CN104673819A - Vector for knocking out L-lactic dehydrogenase 1 gene and construction method of vector - Google Patents
Vector for knocking out L-lactic dehydrogenase 1 gene and construction method of vector Download PDFInfo
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Abstract
The invention discloses a vector for knocking out an L-lactic dehydrogenase 1 gene and a construction method of the vector. The construction method comprises the following steps: completely splicing upstream and downstream DNA fragments of a gene which does not contain an L-lactic dehydrogenase encoding gene by using an overlapping extension process, also cloning the spliced fragments to a sub-vector pMD18-T and transforming the spliced fragments to escherichia coli DH5alpha competent cells, screening constructed sub-cloning vectors by virtue of blue-white spots, performing double enzyme digestion to obtain a target strip, then connecting the target strip to a pG+host9 plasmid, and performing bacterial colony PCR and double enzyme digestion identification to successfully obtain a recombinant vector. According to the vector and the construction method thereof disclosed by the invention, a recombinant plasmid of which the L-lactic dehydrogenase encoding genes are knocked out is constructed by using an overlapping extension PCR process, so that excessive base sequences introduced by enzyme digestion and connection can be avoided; and the recombinant vector disclosed by the invention can be used for producing D-lactic acid with high optical purity.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to utilize the technical battery of Overlap extension PCR to connect LDH 1 upstream and downstream sequence in plant lactobacillus DMDL9010, and by structure subcloning vector for the upstream and downstream fragment of splicing provides restriction enzyme site, it is the plasmid of selection markers that screening simultaneously obtains for building with erythromycin, successfully builds the recombinant vectors for knocking out object bacteria LDH 1 gene.
Background technology
The D-ALPHA-Hydroxypropionic acid of high-optical-purity more than 97% or Pfansteihl are important chiral intermediate and organic synthesis raw material, can be widely used in the chiral synthesize in pharmacy, higher effective and lower toxic pesticide and the field such as weedicide, makeup.D-ALPHA-Hydroxypropionic acid also has vital role in raising poly(lactic acid) thermostability etc.The major cause of the widespread use of current restriction D-ALPHA-Hydroxypropionic acid has: the production cost of (1) D-ALPHA-Hydroxypropionic acid is high, and the material cost of its fermentative production is high, purify and refining cost high; (2) do not screen suitable fermenting and produce the bacterial strain of high optical purity D-lactic acid, because general milk-acid bacteria mainly produces Pfansteihl to ferment; (3) existing known milk-acid bacteria generally can not utilize pentose, and such as wood sugar, ribose and pectinose are as carbon source.Microbe fermentation method is one of main method preparing D-ALPHA-Hydroxypropionic acid at present, because the physico-chemical property of D-ALPHA-Hydroxypropionic acid is extremely similar to Pfansteihl, utilize chemical process to be difficult to two kinds of isomerss to separate, the key of therefore producing D-ALPHA-Hydroxypropionic acid is to filter out high optical purity D-lactic acid high-yield strains.
Confirm at present, by LDH gene knockout, can the D-ALPHA-Hydroxypropionic acid of direct production high-optical-purity.Traditional structure knocks out the method for the recombinant vectors of LDH 1 gene, needs to introduce restriction enzyme site between junction fragment, and enzyme to cut connection more consuming time, efficiency is low; PCR is difficult to successfully the restructuring being shorter than 100bp and long tens fragments.
Summary of the invention
For obtaining more highly purified D-ALPHA-Hydroxypropionic acid, the invention provides a kind of method that LDH encoding gene knocks out, and provide a kind of method of more effective gene order splicing, mainly utilize the method for Overlap extension PCR to obtain the upstream and downstream splicing fragment of LDH encoding gene in a large number, then splicing fragment is cloned into pG
+the recombinant vectors obtained for knocking out plant lactobacillus DMDL9010 is built in host9 plasmid vector.
To achieve these goals, present invention employs following technical scheme:
For knocking out a construction process for the carrier of LDH 1 gene, comprise the steps:
(1) from NCBI, find the position of LDH 1 encoding gene and the DNA sequence dna of upstream and downstream 800 ~ 1000bp thereof, then determine conserved sequence design primer and introduce overlapping fragments, extract the DNA of plant lactobacillus (Lactobacillus plantarum) DMDL 9010, and as template, amplification obtains the upstream and downstream DNA fragmentation of this gene not containing LDH encoding gene (as SEQ ID NO.2, shown in SEQ ID NO.3), the upstream and downstream DNA fragmentation (as shown in SEQ ID NO.1) utilizing high-fidelity enzyme to carry out Overlap extension PCR to connect this gene, the deposit number of described plant lactobacillus DMDL is CGMCC NO.5172,
(2) after 3 ' of above-mentioned junction fragment end being carried out adding A reaction and pMD18-T carrier carry out ligation and be transformed in intestinal bacteria DH 5 α competent cell, finally obtain subcloning vector, then subcloning vector is prepared in a large number, carry out Xho I and Pst I double digestion, overlapping fragments introduces sticky end;
(3) by the pG through Xho I and the process of Pst I double digestion
+the overlapping fragments that host9 plasmid and step (2) obtain carries out ligation, then, after forwarding this connection product to bacillus coli DH 5 alpha blue hickie screening, a series of positive clone identification experiment (PCR qualification and double digestion qualification) is carried out;
(4) from four kinds of intestinal bacteria, screening obtains host e. coli MC1061 and TG1 for building with erythromycin the recombinant vectors being mark, obtains the erythromycin concentration 500 μ g/ml of the most applicable screening simultaneously.
(5) after utilizing Pst I, XhoI toolenzyme to be cut by above-mentioned subcloning vector enzyme must object fragment containing sticky end simultaneously with by the pG of double digestion
+host9 connects, then proceeds in intestinal bacteria MC1061 or TG1 through thermal transition, utilizes erythromycin to carry out screening positive engineering bacteria;
(6) carry out bacterium colony PCR qualification to above-mentioned engineering bacteria, positive bacteria extracts plasmid through fermentation culture, then carries out double digestion qualification, obtains recombinant vectors.
The condition of described Overlap extension PCR is: single step Overlap extension PCR, and template concentrations is 0.4ng/ μ l, and the overlapping fragments introducing primer is 15bp.
Described in step (1), primer is as shown in table 1.
The primer of the upstream and downstream DNA fragmentation of table 1 LDH 1 encoding gene
The present invention is separated and obtains the higher wild-type plant Bacterium lacticum DMDL9010 of lactic acid production from fermented vegetables, intends adopting overlap extension pcr to connect two recombinant fragments, builds the homologous recombination vector that can be applicable to plant lactobacillus.Utilize this homologous recombination vector can with the encoding gene generation homologous double-crossover of LDH 1 on plant lactobacillus karyomit(e), realize knocking out of LDH 1, for the expression realizing foreign gene further integrated in plant lactobacillus lays the foundation.The structure utilizing Overlap extension PCR to carry out recombinant vectors can greatly reduce the access times of restriction endonuclease and ligase enzyme, saves cost and time, the non-essential nucleotide sequence added when Overlap extension PCR can avoid enzyme to cut simultaneously.Overlap-extension PCR does not have particular requirement to splicing site and neighbouring sequence thereof, as long as just can conveniently PCR operation according to the good overlapping primers of principle design.
Compared with prior art, tool has the following advantages and beneficial effect in the present invention:
(1) the present invention builds by the method for Overlap extension PCR the recombinant plasmid knocking out LDH encoding gene, can effectively connect two DNA fragmentations in vitro, unnecessary non-target DNA sequence can not be introduced in centre, the DNA sequence dna of long segment can be obtained, avoid enzyme and cut the unnecessary base sequence of connection introducing.
(2) the present invention can improve the successful probability of gene splicing by the splicing reaction condition optimizing the upstream and downstream sequence of this encoding gene, can be applied to other engineered fields.
(3) recombinant vectors of the present invention is for the production of the D-ALPHA-Hydroxypropionic acid of high-optical-purity.
Plant lactobacillus DMDL 9010 is open in patent CN102978134A, belong to prior art, its preservation information is: plant lactobacillus DMDL 9010 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 19th, 2011, be called for short: CGMCC, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number: CGMCC NO.5172.
Accompanying drawing explanation
Fig. 1 is the extraction of plant lactobacillus DMDL9010DNA and the electrophorogram of high-fidelity enzymatic amplification ldhL1 upstream and downstream DNA fragmentation, and a is DNA electrophorogram, and b is ldhL1 upstream and downstream DNA fragmentation electrophorogram;
Fig. 2 is the electrophorogram of distribution Overlap extension PCR and single step Overlap extension PCR amplified fragments, and a is single step Overlap extension PCR electrophorogram, and b is distribution Overlap extension PCR electrophorogram (the UP-DOWN fragment of wherein 1,2-single step Overlap extension PCR splicing; The UP-DOWN fragment of 4,5-substep Overlap extension PCR splicing; 3,6-DNA standard substance DL2000 and DL10000);
Fig. 3 be up and the down fragment of different concns Overlap extension PCR is affected electrophorogram (1,2,4,5, the add-on of 6-UP and DOWN is respectively 10ng, 20ng, 30ng, 40ng, 50ng; 3,7-is DNA standard substance DL 10000);
Fig. 4 is that four kinds of intestinal bacteria heat shocks transform pG
+colony growth condition diagram after host9, a, b, c, d are respectively e. coli bl21, intestinal bacteria MC1061, bacillus coli DH 5 alpha e. coli tg1, wherein, e. coli tg1 and MC1061 flat board have obvious single bacterium colony, and bacillus coli DH 5 alpha and BL21 do not have single colony growth;
Fig. 5 is the electrophorogram of double digestion qualification recombinant plasmid pMD-up-down;
Fig. 6 is the electrophorogram of bacterium colony PCR and double digestion qualification recombinant plasmid pGH-up-down, and a is that bacterium colony PCR identifies electrophorogram, and b is that double digestion qualification electrophorogram (wherein 1,2,3 is respectively the target fragment that pcr amplification obtains; 4 is DNA standard substance DL10000; 5 is the pG after single endonuclease digestion
+host9; 6,7,8 is the DNA band after double digestion; 9 is DNA standard substance DL10000).
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but application claims protection domain is not limited thereto.
The extraction of the genomic preparation of embodiment 1 plant lactobacillus DM9010 and recombinant plasmid.Comprise the steps:
(1) centrifugal and collecting precipitation is carried out to the incubated overnight plant lactobacillus bacterium liquid of 1.5ml, add the N,O-Diacetylmuramidase of 0.6mL20mg/ml, after putting upside down mixing 5 ~ 10 times, put 37 DEG C of insulation 40min;
The centrifugal 10min of (2) 12,000rpm room temperature, carefully abandons supernatant liquor.
(3) 150 μ L SP Buffer (containing RNase A1) fully suspended bacterial precipitation is added.
(4) add the Lysozyme solution of 20 μ L, after Homogeneous phase mixing, room temperature leaves standstill 5min.
(5) add the EDTA Buffer of 30 μ L, after Homogeneous phase mixing, room temperature leaves standstill 5min.
(6) the Solution A of 200 μ L is added, 65 DEG C of insulation 10min after concuss.
(7) the Solution B of 400 μ L is added, concuss 15s.
(8) add the Solution C of 650 μ L, turn upside down Homogeneous phase mixing.
The centrifugal 1min of (9) 12,000rpm.
(10) first discard upper organic phase, then aqueous phase solution (colourless lower floor) is moved into the Filter Cup be placed in advance on 1.5mL centrifuge tube, the centrifugal 1min of 12,000rpm.
(11) abandon Filter Cup, in filtrate, add the DB Buffer of 450 μ L, mix.
(12) the Spin Column in test kit is placed on Collection Tube.
(13) be transferred in Spin Column by the mixed solution of above-mentioned (11), the centrifugal 1min of 12,000rpm, abandons filtrate.
(14) be added in Spin Column by the Rinse A of 500 μ L, the centrifugal 1min of 12,000rpm, abandons filtrate.
(15) be added in Spin Column by the Rinse B of 700 μ L, the centrifugal 1min of 12,000rpm, abandons filtrate.
(16) repetitive operation step (15).
(17) Spin Column is placed on Collection Tube, the centrifugal 1min of 12,000rpm.
(18) be placed in by Spin Column on the centrifuge tube of new 1.5mL, add the sterile purified water of 60 μ L in the central authorities of Spin Column film, room temperature leaves standstill 1min.
The centrifugal 1min eluted dna of (19) 12,000rpm.
(20), after extraction terminates, the sepharose with 1% carries out electrophoresis detection to DNA, DNA is stored in-20 DEG C of refrigerators.Plasmid Mini Kit specification sheets with reference to Takara company extracts plasmid DNA (alkaline lysis), carries out agarose gel electrophoresis detection ,-20 DEG C of Refrigerator stores to the DNA of plasmid.
The upstream and downstream fragment primer design of embodiment 2 plant lactobacillus DM9010L-serum lactic dehydrogenase 1 gene and pcr amplification, concrete operations are as follows:
(1) according to the gene sequencing of the coding LDH 1 reported, BLAST is carried out to plant lactobacillus WCFS1, ST-III, the JDM1, ZJ316, the P8,16 that carry out gene order-checking in NCBI, find the position of LDH 1 encoding gene and upstream and downstream thereof to be about the DNA sequence dna of 1000bp, then determine that conserved sequence designs primer and introduces the overlapping fragments of restriction enzyme site and 15bp.
(2) pcr amplification upstream DNA fragment 978bp, segments downstream 870bp, determine that the reaction system of 50 μ l is as follows: plant lactobacillus DNA profiling 5 μ l, 5 × PCR PrimerSTAR, Buffer (Mg
2+plus) 10 μ l, dNTP Mixture 4 μ l, primer each 1 μ l, PrimerSTAR HS DNA Polymerase 0.5 μ l, adds water to 50 μ l.Reaction conditions is as follows: 94 DEG C of denaturation 4min; 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, totally 30 circulations; 72 DEG C extend 5min. and detect PCR primer 1% agarose gel electrophoresis.Glue reclaims purifying object fragment ,-20 DEG C of preservations.Pcr amplification result as shown in Fig. 1 (B) figure, the size of fragment and the in the same size of expection and band is comparatively limpid in sight.
Embodiment 3 Overlap extension PCR connects the condition optimizing of upstream and downstream DNA fragmentation.Concrete operations are as follows:
(1) substep Overlap extension PCR reaction process is divided into three steps: (a) utilizes high-fidelity DNA polymerase to increase respectively upstream and downstream fragment, cuts glue and reclaims.B () carries out overlap-extension PCR to upstream and downstream DNA fragmentation, in reaction process, the high-temperature denatured upstream and downstream DNA fragmentation that makes becomes strand, and then annealing makes the overlapping region (30bp) of upstream and downstream DNA fragmentation match, and template extends each other.Reaction system is as follows: 5 × PCR PrimerSTAR, Buffer (Mg
2+plus) 10 μ l, dNTP Mixture 4 μ l, upstream DNA fragment 2 μ l (about 20ng), downstream DNA fragment 2 μ l (about 20ng), PrimerSTAR HS DNA Polymerase 0.5 μ l, add water 50 μ l.Extension condition: 94 DEG C of denaturation 4min; 94 DEG C of 30s, 55 DEG C of 1min, 72 DEG C of 2min, totally 10 circulations; 72 DEG C extend 5min.C () for template with the upstream and downstream fragment be spliced into, adds template two ends primer and carries out PCR and be obtained by reacting a large amount of splicing fragments.In second step 50 μ l system, add primer Lps-F1, each 1 μ l of Lps-F2, reaction conditions is: 94 DEG C of denaturation 4min; 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 2min, totally 30 circulations; 72 DEG C extend 5min.
(2) single step Overlap extension PCR reaction process is divided into two steps: (a) to increase upstream and downstream fragment respectively with high-fidelity DNA polymerase, cuts glue and reclaims.B the 2nd step above and the 3rd step combine by (), reaction system is: 5 × PCR PrimerSTAR, Buffer (Mg
2+plus) 10 μ l, dNTP Mixture 4 μ l, upstream DNA fragment 2 μ l (about 10ng), downstream DNA fragment 2 μ l (about 10ng), each 1 μ l of primer Lps-F1, Lps-F2, PrimerSTAR HS DNA Polymerase 0.5 μ l, add water 50 μ l.Reaction conditions is: 94 DEG C of denaturation 4min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min, totally 30 circulations; 72 DEG C extend 5min.Be optimized Overlap extension PCR template amount and annealing temperature, be 1:1 according to the concentration ratio of upstream and downstream DNA fragmentation simultaneously, and in the system of 50 μ l, the add-on of upstream and downstream DNA fragmentation is respectively 10ng, 20ng, 30ng, 40ng, 50ng.
Substep Overlap extension PCR and single step Overlap extension PCR, amplification is shown in Fig. 2, the Overlap extension PCR electrophorogram that comparison two kinds is different, can see that single exchange process increases out a little dark than the band of substep exchange process at the DNA band of about 2000bp, namely the operation steps of low 1 the mono-exchange process of of concentration is comparatively simple and save time, the various factors affecting amplification efficiency is less, be conducive to next step and optimize Overlap extension PCR amplification condition, the DNA band hangover that simultaneously two splicings obtain is all relatively more serious, and PCR condition needs to be optimized.The method of single step Overlap extension PCR is adopted to be optimized the consumption of UP fragment and DOWN fragment in the system of amplification, electrophoresis result is shown in Fig. 3, add-on with upstream and downstream DNA fragmentation constantly increases, the brightness flop that the conditions of streaking of DNA band is more and more serious and splice the UP-DOWN band (near 2000bp) obtained is not clearly, the amplification efficiency impact of add-on on Overlap extension PCR of pattern of descriptive parts DNA is not clearly, but excessive template DNA can cause the generation of conditions of streaking, simultaneously also many template DNAs of loss.Locate outside UP-DOWN DNA band in electrophorogram, a nonspecific DNA band is also had at about 1000bp, brightness and object fragment similar, find that this DNA band is that primer residual in upstream and downstream DNA fragmentation causes through checking, therefore when cutting glue and reclaiming DNA band, the time of electrophoresis will, more than 1h, make primer fully separate with PCR primer, reduces non-specific amplification.
Embodiment 4 is for building the screening of recombinant vectors colibacillus engineering.Concrete operations are as follows:
(1) erythromycin of different concns gradient is tested to 4 kinds of colibacillary inhibitions.Colibacillus DH5 α, MC1061, BL21, TG1 that-80 DEG C of glycerine are preserved are carried out being diluted to 10 respectively
-5, 10
-6, 10
-7dilution bacterium liquid, then respectively get these 3 kinds dilution bacterium liquid 100 μ l and be applied on LB flat board, at 37 DEG C, incubated overnight is separated single bacterium colony.Single bacterium colony is inoculated into respectively in LB liquid nutrient medium, in 37 DEG C, bacterium liquid is obtained after 180rpm incubated overnight, bacterium liquid is applied to and is respectively 100 μ g/ml containing erythromycin concentration respectively, 200 μ g/ml, 300 μ g/ml, 400 μ g/ml, on the LB flat board of 500 μ g/ml, incubated overnight is inverted in 37 DEG C, observe four kinds of different bacillus coli DH 5 alphas, MC1061, BL21, TG1 is at the upgrowth situation of the erythromycin of different concns gradient, along with the raising of erythromycin concentration in resistant panel, four kinds of colibacillary growths have delay, but find after cultivation 24h, maximum concentration erythromycin flat board on these four kinds of colibacillary growing states still fine, and be paved with whole flat board.
(2) CaCl is used according in " molecular cloning guide " (third edition)
2prepare the method for competent escherichia coli cell.First the intestinal bacteria inoculating incubated overnight are to LB substratum, and by spectrophotometric determination bacterium liquid OD value to 0.35-0.40, Bacillus coli cells uses the MgCl of 20mmol after ice bath
2-CaCl
2mixed solution is resuspended rear centrifugal, the CaCl of cell precipitation 20mmol
2resuspended.The competent escherichia coli cell getting 200 μ l joins the 1.5ml centrifuge tube of precooling, adds the pG of 1.5 μ l (about 15ng) simultaneously
+host9 plasmid, mixing also places 30min on ice.The transformation mixture of precooling is put into heat shock 90s in the water-bath of 42 DEG C, take out ice bath 1-2min immediately, add the LB substratum of 800ml 37 DEG C of preheatings, the substratum of centrifugal segregation 800ml after 37 DEG C of cultivation 1h, it is the resistant panel of 500 μ g/ml that resuspended bacterium liquid is coated containing erythromycin concentration, incubated overnight.Arrange control group, replace plasmid with sterilized water, its operating process is identical simultaneously.Very consistent through cultivating colony growth on the flat board finding that e. coli bl21 and DH5 α grow, obviously do not distinguish, and the flat board of intestinal bacteria MC1061, TG1 growth has larger white colony to grow out, the bacterium colony being different from other clearly.From two flat boards, the single bacterium colony of picking 5 extracts plasmid after cultivating respectively, and electrophoresis detection is determined containing pG
+the positive bacteria of host9.Therefore intestinal bacteria MC1061, TG1 can as the engineering bacterias building knockout carrier.
The construction process of embodiment 5 homologous recombination vector, method is as follows:
(1) A reaction is added to the DNA fragmentation of splicing, determine that the reaction system of 10 μ l is 10 × Taq Buffer 1 μ l, dATP 1 μ l, DNA profiling 7.5 μ l, Taq polysaccharase 0.5 μ l; Reaction conditions is 72 DEG C and carries out 20min, reclaims DNA fragmentation with cleaning agents box.
(2) UP-DOWN fragment and the pMD18-T carrier hybrid reaction of A will be added according to the method for the product description of Takara.
(3) mixed reaction solution is transformed in intestinal bacteria DH 5 α, coats in the LB flat board containing X-gal and penbritin and carry out blue hickie screening, choose white colony and cultivate and extract plasmid.Pcr amplification qualification is carried out to plasmid, obtains the positive bacteria containing plasmid, first carry out single endonuclease digestion with Xho I, cut with Pst I enzyme after reclaiming single endonuclease digestion fragment, the enzyme system of cutting is 20 μ l systems, and plasmid consumption is no more than 1 μ g again, cut glue and reclaim object fragment, double digestion result as shown in Figure 5.
(4) with the pG cut through Xho I, Pst I substep enzyme
+host9 connects, the ligation system of 10 μ l: 10 × T4Ligase Buffer, UP-DOWN 6 μ l (about 60ng), pG
+host92 μ l (about 20ng), T4Ligase 1 μ l, wherein the mol ratio of vector plasmid and DNA fragmentation is 1:6.5, in 4 DEG C of connections of spending the night.
(5) be then transformed in e. coli tg1, coating containing erythromycin is on the LB flat board of 500 μ g/ml, incubated overnight screening positive bacteria.
(6) bacterium colony PCR identifies positive bacterium colony, and method is as follows: the main mixed solution preparing pcr amplification, is specially: 10 × Taq Buffer 125 μ l, dNTP 100 μ l, Lps-F125 μ l, Lps-F225 μ l, sterilized water 975 μ l; Get single bacterium colony with the sterilizing rifle choicest of 20 μ l and to contain piping and druming mixing in the substratum of 500 μ g/ml erythromycin to 10 μ l, then get the main mixed solution of 1 μ l bacterium liquid to 25 μ l to boiling water bath 10min in PCR pipe; The centrifugal 1min of 12000rpm and ice bath cooling, add Taq polysaccharase, pcr amplification condition is as embodiment 2, amplification is shown in Fig. 6 (A), the size of amplified band and the in the same size of overlap-extension PCR fragment, and band is limpid in sight, be initially identified as the positive bacteria containing recombinant plasmid, check order to the fragment that pcr amplification obtains, sequencing result is as follows simultaneously, can determine the consistent of fragment upstream and segments downstream connecting portion sequence and design from sequence.Cultivate the positive bacterium colony of PCR qualification simultaneously and extract plasmid and carry out double digestion qualification, the same step of endonuclease reaction system (3), result is as shown in Fig. 6 (B), it is consistent that double digestion obtains the increase clip size that obtains of ground object fragment and Overlap extension PCR, the size of linear empty plasmid fragment also with contrast clip size and conform to.Through bacterium colony PCR and double digestion qualification, successfully build and knock out plasmid pGH-up-down.
Claims (3)
1., for knocking out a construction process for the carrier of LDH 1 gene, it is characterized in that, comprise the steps:
(1) from NCBI, find the position of LDH 1 encoding gene and the DNA sequence dna of upstream and downstream 800 ~ 1000bp thereof, then determine conserved sequence design primer and introduce overlapping fragments, extract the DNA of plant lactobacillus (Lactobacillus plantarum) DMDL 9010, and as template, amplification obtains not containing the upstream and downstream DNA fragmentation of this gene of LDH encoding gene, the upstream and downstream DNA fragmentation utilizing high-fidelity enzyme to carry out Overlap extension PCR to connect this gene; The deposit number of described plant lactobacillus DMDL is CGMCC NO.5172;
(2) after 3 ' of above-mentioned junction fragment end being carried out adding A reaction and pMD18-T carrier carry out ligation and be transformed in intestinal bacteria DH 5 α competent cell, finally obtain subcloning vector, then subcloning vector is prepared in a large number, carry out Xho I and Pst I double digestion, overlapping fragments introduces sticky end;
(3) by the pG through Xho I and the process of Pst I double digestion
+the overlapping fragments that host9 plasmid and step (2) obtain carries out ligation, then this is connected product and forwards in intestinal bacteria MC1061 or TG1, in the dull and stereotyped enterprising screening positive transformants bacterium of Erythromycinresistant, obtains recombinant vectors.
2. according to claim 1 construction process, it is characterized in that, the condition of described Overlap extension PCR is: single step Overlap extension PCR, and template concentrations is 0.4ng/ μ l, and introducing the overlapping fragments of primer is 15bp.
3. the carrier of method structure described in claim 1 or 2.
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| CN109385389A (en) * | 2017-08-08 | 2019-02-26 | 光明乳业股份有限公司 | A kind of lactobacillus plantarum ST-III and preparation method thereof lacking ldhA gene |
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