WO2013139193A1 - 一种克服靶细菌限制修饰障碍导入外源dna的方法 - Google Patents

一种克服靶细菌限制修饰障碍导入外源dna的方法 Download PDF

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WO2013139193A1
WO2013139193A1 PCT/CN2013/071730 CN2013071730W WO2013139193A1 WO 2013139193 A1 WO2013139193 A1 WO 2013139193A1 CN 2013071730 W CN2013071730 W CN 2013071730W WO 2013139193 A1 WO2013139193 A1 WO 2013139193A1
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bce
dna
sequence
nham
plasmid
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温廷益
张国强
邓爱华
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中国科学院微生物研究所
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Definitions

  • the present invention relates to the field of biotechnology, and more particularly to a method for introducing foreign DNA by overcoming a target bacterial restriction modification disorder. Background technique
  • the bacterial restriction modification system consists of a restriction enzyme (restriction enzyme) that specifically recognizes and cleaves DNA, and a DNA methyltransferase that can add a methyl modification to the base of the DNA, thereby preventing the restriction enzyme pair. DNA cutting.
  • the restriction modification system can selectively degrade the foreign DNA invading the bacteria to achieve the purpose of self-protection of the bacteria. According to the subunit composition, cleavage site, sequence specificity and cofactor characteristics of the modified system, it is divided into four types.
  • the restriction enzyme subunits of the type I, II, and III restriction modification systems recognize and cleave unmethylated DNA, but if the DNA is first recognized and modified by the methyltransferase subunit, the restriction enzyme cannot complete the cleavage.
  • the type IV restriction modification system has only a restriction enzyme composition and does not contain a methyltransferase, which recognizes and cleaves DNA having an exogenous methylation pattern and, therefore, is a methylation-dependent restriction enzyme.
  • recent studies have shown that the DNA backbone of the phosphorothioate-modifying enzyme and its corresponding restriction enzymes are a new class of restriction modification systems (Nucleic Acids Research, 38, 7133-7141).
  • This complex modification-cutting mode greatly protects the safety of the bacteria's own DNA, and is the main means by which bacteria can effectively prevent the invasion of foreign DNA such as phage and DNA released by dead bacteria in the environment. At the same time, this has become a major obstacle to the use of molecular biology to introduce foreign DNA into bacteria and achieve genetic manipulation. Genetic manipulation involving multiple sets of bacteria that limit the modification system is particularly difficult.
  • the first type of strategy is the strategy of modifying exogenous DNA, including in vitro modifications and E. coli modifications. Transformation of Helicobacter pylori, Bacillus cereus, and Bacillus cerevisiae can be achieved by in vitro modification of exogenous DNA using target bacterial crude protein extract (containing DNA methyltransferase) (Molecular Microbiology, 37, 1066-1074 , Applied and Environmental Microbiology, 74, 7817-7820); or cloning and expression of the target bacterial DNA methyltransferase in E.
  • target bacterial crude protein extract containing DNA methyltransferase
  • the second type of method is the method of inactivating a limiting modification system, including the use of physical means of inactivation and gene knockout.
  • the target bacterial restriction enzyme was temporarily inactivated by post-transformation heating, and the conversion of the exogenous plasmid to C.
  • Some of the protocols can modify the foreign DNA molecule in vitro by using the target bacterial DNA methyltransferase, but The in vitro modification of the crude protein extract is inefficient, and the restriction enzyme also degrades part of the plasmid while modifying; the in vivo modification scheme does not deactivate the methylation of the E. coli DNA methyltransferase to the foreign DNA molecule.
  • coli methylation pattern is a type II restriction system that easily activates target bacteria; many bacterial restriction enzymes are non-thermally sensitive and cannot be temporarily inactivated by heating; if the bacteria contain multiple sets of restriction modification systems, It is time-consuming and labor-intensive to knock out the restriction enzyme genes one by one, and knocking out the restriction enzymes also causes the target bacteria to be susceptible to phage infection, which is extremely unfavorable for the construction of industrial microbial fermentation strains; the versatility of the technology is not strong, and may only be applied to one or a few Bacteria
  • the method provided by the invention comprises the following steps: 1) co-expressing all DNA methyltransferase encoding genes in a target bacterial genome in E. coli deleted by the self-limiting modification system to obtain recombinant bacteria A;
  • step 1) the DNA methyltransferase encoding gene in the target bacterial genome is co-expressed in E. coli deleted by the self-limiting modification system to transfer all DNA methyl groups in the target bacterial genome.
  • the enzyme-encoding gene is introduced into the Escherichia coli in which the self-limiting modification system is deleted by a recombinant vector;
  • Step 2) includes the following steps:
  • the recombinant vector is inserted into the expression plasmid to obtain all the DNA methyltransferase coding genes, and a recombinant vector which co-expresses all DNA methyltransferases is obtained;
  • Each of the DNA methyltransferase encoding genes in the above recombinant vector may use a respective promoter or share a promoter (a structure constituting an operon).
  • the method of inducing culture is temperature induction, or induction using an inducer such as arabinose, IPTG, xylose or rhamnose.
  • the recombinant bacteria B is contained in the final concentration of 0.2% (quality 100%). Inducing culture in a liquid medium of arabinose;
  • the induced culture temperature is 25 ° C - 37 ° C, and the induction culture time is 3-24 hours;
  • the temperature for inducing the culture is preferably 30 ° C, and the time for inducing the culture is preferably 12 hours.
  • the target bacterium is a eubacteria or archaea containing a restriction modification system
  • the eubacteria or archaea containing the restriction modification system may be, but not limited to, Bacillus amyloliquefaciens
  • the Escherichia coli lacking the self-limiting modification system may be, but not limited to, Escherichia coll EC135 CGMCC NO. 5925; its restriction modification system genotype is mcrA ⁇ ⁇ mrr-hsdRMS-mcrB ⁇ dcm:: FRT ⁇ dam :: FRT.
  • the exogenous DNA molecule may be but is not limited pAD123, pAD43_25, pMK3, pMK4, pHCMC02, pHCMC04, pDG148StuI, pWYE748 or pBBRl- MCS5- P Nh - 345. - GFP.
  • all of the DNA methyltransferase encoding genes in the Bacillus amyloliquefaciens TA208 are BAMTA208-06525, BAMTA208_6715, BAMTA208_19835 and BAMTA208_16660; the nucleotide sequences of the BAMTA208_06525, BAMTA208_6715, BAMTA208_19835 and BAMTA208_16660 are in turn For sequence 2, sequence 3, sequence 4 and sequence 5 in the sequence listing;
  • a CC 10987 are BCE_0393, BCE_4605, BCE_5606, BCE_5607, BCE_0365 and BCE_0392; said BCE_0393, BCE_4605, BCE_5606, BCE_5607, BCE_0365 and BCE_0392
  • the nucleotide sequence is sequence 6, sequence 7, sequence 8, sequence 9, sequence 10 and sequence 11 in the sequence listing
  • the Hans nitrifying bacteria (i All DNA methyltransferase encoding genes in hamburgensis) lU are Nham_0569, Nham_0582, Nham_0803 and Nham_3225 ; the nucleotide sequences of Nham_0569, Nham_0582, Nham_0803 and Nham_3225 are sequence 12, sequence 13, sequence 14 and Sequence 15.
  • Also included in the above method is the step of determining all of the DNA methyltransferase encoding genes in the target bacterial genome:
  • (C) separately preparing the genomic DNA (including chromosomal DNA and expression vector) of Escherichia coli transferred to the expression vector, and detecting whether the DNA has been modified by methylation, and if it is determined that the DNA has been methylated, the prediction is predicted.
  • DNA methyltransferase does have a DNA methylation modification function, and its protein is active in Escherichia coli. Detection methods include high performance liquid chromatography and DNA dot hybridization.
  • DNA methyltransferases include type I, type I I and type I I methyltransferases, and type I DNA methyltransferases should include a methyl transfer subunit and a DNA recognition subunit.
  • the inducible expression vector can be either a low copy or a high copy vector, but should be compatible with the shuttle/integration plasmid that transforms the target bacteria.
  • the promoter of methyltransferase may be its own promoter, or may be an inducible promoter in E. coli, including but not limited to arabinose-inducible promoter, IPTG-inducible promoter and rhamnose induction. Promoter.
  • the methyltransferase induction temperature is from 8 ° C to 43 ° C.
  • the Escherichia coli in which the self-limiting modification system is deleted is a known restriction enzyme, and the DNA methyltransferase is completely deficient in Escherichia coli, and these enzymes include, but are not limited to, Dam, Dcm, EcoKI, Mrr, McrA and P Mrr.
  • Another object of the invention is to provide an Escherichia coli ⁇ Escherichia coll) EC135.
  • the Escherichia coli provided by the present invention has the accession number CGMCC No. 5925.
  • the strain EC135 was deposited on March 21, 2012 at the General Microbiology Center of the China Microbial Culture Collection Management Committee (CGMCC, Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101).
  • CGMCC China Microbial Culture Collection Management Committee
  • the accession number is CGMCC No. 5925, which is classified as Escherichia colD.
  • the method of transforming the target bacteria varies depending on the selected target bacteria, including but not limited to chemical transformation, junction transfer, electroporation, and protoplast transformation.
  • a plurality of methyltransferase genes can be assembled using Saccharomyces cerevisiae, and the construction of the co-expression plasmid can be completed within one week, which accelerates the process of overcoming the target bacterial restriction modification disorder;
  • the target bacterial transformant obtained by the method, the restriction modification system is consistent with the starting strain. This method does not cause damage to the original limiting modification system of the target bacteria;
  • the invention has strong versatility and has been successfully applied to two strains of Bacillus, one Gram-negative, chemically capable of self-supporting
  • the genetic manipulation of bacteria is expected to extend to more bacterial species.
  • the invention is of great significance for the construction of a refractory bacterial genetic operating system containing multiple sets of restriction modification systems.
  • Figure 1 shows the results of PCR detection of T0P10 tfcffl gene knockout.
  • Figure 2 shows the results of PCR detection of EC135 t affl gene knockout
  • Figure 3 shows the results of PCR amplification of B. amyloliquefaciens TA208 DNA methyltransferase gene
  • Figure 4 shows the detection of Bacillus amyloliquefaciens TA208 DNA methyltransferase activity by dot hybridization
  • Figure 5 shows the plasmid map of pWYE724
  • Figure 6 shows the EcoIN single-cut detection pM.
  • Figure 7 shows the pM. Bam plasmid map.
  • Figure 8 shows the transformation efficiency of the shuttle plasmid prepared by different hosts against Bacillus amyloliquefaciens TA208 (* not detected).
  • Figure 9 shows the results of PCR validation of B. amyloliquefaciens TA208 3 ⁇ 43 ⁇ 40 gene knockout.
  • Figure 10 shows the growth of Bacillus amyloliquefaciens TA208 and BS043 in ⁇ , MM+ 5-FU medium.
  • Figure 11 shows the DNA methyltransferase activity of Bacillus cereus ATCC 10987 by dot hybridization
  • Figure 12 shows HindV l l single cut detection pM. Bee electropherogram
  • Figure 13 shows the pM. Bee plasmid map.
  • Figure 14 shows the transformation efficiency of shuttle plasmids prepared by different hosts against Bacillus cereus ATCC 10987 (* not detected)
  • Figure 15 shows the X14 DNA methyltransferase activity of Hans nitrifying bacteria by dot hybridization
  • Figure 16 is a BanM single cut detection pM. Nham electropherogram
  • Figure 17 shows the pM. Nham plasmid map.
  • Figure 18 shows the expression of green fluorescent protein in Hans nitrifying bacteria X14
  • the following examples are provided to facilitate a better understanding of the invention but are not intended to limit the invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, were purchased from a conventional biochemical reagent store. In the quantitative tests in the following examples, three replicate experiments were set, and the results were averaged.
  • Escherichia coli T0P10 (Beijing Quanjin Biotechnology Co., Ltd., product No. CD101) was used as the starting strain, and the tfcffl gene (Dcm methylase encoding gene) was knocked out in turn to mutate the chromosome re gene into wild type and knock out t affl.
  • the gene (Dam methylase encoding gene) is obtained from Escherichia coli EC135, as follows:
  • Competent cells of the T0P10 strain were prepared, and plasmid pKD46 (purchased from Col i Genetic Stock Center, hereinafter referred to as CGSC, No. 7739) was transformed into the T0P10 strain, and ampicillin-resistant transformants were screened at 30 ° C to obtain T0P10/pKD46.
  • CGSC Col i Genetic Stock Center
  • the pKD3 plasmid (purchased from CGSC, No. 7631) was used as a template to amplify the chloramphenicol resistance gene, and 1166 bp of the gel was recovered to obtain a chloramphenicol resistance gene PCR product.
  • the T0P10/pKD46 strain was cultured in LB medium at 30 ° C at 0D 6 . .
  • 5 ⁇ L of the recovered chloramphenicol resistance gene PCR product was used to transform 4Q ⁇ L of competent cells, and after 1 hour of recovery at 30 ° C, The resuscitation product was spread on an LB plate containing 3 ⁇ g/mL chloramphenicol and cultured overnight at 37 °C. Recombinants were selected and colony PCR was performed using primers TO064 and TO065.
  • the size of the positive recombinant PCR product should be 1816 bp, while the original gene size was 1980 bp.
  • T0P10 tfcffl : : CmR strain was selected to prepare the electroporation competent state, and the pCP20 (purchased from the US CGSC, No. 7629) plasmid was transformed into the T0P10 tfcffl : : CmR strain, and the ampicillin resistant recombinant was selected.
  • Single colonies were grown to 0D 6 at 30 ° C in LB medium without antibiotics. . The temperature was 0.2 and then the temperature was raised to 42 ° C overnight.
  • the positive recombinant amplification product of the chloramphenicol resistance gene elimination should be 886 bp in size (Fig. 1), and the positive recombinant is named ⁇ 0 ⁇ 10 ⁇ tfcffl: : FRT.
  • the mutation re ⁇ gene must be returned before knocking out the ⁇ » gene.
  • the wild type re gene (1236 bp) was amplified using the chromosomal DNA of Escherichia coli W3110 (purchased from CGSC, USA No.
  • pKOV-recA was transformed into pKOV-re into TOP 10 ⁇ dcm: : FRT strain, chloramphenicol resistant transformants were screened at 30 °C, and transformants were picked and then in liquid LB medium at 42 °C. The overnight culture was carried out, and an LB plate containing chloramphenicol was applied to integrate pKOV-re into the chromosome recAl site. Colony PCR was performed using primers TO255 and TO256. If there is a PCR band greater than 7Kb, pKOV-re has been integrated into the chromosome.
  • the correct recombinants were picked, cultured at 42 °C, and applied to LB plates containing 10% sucrose. Single colonies were streaked on LB plates containing chloramphenicol and chloramphenicol, and passaged, and chloramphenicol was selected. Sensitive colonies. After amplification of the recA gene by primers TO255 and TO256, the base of the 482th gene of the i ⁇ cA gene should be G, and the strain of T0P10 is A.
  • the t affl gene was knocked out in the same manner as the tfcffl knockout step.
  • the chloramphenicol resistance gene amplification primers used were TO087 and TO088, and the outer detection primers were WB062 and TO063.
  • the size of the amplified product after gene knockout was 740 bp, and the original gene size was 1356 bp (Fig. 2).
  • the obtained strain with tfcffl t affl deletion and re-reversion mutation is EC135.
  • the above-mentioned strain EC135 was deposited on March 21, 2012 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC, Address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China) Zip code 100101), the accession number is CGMCC No. 5925, and its classification is named Escherichia colf.
  • the PCR primer sequences used are shown in Table 1.
  • WB582 GTTTAAAGAAGAATTAGCTATTGAGAGGAGGGAGAGTGATATTATGATAT
  • the strain TA208 encodes the DNA methyltransferase gene.
  • strain TA208 has been determined and is available under GenBank accession number CP002627. There are five putative genes encoding DNA methyltransferase on the chromosome. The locus_tag of the gene are BAMTA208_06525, BAMTA208_6715, BAMTA208_14440, BAMTA208_19835 and BAMTA208_16660, respectively. The PCR amplification results of the five gene fragments are shown in Figure 3.
  • strain TA208 encodes the DNA methyltransferase gene verification
  • the above five genes were cloned into the pBAD43 plasmid (Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. LM Guzman, D Bel in' MJ Carson, J Beckwith. Journal of Bacteriology, 1995 177 : 4121 -30, the public can obtain the pBAD43 plasmid containing BAMTA208_06525 between Afcol and 3 ⁇ 4al, fco/a and 3 ⁇ 4al, fco/a and 3 ⁇ 4al, f and Sail, EcoRi and S a ll sites.
  • pBAD43 plasmid containing BAMTA208_6715 pBAD43 plasmid containing BAMTA208_14440, pBAD43 plasmid containing BAMTA208_19835, and pBAD43 plasmid containing BAMTA208_16660; and then transforming 5 plasmids into Escherichia coli EC135, respectively Group bacteria 1-5.
  • the plasmid from which the recombinant bacteria 1-5 was extracted was sent to the sequencing and verified correctly, so the recombinant strain was a positive recombinant strain.
  • Dot blot validation verification methyltransferase enzyme expression induced by recombinant positive strain 1-5 (cultured in LB medium to 0D 6 .. After a final concentration of 0.2 to 0.2% (mass percentage The arabinose was induced at 30 °C for 12 hours), and then the total DNA was extracted with DNeasy Blood and Ti ssue Kit (Qiagen) to obtain DNA1_DNA5.
  • the DNA1-DNA5 obtained above was boiled for 3 min, respectively, and denatured into a single strand, and then inserted into an ice water mixture to be quenched.
  • a negative control was performed with EC135/pBAD43 (pBAD43 transferred to EC135).
  • the total DNA of EC135/pBAD43 and the five samples of DNA1-DNA5 were placed on a Protran BA85 nitrocellulose membrane (Whatman) at 450 ng, 150 ng and 50 ng, respectively, and three membranes were repeated. After 2 minutes of UV crosslinking, the membrane was placed in 5% skim milk powder prepared with TBST (200 mM NaCl, 0.1% Tween20, 50 mM Tris-HCl, pH 7.4), and blocked at room temperature (25 °C) for 1 hour. .
  • Fig. 4 The results are shown in Fig. 4 (m6A/m4C/m5C in the figure are the results of hybridization using different antibodies), and it can be seen that BAMTA208_06525, BAMTA208_6715, BAMTA208_19835 and BAMTA208_16660 have DNA methylation modification activity, and are DNA methyltransferases. gene.
  • the pBAD43 plasmid containing BAMTA208_06525 (SEQ ID NO: 2), the PBAD43 plasmid containing BAMTA208_6715 (SEQ ID NO: 3), the pBAD43 plasmid containing BAMTA208_19835 (SEQ ID NO: 4), and the pBAD43 plasmid containing BAMTA208_16660 (SEQ ID NO: 5) were used as templates, and the primer used for BAMTA208_06525 was WB325 and WB475 (size 839 bp, Figure 3), primers used for BAMTA208_6715 were WB476 and WB477 (size 1512 bp, Figure 3), primers used for BAMTA208-19835 were WB478 and WB479 (size 1794 bp, Figure 3), and primers used for BAMTA208_16660 were WB480 and WB326 (size 1272 bp, Figure 3), primer sequences as shown in Table 1, were subject
  • the four PCR products were separately subjected to gelation recovery, the four PCR products were mixed in equal amounts to a total volume of 50 ⁇ to obtain a total PCR product.
  • Transformants were selected on a complete synthetic medium (SC trp -, Beijing Panoroke) plate without the addition of tryptophan. Single colonies were picked into YPD medium, and yeast plasmids were extracted by glass beads (Nucleic Acids Research, 20, 3790). The plasmid was transformed into E. coli T0P10, and the transformants were screened with LB plates containing 100 g/mL spectinomycin. The plasmid was extracted and EcoW was cleaved. The correct recombinant plasmid should produce four bands of 8515 bp, 3633 bp, 1018 bp and 571 bp (Fig. 6). Shown), named pM. Bam (structure diagram as shown 7)), sent the sequencing results correctly.
  • the plasmid pM.Bam was transformed into the strain EC135 obtained in Example 1, and confirmed by the above method, the correct transformant was EC135/pM.Bam, which was accurately simulated by the DNA methylation pattern of B. amyloliquefaciens TA208. Host.
  • the shuttle plasmid pAD43-25 (7262 bp, purchased from Bacillus Genetic Stock Center, hereinafter, BGSC, product number ECE166) was transformed into the above-obtained EC135/pM. Bam to obtain recombinant bacteria, respectively.
  • the plasmid here includes pM. Bam and shuttle plasmid, but pWYE724 is a low copy plasmid, the copy number of pM.
  • Bam is about 5 cells/cell, and the copy number of the shuttle plasmid is about 300 /cell), transformation of Bacillus amyloliquefaciens TA208 (Complete sequence sequence of Bacillus amyloliquefaciens TA208, a strain for industrial production of guanosine and ribavirin, Guoqiang Zhang, Aihua Deng, Qingyang Xu, Yong Liang, Ning Chen, Tingyi Wen, Journal of Bacteriology, 193 (12): 3142-3143), Method as described in Analytical Biochemistry, 409, 130-137, gives TA208/pAD43_25 (EC135/pM. Bam).
  • the shuttle plasmid PAD43-25 was transferred to T0P10 and EC135 in the same manner, and then modified into TA208 to obtain TA208/pAD43-25 (T0P10) and TA208/pAD43_25 (EC135), respectively.
  • the shuttle plasmid pAD123 (5952 bp, purchased from BGSC, USA, numbered ECE165), pMK3 (7214 bp, purchased from BGSC, USA, numbered ECE15), pMK4 (5585 bp, purchased from BGSC, USA, numbered ECE16), pHCMC02 (6866 bp, was used in the same manner. Purchased from US BGSC, number ECE188), pHCMC04 (8089bp, purchased from American BGSC, number ECE189), pDG148StuI (8272bp, purchased from American BGSC, number ECE145) were transferred to EC135/pM. Bam, T0P10 and EC135 were modified. Transfer to TA208, respectively, as follows:
  • TA208/ pAD123 (EC135/pM. Bam), TA208/ pAD123 (T0P10), TA208/ pAD123 (EC135), TA208/ pMK3 (EC135/pM. Bam), TA208/ pMK3 (T0P10), TA208/ pMK3 (EC135), TA208/ pMK4 (EC135/pM. Bam), TA208/ pMK4 (T0P10), TA208/ pMK4 (EC135), TA208/pHCMC02 (EC135/pM.
  • TA208/pAD43-25 (EC135/pM. Bam), TA208/pAD43-25 (T0P10) and TA208/pAD43-25 (EC135)
  • TA208/ pAD123 (EC135/pM. Bam)
  • TA208/ pAD123 (T0P10)
  • TA208 / pAD123 (EC135)
  • TA208/ pMK3 ( EC135/pM. Bam )
  • TA208/ pMK3 T0P10
  • TA208/ pMK3 ( EC135 )
  • TA208/ pMK4 (EC135/pM.
  • the conversion rate was: single colony number (CFU) X (volume of resuscitation fluid after conversion / volume of coated bacterial solution) X resuscitation solution dilution ratio / amount of shuttle plasmid used for transformation (g).
  • the conversion rate of TA208/pAD43-25 (T0P10) is 0;
  • the conversion rate of TA208/pAD43-25 (EC135) was 2 ⁇ 4 ⁇ 1 ⁇ 6 X 10 3 CFU/ ⁇ g DNA;
  • the conversion rate of TA208/pAD123 (EC135/pM. Bam) was 9. 1 ⁇ 4. 3 X 10 5 CFU/ ⁇ g DNA;
  • the conversion rate of TA208/ pAD123 (T0P10) is 0;
  • the conversion rate of TA208/ pAD123 (EC135) is 0;
  • the conversion rate of TA208/pMK3 (EC135/pM. Bam) was 2. 2 ⁇ 0 ⁇ 5 X 10 5 CFU/g DNA;
  • the conversion rate of TA208/ pMK3 (T0P10) is 0;
  • the conversion rate of TA208/pMK3 (EC135) was 4. 5 X lO FU/ ⁇ g DNA;
  • the conversion rate of TA208/ pMK4 (T0P10) is 0;
  • the conversion of TA208/pMK4 (EC135) is 3. 1 X 10 2 CFU/ ⁇ g DNA;
  • the conversion of TA208/pHCMC02 (EC135/pM. Bam) is 2.0 ⁇ 0.33 X 10 6 CFU / ⁇ g DNA;
  • the conversion rate of TA208/pHCMC02 (T0P10) is 0;
  • the conversion rate of TA208/pHCMC02 (EC135) was 6. 3 X lO FU/ ⁇ g DNA;
  • the conversion rate of TA208/pHCMC 04 (EC135/pM. Bam) was 1. 9 ⁇ 0 ⁇ 1 X 10 6 CFU/ ⁇ g DNA;
  • the conversion rate of TA208/pHCMC 04 (T0P10) is 0;
  • the conversion rate of TA208/pHCMC 04 (EC135) is 1 ⁇ 5 X lO FU/ ⁇ g DNA;
  • the conversion rate of TA208/pDG148StuI (EC135/pM. Bam) was 9. 7 ⁇ 3. 2 X 10 4 CFU/ ⁇ g DNA; the conversion rate of TA208/ pDG148StuI (T0P10) was 0;
  • the conversion rate of TA208/pDG148StuI (EC135) is 0;
  • the plasmid prepared by T0P10 could not transform strain TA208, and the plasmid conversion rate prepared in EC135 strain was 0 ⁇ 10 3 CFU/Vg DNA level, while the conversion rate of shuttle plasmid prepared in EC135/pM. Bam was 10 4 ⁇ 10 6 CFU/Vg DNA level. .
  • the plasmids of the single colonies of each of the bacteria were extracted and compared with the plasmids before the transfer, and the results were identical in size, indicating that these strains were positive plasmids for transferring into foreign DNA molecules.
  • the integrated plasmid is introduced into the target bacteria.
  • the integrated plasmid PWYE748 containing the 3 ⁇ 4o gene homology arm and the chloramphenicol resistance gene was prepared as follows: primers WB607 and WB608 were used as primers, and TA208 chromosomal DNA was used as a template to amplify the 641 bp homologous arm of the 3 ⁇ 43 ⁇ 4o gene; WB609, WB610 As a primer, the plasmid pMK4 was used as a template to amplify the chloramphenicol resistance gene of 906 bp .
  • TA208 chromosomal DNA was used as a template to amplify the 669 bp downstream of the 3 ⁇ 43 ⁇ 40 gene. After the three PCR products were recovered, Take 1 each The ⁇ L was mixed and used as a template, and a 2216 bp fragment was obtained by PCR using TO607 and WB612 as primers. The PCR product was cloned into the PMD19-T plasmid (Taraka, Cat. No. D106A), and the resulting plasmid was the integration plasmid pWYE748.
  • the integrated plasmid PWYE748 was modified in EC135/pM.Bam, and then the strain TA208 was transformed and the chloramphenicol-resistant transformant was selected.
  • the primers TO605 and TO606 were amplified by PCR, and the genomic DNA of the transformant DNA and the wild type TA208 was used as a template.
  • the amplification result is shown in Fig. 9.
  • the transformant amplification size was 2375 bp (the chloramphenicol resistance gene was inserted into the 3 ⁇ 43 ⁇ 4o).
  • the gene), and the original chloramphenicol resistance gene of wild type TA208 was 2049 bp, which proved to be transferred to a foreign gene, and the integration was successful, and the transformant was named BS043.
  • the 3 ⁇ 4o gene encodes a uracil phosphoribosyltransferase that converts 5-fluorouracil to 5-fluorouracil monophosphate and is ultimately metabolized to the toxic metabolite 5-fluorouracil diphosphate, which strongly inhibits the activity of thymidylate synthase , causing the death of the bacteria.
  • BS043 introduced a foreign gene by the following method:
  • the strain BS043 was grown on MM medium without adding and adding 10 ⁇ 5_fluorouracil (Sigma, Cat. No. F6627) (see Molecular Microbiology, 46, 25-36. for the composition of the medium). 100 mg/L of adenosine was added, and strain TA208 was used as a control. The results are shown in Fig. 10. It can be seen that BS043 can be grown on sputum medium supplemented with 10 ⁇ 5-fluorouracil, indicating that the foreign gene was transferred and the integration was successful.
  • Example 3 Exogenous DNA Molecules Overcome Limiting Modification Disorders Introducing Bacillus cereus ( C ii « cereus) A CC 10987
  • the whole genome sequence of the strain ATCC 10987 has been published, and the GenBank accession number is AE017194. There are 9 putative genes encoding DNA methyltransferase on the chromosome.
  • the locus_tag of the gene are BCE_0841_BCE_0842, BCE_0839-BCE_0842, BCE_0365, BCE_0392, BCE_0393, BCE_4605, BCE_5606, BCE_5607 and BCE_1018.
  • BCE_0841— BCE_0842, BCE_0839— BCE_0842, BCE_0365, BCE_0392, BCE_0393, BCE_4605 BCE—5606, BCE—5607 and BCE—1018 were cloned into pBAD43 plasmids Nhel and Kpnl, Nco l and Kpnl, Nhel and Kpnl, Nhel and Kpnl, respectively.
  • Nhel and Kpnl Between Nhel and Kpnl, Nhel and Kpnl, Nhel and Kpnl, Nhel and Xbal, Nhel and Kpnl sites, a pBAD43 plasmid containing BCE_0841-BCE_0842, a PBAD43 plasmid containing BCE_0839_BCE_0842, and a pBAD43 plasmid containing BCE_0365 were obtained.
  • the above plasmids were separately transformed into Escherichia coli EC135 to obtain recombinant bacteria 1-9.
  • the plasmid from which the recombinant strain 1-9 was extracted was sent to the sequencing and verified correctly, so the recombinant strain was a positive recombinant strain.
  • DNA1_DNA9 was obtained.
  • the DNA1-DNA9 obtained above was boiled for 3 min to be denatured into a single strand and then quenched by inserting into an ice water mixture. EC135/pBAD43 was used as a negative control.
  • the total DNA of EC135/pBAD43 and 9 samples of DNA1-DNA9 were each placed on a Protran BA85 nitrocellulose membrane (Whatman) at 450 ng, 150 ng and 50 ng points, and three membranes were repeated. After 2 minutes of UV cross-linking, the membrane was placed in 5% skim milk powder prepared with TBST (200 mM NaCl, 0.1% Tween 20, 50 mM Tris-HCl, pH 7.4), and blocked at room temperature for 1 hour.
  • TBST 200 mM NaCl, 0.1% Tween 20, 50 mM Tris-HCl, pH 7.4
  • the six PCR products were separately recovered by gelation, the six PCR products were mixed in equal proportions and concentrated to a total volume of 50 ⁇ to obtain a total PCR product.
  • the plasmid pM. Bce was transformed into the strain EC135 obtained in Example 1, and the correct transformant was EC135/pM. Bce according to the above method, and the DNA methylation pattern of Bacillus cereus ATCC 10987 was accurately simulated. Host.
  • EC135/pM EC135/pM.
  • Bee was used at a final concentration of 0.2% arabinose for induction at 30 °C for 12 hours, methyltransferase gene expression, total DNA extraction, strain ATCC
  • the chromosomal DNA of 10987 was detected by point hybridization according to the previous method. The results showed that there was no significant difference between the hybridization results of EC135/pM. Bee and ATCC 10987, indicating that methyltransferase was expressed, indicating that EC135/pM. Bce was co-expressed. Recombinant bacteria encoding all DNA methyltransferase genes.
  • the shuttle plasmid overcomes the restriction of the modification of the barrier into the target bacteria
  • the shuttle plasmid was transferred to EC135/pM. Bce, EC135 and TOP10, respectively, and arabinose induction (method see above) was followed by extraction of the plasmid, and the strain ATCC 10987 was transformed, respectively.
  • the method was the same as in Example 2, and the shuttle plasmid was pAD43_25. pAD123, pMK3, pMK4, and pHCMC02 were obtained as follows:
  • ATCC 10987/ pAD43-25 (EC135/pM. Bce), ATCC 10987/ pAD43-25 (TOPIO), ATCC 10987/ pAD43-25 (EC135), ATCC 10987/ pAD123 (EC135/pM. Bce), ATCC 10987/ pAD123 (TOPIO), ATCC 10987/ pAD123 (EC135), ATCC 10987/ pMK3 (EC135/pM. Bce), ATCC 10987/ pMK3 (TOPIO), ATCC 10987/ pMK3 (EC135), ATCC 10987/ pMK4 (EC135/pM.
  • Bce ATCC 10987/ pMK4 (TOPIO), ATCC 10987/ pMK4 (EC135), ATCC 10987/pHCMC02 (EC135/pM. Bce), ATCC 10987/pHCMC02 (TOPIO), ATCC 10987/pHCMC02 (EC135);
  • the conversion rate was: single colony number (CFU) X (volume of resuscitation fluid after conversion / volume of coated bacterial solution) X resuscitation solution dilution ratio / amount of shuttle plasmid used for transformation (g).
  • the conversion rate of ATCC 10987/ pAD43-25 (EC135/pM. Bee) was 3.2 ⁇ 1.3X 10 5 CFU / ⁇ g DNA;
  • the conversion rate of ATCC 10987/ pAD43-25 was 8.3 ⁇ 0.7X 10 3 CFU / ⁇ g DNA;
  • the conversion rate of ATCC 10987/ pAD43-25 (EC135) was 7.1 ⁇ 2.9 X 10 3 CFU/ ⁇ g DNA;
  • the conversion rate of ATCC 10987/ pAD123 (EC135/pM. Bee) was 9.0 ⁇ 1.4X 10 5 CFU/ ⁇ g DNA;
  • the conversion rate of ATCC 10987/ pAD123 was 8.7 ⁇ 0.8X 10 3 CFU/ ⁇ g DNA;
  • the conversion rate of ATCC 10987/ pAD123 (EC135) was 5.7 ⁇ 3.3X 10 3 CFU/ ⁇ g DNA;
  • the conversion rate of ATCC 10987/ pMK3 (EC135/pM. Bee) was 8.4 ⁇ 2.2 ⁇ 10 5 CFU/ ⁇ g DNA;
  • the conversion rate of ATCC 10987/ pMK3 was 8.7 ⁇ 2.7X 10 2 CFU/ ⁇ g DNA;
  • the conversion rate of ATCC 10987/ pMK3 (EC135) was 1.2 ⁇ 0 ⁇ 6 ⁇ 10 3 CFU/ ⁇ g DNA;
  • the conversion rate of ATCC 10987/ pMK4 (EC135/pM. Bee) was 2.3 ⁇ 0.2 ⁇ 10 7 CFU/ ⁇ g DNA;
  • the conversion rate of ATCC 10987/ pMK4 was 3.3 ⁇ 0.4X 10 5 CFU/ ⁇ g DNA;
  • the conversion rate of ATCC 10987/ pMK4 (EC135) was 3.5 ⁇ 1.4X 10 5 CFU/ ⁇ g DNA;
  • the conversion rate of ATCC 10987/pHCMC02 (EC135/pM. Bee) was 2.5 ⁇ 1.5 ⁇ 10 5 CFU/ ⁇ g DNA;
  • the conversion rate of ATCC 10987/ pHCMC02 (T0P10) is 0;
  • the conversion rate of ATCC 10987/pHCMC02 (EC135) is 0;
  • the plasmid conversion rate prepared in EC135/pM. Bee is 10 2 to 10 3 times the plasmid conversion rate prepared in TOP10 and EC135.
  • the plasmids of the single colonies of each of the bacteria were extracted and compared with the plasmids before the transfer, and the results were identical in size, indicating that these strains were positive plasmids for transferring into foreign DNA molecules.
  • Example 4 Exogenous DNA molecules overcome the restriction modification disorder and introduce Hans nitrifying bacteria (iir 0 1 ⁇ 2 C ter hamburgensis) X14
  • Nham_0569, Nham_0582, Nham_0803, Nham_0842, Nham_1185, Nham_1353, Nham_2515, Nham_3225, Nham_3845 and Nham_4499 were cloned into pBAD43 plasmids, EcoRI and KpnI, EcoRI and Kpnl, Nhel and Kpnl, Nhel and Kpnl, EcoRI and Kpnl, EcoRI, respectively.
  • the plasmid extracted from the recombinant strain 1-10 was sent to the sequencing and verified correctly, so the recombinant strain was a positive recombinant strain.
  • DNA1_DNA10 was obtained.
  • the DNA1-DNA10 obtained above was boiled for 3 min to be denatured into a single strand and then quenched by inserting into an ice water mixture. EC135/pBAD43 was used as a negative control.
  • the total DNA of EC135/pBAD43 and 10 samples of DNA1-DNA10 were each placed on a Protran BA85 nitrocellulose membrane (Whatman) at 450 ng, 150 ng, and 50 ng, respectively, and three membranes were repeated. After 2 minutes of UV crosslinking, the membrane was placed in 5% skim milk powder prepared with TBST (200 mM NaCl, 0.1% Tween 20, 50 mM Tris-HCl, pH 7.4), and blocked at room temperature for 1 hour.
  • TBST 200 mM NaCl, 0.1% Tween 20, 50 mM Tris-HCl, pH 7.4
  • Fig. 15 The results are shown in Fig. 15 (m6A/m4C/m5C in the figure are the results of hybridization using different antibodies), and it can be seen that Nham_0569, Nham_0582, Nham_0803 and Nham_3225 have DNA methylation modification activity and are DNA methyltransferases. gene.
  • the four PCR products were separately subjected to gelation recovery, the four PCR products were mixed in equal proportions and concentrated to a total volume of 50 ⁇ to obtain a total PCR product.
  • the pM. Nham was transformed into the strain EC135 obtained in Example 1, and the correct transformant was EC135/pM. Nham according to the above method, and the DNA methylation pattern of Han's nitrifying bacteria X14 was accurately simulated. .
  • the shuttle plasmid overcomes the restriction of the modification of the barrier into the target bacteria
  • Plasmid pBBRl-MCS5-P Nh — 345 The specific construction method of GFP was as follows: TO654 and TO655 were used as primers, and the X17 genomic DNA was used as a template to amplify the 216 bp promoter fragment of Nham_3450 gene; WB656 and WB650 were used as primers, and pAD123 was used as a template to amplify GFP gene 717 bp; After the gel was recovered, 1 ⁇ L of each was used as a template, and PCR was amplified by using TO654 and WB650 as primers.
  • the 933 bp PCR product was obtained and cloned into pBBR1-MCS5 (Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying Different antibiotic-resistance cassettes.
  • pBBR1-MCS5 Flu new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying Different antibiotic-resistance cassettes.
  • -GFP is a broad-host plasmid containing the green fluorescent protein-encoding gene under the control of Nham_3450, which is transferred to EC135/pM. Nham. After arabinose induction, the plasmid is transformed and transformed into strain X14.
  • the transformation method of strain X14 is as follows: DSM756a medium (1.5 g yeast extract, 1. 5 g peptone, 2 g NaN0 2 ,
  • the cells were washed into 100 mL of 756a medium, resuscitated at 28 ° C for 1 day, and then incubated with a final concentration of 20 ⁇ g / mL of gentamicin for 1 day.
  • the resuscitation mixture was transferred to fresh 756a medium containing 20 ⁇ g/mL gentamicin at a volume ratio of 1/100.
  • Medium 0D 6 . .
  • After being 0.1 transfer again in the same manner until the cells grow to 0D 6 . . 0. 1, X14/ pBBRl-MCS5-P Nh stii_ 3450 -GFP (EC135/pM.

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Abstract

本发明公开了一种克服靶细菌限制修饰障碍导入外源DNA的方法。本发明提供的方法，包括如下步骤：1）将靶细菌基因组中所有DNA甲基转移酶编码基因在自身限制修饰系统缺失的大肠杆菌中共表达，得到重组菌A；2）将外源DNA分子导入所述重组菌A进行体内修饰，得到甲基化修饰的外源DNA分子；3）将所述甲基化修饰的外源DNA分子导入所述靶细菌中。本发明的实验证明，本发明与现有的克服细菌限制修饰障碍，实现遗传操作的方法相比转化率高。

Description

一种克服靶细菌限制修饰障碍导入外源 DNA的方法 技术领域
本发明涉及生物技术领域，尤其涉及一种克服靶细菌限制修饰障碍导入外源 DNA的方法。 背景技术
细菌限制修饰系统由限制性内切酶 （限制酶） 与 DNA甲基转移酶组成， 前者能够特异性 识别并切割 DNA， 后者能够向 DNA的碱基添加一个甲基修饰， 从而防止限制酶对 DNA的切割。 限制修饰系统能够选择性的降解侵入细菌内部的外源 DNA， 实现细菌自我保护的目的。 根据 限制修饰系统的亚基组成、 切割位点、 序列特异性与辅因子特征， 将其划分为四大类型。 I 型、 II型和 III型限制修饰系统的限制酶亚基能够识别并切割未甲基化的 DNA， 但如果 DNA 首先被甲基转移酶亚基识别并修饰后， 限制酶即不能完成切割。 IV型限制修饰系统只有限制 酶组成， 不含甲基转移酶， 它识别并切割具有外源甲基化模式的 DNA， 因此， 是一种甲基化 依赖的限制酶。 另外， 最近研究表明 DNA骨架的磷硫酰修饰酶及其对应的限制酶是一类新型 的限制修饰系统 （Nucleic Acids Research, 38， 7133-7141 )。 这种复杂的修饰 -切割模式， 极大的保护了细菌自身 DNA的安全性，是细菌作为有效防止噬菌体、环境中死细菌释放的 DNA 等外源 DNA入侵的主要手段。 同时， 这也成为现今利用分子生物学手段将外源 DNA导入细菌、 实现遗传操作的主要障碍。 含有多套限制修饰系统细菌的遗传操作尤为困难。
迄今为止研究人员发明了两类技术克服限制修饰障碍。 第一类策略即修饰外源 DNA的策 略， 包括体外修饰和大肠杆菌体内修饰。 如利用靶细菌粗蛋白抽提物（含有 DNA甲基转移酶） 体外修饰外源 DNA，能够实现对幽门螺杆菌、蜡样芽胞杆菌和韦氏芽胞杆菌的转化（Molecular Microbiology, 37， 1066-1074， Applied and Environmental Microbiology, 74， 7817-7820)； 或在大肠杆菌中克隆表达靶细菌 DNA甲基转移酶，体内修饰外源质粒 DNA，如在大肠杆菌 T0P10 中克隆表达青春棒杆菌的两个 DNA甲基转移酶， 进行穿梭质粒的体内修饰， 实现了青春棒杆 菌的遗传转化 （Nucleic Acids Research, 37, e3)。 第二类方法即灭活限制修饰系统的方法， 包括利用物理手段灭活和基因敲除。 利用转化后加热的方法暂时性灭活靶细菌限制酶， 外源 质粒对谷氨酸棒杆菌转化率提高至 108 CFU/ μ g DNA ( Microbial Biotechnology, 52， 541-545)； 敲除丙酮丁醇梭菌 CAC1502基因后可以使其接受非甲基化质粒 DNA (PLoS ONE, 5， e9038 ) ; 但也有报道表明敲除 Saul 限制性内切酶不足以使金黄色葡萄球菌接受外源 DNA (Applied and Environmental Microbiology, 75， 3034-3038)。
虽然上述技术可以在一定程度上提高外源 DNA对靶细菌的转化率， 但是还存在以下问题 和不足之处： 部分方案虽然能够利用靶细菌 DNA甲基转移酶在体外修饰外源 DNA分子， 但利 用粗蛋白抽提物的体外修饰效率较低， 且在修饰的同时限制酶也会降解部分质粒； 体内修饰 方案未解除大肠杆菌自身 DNA甲基转移酶对外源 DNA分子的甲基化， 这种带有大肠杆菌甲基 化模式的 DNA易激活靶细菌的 II I型限制系统； 许多细菌的限制酶为非热敏感性， 不能利用 加热手段暂时灭活； 如果细菌含有多套限制修饰系统时， 逐个敲除限制酶基因费时费力， 同 时敲除限制酶还造成靶细菌易受噬菌体感染， 对工业微生物发酵菌种的构建极为不利； 技术 的通用性不强， 可能仅适用于一种或少数几种细菌
发明内容
本发明的一个目的是提供一种将外源 DNA分子导入靶细菌的方法。
本发明提供的方法， 包括如下步骤： 1 )将一株靶细菌基因组中所有的 DNA甲基转移酶编码基因在自身限制修饰系统缺失的大 肠杆菌中共表达， 得到重组菌 A;
2 )将外源质粒 DNA分子导入所述重组菌 A进行体内修饰， 得到甲基化修饰的外源 DNA质 粒分子；
3 ) 将所述甲基化修饰的外源质粒 DNA分子导入所述靶细菌中。
上述方法中， 步骤 1 ) 中， 所述将靶细菌基因组中所有的 DNA 甲基转移酶编码基因在自 身限制修饰系统缺失的大肠杆菌中共表达为将所述靶细菌基因组中所有的 DNA甲基转移酶编 码基因通过重组载体导入所述自身限制修饰系统缺失的大肠杆菌内；
步骤 2 ) 包括如下步骤：
A) 将所述外源质粒 DNA分子导入所述重组菌 A中， 得到重组菌 B;
B) 诱导培养所述重组菌 B， 得到诱导后重组菌 B;
C) 提取所述诱导后重组菌 B的质粒 DNA， 即得到甲基化修饰的外源质粒 DNA分子。
上述方法中， 步骤 1 ) 中， 所述重组载体为将所述所有 DNA 甲基转移酶编码基因均插入 表达质粒中， 得到共表达所有 DNA甲基转移酶的重组载体；
上述重组载体中的每个 DNA甲基转移酶编码基因可以使用各自的启动子或共用一个启动 子 （构成操纵子的结构）。
步骤 2 )的 B)中，所述诱导培养采用的方法为温度诱导，或使用诱导剂如阿拉伯糖、 IPTG、 木糖或鼠李糖诱导。
上述方法中， 步骤 2 ) 的 B) 中， 所述诱导培养为将重组菌 B在诱导条件下培养； 最优诱导条件为将所述重组菌 B在含有终浓度为 0. 2% (质量百分含量） 的阿拉伯糖的液 体培养基中诱导培养；
所述诱导培养的温度为 25°C-37°C， 所述诱导培养的时间为 3-24小时；
所述诱导培养的温度优选为 30°C， 所述诱导培养的时间优选为 12小时。
上述方法中， 所述靶细菌为含有限制修饰系统的真细菌或古生菌， 所述含有限制修饰系 统的真细菌或古生菌可以是但不局限于解淀粉芽胞杆菌 i Bacillus amyloliquefaciens)
TA208、 蜡样芽胞杆菌 Bacillus cereus) A CC 10987 或汉氏硝化细菌 (M trobacter hamburgensis) X14;
所述自身限制修饰系统缺失的大肠杆菌可以是但不局限于大肠杆菌 (Escherichia coll) EC135 CGMCC NO. 5925； 其限制修饰系统基因型为 mcrA Δ {mrr-hsdRMS-mcrB Δ dcm:： FRT Δ dam:： FRT。
上述方法中， 所述外源 DNA 分子可以是但不局限于 pAD123、 pAD43_25、 pMK3、 pMK4、 pHCMC02、 pHCMC04、 pDG148StuI、 pWYE748或 pBBRl- MCS5- PNh— 345。- GFP。
上述方法中， 所述解淀粉芽胞杆菌 (bacillus amyloliquefaciens) TA208 中所有 DNA 甲基转移酶编码基因为 BAMTA208— 06525 、 BAMTA208_6715 、 BAMTA208_19835 和 BAMTA208_16660;所述 BAMTA208_06525、 BAMTA208_6715、 BAMTA208_19835和 BAMTA208_16660 的核苷酸序列依次为序列表中的序列 2、 序列 3、 序列 4和序列 5;
所述蜡样芽胞杆菌（ aci i« cereus) A CC 10987 中所有 DNA 甲基转移酶编码基因为 BCE_0393、 BCE_4605、 BCE_5606、 BCE_5607、 BCE_0365和 BCE_0392;所述 BCE_0393、 BCE_4605、 BCE_5606、 BCE_5607、 BCE_0365和 BCE_0392的核苷酸序列依次为序列表中的序列 6、序列 7、 序列 8、 序列 9、 序列 10和序列 11 ; 所述汉氏硝化细菌（ i
Figure imgf000004_0001
hamburgensis) lU中所有 DNA 甲基转移酶编码基因为 Nham_0569、 Nham_0582、 Nham_0803和 Nham_3225; 所述 Nham_0569、 Nham_0582、 Nham_0803 和 Nham_3225的核苷酸序列依次为序列表中的序列 12、 序列 13、 序列 14和序列 15。
在上述方法前还包括确定靶细菌基因组中所有 DNA甲基转移酶编码基因的步骤：
( a) 通过同源序列比对等方法， 预测靶细菌中编码 DNA甲基转移酶的基因；
(b )将预测的所有编码 DNA甲基转移酶的基因分别连入可诱导的表达载体， 再转入自身 限制修饰系统缺失的大肠杆菌中， 然后分别诱导培养。
( C)分别制备上述转入表达载体的大肠杆菌的基因组 DNA (包括染色体 DNA与表达载体）， 检测 DNA是否含已被甲基化修饰， 如果确定 DNA已被甲基化修饰， 即证明预测的 DNA甲基转 移酶确实具有 DNA甲基化修饰功能， 并且其蛋白在大肠杆菌中具有活性。 检测方法包括高效 液相色谱法和 DNA点杂交方法。
上述 DNA甲基转移酶， 包括 I型、 I I型与 I I I型甲基转移酶， I型 DNA甲基转移酶应包 括甲基转移亚基与 DNA识别亚基。
其中所述可诱导的表达载体， 可以是低拷贝， 也可以是高拷贝载体， 但应与转化靶细菌 的穿梭 /整合质粒能够相容。 甲基转移酶的启动子可以是其自身的启动子， 也可以是在大肠杆 菌中可诱导型的启动子， 包括但不局限于阿拉伯糖诱导启动子、 IPTG诱导型启动子和鼠李糖 诱导启动子。 甲基转移酶诱导温度为 8°C-43°C。
其中所述自身限制修饰系统缺失的大肠杆菌为已知的限制酶、 DNA 甲基转移酶完全缺失 的大肠杆菌， 这些酶包括但不局限于 Dam、 Dcm、 EcoKI、 Mrr、 McrA禾 P Mrr。
本发明的另一个目的是提供一株大肠杆菌 ^ Escherichia coll) EC135。
本发明提供的大肠杆菌， 其保藏编号为 CGMCC NO. 5925。
菌株 EC135于 2012年 3月 21 日保藏于中国微生物菌种保藏管理委员会普通微生物中心 (简称 CGMCC, 地址： 北京市朝阳区北辰西路 1号院 3号， 中国科学院微生物研究所， 邮编 100101 )， 保藏号为 CGMCC No. 5925， 其分类命名为大肠埃希氏菌 Escherichia colD。
其中所述转化靶细菌的方法， 因选择的靶细菌不同而异， 包括但不局限于化学转化法、 接合转移、 电转化法及原生质体转化法。
本发明的实验证明， 本发明与现有的克服细菌限制修饰障碍， 实现遗传操作的方法相比， 优点在于：
1. 使用一株自身限制修饰系统完全缺失的大肠杆菌作为宿主， 一方面克服了大肠杆菌修 饰模式 （Dam、 Dcm和 EcoKI )对靶细菌 IV型限制修饰系统的激活作用， 另一方面， 大肠杆菌 Mrr、 McrA和 McrBC的缺失有利于靶细菌 DNA甲基转移酶的克隆表达；
2. 对靶细菌所有的 DNA甲基转移酶进行共表达， 真正实现靶细菌 DNA甲基化模式的精确 模拟；
3. 使用的是在大肠杆菌体内 DNA甲基化修饰的方式， 在培养细菌的过程中同时完成， 无 需额外的体外反应， 无需添加甲基化反应底物， 方便快捷；
4. 在本发明中可使用酿酒酵母组装多个甲基转移酶基因， 共表达质粒的构建可在一周内 完成， 加速了靶细菌限制修饰障碍克服的过程；
5. 利用本方法得到的靶细菌转化子， 其限制修饰系统与出发菌株一致。 本方法不会对靶 细菌原有的限制修饰系统造成损伤；
6. 本发明具有很强的通用性， 已成功应用于两株芽胞杆菌， 一株革兰氏阴性、 化能自养 细菌的遗传操作， 并有望扩展至更多的细菌种属。
本发明对于含有多套限制修饰系统的顽固型细菌遗传操作系统构建具有重要意义。
附图说明
图 1为 T0P10 tfcffl基因敲除 PCR检测结果
图 2为 EC135 t affl基因敲除 PCR检测结果
图 3为解淀粉芽胞杆菌 TA208 DNA甲基转移酶基因 PCR扩增结果
图 4为点杂交检测解淀粉芽胞杆菌 TA208 DNA甲基转移酶活性
图 5为 pWYE724质粒图谱
图 6为 EcoIN单切检测 pM. Bam电泳图
图 7为 pM. Bam质粒图谱
图 8为不同宿主制备的穿梭质粒对解淀粉芽胞杆菌 TA208的转化效率 （* 未检测到） 图 9为解淀粉芽胞杆菌 TA208 ¾¾0基因敲除 PCR验证结果
图 10为解淀粉芽胞杆菌 TA208和 BS043在匪、 MM+ 5-FU培养基生长情况
图 11为点杂交检测蜡样芽胞杆菌 ATCC 10987 DNA甲基转移酶活性
图 12为 HindV l l单切检测 pM. Bee电泳图
图 13为 pM. Bee质粒图谱
图 14为不同宿主制备的穿梭质粒对蜡样芽胞杆菌 ATCC 10987的转化效率 （ * 未检测 到）
图 15为点杂交检测汉氏硝化细菌 X14 DNA甲基转移酶活性
图 16为 BanM单切检测 pM. Nham电泳图
图 17为 pM. Nham质粒图谱
图 18为绿色荧光蛋白在汉氏硝化细菌 X14中的表达
具体实施方式
以下的实施例便于更好地理解本发明， 但并不限定本发明。 下述实施例中的实验方法， 如无特殊说明， 均为常规方法。 下述实施例中所用的试验材料， 如无特殊说明， 均为自常规 生化试剂商店购买得到的。 以下实施例中的定量试验， 均设置三次重复实验， 结果取平均值。
实施例 1、 限制修饰系统完全缺失的大肠杆菌 EC135的构建
以大肠杆菌 T0P10 (北京全式金生物技术有限公司， 货号为 CD101 )为出发菌株， 依次敲 除 tfcffl基因（Dcm甲基化酶编码基因），将染色体 re 基因突变为野生型，敲除 t affl基因（Dam 甲基化酶编码基因） 得到大肠杆菌 EC135, 具体如下：
制备 T0P10菌株的感受态细胞， 将质粒 pKD46 (购自美国 Col i Genetic Stock Center, 以下简写为 CGSC， 编号 7739 ) 转化入 T0P10菌株， 30°C筛选氨苄青霉素抗性转化子， 得到 T0P10/ pKD46。
利用引物 WB089与 TO090, 以 pKD3质粒 （购自美国 CGSC, 编号 7631 ) 为模板扩增氯霉 素抗性基因， 切胶回收 1166bp， 得到氯霉素抗性基因 PCR产物。
将 T0P10/ pKD46菌株在 LB培养基中 30°C培养， 在 0D6。。为 0. 2时加入终浓度 lOOmM的阿 拉伯糖诱导 1小时， 制备感受态后利用 5 μ L 回收的氯霉素抗性基因 PCR产物转化 4Q μ L感 受态细胞， 30°C复苏 1小时后将复苏产物涂布于含有 3^ μ g/mL氯霉素的 LB平板， 37°C培养 过夜。 挑选重组子， 利用引物 TO064和 TO065进行菌落 PCR鉴定。 阳性重组子 PCR产物大小 应为 1816bp， 而原始基因大小为 1980bp。 挑取阳性重组子， 在 LB液体培养基中 42 °C连续培 养三代， 以消除 PKD46质粒， 得到 TOP 10 cfcffl: : CmR菌株。
稀释涂布平板后挑取单菌落 T0P10 tfcffl: : CmR菌株制备电转感受态， 将 pCP20 (购自美国 CGSC, 编号 7629 ) 质粒转化入 T0P10 tfcffl: : CmR菌株， 筛选氨苄青霉素抗性重组子后挑单菌 落在不含抗生素的 LB培养基中 30 °C培养至 0D6。。为 0. 2， 然后提高温度至 42 °C培养过夜。 稀 释菌液后涂布无抗性的 LB平板， 挑取单菌落利用引物 TO064和 TO065进行菌落 PCR鉴定。氯 霉素抗性基因消除的阳性重组子扩增产物应为大小应为 886bp (图 1 )， 将阳性重组子命名为 Τ0Ρ10 Δ tfcffl: ： FRT。
由于 dam与 re 双突变是致死基因型，因此在敲除 τ»基因之前要先回复突变 re ^基 因。 利用引物 TO253和 TO254, 以大肠杆菌 W3110 (购自美国 CGSC, 编号 4474 ) 染色体 DNA 为模板扩增野生型 re 基因 （1236bp)， 连接至载体 pKOV (购自美国 Addgene，货号 #25769 ) 的 No tY与 BanM位点， 得到 pKOV-recA 将 pKOV-re 转化入 TOP 10 Δ dcm: ： FRT菌株， 30 °C 筛选氯霉素抗性转化子， 挑取转化子后在 42 °C液体 LB培养基中培养过夜， 并涂布含有氯霉 素的 LB平板，使 pKOV-re 整合至染色体 recAl位点。得到单菌落后利用引物 TO255和 TO256 进行菌落 PCR， 如果有大于 7Kb的 PCR条带， 即说明 pKOV-re 已整合至染色体。 挑取正确 的重组子， 42 °C培养后涂布于含有 10%蔗糖的 LB平板， 将单菌落在含有氯霉素和不含氯霉素 的 LB平板上划线同时传代， 挑选氯霉素敏感的菌落。 利用引物 TO255和 TO256扩增 recA基 因后测序， re> +重组子 i^cA基因第 482位碱基应为 G， 而 T0P10菌株为 A。
t affl基因的敲除与 tfcffl敲除步骤相同，所用的氯霉素抗性基因扩增引物为 TO087和 TO088 , 外侧检测引物为 WB062和 TO063。基因敲除后扩增产物大小为 740bp，原始基因大小为 1356bp (图 2)。
经过上述三步遗传操作， 所得到的 tfcffl t affl缺失、 re 回复突变的菌株即为 EC135。 上述提到的菌株 EC135于 2012年 3月 21日保藏于中国微生物菌种保藏管理委员会普通 微生物中心 （简称 CGMCC, 地址： 北京市朝阳区北辰西路 1号院 3号， 中国科学院微生物研 究所， 邮编 100101 )， 保藏号为 CGMCC No. 5925， 其分类命名为大肠埃希氏菌 (Escherichia colf 。
所用到的 PCR引物序列见表 1。
表 1、 本发明所使用的 PCR引物
Figure imgf000006_0001
WB064 TGCTGAAGCTACCGCAAACCATG
WB065 GCACTCCCAGACAATCAATACGC
WB253 ATAAGAATGCGGCCGCCACTTGATACTGTATGAGCATACAG
WB254 CGCGGATCCCGGGATGTTGATTCTGTCATGGCAT
ATTACCCGGCGGGAATGCTTCAG
Figure imgf000006_0002
WB062 GGCCGATCTGAAGTAATCAAGGT
WB063 TCCAGATAGCTCAGAGGTGTCGC
WB325 ATGCCATAGCATTTTTATCC
WB475 CGTAGTTTATTCATGAATTCCTCCTTCAACTATGTACTTGAGGTAATCGA
WB476 TCGATTACCTCAAGTACATAGTTGAAGGAGGAATTCATGAATAAACTACG
WB477 TTATTGCTGTTCATGAATTCCTCCTTTATTCAGATTCTTTATTATCGTAT
WB478 ATACGATAATAAAGAATCTGAATAAAGGAGGAATTCATGAACAGCAATAA
WB479 GAAAAAAAACGCATGAATTCCTCCTTTATTCTAAATCTAATAATTCATTT WB480 AAATGAATTATTAGATTTAGAATAAAGGAGGAATTCATGCGTTTTTTTTC
WB326 GATTTAATCTGTATCAGG
WB325
WB575 ATACAGTTCATATGTCTTACCTCCTTTAATCGGCGGTATTTTGTGTAGAT
WB576 ATCTACACAAAATACCGCCGATTAAAGGAGGTAAGACATATGAACTGTAT
WB577 TTTAAACATATAACACTTTCCTCCTTTACGCTTCTAATGTCTCTCGAATG
WB578 CATTCGAGAGACATTAGAAGCGTAAAGGAGGAAAGTGTTATATGTTTAAA
WB579 AATCTATCATTTAAAAACACCTCTTGTCTACTCAACTAACATTAAGTAGA
WB580 TCTACTTAATGTTAGTTGAGTAGACAAGAGGTGTTTTTAAATGATAGATT
WB581 ATATCATAATATCACTCTCCCTCCTCTCAATAGCTAATTCTTCTTTAAAC
WB582 GTTTAAAGAAGAATTAGCTATTGAGAGGAGGGAGAGTGATATTATGATAT
WB583 CTATCAACATACTTTTCCACCGCCTTCATTCTTTAATACTTGGCTCTACG
WB584 CGTAGAGCCAAGTATTAAAGAATGAAGGCGGTGGAAAAGTATGTTGATAG
WB326
WB325
WB585 TCGATTCCGTGCATGAATTCCTCCTTTATGCCGCAAGTCTCCGGGCGGCG
WB586 CGCCGCCCGGAGACTTGCGGCATAAAGGAGGAATTCATGCACGGAATCGA
WB587 GGATGTTATGCATACGACACCTCCTTCAGAGACTACGCACGTCGAGAATG
WB588 CATTCTCGACGTGCGTAGTCTCTGAAGGAGGTGTCGTATGCATAACATCC
WB589 CGCGACACACCCATGAATTCCTCCTTCATTTGCCACCTCCATCGGTAGAT
WB590 ATCTACCGATGGAGGTGGCAAATGAAGGAGGAATTCATGGGTGTGTCGCG
WB326
WB607 CAAGGCGGACCGCTTATGCATG
WB608 CTTTAGTTGAAGCAAATACGTAAACCTTTCCCAT
WB609 TTTACGTATTTGCTTCAACTAAAGCACCCATTAGTTC
WB610 AGTCTGTCACCCAACCTTCTTCAACTAACGGGGCAGG
WB611 TTGAAGAAGGTTGGGTGACAGACTGTACGGAAC
WB612 TCCCGAGTGATCGTATGGAC
WB605 AACACTTCGCGGACCGCGCG
WB606 TGCCACACTGACTTTGTCGG
WB654 TACGCGTCGACCGCTGATCACACGATAGTCGGCG
WB655 TCCTTTACTCATGATCCCTCGTCCTCAGATCCAT
WB656 GGACGAGGGATCATGAGTAAAGGAGAAGAACTT
WB650 TGCAACTGCAGTTATTTGTATAGTTCATCCAT
实施例 2、 外源 DNA 分子克服限制修饰障碍导入解淀粉芽胞杆菌 Bacillus amyloliquefaciens) TA208
一、 共表达 TA208所有 DNA甲基转移酶编码基因的重组菌的构建
1、 菌株 TA208编码 DNA甲基转移酶基因的获得
1 ) 菌株 TA208编码 DNA甲基转移酶基因的预测
菌株 TA208全基因组序列已经得到测定， 在 GenBank登录号为 CP002627。其染色体上共 存在 5个推定的编码 DNA 甲基转移酶的基因， 基因的 locus_tag分别是 BAMTA208_06525， BAMTA208_6715， BAMTA208_14440， BAMTA208_19835和 BAMTA208_16660。五个基因片段的 PCR 扩增结果如图 3所示。
2 )、 菌株 TA208编码 DNA甲基转移酶基因的验证
将上述五个基因分别克隆至 pBAD43 质粒 （Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. L M Guzman, D Bel in' M J Carson, J Beckwith. Journal of Bacteriology, 1995 177 : 4121-30， 公众可从 中国科学院微生物研究所获得。） 的 Afcol和 ¾al、 fco/a和 ¾al、 fco/a和 ¾al、 f 和 Sail , EcoRi和 Sall位点间，得到含有 BAMTA208_06525的 pBAD43质粒、含有 BAMTA208_6715 的 pBAD43质粒、含有 BAMTA208_14440的 pBAD43质粒、含有 BAMTA208_19835的 pBAD43质粒 和含有 BAMTA208_16660的 pBAD43质粒； 再将 5种质粒分别转化入大肠杆菌 EC135 , 得到重 组菌 1-5。 提取重组菌 1-5的质粒送去测序验证正确， 因此重组菌为阳性重组菌。 点杂交验证： 将验证为阳性的重组菌 1-5诱导甲基转移酶表达 （在 LB培养基中培养至 0D6。。为 0. 2后加入终浓度为 0. 2% (质量百分含量） 的阿拉伯糖， 30 °C诱导 12小时）， 然后用 DNeasy Blood and Ti ssue Kit (Qiagen)提取总 DNA，得到 DNA1_DNA5。将上述得到的 DNA1-DNA5 分别煮沸 3min变性为单链后插入冰水混合物淬火。 以 EC135/pBAD43 ( pBAD43转入 EC135 ) 为阴性对照。
将 EC135/pBAD43的总 DNA和 5个样品 DNA1-DNA5分别均按 450ng、 150ng和 50ng点至 Protran BA85硝酸纤维素膜 （Whatman ) 上， 重复点三张膜。 紫外交联 2分钟后将膜放入用 TBST ( 200 mM NaCl , 0. l% Tween20 , 50 mM Tri s-HCl , pH7. 4 )配制的 5%脱脂奶粉， 室温（25 °C ) 封闭 1小时。然后将三张膜放入杂交袋，分别加入 10 mL 1： 10000稀释的兔抗 N6mA血清（New England Biolabs ) , 10 mL 1： 10000稀释的兔抗 N4mC血清 ( New England Biolabs ) , 10 mL 1： 20000稀释的鼠抗 5mC单克隆抗体 （Zymo Research, 货号 A3001_50)。 室温 （25 °C ) 孵育 1小时后用 TBST洗膜 5次， 将膜放入杂交袋后加入相应的辣根过氧化物酶标记的羊抗兔二抗
(美国 Jackson ImmunolResearch，货号 111-035-003 ) 或羊抗鼠二抗 （美国 Zymed, 货号 81-6520)， 稀释比例为 1 : 10000。 室温孵育 1小时后用 TBST洗膜 5次。 取 ECL试剂 A、 B液
(Amersham, 货号 RPN2232 )各 0. 5mL， 混匀后均匀滴加在膜表面。 暗室中将荧光信号曝光于 X光片。
结果如图 4所示 （图中的 m6A/m4C/m5C分别为使用不同抗体的杂交结果）， 可以看出， BAMTA208_06525， BAMTA208_6715， BAMTA208_19835和 BAMTA208_16660具有 DNA甲基化修 饰活性， 为 DNA甲基转移酶基因。
2、 共表达所有 DNA甲基转移酶编码基因的重组菌的获得
1 ) 构建
分别以含有 BAMTA208_06525 (序列 2 ) 的 pBAD43质粒、 含有 BAMTA208_6715 (序列 3 ) 的 PBAD43质粒、 含有 BAMTA208_19835 (序列 4 ) 的 pBAD43质粒和含有 BAMTA208_16660 (序 列 5 ) 的 pBAD43质粒为模板， BAMTA208_06525所用引物为 WB325和 WB475 (大小为 839bp， 图 3)， BAMTA208_6715所用引物为 WB476和 WB477 (大小为 1512bp , 图 3)， BAMTA208—19835 所用引物为 WB478和 WB479 (大小为 1794bp， 图 3)， BAMTA208_16660所用引物为 WB480和 WB326 (大小为 1272bp， 图 3)， 引物序列如表 1所示， 进行 PCR扩增， 分别得到 4个 PCR产 物。
将 4个 PCR产物分别切胶回收后，再将 4个 PCR产物等量混合浓缩至总体积 50μί，得到 PCR总产物。
EcoRi和 Sall双酶切 500 ng pWYE724质粒 （质粒图谱见图 5， 其序列为序列 1 )， 切胶 回收后与上述 PCR总产物混合， 然后用醋酸锂转化法（Methods in Enzymology, 350， 87-96. ) 转化酉良酒酵母 DAY414 (Mds3 regulates morphogenesi s in Candida albicans through the TOR pathway, Zacchi LF, Gomez-Raja J, Davi s DA, Molecular and Cel lular Biology, 2010, 30 : 3695-3710， 公众可从中国科学院微生物研究所获得）。 在不添加色氨酸的完全合成培养 基 （SC trp -， 北京泛诺基公司） 平板上筛选转化子。 挑取单菌落入 YPD培养基， 玻璃珠法提 取酵母质粒 （Nucleic Acids Research, 20, 3790)。 将质粒转化入大肠杆菌 T0P10 , 用含有 lOO g/mL壮观霉素的 LB平板筛选转化子， 提取质粒后 EcoW单切， 正确的重组质粒应产生 8515bp、 3633bp、 1018bp及 571bp四条带 (如图 6所示）， 命名为 pM. Bam (结构示意图如图 7所示）， 送去测序结果正确。
将质粒 pM.Bam转化入由实施例 1得到的菌株 EC135, 按照上述的方法酶切验证， 正确的 转化子即为 EC135/pM.Bam， 为解淀粉芽胞杆菌 TA208 DNA甲基化模式精确模拟的宿主。
2) 验证
为检测甲基化模拟的有效性， 利用终浓度为 0.2%的阿拉伯糖诱导在 30°C诱导 12小时， 使 EC135/pM. Bam中的甲基转移酶基因表达， 提取总 DNA， 使用菌株 TA208的染色体 DNA为对 照按照上述 1的方法进行点杂交检测，结果为 EC135/pM. Bam与 TA208的杂交结果无显著差异， 说明甲基转移酶均得到表达，说明 EC135/pM. Bam为共表达所有 DNA甲基转移酶编码基因的重 组菌。
二、 外源 DNA分子克服限制修饰障碍导入靶细菌
1、 穿梭质粒导入克服限制修饰障碍导入靶细菌
A、 导入
1) 将穿梭质粒 pAD43-25 (7262bp, 购自美国 Bacillus Genetic Stock Center, 以下 简写为 BGSC， 货号 ECE166) 转化入上述一得到的 EC135/pM. Bam， 分别得到重组菌。
2)、 将重组菌经阿拉伯糖诱导 （使用终浓度为 0.2%的阿拉伯糖在 30°C诱导 12小时）， 得到诱导后重组菌。
3)、提取诱导后重组菌的质粒（这里的质粒包括 pM. Bam和穿梭质粒， 但是 pWYE724为低 拷贝质粒， pM. Bam拷贝数为 5个左右 /细胞， 穿梭质粒的拷贝数为 300个左右 /细胞）， 转化 角牟淀粉芽胞杆菌 ( Bacillus amyloliquefaciens ) TA208 ( Complete genome sequence of Bacillus amyloliquefaciens TA208, a strain for industrial production of guanosine and ribavirin, Guoqiang Zhang, Aihua Deng, Qingyang Xu, Yong Liang, Ning Chen, Tingyi Wen, Journal of Bacteriology, 193 (12): 3142-3143)， 方法如 Analytical Biochemistry, 409， 130-137记载， 得到 TA208/pAD43_25 (EC135/pM. Bam)。
采用同样的方法将穿梭质粒 PAD43-25分别转入 T0P10和 EC135, 经修饰后再转入 TA208 中， 分别得到 TA208/ pAD43-25 (T0P10)和 TA208/pAD43_25 (EC135)。
采用同样的方法将穿梭质粒 pAD123(5952bp，购自美国 BGSC，编号 ECE165)、pMK3(7214bp， 购自美国 BGSC，编号 ECE15)、 pMK4 (5585bp，购自美国 BGSC，编号 ECE16)、 pHCMC02 (6866bp， 购自美国 BGSC，编号 ECE188)、 pHCMC04 (8089bp,购自美国 BGSC，编号 ECE189)、 pDG148StuI (8272bp, 购自美国 BGSC， 编号 ECE145)分别转入 EC135/pM. Bam、 T0P10和 EC135经修饰后 再转入 TA208中， 分别得到如下：
TA208/ pAD123 (EC135/pM. Bam)、 TA208/ pAD123 (T0P10)、 TA208/ pAD123 (EC135)、 TA208/ pMK3 (EC135/pM. Bam)、 TA208/ pMK3 (T0P10)、 TA208/ pMK3 (EC135)、 TA208/ pMK4 (EC135/pM. Bam)、 TA208/ pMK4 (T0P10)、 TA208/ pMK4 (EC135)、 TA208/ pHCMC02 (EC135/pM. Bam)、 TA208/ pHCMC02 (T0P10)、 TA208/ pHCMC02 (EC135)、 TA208/ pHCMC 04 (EC135/pM. Bam)、 TA208/ pHCMC 04 (T0P10)、 TA208/ pHCMC 04 (EC135)、 TA208/pDG148StuI (EC135/pM. Bam)、 TA208/ pDG148StuI (T0P10)、 TA208/pDG148StuI (EC135)。
B、 转化率计算
TA208/pAD43-25 (EC135/pM. Bam)、 TA208/pAD43- 25 (T0P10)和 TA208/pAD43- 25 (EC135)、 TA208/ pAD123 (EC135/pM. Bam)、 TA208/ pAD123 (T0P10)、 TA208/ pAD123 (EC135)、 TA208/ pMK3 ( EC135/pM. Bam )、 TA208/ pMK3 (T0P10)、 TA208/ pMK3 ( EC135 )、 TA208/ pMK4 ( EC135/pM. Bam )、 TA208/ pMK4 (T0P10) 、 TA208/ pMK4 ( EC135 )、 TA208/ pHCMC02 (EC135/pM. Bam)、 TA208/ pHCMC02 (T0P10)、 TA208/ pHCMC02 (EC135 )、 TA208/ pHCMC 04 (EC135/pM. Bam)、 TA208/ pHCMC 04 (TOP10)、 TA208/ pHCMC 04 (EC135)、 TA208/pDG148StuI (EC135/pM. Bam)、 TA208/ pDG148StuI (T0P10)、 TA208/pDG148StuI (EC135 ) 转化后涂布的 平板上单菌落的个数， 根据所使用的穿梭质粒 DNA的量 （pM. Bam拷贝数太低， 所以加入质粒 的总量即为穿梭质粒的 DNA量） 计算转化率。 随机挑取 4个转化子提取质粒 DNA验证。 实验 重复三次， 结果取平均值士标准差。
转化率为： 单菌落数 （CFU) X (转化后复苏液的体积 /所涂布菌液的体积） X复苏液稀 释倍数 /转化所用的穿梭质粒的量 （ g)。
结果如图 8所示， 可以看出，
TA208/pAD43-25 (EC135/pM. Bam) 的转化率为 6. 4 ± 2. 6 X 105CFU/ μ g DNA;
TA208/pAD43-25 (T0P10) 的转化率为 0;
TA208/pAD43-25 (EC135 ) 的转化率为 2· 4 ± 1· 6 X 103CFU/ μ g DNA;
TA208/ pAD123 (EC135/pM. Bam) 的转化率为 9. 1 ± 4. 3 X 105CFU/ μ g DNA;
TA208/ pAD123 (T0P10) 的转化率为 0;
TA208/ pAD123 (EC135 ) 的转化率为 0;
TA208/ pMK3 (EC135/pM. Bam) 的转化率为 2. 2 ± 0· 5 X 105CFU/ g DNA;
TA208/ pMK3 (T0P10) 的转化率为 0;
TA208/ pMK3 (EC135 ) 的转化率为 4. 5 X lO FU/ μ g DNA;
TA208/ pMK4 (EC135/pM. Bam) 的转化率为 3. 0 ± 0. 4 X 106CFU/ μ g DNA;
TA208/ pMK4 (T0P10) 的转化率为 0;
TA208/ pMK4 (EC135 ) 的转化率为 3. 1 X 102CFU/ μ g DNA;
TA208/ pHCMC02 (EC135/pM. Bam) 的转化率为 2. 0 ± 0. 3 X 106CFU/ μ g DNA;
TA208/ pHCMC02 (T0P10) 的转化率为 0;
TA208/ pHCMC02 (EC135 ) 的转化率为 6. 3 X lO FU/ μ g DNA;
TA208/ pHCMC 04 (EC135/pM. Bam) 的转化率为 1· 9 ± 0· 1 X 106CFU/ μ g DNA;
TA208/ pHCMC 04 (T0P10) 的转化率为 0;
TA208/ pHCMC 04 (EC135 ) 的转化率为 1· 5 X lO FU/ μ g DNA;
TA208/pDG148StuI ( EC135/pM. Bam) 的转化率为 9. 7 ± 3. 2 X 104CFU/ μ g DNA; TA208/ pDG148StuI (T0P10)的转化率为 0;
TA208/pDG148StuI (EC135 ) 的转化率为 0;
T0P10 制备的质粒不能转化菌株 TA208， EC135 菌株中制备的质粒转化率在 0〜 103CFU/VgDNA水平， 而 EC135/pM. Bam中制备的穿梭质粒转化率在 104〜106CFU/VgDNA水平。
同时提取每种菌的单菌落的质粒和未转入前的质粒进行电泳或测序比较， 结果大小一 致， 说明这些菌株都为转入外源 DNA分子的阳性质粒。
2、 整合型质粒导入靶细菌
含有 ¾o基因同源臂、 氯霉素抗性基因的整合型质粒 PWYE748 制备方法如下： 以引物 WB607、WB608为引物， TA208染色体 DNA为模板 PCR扩增 ¾¾o基因上游同源臂 641bp;以 WB609、 WB610为引物，质粒 pMK4为模板扩增氯霉素抗性基因 906bp; 以 WB61 U WB612为引物， TA208 染色体 DNA为模板 PCR扩增 ¾¾0基因下游同源臂 669bp; 将三段 PCR产物切胶回收后， 各取 1 μ L混合后作为模板， 以 TO607、 WB612为引物再次 PCR得到 2216bp的片段， 将 PCR产物克 隆入 PMD19-T质粒 （Taraka, 货号 D106A) , 所得质粒即为整合质粒 pWYE748。
采用上述 1的转化方法， 将整合型质粒 PWYE748在 EC135/pM. Bam内修饰后， 再转化菌 株 TA208并筛选氯霉素抗性转化子。
用引物 TO605和 TO606 PCR扩增鉴定， 以转化子的 DNA和野生型 TA208的基因组 DNA为 模板， 扩增结果如图 9所示， 转化子扩增大小为 2375bp (氯霉素抗性基因插入 ¾¾o基因）， 而野生型 TA208原始的氯霉素抗性基因大小为 2049bp， 证明转入外源基因， 整合成功， 将该 转化子命名为 BS043。
¾o基因编码尿嘧啶磷酸核糖转移酶， 可将 5-氟尿嘧啶转化为 5-氟尿嘧啶核苷单磷酸， 最终代谢为有毒的代谢产物 5-氟尿嘧啶核苷二磷酸， 可强烈抑制胸苷酸合成酶的活性， 造成 菌体死亡。
进一步证明 BS043是否导入了外源基因， 方法为： 菌株 BS043在不添加和添加 10μΜ5_ 氟尿嘧啶（Sigma,货号 F6627 )的 MM培养基上生长（培养基成分见 Molecular Microbiology, 46， 25-36.，并添加 100mg/L的腺苷）， 以菌株 TA208为对照。 结果如图 10所示， 可以看出， BS043可在添加 10μΜ5-氟尿嘧啶的匪培养基上生长， 说明转入外源基因， 整合成功。
而在 EC135及 T0P10中提取的整合质粒 pWYE748转化 ΤΑ208多次均未得到转化子。 实施例 3、 外源 DNA 分子克服限制修饰障碍导入蜡样芽胞杆菌（ Ci i« cereus) A CC 10987
一、 共表达 ATCC 10987所有 DNA甲基转移酶编码基因的重组菌的构建
1、 菌株 ATCC 10987编码 DNA甲基转移酶基因的获得
1 ) 菌株 ATCC 10987编码 DNA甲基转移酶基因的预测
菌株 ATCC 10987全基因组序列已经公布， GenBank登录号为 AE017194。 其染色体上共 存在 9个推定的编码 DNA甲基转移酶的基因， 基因的 locus_tag分别是 BCE_0841_BCE_0842， BCE_0839-BCE_0842, BCE_0365， BCE_0392, BCE_0393， BCE_4605, BCE_5606， BCE_5607 和 BCE_1018。
2 )、 菌株 ATCC 10987编码 DNA甲基转移酶基因验证
将 BCE_0841— BCE_0842， BCE_0839— BCE_0842， BCE_0365， BCE_0392， BCE_0393， BCE_4605 BCE—5606, BCE—5607和 BCE—1018分别克隆至 pBAD43质粒 Nhel和 Kpnl、 Nco l和 Kpnl、 Nhel 禾口 Kpnl、 Nhel禾口 Kpnl、 Nhel禾口 Kpnl、 Nhel禾口 Kpnl、 Nhel禾口 Kpnl、 Nhel禾口 Xbal、 Nhel禾口 Kpnl 位点间， 得到含有 BCE_0841-BCE_0842 的 pBAD43质粒， 含有 BCE_0839_BCE_0842 的 PBAD43质粒， 含有 BCE_0365的 pBAD43质粒， 含有 BCE_0392的 pBAD43质粒， 含有 BCE_0393 的 pBAD43质粒， 含有 BCE_4605的 pBAD43质粒， 含有 BCE_5606的 pBAD43质粒， 含有 BCE_5607 的 pBAD43质粒和 含有 BCE_1018的 pBAD43质粒。
再将上述质粒分别转化入大肠杆菌 EC135 , 得到重组菌 1-9。
提取重组菌 1-9的质粒送去测序验证正确， 因此重组菌为阳性重组菌。
点杂交验证：
将验证为阳性的重组菌 1-9诱导甲基转移酶表达 (使用终浓度为 0. 2%的阿拉伯糖在 30 °C 诱导 12小时） 后， 用 DNeasy Blood and Ti ssue Kit (Qiagen)提取总 DNA, 得到 DNA1_DNA9。 将上述得到的 DNA1-DNA9煮沸 3min变性为单链后插入冰水混合物淬火。 以 EC135/pBAD43为 阴性对照。 将 EC135/pBAD43的总 DNA和 9个样品 DNA1-DNA9分别均按 450ng、 150ng和 50ng点至 Protran BA85硝酸纤维素膜 （Whatman) 上， 重复点三张膜。 紫外交联 2分钟后将膜放入用 TBST ( 200 mM NaCl , 0. 1% Tween20, 50 mM Tris-HCl , pH7. 4) 配制的 5%脱脂奶粉， 室温封 闭 1小时。然后将三张膜放入杂交袋， 分别加入 lO mL l : 10000稀释的兔抗 N6mA血清， 10 mL 1： 10000稀释的兔抗 N4mC血清， 10 mL 1： 20000稀释的鼠抗 5mC单克隆抗体。 室温孵育 1 小时后用 TBST洗膜 5次，将膜放入杂交带后加入相应的羊抗兔或羊抗鼠二抗，稀释比例为 1 : 10000。 室温孵育 1小时后用 TBST洗膜 5次。 取 ECL试剂 A、 B液各 0. 5mL， 混匀后均匀滴加 在膜表面。 暗室中将荧光信号曝光于 X光片。
结果如图 11 (图中的 m6A/m4C/m5C分别为使用不同抗体的杂交结果），可以看出， BCE_0393， BCE_4605， BCE_5606， BCE_5607， BCE_0365和 BCE_0392具有 DNA甲基化修饰活性， 为 DNA 甲基转移酶基因。
2、 共表达所有 DNA甲基转移酶编码基因的重组菌的获得
1 ) 构建
分别以含有 BCE_0393 (序列 6 ) 的 pBAD43质粒、 含有 BCE_4605 (序列 7 ) 的 pBAD43质 粒、 含有 BCE_5606 (序列 8 ) 的 pBAD43质粒、 含有 BCE—5607 (序列 9 ) 的 pBAD43质粒、 含 有 BCE_0365 (序列 10 ) 的 pBAD43质粒、 含有 BCE_0392 (序列 11 ) 的 pBAD43质粒为模板， BCE_0393所用引物为 WB325和 WB575 (大小为 2160bp ) ， BCE_4605为 WB576和 WB577 (大小 为 1102bp ) ， BCE_5606为 WB578和 WB579 (大小为 1345bp ) ， BCE_5607为 WB580和 WB581 (大小为 1316bp)， BCE_0365为 WB582和 WB583(大小为 1071bp)， BCE_0392为 WB584和 WB326 (大小为 1257bp)， 引物序列如表 1所示， 进行 PCR扩增， 分别得到 6个 PCR产物。
将 6个 PCR产物分别切胶回收后， 再将 6个 PCR产物等比例混合， 浓缩至总体积 50μί， 得到 PCR总产物。
EcoRi和 Sail双酶切 500 ng pWYE724质粒， 切胶回收后与上述 PCR总产物混合， 然后 用醋酸锂转化法 （Methods in Enzymology, 350， 87-96. ) 转化酿酒酵母 DAY414。 在不添加 色氨酸的完全合成培养基 （SC trp-, 北京泛诺基公司） 平板上筛选转化子， 挑取单菌落入 YPD培养基， 玻璃珠法提取酵母质粒 （Nucleic Acids Research, 20, 3790)。 将质粒转化入 大肠杆菌 T0P10, 用含有 10(^g/mL壮观霉素的 LB平板筛选转化子， 提取质粒后 Hindi l l单 切， 正确的重组质粒应产生 6544bp、 4237bp、 3642bp、 1315bp及 827bp五条带 （如图 12所 示）， 命名为 pM. Bce (结构示意图如图 13所示）， 送去测序结果正确。
将质粒 pM. Bce转化入由实施例 1得到的菌株 EC135, 按照上述的方法酶切验证， 正确的 转化子即为 EC135/pM. Bce， 为蜡样芽胞杆菌 ATCC 10987 DNA甲基化模式精确模拟的宿主。
2 ) 验证
为检测甲基化模拟的有效性， 将 EC135/pM. Bee使用终浓度为 0. 2%阿拉伯糖诱导在 30°C 诱导 12小时， 使甲基转移酶基因表达， 提取总 DNA， 使用菌株 ATCC 10987的染色体 DNA为 对照按照前面的方法进行点杂交检测， 结果为 EC135/pM. Bee与 ATCC 10987的杂交结果无显 著差异， 说明甲基转移酶均得到表达， 说明 EC135/pM. Bce为共表达所有 DNA甲基转移酶编码 基因的重组菌。
二、 穿梭质粒克服限制修饰障碍导入靶细菌
将穿梭质粒分别转入 EC135/pM. Bce、 EC135和 T0P10, 阿拉伯糖诱导 （方法见前面） 后 提取质粒， 分别转化菌株 ATCC 10987， 方法同实施例 2 的一， 上述穿梭质粒为 pAD43_25、 pAD123、 pMK3、 pMK4、 pHCMC02, 分别得到如下：
ATCC 10987/ pAD43-25 (EC135/pM. Bce)、 ATCC 10987/ pAD43- 25 (TOPIO)、 ATCC 10987/ pAD43-25 (EC135)、 ATCC 10987/ pAD123 (EC135/pM. Bce)、 ATCC 10987/ pAD123 (TOPIO)、 ATCC 10987/ pAD123 (EC135)、 ATCC 10987/ pMK3 (EC135/pM. Bce)、 ATCC 10987/ pMK3 (TOPIO)、 ATCC 10987/ pMK3 (EC135)、 ATCC 10987/ pMK4 (EC135/pM. Bce)、 ATCC 10987/ pMK4 (TOPIO)、 ATCC 10987/ pMK4 (EC135)、 ATCC 10987/ pHCMC02 (EC135/pM. Bce)、 ATCC 10987/ pHCMC02 (TOPIO)、 ATCC 10987/ pHCMC02 (EC135);
转化后涂布的平板上单菌落的个数， 根据所使用的穿梭质粒 DNA的量 （pM. Bee拷贝数 为 5个左右 /细胞， 穿梭质粒的拷贝数为 300个左右 /细胞， 所以加入质粒的总量即为穿梭质 粒的 DNA量） 计算转化率。 随机挑取 4个转化子提取质粒 DNA验证。 实验重复三次， 结果取 平均值士标准差。
转化率为： 单菌落数 （CFU) X (转化后复苏液的体积 /所涂布菌液的体积） X复苏液稀 释倍数 /转化所用的穿梭质粒的量 （ g)。
结果如图 14所示， 可以看出，
ATCC 10987/ pAD43-25 (EC135/pM. Bee) 的转化率为 3.2± 1.3X 105CFU/ μ g DNA;
ATCC 10987/ pAD43-25 (T0P10) 的转化率为 8.3±0.7X 103CFU/ μ g DNA;
ATCC 10987/ pAD43-25 (EC135) 的转化率为 7.1 ±2.9 X 103CFU/ μ g DNA;
ATCC 10987/ pAD123 (EC135/pM. Bee) 的转化率为 9.0± 1.4X 105CFU/ μ g DNA;
ATCC 10987/ pAD123 (T0P10) 的转化率为 8.7±0.8X 103CFU/ μ g DNA;
ATCC 10987/ pAD123 (EC135) 的转化率为 5.7±3.3X 103CFU/ μ g DNA;
ATCC 10987/ pMK3 (EC135/pM. Bee) 的转化率为 8.4±2.2X 105CFU/ μ g DNA;
ATCC 10987/ pMK3 (T0P10) 的转化率为 8.7±2.7X 102CFU/ μ g DNA;
ATCC 10987/ pMK3 (EC135) 的转化率为 1.2±0· 6X 103CFU/ μ g DNA;
ATCC 10987/ pMK4 (EC135/pM. Bee) 的转化率为 2.3±0.2X 107CFU/ μ g DNA;
ATCC 10987/ pMK4 (T0P10) 的转化率为 3.3±0.4X 105CFU/ μ g DNA;
ATCC 10987/ pMK4 (EC135) 的转化率为 3.5± 1.4X 105CFU/ μ g DNA;
ATCC 10987/ pHCMC02 (EC135/pM. Bee) 的转化率为 2.5± 1.5X 105CFU/ μ g DNA;
ATCC 10987/ pHCMC02 (T0P10) 的转化率为 0;
ATCC 10987/ pHCMC02 (EC135) 的转化率为 0;
可以看出， EC135/pM. Bee中制备的质粒转化率是 T0P10和 EC135中制备的质粒转化率的 102〜103倍。
同时提取每种菌的单菌落的质粒和未转入前的质粒进行电泳或测序比较， 结果大小一 致， 说明这些菌株都为转入外源 DNA分子的阳性质粒。
实施例 4、 外源 DNA 分子克服限制修饰障碍导入汉氏硝化细菌（ iir0½Cter hamburgensis) X14
一、 共表达 X14所有 DNA甲基转移酶编码基因的重组菌的构建
1、 菌株 X14编码 DNA甲基转移酶基因的获得
1) 菌株 X14编码 DNA甲基转移酶基因的预测
菌株 X14全基因组序列已经公布， GenBank登录号为 CP000319, CP000320, CP000321 CP000322。其基因组中共存在 10个推定的编码 DNA甲基转移酶的基因， 基因的 locus_tag分 别是 Nham_0569， Nham_0582， Nham_0803， Nham_0842， Nham_1185， Nham_1353， Nham_2515， Nham_3225， Nham_3845和 Nham_4499。
2)、 菌株 X14编码 DNA甲基转移酶基因的验证
将 Nham_0569， Nham_0582， Nham_0803， Nham_0842， Nham_1185， Nham_1353， Nham_2515， Nham_3225， Nham_3845和 Nham_4499分别克隆至 pBAD43质粒的 EcoRI和 KpnI、EcoRI和 Kpnl、 Nhel禾口 Kpnl、 Nhel禾口 Kpnl、 EcoRI禾口 Kpnl、 EcoRI禾口 Kpnl、 Nhel禾口 Kpnl、 EcoRI禾口 Kpnl、 Nhel和 Kpnl、 EcoRI和 Kpnl位点， 得到含有 Nham_0569的 pBAD43质粒， 含有 Nham_0582的 PBAD43质粒， 含有 Nham_0803的 pBAD43质粒， 含有 Nham_0842的 pBAD43质粒， 含有 Nham_l 185 的 pBAD43质粒， 含有 Nham_1353 的 pBAD43质粒， 含有 Nham_2515 的 pBAD43质粒， 含有 Nham_3225的 pBAD43质粒， 含有 Nham_3845的 pBAD43质粒和含有 Nham_4499的 pBAD43质粒。 再将上述质粒分别转入大肠杆菌 EC135, 得到重组菌 1-10。
提取重组菌 1-10的质粒送去测序验证正确， 因此重组菌为阳性重组菌。
点杂交验证：
将验证为阳性的重组菌 1-10诱导甲基转移酶表达 （使用终浓度为 0. 2%阿拉伯糖诱导在 30°C诱导 12小时）后，用 DNeasy Blood and Tissue Kit (Qiagen)提取总 DNA，得到 DNA1_DNA10。 将上述得到的 DNA1-DNA10煮沸 3min变性为单链后插入冰水混合物淬火。以 EC135/pBAD43为 阴性对照。
将 EC135/pBAD43的总 DNA和 10个样品 DNA1-DNA10分别均按 450ng、 150ng和 50ng点 至 Protran BA85硝酸纤维素膜 （Whatman) 上， 重复点三张膜。 紫外交联 2分钟后将膜放入 用 TBST ( 200 mM NaCl , 0. 1% Tween20, 50 mM Tris-HCl , pH7. 4) 配制的 5%脱脂奶粉， 室温 封闭 1小时。 然后将三张膜放入杂交袋， 分别加入 10 mL 1： 10000稀释的兔抗 N6mA血清， 10 mL 1： 10000稀释的兔抗 N4mC血清， 10 mL 1： 20000稀释的鼠抗 5mC单克隆抗体。 室温 孵育 1小时后用 TBST洗膜 5次， 将膜放入杂交带后加入相应的羊抗兔或羊抗鼠二抗， 稀释比 例为 1 : 10000。 室温孵育 1小时后用 TBST洗膜 5次。 取 ECL试剂 A、 B液各 0. 5mL， 混匀后 均匀滴加在膜表面。 暗室中将荧光信号曝光于 X光片。
结果如图 15所示 （图中的 m6A/m4C/m5C分别为使用不同抗体的杂交结果）， 可以看出， Nham_0569, Nham_0582, Nham_0803和 Nham_3225具有 DNA甲基化修饰活性， 为 DNA甲基转 移酶基因。
达所有 DNA甲基转移酶编码基因的重组菌的获得 分别以含有 Nham—0569 (序列 12)、 含有 Nham—0582 (序列 13 ) 的 pBAD43质粒、 含有 Nham_0803 (序列 14) 的 pBAD43质粒和含有 Nham_3225 (序列 15 ) 的 pBAD43质粒为模板， Nham_0569所用引物为 WB325和 WB585 (大小为 648bp ) ， Nham_0582为 WB586和 WB587 (大 小为 1983bp )， Nham_0803为 WB588和 WB589 (大小为 1317bp)， Nham_3225为 WB590和 WB326 (大小为 1131bp)， 引物序列如表 1所示， 进行 PCR扩增， 分别得到 4个 PCR产物。
将 4个 PCR产物分别切胶回收后，再将 4个 PCR产物等比例混合，并浓缩至总体积 50μί， 得到 PCR总产物。
EcoRI和 Sail双酶切 500 ng pWYE724质粒， 切胶回收后与上述 PCR总产物混合， 然后 用醋酸锂转化法 （Methods in Enzymology, 350， 87-96. ) 转化酿酒酵母 DAY414。 在不添加 色氨酸的完全合成培养基 （SC trp-, 北京泛诺基公司） 平板上筛选转化子， 挑取单菌落入 YPD培养基， 玻璃珠法提取酵母质粒 （Nucleic Acids Research, 20, 3790)。 将质粒转化入 大肠杆菌 T0P10, 用含有 10(^g/mL壮观霉素的 LB平板筛选转化子， 提取质粒后 BanM单切， 正确的重组质粒应产生 8217bp、 3848bp、 1003bp和 343bp四条带 （如图 16所示）， 命名为 pM. Nham (结构示意图如如图 17所示）， 送去测序结果正确。
将 pM. Nham转化入由实施例 1得到的菌株 EC135, 按照上述的方法酶切验证， 正确的转 化子即为 EC135/pM. Nham, 为汉氏硝化细菌 X14的 DNA甲基化模式精确模拟宿主。
2 ) 验证
为检测甲基化模拟的有效性， 阿拉伯糖诱导 （使用终浓度为 0. 2%阿拉伯糖诱导在 30°C 诱导 12小时） EC135/pM. Nham中的甲基转移酶基因表达， 提取总 DNA， 使用菌株 X14的染色 体 DNA为对照按照前面的方法进行点杂交检测， 结果为 EC135/pM. Nham与菌株 X14的杂交结 果无显著差异， 说明甲基转移酶均得到表达， 说明 EC135/pM. Nham为共表达所有 DNA甲基转 移酶编码基因的重组菌。
二、 穿梭质粒克服限制修饰障碍导入靶细菌
质粒 pBBRl-MCS5-PNh — 345。-GFP具体构建方法为： 以 TO654、 TO655为引物， 菌株 X14基因 组 DNA为模板扩增 Nham_3450基因启动子片段 216bp; 以 WB656、 WB650为引物， pAD123为模 板扩增 GFP基因 717bp; 将两个 PCR产物切胶回收后各取 1 μ L作为模板， 以 TO654, WB650 为引物再次 PCR扩增， 得到 933bp的 PCR产物后克隆入 pBBRl-MCS5 (Four new derivatives of the broad-host-range cloning vector pBBRlMCS, carrying different antibiotic-resistance cassettes. Kovach, Michael E. Elzer, Phi l ip H. Steven Hi l l, D. Robertson, Gregory T. Farris, Michael A. Roop I i， R. Martin Peterson, Kenneth M. . 1995， Gene 166 ( 1 ) ： 175-176， 公众可从中国科学院微生物研究所获得） 质粒的 I 和 Ps tl位点， 并进行测序验证。
质粒 pBBRl-MCS5-PNh — 345。-GFP是一个广宿主质粒， 含有 Nham_3450操控下的绿色荧光蛋 白编码基因， 将其转入 EC135/pM. Nham, 阿拉伯糖诱导后提取质粒， 转化菌株 X14， 菌株 X14 的转化方法如下： 将菌株在 DSM756a培养基 ( 1. 5 g yeast extract, 1. 5 g peptone, 2 g NaN02,
0. 55 g sodium pyruvate, 1 mL trace element solution (33. 8 mg MnS04-H20, 49. 4 mg H3B03, 43. 1 mg ZnS04-7H20, 37. 1 mg (N ) 6Mo7024， 97. 3 mg FeS04-7H20 and 25 mg CuS04-5H20 in 1 L deionized water) and 100 mL stock solution (0. 07 g CaC03, 5 g NaCl, 0. 5 g MgS04-7H20,
1. 5 g KH2P04 in 1 L deionized water) in 1 L deionized water, pH 7. 4) ) 中培养至 OD600 为 0. 1， 冰浴 10min， 4°C 8000rpm离心 lOmin收集菌体， 并用预冷的 10%甘油洗涤菌体 4次， 后重悬于 1/1000原始培养体积的 10%甘油。 取 90 μ L细胞， 与 150 ng质粒混匀后加入 1讓 的电转杯， 1200V电击一次（ECM399电转仪）。将细胞洗入 100mL756a培养基， 28°C复苏 1天， 然后加入终浓度 20 μ g/mL的庆大霉素继续复苏 1天。以 1/100的体积比将复苏混合物转接入 含有 20 μ g/mL庆大霉素的新鲜 756a培养基。 培养基 0D6。。为 0. 1后， 以相同的方法再转接一 次， 直至菌体生长至 0D6。。为 0. 1， 得到 X14/ pBBRl-MCS5-PNh„_3450-GFP (EC135/pM. Nham); 分别取 X14/ pBBRl-MCS5-PNh„— 345。-GFP (EC135/pM. Nham) 培养液 10 μ L， 涂片后置于荧光 显微镜下观察， 以 X14为对照。
结果如图 18所示， A为 X14, B为 X14/ pBBRl-MCS5_PNh— 345。_GFP (EC135/pM. Nham), 可 以看出， X14/ pBBRl-MCS5-PNh„_345o-GFP (EC135/pM. Nham) 细菌在 488 nm激发光下发出绿色 荧光， 而未转化的细菌 X14没有荧光。 说明转入外源基因。
取 10mL X14/ pBBRl-MCS5-PNh„_3450-GFP (EC135/pM. Nham) 菌液， 用细菌 DNA提取试剂盒 (天根， DP302-02) 提取总 DNA， 然后取 5 μ L DNA转化大肠杆菌 T0P10, 筛选庆大霉素抗性 转化子， 提取质粒验证后与原始质粒 pBBRl-MCS5-PNham_3450-GFP大小一致（5701bp)， 说明 X14/ pBBRl-MCS5-P -GFP (EC135/pM. Nham) 中的外源 DNA分子 pBBRl- MCS5- PNh— 345。- GFP 没有损失， 是克服限制修饰障碍完全转入 X14中。
而 Carsiotis, M.等艮道 (Genetic engineering of enhanced microbial nitrification. Carsiotis, M. and Khanna, S. US Environmental Protection Agency, Risk Reduction Engineering Laboratory. 1989)， 提取自大肠杆菌普通宿主的质粒 DNA不能实现菌株 X14的 遗传转化， 因此本专利报道的方法具有较大优势。

Claims

权 利 要 求
1、 一种将外源 DNA分子导入靶细菌的方法， 包括如下步骤：
1 )将一株靶细菌基因组中所有的 DNA甲基转移酶编码基因在自身限制修饰系统缺失的大 肠杆菌中共表达， 得到重组菌 A;
2 ) 将外源质粒 DNA分子导入所述重组菌 A进行体内修饰， 得到甲基化修饰的外源质粒 DNA分子；
3 ) 将所述甲基化修饰的外源质粒 DNA分子导入所述靶细菌中。
2、 根据权利要求 1所述的方法， 其特征在于：
步骤 1 ) 中， 所述将靶细菌基因组中所有的 DNA 甲基转移酶编码基因在自身限制修饰系 统缺失的大肠杆菌中共表达为将所述靶细菌基因组中所有的 DNA甲基转移酶编码基因通过重 组载体导入所述自身限制修饰系统缺失的大肠杆菌内；
步骤 2 ) 包括如下步骤：
A) 将所述外源质粒 DNA分子导入所述重组菌 A中， 得到重组菌 B;
B) 诱导培养所述重组菌 B， 得到诱导后重组菌 B;
C) 提取所述诱导后重组菌 B的质粒 DNA， 即得到甲基化修饰的外源质粒 DNA分子。
3、 根据权利要求 1或 2所述的方法， 其特征在于：
步骤 1 ) 中， 所述重组载体为将所述所有 DNA 甲基转移酶编码基因均插入表达质粒中， 得到表达所有 DNA甲基转移酶的重组载体；
步骤 2 )的 B)中，所述诱导培养采用的方法为温度诱导，或使用诱导剂如阿拉伯糖、 IPTG、 木糖或鼠李糖诱导。
4、 根据权利要求 1-3中任一所述的方法， 其特征在于：
步骤 2 ) 的 B) 中， 所述诱导培养为将重组菌 B在诱导条件下培养；
最优诱导条件为将所述重组菌 B在含有终浓度为 0. 2% (质量百分含量） 的阿拉伯糖的液 体培养基中诱导培养；
所述诱导培养的温度为 25°C-37°C， 所述诱导培养的时间为 3-24小时；
所述诱导培养的温度优选为 30°C， 所述诱导培养的时间优选为 12小时。
5、 根据权利要求 1-4中任一所述的方法， 其特征在于：
所述靶细菌为含有限制修饰系统的真细菌或古生菌， 所述含有限制修饰系统的真细菌或 古生菌可以是但不局限于解淀粉芽胞杆菌 (bacillus amyloliquefaciens TA208、 蜡样芽胞 杆菌（ aci iAs cereus) ATCC 10987或汉氏硝化细菌（/Vi i r^acier hamburgensis) \ ;
所述自身限制修饰系统缺失的大肠杆菌可以是但不局限于大肠杆菌 (Escherichia coll) EC135 CGMCC No. 5925。
6、根据权利要求 1-5中任一所述的方法， 其特征在于： 所述外源 DNA分子可以是但不局 限于 pAD123、pMK3、pMK4、pHCMC02、pHCMC04、pDG148StuI、pWYE748或 pBBRl-MCS5_PNh — 345。_GFP。
7、 根据权利要求 1-6中任一所述的方法， 其特征在于：
所述解淀粉芽胞杆菌 i Bacillus amyloliquefaciens) TA208中所有 DNA 甲基转移酶编 码基因为 BAMTA208_06525、 BAMTA208_6715、 BAMTA208_19835 禾 P BAMTA208_16660； 所述 BAMTA208_06525、 BAMTA208_6715、 BAMTA208_19835和 BAMTA208_16660的核苷酸序列依次为 序列表中的序列 2、 序列 3、 序列 4和序列 5; 所述蜡样芽胞杆菌（ aci i« cereus) A CC 10987 中所有 DNA 甲基转移酶编码基因为 BCE_0393、 BCE_4605、 BCE_5606、 BCE_5607、 BCE_0365和 BCE_0392;所述 BCE_0393、 BCE_4605、 BCE_5606、 BCE_5607、 BCE_0365和 BCE_0392的核苷酸序列依次为序列表中的序列 6、序列 7、 序列 8、 序列 9、 序列 10和序列 11 ;
所述汉氏硝化细菌（ i
Figure imgf000018_0001
hamburgensis) lU中所有 DNA 甲基转移酶编码基因为 Nham_0569、 Nham_0582、 Nham_0803和 Nham_3225; 所述 Nham_0569、 Nham_0582、 Nham_0803 和 Nham_3225的核苷酸序列依次为序列表中的序列 12、 序列 13、 序列 14和序列 15。
8、 大肠杆菌 (Escherichia coll) EC135, 其保藏编号为 CGMCC No. 5925。
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