as shown in SEQIDNo.1.The promotor of fluorescence protein gene can be selected, the strong promoter that preferred recipient cell contains self according to the kind of recipient cell.Base sequence shown in SEQIDNo.2 is a part for Pyricularia oryzae H3 promoter sequence, and Pyricularia oryzae H3 promoter sequence is a strong promoter, all has strong startup effect in the mycelia of Pyricularia oryzae, spore and appressorium stage.Utilize H3 promotor to start fluorescence protein gene to express as negative selection markers, the screening efficiency of transformant can be improved.

The construction process of described Escherichia coli-yeast-agrobacteshuttle shuttle expression vector, comprising:

First H3 promoter sequence and fluorescence protein gene are inserted in described plant expressing vector successively, obtain initial recombinant vectors;

Again by cerevisiae dna replicon, yeast screening assay gene and initial recombinant vectors mixing, transformed yeast cell, obtains described Escherichia coli-yeast-agrobacteshuttle shuttle expression vector after yeast homologous restructuring.

Present invention also offers the recombinant bacterial strain containing described Escherichia coli-yeast-agrobacteshuttle shuttle expression vector.Described recombinant bacterial strain is yeast or intestinal bacteria.

Present invention also offers described Escherichia coli-yeast-agrobacteshuttle shuttle expression vector to the application in fungi or transforming plant cells DNA.Utilize this shuttle expression carrier can realize the high frequency zone of the rapid build of the recombinant vectors containing goal gene, the rapid conversion in fungi or vegetable cell and mutant.

Particularly, described application comprises:

(1) by goal gene transformed yeast cell together with described Escherichia coli-yeast-agrobacteshuttle shuttle expression vector, recombinant expression vector is obtained through yeast homologous restructuring;

(2) by Agrobacterium_mediated method, described recombinant expression vector is proceeded in fungi or vegetable cell, and on selective medium picking transformant.

When for gene knockout, upstream homologous sequence and the downstream homologous sequence of gene to be knocked out is cloned from recipient cell genome, the Escherichia coli-yeast-agrobacteshuttle shuttle expression vector mixing that this upstream homologous sequence and downstream homologous sequence, suitable resistant gene and enzyme are cut through, transformed yeast competent cell; The gene knockout carrier containing gene knockout structure (the downstream homologous sequence of the upstream homologous sequence-resistant gene-gene to be knocked out of gene to be knocked out) is obtained through yeast homologous restructuring; By this gene knockout carrier transformation Agrobacterium, be then transformed in recipient cell through ATMT method, and on selective medium picking transformant; Under fluorescent microscope, fluorescigenic transformant is the transformant of radom insertion, and not fluorescent transformant is knock out mutants body.

When for genetic expression, clone's goal gene and promotor thereof, or the coding region, the in addition PCR that only clone goal gene clone the suitable promotor in other sources; Then the Escherichia coli-yeast-agrobacteshuttle shuttle expression vector mixing will cut through with the goal gene of promotor or the coding region of goal gene and appropriate promoters, suitable resistant gene and enzyme, transformed yeast competent cell; The expression vector containing goal gene is obtained through yeast homologous restructuring; By this expression vector transformation Agrobacterium, be then transformed in recipient cell through ATMT method, and on selective medium picking transformant; Extract the total protein of transformant (plant or fungi), according to the characteristic of target protein, adopt the method such as Westernblot or enzyme activity determination qualification goal gene whether to express and expression amount.

Although Escherichia coli-yeast-agrobacteshuttle shuttle expression vector of the present invention can be used for transfering DNA in fungi or vegetable cell, but due to the singularity of vegetable cell, cause DNA recombination efficiency very low, therefore this Escherichia coli-yeast-agrobacteshuttle shuttle expression vector can only be used for the genetic expression that realizes in vegetable cell, cannot be used for the gene knockout realized in vegetable cell.

Compared with prior art, beneficial effect of the present invention is:

Escherichia coli-yeast-agrobacteshuttle shuttle expression vector of the present invention be in the world reported first, can copy in yeast, intestinal bacteria and Agrobacterium simultaneously, breed and the plasmid of stable existence, this shuttle expression carrier directly can transform fungi or vegetable cell by agriculture bacillus mediated mode, shorten flow process and the time of genetic manipulation, easy to use, working efficiency is high, and preparation process is easy, quick, efficient.

Accompanying drawing explanation

Fig. 1 is the plasmid map of pCAMBIA1300 carrier;

Fig. 2 is the plasmid map of pKO1A carrier;

Fig. 3 is the plasmid map of pKO1A-RED carrier;

Fig. 4 is the plasmid map of pKO1B carrier;

Fig. 5 is the plasmid map of pKO1B-RED carrier;

Fig. 6 is the plasmid map of pKO1B-HIS3 carrier;

Fig. 7 is the plasmid map of pKO1B-LEU2 carrier;

Fig. 8 is the plasmid map of pKO1B-LYS2 carrier;

Fig. 9 is the plasmid map of pKO1B-TRP1 carrier;

Figure 10 is the plasmid map of pKO1B-MET15 carrier;

Figure 11 is the plasmid map of pKO1B-ADE2 carrier;

Figure 12 is the plasmid map of pKO1B-ADE1 carrier;

Figure 13 is the result figure using green fluorescent label screening-gene to knock out mutant;

Figure 14 is electrophoresis result figure knock out mutants body being carried out to PCR checking.

Embodiment

Embodiment 1

The construction process of Escherichia coli-yeast-agrobacteshuttle shuttle expression vector pKO1B or pKO1B-RED, comprising:

1 builds the E. coli-agrobacterium shuttle expression carrier carrying H3 promotor and fluorescence protein gene

(1) Pyricularia oryzae H3 promotor is obtained

With pKD5 plasmid for template, utilize primer a1/a2 to carry out high-fidelity PCR amplification, obtain the partial sequence (length is 1.2kb) of H3 promoter sequence (length is 1.5kb), the base sequence of primer a1/a2 is as follows:

Upstream primer a1:5 '-TT cTCGAGgTGTATTTTCTTTCTGCTCG-3 ' (underscore place is XhoI restriction enzyme site);

Downstream primer a2:5 '-TT gAATTCgGCCATTGTGATTGATTTGT-3 ' (underscore place is EcoRI restriction enzyme site).

PCR reaction system is: pKD5DNA10ng, high-fidelity DNA polymerase 0.5 μ L, dNTP(50mM) 0.4 μ L, each 0.5 μ L of upstream and downstream primer, 10xPCR damping fluid 5 μ L, adds water to 50 μ L.

PCR reaction conditions is: 94 DEG C 3 minutes, 35 circulations (94 DEG C 30 seconds, 58 DEG C 1 minute, 72 DEG C 1 minute 20 seconds), 72 DEG C 10 minutes.

PCR primer, through agarose electrophoresis, cuts the object band of 1.2kb size, and reclaim purifying through glue, be connected on pGEM-T carrier, transformation of E. coli competent cell DH5 α, the bacterium colony that picking amine benzyl flat board grows is cultivated, and extraction plasmid, enzyme cut qualification.

Enzyme is cut the correct H3 promotor of qualification inserts between XhoI and the EcoRI site of pCAMBIA1300 carrier (as shown in Figure 1) after XhoI and EcoRI double digestion, restructuring pCAMBIA1300 carrier called after pKO10 after digestion verification.

(2) green fluorescence protein gene is inserted

With pEGFP plasmid for template, utilize primer b1/b2 to carry out pcr amplification, obtain the GFP gene fragment of 0.7kb, the base sequence of primer b1/b2 is:

Upstream primer b1:5 '-AT gAATTCaTGGTGAGCAAGGGCGAGGA-3 ' (underscore place is EcoRI restriction enzyme site);

Downstream primer b2:5 '-AT gAGCTCtTACTTGTACAGCTCGTCCA-3 ' (underscore place is SacI restriction enzyme site);

PCR reaction system is: pEGFPDNA10ng, high-fidelity DNA polymerase 0.5 μ L, dNTP (50mM) 0.4 μ L, and each 0.5 μ L of upstream and downstream primer, 10xPCR damping fluid 5 μ L, adds water to 50 μ L.

PCR reaction conditions is: 94 DEG C 3 minutes, 35 circulations (94 DEG C 30 seconds, 56 DEG C 30 seconds, 72 DEG C 40 seconds), 72 DEG C 10 minutes.

PCR primer reclaims purifying rear clone to pGEM-T carrier, transformation of E. coli competent cell DH5 α through agarose electrophoresis, glue, and the bacterium colony that picking amine benzyl flat board grows is cultivated, and extraction plasmid, enzyme cut qualification.

Enzyme cuts the correct restructuring pGEM-T carrier EcoRI of qualification and SacI enzyme is cut, and inserts between EcoRI and the SacI site of pKO10 carrier by endonuclease bamhi (GFP gene fragment); Restructuring pKO10 vector competent escherichia coli cell DH5 α, the bacterium colony that picking amine benzyl flat board grows is cultivated, and extraction plasmid, enzyme cut qualification, by carrier called after pKO1A(correct for checking as shown in Figure 2).

(3) red fluorescent protein gene is inserted

With pDsRED2 plasmid for template, utilize primer c1/c2 to carry out pcr amplification, obtain the DsRED2 gene fragment of 0.7kb, primer c1(upstream primer)/c2(downstream primer) base sequence be:

c1：5’-AA GAATTCATGGCCTCCTCCGAGAACGTCATC-3’；

c2：5’-AA GAGCTCCAGGAACAGGTGGTGGCG-3’；

PCR reaction system is: pDsRED2DNA10ng, high-fidelity DNA polymerase 0.5 μ L, dNTP (50mM) 0.4 μ L, and each 0.5 μ L of upstream and downstream primer, 10xPCR damping fluid 5 μ L, adds water to 50 μ L.

PCR reaction conditions is: 94 DEG C 3 minutes, 35 circulations (94 DEG C 30 seconds, 56 DEG C 30 seconds, 72 DEG C 40 seconds), 72 DEG C 10 minutes.

PCR primer reclaims purifying rear clone to pGEM-T carrier, transformation of E. coli competent cell DH5 α through agarose electrophoresis, glue, and the bacterium colony that picking amine benzyl flat board grows is cultivated, and extraction plasmid, enzyme cut qualification.

Enzyme cuts the correct restructuring pGEM-T carrier EcoRI of qualification and SacI enzyme is cut, and inserts between EcoRI and the SacI site of pKO10 carrier by endonuclease bamhi (DsRED gene fragment); Restructuring pKO10 vector competent escherichia coli cell DH5 α, the bacterium colony that picking amine benzyl flat board grows is cultivated, and extraction plasmid, enzyme cut qualification, by carrier called after pKO1A-RED(correct for checking as shown in Figure 3).

2 build Escherichia coli-yeast-agrobacteshuttle shuttle expression vector

With pYES2 plasmid for template, utilize primer d1/d2 to carry out pcr amplification, obtain the DNA fragmentation of 2micro2_origin and the URA3 gene of 2.9kb, the base sequence of primer d1/d2 is:

Upstream primer d1:5 '-GTATCGACGGAGCCGATTTTGAAA

cCGCGGgGAACAACACTCAACCCTA-3 ' (underscore place is SacII restriction enzyme site);

Downstream primer d2:5 '-ACAGAGCGTTGCTGCCTGTGATCA

cCGCGGtTCGATGTAACCCACTCG-3 ' (underscore place is SacII restriction enzyme site);

PCR reaction system is: pYES2DNA10ng, high-fidelity DNA polymerase 0.5 μ L, dNTP (50mM) 0.4 μ L, and each 0.5 μ L of upstream and downstream primer, 10xPCR damping fluid 5 μ L, adds water to 50 μ L.

PCR reaction conditions is: 94 DEG C 3 minutes, 35 circulations (94 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 3 minutes), 72 DEG C 10 minutes.

PCR primer is after electrophoresis checking is correct, get 4 μ LPCR products and 100ngSacII enzyme cut after pKO1A carrier or the mixing of pKO1A-RED carrier, use the competent cell of commercial kit (S.c.EasyCompTransformationKit) the transformed saccharomyces cerevisiae bacterial strain FY834 of Invitrogen company;

Transformed yeast cell is coated in SC-URA and screens on flat board, cultivates 3 days for 30 DEG C; Get the yeast cell on SC-URA screening flat board with aseptic washing, use yeast plasmid to extract test kit and extract yeast plasmid; The bacterium colony that plasmid transformation escherichia coli competent cell DH5 α, picking kantlex LB flat board grows is cultivated, and extracts plasmid, order-checking qualification.To verify that correct carrier called after pKO1B(is as shown in Figure 4) or pKO1B-RED(is as shown in Figure 5).

Embodiment 2

The construction process of Escherichia coli-yeast-agrobacteshuttle shuttle expression vector pKO1B-HIS3, comprising:

1 according to embodiment 1 step 1-(1) method of (2) builds pKO1A carrier;

2 build pKO1B-HIS3

(1) 2micro2_originDNA fragment is obtained

With pYES2 plasmid for template, utilize primer e1/e2 to carry out pcr amplification, obtain the 2micro2_originDNA fragment of 1.1kb, the base sequence of primer e1/e2 is:

Upstream primer e1:5 '-GTATCGACGGAGCCGATTTTGAAA cCGCGG

GCTAGCTTTTCAATTCAATTCATC-3 ' (underscore place is SacII restriction enzyme site);

Downstream primer e2:5 '-TTCGATGTAACCCACTCGTGCA-3 ';

PCR reaction system is: pYES2DNA10ng, high-fidelity DNA polymerase 0.5 μ L, dNTP (50mM) 0.4 μ L, and each 0.5 μ L of upstream and downstream primer, 10xPCR damping fluid 5 μ L, adds water to 50 μ L.

PCR reaction conditions is: 94 DEG C 3 minutes, 35 circulations (94 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 1 minute 10 seconds), 72 DEG C 10 minutes.

The PCR primer (2micro2_originDNA fragment) obtained is through electrophoresis checking, and glue reclaims purifying.

(2) HIS3 gene fragment is obtained

Utilize the PCR primer of HIS3, obtain the DNA fragmentation of the long HIS3 gene of 1.3kb from pTAL3_His carrier through PCR,

Upstream primer f1:5 '-ATCAGTTGGGTGCACGAGTGGGTTACAT

CGAAGTTCGTATAAATACTTACTGACA-3’；

Downstream primer f2:5 '-ACAGAGCGTTGCTGCCTGTGATCA cCGCGG

CTTCATTCCGTAACTCTTCTACCT-3 ' (underscore place is SacII restriction enzyme site);

PCR reaction system is: pTAL3_HisDNA10ng, high-fidelity DNA polymerase 0.5 μ L, dNTP (50mM) 0.4 μ L, and each 0.5 μ L of upstream and downstream primer, 10xPCR damping fluid 5 μ L, adds water to 50 μ L.

PCR reaction conditions is: 94 DEG C 3 minutes, 35 circulations (94 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 1 minute 20 seconds), 72 DEG C 10 minutes.

The PCR primer (DNA fragmentation of HIS3 gene) obtained is through electrophoresis checking, and glue reclaims purifying.

Respectively get the above-mentioned 2 kinds of PCR primer of 4 μ L (2micro2_originDNA fragment and HIS3 gene fragment) and 100ngSacII enzyme cut after the mixing of pKO1A carrier, use the competent cell of commercial kit (S.c.EasyCompTransformationKit) the transformed saccharomyces cerevisiae bacterial strain FY834 of Invitrogen company.Transformed yeast cell is coated in SC-HIS and screens on flat board, cultivates 3 days for 30 DEG C; The yeast cell on SC-HIS screening flat board is got with aseptic washing, use yeast plasmid to extract test kit and extract yeast plasmid, the bacterium colony that plasmid transformation escherichia coli competent cell DH5 α, picking kantlex LB flat board grows is cultivated, extract plasmid, order-checking qualification.By carrier called after pKO1B-HIS3(correct for checking as shown in Figure 6).

Embodiment 3

The construction process of Escherichia coli-yeast-agrobacteshuttle shuttle expression vector pKO1B-LEU2, comprising:

1 according to embodiment 1 step 1-(1) method of (2) builds pKO1A carrier;

2 build pKO1B-LEU2

(1) according to embodiment 2 step 2-(1) method obtain 2micro2_originDNA fragment;

(2) LEU2 gene fragment is obtained

With pTAL4_Leu plasmid for template, utilize primer g1/g2 to carry out pcr amplification, obtain the LEU2 gene fragment that 2.2kb is long, the base sequence of primer g1/g2 is:

Upstream primer g1:5 '-ATCAGTTGGGTGCACGAGTGGGTTACAT

CGAATCATATAATTTCTGTTGAATTAC-3’；

Downstream primer g2:5 '-ACAGAGCGTTGCTGCCTGTGATCA cCGCGG

CGTCTACCCTATGAACATATTCCA-3 ' (underscore place is SacII restriction enzyme site);

PCR reaction system is: pTAL4_LeuDNA10ng, high-fidelity DNA polymerase 0.5 μ L, dNTP (50mM) 0.4 μ L, and each 0.5 μ L of upstream and downstream primer, 10xPCR damping fluid 5 μ L, adds water to 50 μ L.

PCR reaction conditions is: 94 DEG C 3 minutes, 35 circulations (94 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 2 minutes 20 seconds), 72 DEG C 10 minutes.

The PCR primer (LEU2 gene fragment) obtained is through electrophoresis checking, and glue reclaims purifying.

Respectively get the above-mentioned 2 kinds of PCR primer of 4 μ L (2micro2_originDNA fragment and LEU2 gene fragment) and 100ngSacII enzyme cut after the mixing of pKO1A carrier, use the competent cell of commercial kit (S.c.EasyCompTransformationKit) the transformed saccharomyces cerevisiae bacterial strain FY834 of Invitrogen company; Transformed yeast cell is coated in SC-LEU and screens on flat board, cultivates 3 days for 30 DEG C; The yeast cell on SC-LEU screening flat board is got with aseptic washing, use yeast plasmid to extract test kit and extract yeast plasmid, the bacterium colony that plasmid transformation escherichia coli competent cell DH5 α, picking kantlex LB flat board grows is cultivated, extract plasmid, order-checking qualification.By carrier called after pKO1B-LEU2(correct for checking as shown in Figure 7).

Embodiment 4

The construction process of Escherichia coli-yeast-agrobacteshuttle shuttle expression vector pKO1B-LYS2, comprising:

1 according to embodiment 1 step 1-(1) method of (2) builds pKO1A carrier;

2 build pKO1B-LYS2

(1) according to embodiment 2 step 2-(1) method obtain 2micro2_originDNA fragment;

(2) LYS2 gene fragment is obtained

With pRS307 plasmid for template, utilize primer h1/h2 to carry out pcr amplification, obtain the LYS2 gene fragment of 5.0kb; The base sequence of primer h1/h2 is:

Upstream primer h1:5 '-ATCAGTTGGGTGCACGAGTGGGTTACAT

CGAATCTCCTAATTCATATTTAATTATTGT-3’；

Downstream primer h2:5 '-ACAGAGCGTTGCTGCCTGTGATCA cCGCGG

GCCCTGTAGCGGCGCATTAAGCGC-3 ' (underscore place is SacII restriction enzyme site);

PCR reaction system is: pRS307DNA10ng, high-fidelity DNA polymerase 0.5 μ L, dNTP (50mM) 0.4 μ L, and each 0.5 μ L of upstream and downstream primer, 10xPCR damping fluid 5 μ L, adds water to 50 μ L.

PCR reaction conditions is: 94 DEG C 3 minutes, 35 circulations (94 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 5 minutes 10 seconds), 72 DEG C 10 minutes.

The PCR primer (LYS2 gene fragment) obtained is through electrophoresis checking, and glue reclaims purifying.

Respectively get the above-mentioned 2 kinds of PCR primer of 4 μ L (2micro2_originDNA fragment and LYS2 gene fragment) and 100ngSacII enzyme cut after the mixing of pKO1A carrier, use the competent cell of commercial kit (S.c.EasyCompTransformationKit) the transformed saccharomyces cerevisiae bacterial strain FY834 of Invitrogen company.Transformed yeast cell is coated in SC-LYS and screens on flat board, cultivates 3 days for 30 DEG C; The yeast cell on SC-LYS screening flat board is got with aseptic washing, use yeast plasmid to extract test kit and extract yeast plasmid, the bacterium colony that plasmid transformation escherichia coli competent cell DH5 α, picking kantlex LB flat board grows is cultivated, extract plasmid, order-checking qualification.By carrier called after pKO1B-LYS2(correct for checking as shown in Figure 8).

Embodiment 5

The construction process of Escherichia coli-yeast-agrobacteshuttle shuttle expression vector pKO1B-TRP1, comprising:

1 according to embodiment 1 step 1-(1) method of (2) builds pKO1A carrier;

2 build pKO1B-TRP1

(1) according to embodiment 2 step 2-(1) method obtain 2micro2_originDNA fragment;

(2) TRP1 gene fragment is obtained

With pYES2.1-TRP1 plasmid for template, utilize primer i1/i2 to carry out pcr amplification, obtain the TRP1 gene fragment that 1.6kb is long, the base sequence of primer i1/i2 is:

Upstream primer i1:5 '-ATCAGTTGGGTGCACGAGTGGGTTACAT

CGAAGATGATCCACTAGTACGGATTAGA-3’；

Downstream primer i2:5 '-ACAGAGCGTTGCTGCCTGTGATCA cCGCGG

TTCGAGCGTCCCAAAACCTTCTCA-3 ' (underscore place is SacII restriction enzyme site);

PCR reaction system is: pYES2.1-TRP1DNA10ng, high-fidelity DNA polymerase 0.5 μ L, dNTP (50mM) 0.4 μ L, and each 0.5 μ L of upstream and downstream primer, 10xPCR damping fluid 5 μ L, adds water to 50 μ L.

PCR reaction conditions is: 94 DEG C 3 minutes, 35 circulations (94 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 1 minute 40 seconds), 72 DEG C 10 minutes.

The PCR primer (TRP1 gene fragment) obtained is through electrophoresis checking, and glue reclaims purifying.

Respectively get the above-mentioned 2 kinds of PCR primer of 4 μ L (2micro2_originDNA fragment and TRP1 gene fragment) and 100ngSacII enzyme cut after the mixing of pKO1A carrier, use the competent cell of commercial kit (S.c.EasyCompTransformationKit) the transformed saccharomyces cerevisiae bacterial strain FY834 of Invitrogen company.Transformed yeast cell is coated in SC-TRP and screens on flat board, cultivates 3 days for 30 DEG C; The yeast cell on SC-TRP screening flat board is got with aseptic washing, use yeast plasmid to extract test kit and extract yeast plasmid, the bacterium colony that plasmid transformation escherichia coli competent cell DH5 α, picking kantlex LB flat board grows is cultivated, extract plasmid, order-checking qualification.By carrier called after pKO1B-TRP1(correct for checking as shown in Figure 9).

Embodiment 6

The construction process of Escherichia coli-yeast-agrobacteshuttle shuttle expression vector pKO1B-MET15, comprising:

1 according to embodiment 1 step 1-(1) method of (2) builds pKO1A carrier;

2 build pKO1B-MET15

(1) according to embodiment 2 step 2-(1) method obtain 2micro2_originDNA fragment;

(2) MET15 gene fragment is obtained

With pXP214 plasmid for template, utilize primer j1/j2 to carry out pcr amplification, obtain the MET15 gene fragment that 1.7kb is long, the base sequence of primer j1/j2 is:

Upstream primer j1:5 '-ATCAGTTGGGTGCACGAGTGGGTTACATCG

AAATCCCCGGGATAACTTCGTATA-3’；

Downstream primer j2:5 '-ACAGAGCGTTGCTGCCTGTGATCA cCGCGG

AGCTCGGTACCCGGGATAACTTCGT-3 ' (underscore place is SacII restriction enzyme site);

PCR reaction system is: pXP214DNA10ng, high-fidelity DNA polymerase 0.5 μ L, dNTP (50mM) 0.4 μ L, and each 0.5 μ L of upstream and downstream primer, 10xPCR damping fluid 5 μ L, adds water to 50 μ L.

PCR reaction conditions is: 94 DEG C 3 minutes, 35 circulations (94 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 1 minute 50 seconds), 72 DEG C 10 minutes.

The PCR primer (MET15 gene fragment) obtained is through electrophoresis checking, and glue reclaims purifying.

Respectively get the above-mentioned 2 kinds of PCR primer of 4 μ L (2micro2_originDNA fragment and MET15 gene fragment) and 100ngSacII enzyme cut after the mixing of pKO1A carrier, use the competent cell of commercial kit (S.c.EasyCompTransformationKit) the transformed saccharomyces cerevisiae bacterial strain FY834 of Invitrogen company.Transformed yeast cell is coated in SC-MET and screens on flat board, cultivates 3 days for 30 DEG C; The yeast cell on SC-MET screening flat board is got with aseptic washing, use yeast plasmid to extract test kit and extract yeast plasmid, the bacterium colony that plasmid transformation escherichia coli competent cell DH5 α, picking kantlex LB flat board grows is cultivated, extract plasmid, order-checking qualification.By carrier called after pKO1B-MET15(correct for the checking obtained as shown in Figure 10).

Embodiment 7

The construction process of Escherichia coli-yeast-agrobacteshuttle shuttle expression vector pKO1B-ADE2, comprising:

1 according to embodiment 1 step 1-(1) method of (2) builds pKO1A carrier;

2 build pKO1B-ADE2

(1) according to embodiment 2 step 2-(1) method obtain 2micro2_originDNA fragment;

(2) ADE2 gene fragment is obtained

With pRSII312 plasmid for template, utilize primer k1/k2 to carry out pcr amplification, obtain the ADE2 gene fragment that 2.3kb is long, the base sequence of primer k1/k2 is:

Upstream primer k1:5 '-ATCAGTTGGGTGCACGAGTGGGTTACATCG

AAGAATTAATTCTTGAATAATACAT-3’；

Downstream primer k2:5 '-ACAGAGCGTTGCTGCCTGTGATCA cCGCGG

TATGTATGAAATTCTTAAAAAAGG-3 ' (underscore place is SacII restriction enzyme site);

PCR reaction system is: pRSII312DNA10ng, high-fidelity DNA polymerase 0.5 μ L, dNTP (50mM) 0.4 μ L, and each 0.5 μ L of upstream and downstream primer, 10xPCR damping fluid 5 μ L, adds water to 50 μ L.

PCR reaction conditions is: 94 DEG C 3 minutes, 35 circulations (94 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 2 minutes 20 seconds), 72 DEG C 10 minutes.

The PCR primer (ADE2 gene fragment) obtained is through electrophoresis checking, and glue reclaims purifying.

Respectively get the above-mentioned 2 kinds of PCR primer of 4 μ L (2micro2_originDNA fragment and ADE2 gene fragment) and 100ngSacII enzyme cut after the mixing of pKO1A carrier, use the competent cell of commercial kit (S.c.EasyCompTransformationKit) the transformed saccharomyces cerevisiae bacterial strain FY834 of Invitrogen company.Transformed yeast cell is coated in SC-ADE and screens on flat board, cultivates 3 days for 30 DEG C; The yeast cell on SC-ADE screening flat board is got with aseptic washing, use yeast plasmid to extract test kit and extract yeast plasmid, the bacterium colony that plasmid transformation escherichia coli competent cell DH5 α, picking kantlex LB flat board grows is cultivated, extract plasmid, order-checking qualification.By carrier called after pKO1B-ADE2(correct for the checking obtained as shown in figure 11).

Embodiment 8

The construction process of Escherichia coli-yeast-agrobacteshuttle shuttle expression vector pKO1B-ADE1, comprising:

1 according to embodiment 1 step 1-(1) method of (2) builds pKO1A carrier;

2 build pKO1B-ADE1

(1) according to embodiment 2 step 2-(1) method obtain 2micro2_originDNA fragment;

(2) ADE1 gene fragment is obtained

With YCpADE1 plasmid for template, utilize primer I 1/I2 to carry out pcr amplification, obtain the ADE1 gene fragment that 1.3kb is long, the base sequence of primer I 1/I2 is:

Upstream primer l1:5 '-ATCAGTTGGGTGCACGAGTGGGTTACAT

CGACTTATTCACGAGTCAGTCTGACT-3’；

Downstream primer l2:5 '-ACAGAGCGTTGCTGCCTGTGATCA cCGCGG

CGATAAAATTTGCTCTGAGAACAT-3 ' (underscore place is SacII restriction enzyme site);

PCR reaction system is: YCpADE1DNA10ng, high-fidelity DNA polymerase 0.5 μ L, dNTP (50mM) 0.4 μ L, and each 0.5 μ L of upstream and downstream primer, 10xPCR damping fluid 5 μ L, adds water to 50 μ L.

PCR reaction conditions is: 94 DEG C 3 minutes, 35 circulations (94 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 1 minute 20 seconds), 72 DEG C 10 minutes.

The PCR primer (ADE1 gene fragment) obtained is through electrophoresis checking, and glue reclaims purifying.

Respectively get the above-mentioned 2 kinds of PCR primer of 4 μ L (2micro2_originDNA fragment and ADE1 gene fragment) and 100ngSacII enzyme cut after the mixing of pKO1A carrier, use the competent cell of commercial kit (S.c.EasyCompTransformationKit) the transformed saccharomyces cerevisiae bacterial strain FY834 of Invitrogen company.Transformed yeast cell is coated in SC-ADE and screens on flat board, cultivates 3 days for 30 DEG C; The yeast cell on SC-ADE screening flat board is got with aseptic washing, use yeast plasmid to extract test kit and extract yeast plasmid, the bacterium colony that plasmid transformation escherichia coli competent cell DH5 α, picking kantlex LB flat board grows is cultivated, extract plasmid, order-checking qualification.By carrier called after pKO1B-ADE1(correct for the checking obtained as shown in figure 12).

The application of embodiment 9pKO1B carrier in Pyricularia oryzae gene knockout

The structure of 1 transcription factor gene (MGG_01833) knockout carrier

(1) with Pyricularia oryzae genomic dna for template, utilize primer m1/m2 to carry out pcr amplification, obtain the fragment upstream of MGG_01833 gene, the base sequence of primer m1/m2 is:

Upstream primer sequence m1:5 '-GCTGTACAAGTAAGAGCTCGGTACCCG

GGGATCGACGCCTTGGTCTGCCGTTGTTA-3’；

Downstream primer sequence m2:5 '-CCGGGAGATGTGGGGCACTGTGGCGTT

GGCACAATGAAAGGATCTGGGGCGAAGTG-3’；

(2) with Pyricularia oryzae genomic dna for template, utilize primer n1/n2 to carry out pcr amplification, obtain the segments downstream of MGG_01833 gene, the base sequence of primer n1/n2 is:

Upstream primer sequence n1:5 '-TTGATTATTGCACGGGAATTGCATGCTC

TCACATCTGAGCATGACAGCGCATAGCT-3’；

Downstream primer sequence n2:5 '-TTAAGTTGGGTAACGCCAGGGTTTTCCC

AGTCACATCCCAAGACACTCGGCAAAGG-3’；

(3) with pBS-SUR plasmid for template, utilize primer o1/o2 to carry out pcr amplification, obtain SUR resistance gene fragment, the base sequence of primer o1/o2 is:

Upstream primer o1:5 '-GTGCCAACGCCACAGTGCC-3 ';

Downstream primer o2:5 '-GTGAGAGCATGCAATTCCC-3 ';

Electrophoresis verifies that whether above-mentioned PCR primer is correct.

(4) XbaI and HindIII double digestion pKO1B carrier is used, get the pKO1B carrier (100ng/1 μ L) that each 4 μ L of above-mentioned 3 kinds of PCR primer and 1 μ L enzyme cut through to join in the competent cell of 50 μ L yeast saccharomyces cerevisiae FY834 bacterial strains, operate according to the step of the test kit (S.c.EasyCompTransformationKit) of Invitrogen company.Transformed yeast cell is coated in SC-URA and screens on flat board, cultivates 3 days for 30 DEG C; The yeast cell on SC-URA screening flat board is got with aseptic washing, use yeast plasmid to extract test kit and extract yeast plasmid, plasmid transformation escherichia coli competent cell DH5 α, the bacterium colony that picking kantlex LB flat board grows is cultivated, extract plasmid, the same to primer by PCR(3) and order-checking qualification.By MGG_01833 gene knockout carrier called after pKO1B-MGG_01833 correct for the checking obtained.

2ATMT method transforms pKO1B-MGG_01833 to Pyricularia oryzae

Carrier pKO1B-MGG_01833 is transformed into agrobacterium strains AGL1 by freeze-thaw method, and then transform Pyricularia oryzae through ATMT method and sprout in spore, concrete steps are as follows:

(1) the single colony inoculation of an Agrobacterium is selected in 5mLLB liquid nutrient medium (containing 50mg/mL kantlex) from the LB flat board (containing 50mg/mL kantlex) of fresh culture, 200rpm, 28 DEG C of incubated overnight;

200-400 μ L nutrient solution was transferred to 5mL containing in the induced fluid substratum (IM) of 50mg/mL kantlex in (2) second days, OD value about 0.15,28 DEG C of cultivations make OD in 5 ~ 6 hours ₆₀₀reach 0.5 ~ 0.6;

(3) collection of rice blast pathogen conidiospore: the conidium washing lower Pyricularia oryzae with 10mL sterile purified water from the CM flat board cultivated about 10 days with blood counting chamber counting after three layers of lens wiping paper filter, and is 10 with sterilized water dilution spore concentration ⁶individual/mL;

(4) the cultured Agrobacterium AGL-1(of 100 μ L is got containing carrier pKO1B-MGG_01833) the rice blast pathogen conidiospore suspension that diluted of bacterium liquid and 100 μ L mixes, mixed solution (200 μ L) is evenly applied to the nitrocellulose filter surface on AIM flat board, AIM flat board containing 200 μm of ol/L Syringylethanones, 22 DEG C of Dual culture 48 hours;

(5) nitrocellulose filter is transferred to containing fungal antibiotic (Sulphadiazine Sodium) with antibiotic selects flat board (DCM) on, containing 100 μ g/mL Sulphadiazine Sodiums, 400mg/mL cephamycin, 60mg/mL Streptomycin sulphate in selective medium, cultivate transformant at plate being placed in 28 DEG C and occur.Transformant is inoculated into and again identifies containing on 100 μ g/mL Sulphadiazine Sodium DCM flat boards.

3 transcription factor genes (MGG_01833) knock out the qualification of mutant

The transformant that picking has Sulphadiazine Sodium (SUR) resistance detects under fluorescent microscope, if transformant has the green fluorescence of GFP genetic expression, then this transformant is the transformant of radom insertion, abandons; If transformant is fluoresced green not, then may be knock out mutants body, choose and transfer to another and cultivate (Figure 13) containing 100 μ g/mL Sulphadiazine Sodium DCM flat boards continue.

Then the genomic dna of the transformant of not fluoresced green is extracted by CTAB method; Detect MGG_01833 in mutant by double PCR whether also to exist:

The MGG_01833 gene internal primer used is:

Upstream sequence p1:5 '-GCATCTCAACGGCAGTATCT-3 ';

Downstream sequence p2:5 '-ACCTGGCACAGCAAACAA-3 ';

The beta-tubulin(positive control used) gene primer is:

Upstream sequence q1:5 '-TTCCGCGCTGTCACCGTTCC-3 ';

Downstream sequence q2:5 '-GGGCCTCCTCCTCGTACTCCTCTT-3 '.

PCR primer is after electrophoresis, if having 2 electrophoretic bands, 1 is 400bp, and another is the band of 540bp, then gene is not knocked; If only have the band of a 540bp, then gene has been knocked (Figure 14).The knock out mutants body obtained can also continue through the methods such as PCR, qPCR, Southernblot, Westernblot at DNA, mRNA or the enterprising step card of protein level.

The application of embodiment 10pKO1B carrier in the genetic expression of Pyricularia oryzae

With the expression of foreign gene GFP gene in Pyricularia oryzae, the expression how realizing foreign protein is described as an example.

1, the structure of GFP expression vector

(1) with pCB1003 plasmid for template, utilize primer r1/r2 to carry out pcr amplification, obtain the DNA fragmentation of the long damp enzyme element resistant gene of 1.4kb, the base sequence of primer r1/r2 is:

Upstream primer sequence r1:5 '-GCTGTACAAGTAAGAGCTCGGTACCCGG

GGATCTAGTGGAGGTCAACAATGAATG-3’；

Downstream primer sequence r2:5 '-TTAAGTTGGGTAACGCCAGGGTTTTCCC

AGTCACTATTCCTTTGCCCTCGGACGA-3’；

Electrophoresis verifies that whether above-mentioned PCR primer is correct.

(2) XbaI and HindIII double digestion pKO1B carrier is used, get the pKO1B carrier (100ng/1 μ L) that above-mentioned a kind of PCR primer 4 μ L and 1 μ L enzyme cut through to join in the competent cell of 50 μ L yeast saccharomyces cerevisiae FY834 bacterial strains, operate according to the step of the test kit (S.c.EasyCompTransformationKit) of Invitrogen company.Transformed yeast cell is coated in SC-URA and screens on flat board, cultivates 3 days for 30 DEG C; The yeast cell on SC-URA screening flat board is got with aseptic washing, use yeast plasmid to extract test kit and extract yeast plasmid, plasmid transformation escherichia coli competent cell DH5 α, the bacterium colony that picking kantlex LB flat board grows is cultivated, extract plasmid, the same to primer by PCR(1) and order-checking qualification.By GFP expression vector called after pKO1B-HPH correct for the checking obtained.

2ATMT method transforms pKO1B-HPH to Pyricularia oryzae

Carrier pKO1B-HPH is transformed into agrobacterium strains AGL1 by freeze-thaw method, then transforms Pyricularia oryzae through ATMT method and sprout in spore:

(1) concrete steps of-(4) are with (1)-(4) of embodiment 9.

(5) nitrocellulose filter is transferred to containing fungal antibiotic (damp enzyme element) with on the selection flat board (CM) of bacteria antibiotic, containing 200 μ g/mL tide enzyme elements, 400mg/mL cephamycin, 60mg/mL Streptomycin sulphate in selective medium, cultivate transformant at plate being placed in 28 DEG C and occur.Transformant is inoculated into and again identifies containing on 200 μ g/mL Sulphadiazine Sodium DCM flat boards.

The protein expression qualification of 3 genetically modified mutants

The transformant that picking has damp enzyme element resistance detects under fluorescent microscope, if transformant fluoresced green, then illustrates that GFP gene is expressed in Pyricularia oryzae.The GFP obtained expresses mutant can also continue through the methods such as RT-PCR and Westernblot at mRNA or the enterprising step card of protein level.

Claims (5)

1. an Escherichia coli-yeast-agrobacteshuttle shuttle expression vector, is characterized in that, it is the binary vector containing cerevisiae dna replicon and yeast screening assay marker gene;

Described binary vector is pCAMBIA1300;

Described cerevisiae dna replicon is 2micro2_origin;

Described yeast screening assay marker gene is URA3, HIS3, LEU2, LYS2, TRP1, MET15, ADE2 or ADE1.

2. Escherichia coli-yeast-agrobacteshuttle shuttle expression vector as claimed in claim 1, is characterized in that, comprise H3 promotor and fluorescence protein gene.

3. containing, for example the recombinant bacterial strain of the arbitrary described Escherichia coli-yeast-agrobacteshuttle shuttle expression vector of claim 1 ~ 2.

4. Escherichia coli-yeast-agrobacteshuttle shuttle expression vector as described in as arbitrary in claim 1 ~ 2 is to the application in fungi or transforming plant cells DNA.

5. apply as claimed in claim 4, it is characterized in that, comprising:

(1) by goal gene transformed yeast cell together with described Escherichia coli-yeast-agrobacteshuttle shuttle expression vector, recombinant expression vector is obtained through yeast homologous restructuring;

(2) by Agrobacterium_mediated method, described recombinant expression vector is proceeded in fungi or vegetable cell, and on selective medium picking transformant.