CN105647955A - Shuttle plasmid pPG based on bacillus and preparation method and application thereof - Google Patents

Shuttle plasmid pPG based on bacillus and preparation method and application thereof Download PDF

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CN105647955A
CN105647955A CN201610066098.XA CN201610066098A CN105647955A CN 105647955 A CN105647955 A CN 105647955A CN 201610066098 A CN201610066098 A CN 201610066098A CN 105647955 A CN105647955 A CN 105647955A
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gfp
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牛秋红
张�林
惠丰立
柯涛
董冰雪
韩雪梅
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Nanyang Normal University
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Abstract

The invention discloses a shuttle plasmid pPG based on a bacillus and a preparation method thereof. The base sequence of the shuttle plasmid pPG is shown as SEQ IN NO.1. The preparation method of the shuttle plasmid pPG comprises the steps that a promoter Pxyl sequence, a gfp sequence and a terminator sequence are obtained through PCR amplification separately, and the promoter Pxyl sequence, the gfp sequence and the terminator sequence serve as materials to prepare a Pxyl-gfp-terminator module sequence; a bacillus expression vector pHY300PLK and the Pxyl-gfp-terminator module sequence are subjected to BamHI and XbaI double enzyme digestion, products obtained through double enzyme digestion are connected through T4DNA ligase, and the universal shuttle plasmid pPG for expressing green fluorescent protein of the bacillus is built. According to the shuttle plasmid pPG based on the bacillus and the preparation method thereof, the shuttle plasmid pPG can be converted to enter host bacteria, stable expression of the green fluorescent protein in the bacillus is achieved, and the shuttle plasmid pPG is suitable for all bacilli.

Description

A kind of based on the shuttle plasmid pPG of bacillus cereus, preparation method and application thereof
Technical field
The invention belongs to biological technical field, be specifically related to a kind of based on the shuttle plasmid pPG of bacillus cereus, preparation method and application thereof.
Background technology
Bacillus cereus multiformity in nature is abundant, distribution is wide, has the biological natures such as high temperature high voltage resistant, strong stress resistance, easy storage, is widely used in the industries such as agricultural, industry, food, feedstuff and medicine. Bacillus can produce much important bioactive substance, it is also possible to suppresses various plants pathogen; Utilize the microbial forage additive that bacillus is made to have increase efficiency of feed utilization, improve breeding performonce fo animals, promote immunity of organism, promote the effect such as animal gastrointestinal tract microecological balance and antibacterial diseases prevention. Day by day ripe in view of the importance of bacillus cereus and molecule clone technology, if bacillus cereus can be carried out fluorescent labeling, it is achieved real-time tracking, will study molecular level to bacillus cereus, it is possible to deepen the understanding to bacillus cereus molecular mechanisms of action.
In prior art, conventional method is for specific bacillus cereus, select wherein specific house-keeping gene, such as cellular morphology maintains protein gene mreB, then at the one of carbon tip fusion expression of green fluorescent protein (greenfluorescentprotein of house-keeping gene, gfp), it is achieved gfp constitutive expression bacterial strain. But, owing to different bacillus genes exists specificity, often change a kind of bacterial strain and all must rebuild plasmid, lack the popularity of plasmid application, add to build and can stablize and the difficulty of Bacillus strain of high level expression gfp.
To sum up, problems of the prior art are, the existing plasmid expressing gfp being applied to bacillus cereus lacks popularity, versatility.
Summary of the invention
The purpose of the embodiment of the present invention is to provide a kind of based on the shuttle plasmid pPG of bacillus cereus, preparation method and application thereof, in order to solve the problem that the existing plasmid expressing gfp being applied to bacillus cereus lacks popularity, versatility.
First purpose of the present invention is to provide a kind of shuttle plasmid pPG based on bacillus cereus, and its base sequence is such as shown in SEQIDNO.1, and described shuttle plasmid pPG has gfp sequence.
Preferably, described shuttle plasmid pPG is prepared from bacillus subtilis 168 (Bacillussubtilis168) bacterial strain, plasmid pSG1170, bacillus cereus expression vector pHY300PLK for raw material.
The preparation method that second purpose of the present invention is to provide a kind of shuttle plasmid pPG based on bacillus cereus, comprises the following steps:
Step 1, it is thus achieved that promoter Pxyl sequence, gfp sequence and terminator terminator sequence, including:
Extract the genomic DNA of bacillus cereus, and with the genomic DNA of described bacillus cereus for template, with Pxyl-F and Pxyl-R for primer, carrying out pcr amplification, then pcr amplification product carries out sepharose electrophoresis, last glue reclaims, obtain the promoter Pxyl sequence of upstream belt BamHI restriction enzyme site, its base sequence is such as shown in SEQIDNO.4, and the base sequence of described Pxyl-F is such as shown in SEQIDNO.2, and the base sequence of described Pxyl-R is such as shown in SEQIDNO.3;
Buy the plasmid pSG1170 containing gfp sequence, and with plasmid pSG1170 for template, with Gfp-F and Gfp-R for primer, carrying out pcr amplification, then pcr amplification product carries out sepharose electrophoresis, last glue reclaims, obtain gfp sequence, its base sequence is such as shown in SEQIDNO.7, and the base sequence of described Gfp-F is such as shown in SEQIDNO.5, and the base sequence of described Gfp-R is such as shown in SEQIDNO.6;
With the genomic DNA of described bacillus cereus for template, with Teminator-F and Teminator-R for primer, carry out pcr amplification, then pcr amplification product is carried out sepharose electrophoresis, last glue reclaims, it is thus achieved that the terminator terminator sequence in downstream belt XbaI enzyme cutting site, and its base sequence is such as shown in SEQIDNO.10, the base sequence of described Teminator-F is such as shown in SEQIDNO.8, and the base sequence of described Teminator-R is such as shown in SEQIDNO.9;
Step 2, builds Pxyl-gfp sequence, including:
Step 2.1, the promoter Pxyl sequence obtained in step 1 and gfp sequence are used XhoI single endonuclease digestion respectively, then single endonuclease digestion product carries out sepharose electrophoresis, and last glue reclaims, it is thus achieved that the XhoI single endonuclease digestion product of promoter Pxyl sequence and the XhoI single endonuclease digestion product of gfp sequence;
Step 2.2, connects the XhoI single endonuclease digestion product of promoter Pxyl sequence obtained in step 2.1 and the XhoI single endonuclease digestion product of gfp sequence through T4DNA ligase, then reclaims through sepharose electrophoresis, it is thus achieved that connect product;
Step 2.3, with the connection product of step 2.2 acquisition for template, with Pxyl-F and Gfp-R for primer, carries out pcr amplification, then pcr amplification product carries out sepharose electrophoresis, and last glue reclaims, it is thus achieved that Pxyl-gfp sequence;
Step 3, builds Pxyl-gfp-teminator sequence of modules
Step 3.1, the terminator terminator sequence obtained in the Pxyl-gfp sequence obtained in step 2.3 and step 1 is used BglII single endonuclease digestion respectively, then single endonuclease digestion product is carried out sepharose electrophoresis, last glue reclaims, it is thus achieved that the BglII single endonuclease digestion product of Pxyl-gfp sequence and the BglII single endonuclease digestion product of terminator terminator sequence;
Step 3.2, connects the BglII single endonuclease digestion product of the BglII single endonuclease digestion product of Pxyl-gfp sequence and terminator terminator sequence through T4DNA ligase, then reclaims through sepharose electrophoresis, it is thus achieved that connect product;
Step 3.3, the connection product obtained with step 3.2, for template, with Pxyl-F and Teminator-R for primer, carries out pcr amplification, and glue reclaims after pcr amplification product carries out sepharose electrophoresis, it is thus achieved that Pxyl-gfp-teminator sequence of modules;
Step 4, builds shuttle plasmid pPG
Step 4.1, the Pxyl-gfp-terminator sequence of modules described bacillus cereus expression vector pHY300PLK and step 3.3 obtained respectively is through BamHI and XbaI double digestion, then double digestion product is carried out sepharose electrophoresis, last glue reclaims, it is thus achieved that the double digestion product of bacillus cereus expression vector pHY300PLK and the double digestion product of Pxyl-gfp-terminator sequence of modules;
Step 4.2, the double digestion product of bacillus cereus expression vector pHY300PLK step 4.1 obtained and the double digestion product of Pxyl-gfp-terminator sequence of modules connect through T4DNA ligase, then reclaim through sepharose electrophoresis, it is thus achieved that shuttle plasmid pPG.
Preferably, the bacterial strain adopted when extracting the genomic DNA of described bacillus cereus is bacillus subtilis 168 (Bacillussubtilis168);
Preferably, described with Pxyl-F and Pxyl-R for primer, the reaction system carrying out pcr amplification includes: 10 �� PCR buffer of 5 �� L, the primer Pxyl-F of 1 �� L, the primer Pxyl-R of 1 �� L, the genomic DNA of bacillus cereus of 5 �� L, the dNTP of 8 �� L, the archaeal dna polymerase of 1 �� L, 29 �� L ddH2O, reacts cumulative volume 50 �� L;
Described with Gfp-F and Gfp-R for primer, the reaction system carrying out pcr amplification includes: 10 �� PCR buffer of 5 �� L, the primer Gfp-F of 1 �� L, the primer Gfp-R of 1 �� L, the plasmid pSG1170 of 5 �� L, the dNTP of 8 �� L, the archaeal dna polymerase of 1 �� L, 29 �� L ddH2O, reacts cumulative volume 50 �� L;
Described with Teminator-F and Teminator-R for primer, the reaction system carrying out pcr amplification includes: 10 �� PCR buffer of 5 �� L, the primer Teminator-F of 1 �� L, the primer Teminator-R of 1 �� L, the genomic DNA of bacillus cereus of 5 �� L, the dNTP of 8 �� L, the archaeal dna polymerase of 1 �� L, 29 �� L ddH2O, reacts cumulative volume 50 �� L;
The program of described pcr amplification is: the first step, and 94 DEG C maintain 4min; Second step, including 30 circulations, the condition of each of which circulation be 95 DEG C maintain 30s, afterwards 55 DEG C maintain 45s, afterwards 72 DEG C maintain 1min; 3rd step, 72 DEG C maintain 10min; 4th step, temperature reduces to 4 DEG C, and PCR program stopped;
Preferably, the reaction system of the promoter Pxyl sequence XhoI single endonuclease digestion of step 2.1 includes: the promoter Pxyl sequence of 39 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the XhoI restriction endonuclease of 2 �� L, 4 �� L ddH2O, reacts cumulative volume 50 �� L; Reaction temperature 37 DEG C, response time 2h;
The reaction system of the gfp sequence XhoI single endonuclease digestion of step 2.1 includes: the gfp sequence of 39 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the XhoI restriction endonuclease of 2 �� L, 4 �� L ddH2O, reacts cumulative volume 50 �� L; Reaction temperature 37 DEG C, response time 2h;
The reaction system of the Pxyl-gfp sequence BglII single endonuclease digestion of step 3.1 includes: the Pxyl-gfp sequence of 39 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the BglII restriction endonuclease of 2 �� L, 4 �� L ddH2O, reacts cumulative volume 50 �� L; Reaction temperature 37 DEG C, response time 2h;
The reaction system of the terminator sequence BglII single endonuclease digestion of step 3.1 includes: the terminator sequence of 39 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the BglII restriction endonuclease of 2 �� L, 4 �� L ddH2O, reacts cumulative volume 50 �� L; Reaction temperature 37 DEG C, response time 2h;
Preferably, the bacillus cereus expression vector pHY300PLK of step 4.1 includes through the reaction system of BamHI and XbaI double digestion: the bacillus cereus expression vector pHY300PLK of 37 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the BamHI restriction endonuclease of 2 �� L, the XbaI restriction endonuclease of 2 �� L, 4 �� L ddH2O, cumulative volume 50 �� L; Reaction temperature 37 DEG C, response time 2h;
The Pxyl-gfp-terminator sequence of modules of step 4.1 includes through the reaction system of BamHI and XbaI double digestion: the Pxyl-gfp-terminator sequence of modules of 37 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the BamHI restriction endonuclease of 2 �� L, the XbaI restriction endonuclease of 2 �� L, 4 �� L ddH2O, cumulative volume 50 �� L; Reaction temperature 37 DEG C, response time 2h;
Preferably, in described step 2.2, the XhoI single endonuclease digestion product of promoter Pxyl sequence and what the XhoI single endonuclease digestion product of gfp sequence connected through T4DNA ligase, coupled reaction system includes: the XhoI single endonuclease digestion product of the Pxyl sequence of 5 �� L, the XhoI single endonuclease digestion product of Pxyl sequence of 5 �� L, 10 �� T4DNA ligase buffer of 2 �� L, the T4DNA ligase of 1 �� L, 7 �� L ddH2O, reacts cumulative volume 20 �� L; Reaction temperature 22 DEG C, the response time is 1h;
In described step 3.2, the coupled reaction system that the BglII single endonuclease digestion product of Pxyl-gfp sequence and the BglII single endonuclease digestion product of terminator terminator sequence connect through T4DNA ligase includes: the BglII single endonuclease digestion product of the Pxyl-gfp sequence of 5 �� L, the BglII single endonuclease digestion product of terminator terminator sequence of 5 �� L, 10 �� T4DNA ligase buffer of 2 �� L, the T4DNA ligase of 1 �� L, 7 �� L ddH2O, reacts cumulative volume 20 �� L; Reaction temperature 22 DEG C, the response time is 1h;
In described step 4.2, the coupled reaction system that the double digestion product of bacillus cereus expression vector pHY300PLK and the double digestion product of Pxyl-gfp-terminator sequence of modules connect through T4DNA ligase includes: the double digestion product of bacillus cereus expression vector pHY300PLK of 5 �� L, the double digestion product of Pxyl-gfp-terminator sequence of modules of 5 �� L, 10 �� T4DNA ligase buffer of 2 �� L, the T4DNA ligase of 1 �� L, 7 �� L ddH2O, reacts cumulative volume 20 �� L; Reaction temperature 22 DEG C, the response time is 1h.
The present invention the 3rd purpose must be to provide a kind of shuttle plasmid pPG based on bacillus cereus application of expressing green fluorescent protein in bacillus cereus.
The invention has the beneficial effects as follows, shuttle plasmid pPG provided by the invention, utilize bacillus cereus expression vector pHY300PLK, choose the promoter Pxyl of the general xylose height efficient expression of bacillus cereus, choose gfp gene order, successfully construct a kind of shuttle plasmid pPG expressing bacillus cereus green fluorescent protein, this Plastid transformation is entered Host Strains, the whole thalline of bacillus cereus can be realized and present stable green fluorescence, it is adaptable to all bacillus cereuss. Further, this Plastid transformation is entered Host Strains, it may be achieved the whole thalline of bacillus cereus presents stable green fluorescence, a good platform has been built in the research of its molecular mechanism.
Accompanying drawing explanation
Fig. 1 is the structure flow chart illustration of shuttle plasmid pPG of the present invention;
The fluorescence microscopy view of Bacillus pumilus-pPG transformant when Fig. 2 is shuttle plasmid pPG feasibility of the present invention checking;
The fluorescence microscopy view of bacillus subtilis-pPG transformant when Fig. 3 is shuttle plasmid pPG feasibility of the present invention checking;
The fluorescence microscopy view of bacillus amyloliquefaciens-pPG transformant when Fig. 4 is shuttle plasmid pPG feasibility of the present invention checking.
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.
The present invention provides a kind of shuttle plasmid pPG, total length 5989bp based on bacillus cereus, and its base sequence is such as shown in SEQIDNO.1, and described shuttle plasmid pPG has gfp sequence.
Based on same inventive concept, the preparation method that the invention provides a kind of shuttle plasmid pPG, but should not be construed as limitation of the present invention.
If not specializing, the conventional means that technological means used in the preparation method of a kind of shuttle plasmid pPG provided by the invention is well known to those skilled in the art, if and conventional means well-known to those skilled in the art includes more than one alternate embodiment, then any one alternate embodiment impacts all without to the result of the present invention.
The preparation of Bacillus pumilus competent cell and conversion in embodiment, preparation and the conversion of bacillus subtilis bacterium competence cell, preparation and the conversion of bacillus amyloliquefaciens competent cell, the extraction of plasmid and digestion with restriction enzyme, the recovery of DNA fragmentation, the connection of linear DNA fragment, pcr amplification reaction etc. are translated " Molecular Cloning: A Laboratory guide " second edition related Sections with reference to Jin Dongyan, Li Mengfeng etc. and are carried out.
As it is shown in figure 1, the preparation method of a kind of shuttle plasmid pPG of the embodiment of the present invention, comprise the following steps:
Step 1, it is thus achieved that the promoter Pxyl sequence of upstream belt BamHI
Step 1.1, buys bacillus subtilis 168 (Bacillussubtilis168) bacterial strain from Southern Yangtze University's Chinese Universities ' industrial microorganism resource and information centre (CICIM-CU), and extracts the genomic DNA of this bacterial strain;
Step 1.2, the genomic DNA obtained in step 1.1 is template, with Pxyl-F (5 '-cgcggatcccatttccccctttgatttttag-3 ') and Pxyl-R (5 '-ccgctcgagtcctttgtttatccaccgaac-3 ') for primer, carry out pcr amplification, the base sequence of described Pxyl-F such as SEQIDNO.2, the base sequence of described Pxyl-R is such as shown in SEQIDNO.3;
Step 1.3, carries out sepharose electrophoresis by the pcr amplification product in step 1.2, carries out glue recovery afterwards, it is thus achieved that the promoter Pxyl sequence of upstream belt BamHI, and overall length is 285bp, and its base sequence is such as shown in SEQIDNO.4.
Step 2, it is thus achieved that gfp sequence
Step 2.1, the plasmid pSG1170 containing gfp is bought from BacillusGeneticStockCenter (BGSC), and with plasmid pSG1170 for template, with Gfp-F (5 '-ccgctcgaggtggttattattcaaattgc-3 ') and Gfp-R (5 '-ggaagatctgagattttctagttcagtcag-3 ') for primer, carry out pcr amplification, the base sequence of Gfp-F is such as shown in SEQIDNO.5, and the base sequence of described Gfp-R is such as shown in SEQIDNO.6;
Step 2.2, carries out sepharose electrophoresis by the pcr amplification product in step 2.1, carries out glue recovery afterwards, it is thus achieved that total length is the gfp gene of 789bp, and its base sequence is such as shown in SEQIDNO.7.
Step 3, pcr amplification obtains the terminator terminator sequence of downstream belt XbaI
Step 3.1, the genomic DNA obtained in step 1.1 is template, with Teminator-F (5 '-ggaagatctacgttcttgccattgctgc-3 ') and Teminator-R (5 '-ctagtctagatcgatatctctgcagtcgcgatgatt-3 ') for primer, carry out pcr amplification, the base sequence of described Teminator-F is such as shown in SEQIDNO.8, and the base sequence of described Teminator-R is such as shown in SEQIDNO.9;
Step 3.2, carries out sepharose electrophoresis by the pcr amplification product in step 3.1, carries out glue recovery afterwards, it is thus achieved that the terminator terminator sequence of downstream belt XbaI, overall length is 105bp, and its base sequence is such as shown in SEQIDNO.10.
Step 4, builds Pxyl-gfp sequence, including,
Step 4.1, the promoter Pxyl sequence obtained in step 1 and gfp sequence are used XhoI single endonuclease digestion respectively, then single endonuclease digestion product carries out sepharose electrophoresis, and last glue reclaims, it is thus achieved that the XhoI single endonuclease digestion product of promoter Pxyl sequence and the XhoI single endonuclease digestion product of gfp sequence;
Step 4.2, connects the XhoI single endonuclease digestion product of promoter Pxyl sequence obtained in step 4.1 and the XhoI single endonuclease digestion product of gfp sequence through T4DNA ligase, then reclaims through sepharose electrophoresis, it is thus achieved that connect product;
Step 4.3, with the connection product of step 4.2 acquisition for template, with Pxyl-F and Gfp-R for primer, carries out pcr amplification, then pcr amplification product carries out sepharose electrophoresis, and last glue reclaims, it is thus achieved that Pxyl-gfp sequence;
Step 5, builds Pxyl-gfp-teminator sequence of modules
Step 5.1, the terminator terminator sequence obtained in the Pxyl-gfp sequence obtained in step 4.3 and step 1.3 is used BglII single endonuclease digestion respectively, then single endonuclease digestion product is carried out sepharose electrophoresis, last glue reclaims, it is thus achieved that the BglII single endonuclease digestion product of Pxyl-gfp sequence and the BglII single endonuclease digestion product of terminator terminator sequence;
Step 5.2, connects the BglII single endonuclease digestion product of Pxyl-gfp sequence obtained in step 5.1 and the BglII single endonuclease digestion product of terminator terminator sequence through T4DNA ligase, then reclaims through sepharose electrophoresis, it is thus achieved that connect product;
Step 5.3, the connection product obtained with step 5.2, for template, with Pxyl-F and Teminator-R for primer, carries out pcr amplification, and glue reclaims after pcr amplification product carries out sepharose electrophoresis, it is thus achieved that Pxyl-gfp-teminator sequence of modules;
Step 6, builds shuttle plasmid pPG
Step 6.1, the Pxyl-gfp-terminator sequence of modules described bacillus cereus expression vector pHY300PLK and step 5.3 obtained respectively is through BamHI and XbaI double digestion, then double digestion product is carried out sepharose electrophoresis, last glue reclaims, it is thus achieved that the double digestion product of bacillus cereus expression vector pHY300PLK and the double digestion product of Pxyl-gfp-terminator sequence of modules;
Step 6.2, the double digestion product of bacillus cereus expression vector pHY300PLK step 4.1 obtained and the double digestion product of Pxyl-gfp-terminator sequence of modules connect through T4DNA ligase, then reclaim through sepharose electrophoresis, it is thus achieved that shuttle plasmid pPG.
It should be noted that pcr amplification reaction system and program in step 1.2, step 2.1, step 3.1 adopt this area conventional means all can be effective, provide below a kind of preferred pcr amplification condition:
The pcr amplification reaction system of step 1.2 includes: 10 �� PCR buffer of 5 �� L, the primer Pxyl-F of 1 �� L, the primer Pxyl-R of 1 �� L, the dNTP of genomic DNA 8 �� L of bacillus cereus of 5 �� L, the archaeal dna polymerase of 1 �� L, 29 �� L ddH2O, reacts cumulative volume 50 �� L;
The reaction system of the pcr amplification of step 2.1 includes: 10 �� PCR buffer of 5 �� L, the primer Gfp-F of 1 �� L, the primer Gfp-R of 1 �� L, the plasmid pSG1170 of 5 �� L, the dNTP of 8 �� L, the archaeal dna polymerase of 1 �� L, 29 �� L ddH2O, reacts cumulative volume 50 �� L;
The reaction system of the pcr amplification of step 3.1 includes: 10 �� PCR buffer of 5 �� L, the primer Teminator-F of 1 �� L, the primer Teminator-R of 1 �� L, the genomic DNA of bacillus cereus of 5 �� L, the dNTP of 8 �� L, the archaeal dna polymerase of 1 �� L, 29 �� L ddH2O, reacts cumulative volume 50 �� L;
The program of the pcr amplification in step 1.2, step 2.1, step 3.1 is: the first step, and 94 DEG C maintain 4min; Second step, including 30 circulations, the condition of each of which circulation be 95 DEG C maintain 30s, afterwards 55 DEG C maintain 45s, afterwards 72 DEG C maintain 1min; 3rd step, 72 DEG C maintain 10min; 4th step, temperature reduces to 4 DEG C, and PCR program stopped.
It should be noted that endonuclease reaction system in step 4.1, step 5.1 and step 6.1, reaction temperature and response time adopt this area conventional means all can be effective, provide below a kind of preferred endonuclease reaction condition:
The reaction system of the promoter Pxyl sequence XhoI single endonuclease digestion of step 4.1 includes: the promoter Pxyl sequence of 39 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the XhoI restriction endonuclease of 2 �� L, 4 �� L ddH2O, reacts cumulative volume 50 �� L; Reaction temperature 37 DEG C, response time 2h;
The reaction system of the gfp sequence XhoI single endonuclease digestion of step 4.1 includes: the gfp sequence of 39 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the XhoI restriction endonuclease of 2 �� L, 4 �� L ddH2O, reacts cumulative volume 50 �� L; Reaction temperature 37 DEG C, response time 2h;
The reaction system of the Pxyl-gfp sequence BglII single endonuclease digestion in step 5.1 includes: the Pxyl-gfp sequence of 39 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the BglII restriction endonuclease of 2 �� L, 4 �� L ddH2O, reacts cumulative volume 50 �� L; Reaction temperature 37 DEG C, response time 2h;
The reaction system of the terminator sequence BglII single endonuclease digestion of step 5.1 includes: the terminator sequence of 39 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the BglII restriction endonuclease of 2 �� L, 4 �� L ddH2O, reacts cumulative volume 50 �� L; Reaction temperature 37 DEG C, response time 2h.
The bacillus cereus expression vector pHY300PLK of described step 6.1 includes through the reaction system of BamHI and XbaI double digestion: the bacillus cereus expression vector pHY300PLK of 37 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the BamHI restriction endonuclease of 2 �� L, the XbaI restriction endonuclease of 2 �� L, 4 �� L ddH2O, cumulative volume 50 �� L; Reaction temperature 37 DEG C, response time 2h;
The Pxyl-gfp-terminator sequence of modules of step 6.1 includes through the reaction system of BamHI and XbaI double digestion: the Pxyl-gfp-terminator sequence of modules of 37 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the BamHI restriction endonuclease of 2 �� L, the XbaI restriction endonuclease of 2 �� L, 4 �� L ddH2O, cumulative volume 50 �� L; Reaction temperature 37 DEG C, response time 2h.
It should be noted that coupled reaction system in step 4.2, step 5.2 and step 6.2, reaction temperature and response time adopt this area conventional means all can be effective, provide below a kind of preferred coupled reaction condition:
In described step 4.2, the coupled reaction system that the XhoI single endonuclease digestion product of promoter Pxyl sequence and the XhoI single endonuclease digestion product of gfp sequence connect through T4DNA ligase includes: the XhoI single endonuclease digestion product of the promoter Pxyl sequence of 5 �� L, the XhoI single endonuclease digestion product of gfp sequence of 5 �� L, 10 �� T4DNA ligase buffer of 2 �� L, the T4DNA ligase of 1 �� L, 7 �� L ddH2O, reacts cumulative volume 20 �� L;Reaction temperature 22 DEG C, the response time is 1h;
In described step 5.2, the coupled reaction system that the BglII single endonuclease digestion product of Pxyl-gfp sequence and the BglII single endonuclease digestion product of terminator terminator sequence connect through T4DNA ligase includes: the BglII single endonuclease digestion product of the Pxyl-gfp sequence of 5 �� L, the BglII single endonuclease digestion product of terminator terminator sequence of 5 �� L, 10 �� T4DNA ligase buffer of 2 �� L, the T4DNA ligase of 1 �� L, 7 �� L ddH2O, reacts cumulative volume 20 �� L; Reaction temperature 22 DEG C, the response time is 1h;
In described step 6.2, the coupled reaction system that the double digestion product of bacillus cereus expression vector pHY300PLK and the double digestion product of Pxyl-gfp-terminator sequence of modules connect through T4DNA ligase includes: the double digestion product of bacillus cereus expression vector pHY300PLK of 5 �� L, the double digestion product of Pxyl-gfp-terminator sequence of modules of 5 �� L, 10 �� T4DNA ligase buffer of 2 �� L, the T4DNA ligase of 1 �� L, 7 �� L ddH2O, reacts cumulative volume 20 �� L; Reaction temperature 22 DEG C, the response time is 1h.
It should be noted that, described Pxyl-F, Pxyl-R, Gfp-F, Gfp-R, Teminator-F and Teminator-R base sequence synthesized by TakaRa company, restriction endonuclease used in the present invention includes XhoI, BglII, BamHI and XbaI, all purchased from TakaRa company, other experiment materials used in the present invention are all purchased from all purchased from TakaRa company.
It should be noted that, difference only one of which XhoI restriction enzyme site in described Pxyl sequence and gfp sequence, difference only one of which BglII restriction enzyme site in described Pxyl-gfp sequence and terminator terminator sequence, only one of which is distinguished in BamHI and XbaI enzyme cutting site in described Pxyl-gfp-terminator sequence of modules, and only one of which is also distinguished in BamHI and XbaI enzyme cutting site on described bacillus cereus expression vector pHY300PLK.
Shuttle plasmid pPG provided by the invention, utilize bacillus cereus expression vector pHY300PLK, choose the promoter Pxyl of the general xylose height efficient expression of bacillus cereus, choose gfp gene order, successfully construct a kind of general shuttle plasmid pPG expressing bacillus cereus green fluorescent protein, this Plastid transformation is entered Host Strains, it may be achieved the whole thalline of bacillus cereus presents stable green fluorescence, it is adaptable to all bacillus cereuss. Further, this Plastid transformation is entered Host Strains, it may be achieved the whole thalline of bacillus cereus presents stable green fluorescence, a good platform has been built in the research of its molecular mechanism.
Further, have chosen common Bacillus pumilus, bacillus subtilis and bacillus amyloliquefaciens in the experiment of this area and carry out feasibility checking, comprise the following steps:
Prepare the competent cell of Bacillus pumilus, bacillus subtilis and bacillus amyloliquefaciens respectively; The shuttle plasmid pPG built is converted respectively in the competent cell of Bacillus pumilus, bacillus subtilis and bacillus amyloliquefaciens, namely builds Bacillus pumilus-pPG transformant, bacillus subtilis-pPG transformant and bacillus amyloliquefaciens-pPG transformant respectively; Bacillus pumilus-pPG transformant, bacillus subtilis-pPG transformant and bacillus amyloliquefaciens-pPG transformant are respectively placed in fluorescence microscopy Microscopic observation. Result shows: Fig. 2,3 and 4 respectively Bacillus pumilus-pPG transformant, bacillus subtilis-pPG transformant and bacillus amyloliquefaciens-pPG transformants fluorescence microscopy view, result shows, each transformant all can be observed obvious fluorescent labeling effect under fluorescence microscope, illustrate that shuttle plasmid pPG of the present invention all can express in each bacillus cell, there is good versatility.
The present invention can be carried out various change and modification without deviating from the spirit and scope of the present invention by those skilled in the art, if these amendments of the present invention and modification belong within the scope of the claims in the present invention and equivalent technologies thereof, then the present invention is also intended to comprise these change and modification.

Claims (9)

1. the shuttle plasmid pPG based on bacillus cereus, it is characterised in that its base sequence is such as shown in SEQIDNO.1, and described shuttle plasmid pPG has gfp sequence.
2. shuttle plasmid pPG according to claim 1, it is characterized in that, described shuttle plasmid pPG is prepared from bacillus subtilis 168 (Bacillussubtilis168) bacterial strain, plasmid pSG1170, bacillus cereus expression vector pHY300PLK for raw material.
3. the preparation method of the shuttle plasmid pPG described in a claim 1, it is characterised in that comprise the following steps:
Step 1, it is thus achieved that promoter Pxyl sequence, gfp sequence and terminator terminator sequence, including:
Extract the genomic DNA of bacillus cereus, and with the genomic DNA of described bacillus cereus for template, with Pxyl-F and Pxyl-R for primer, carrying out pcr amplification, then pcr amplification product carries out sepharose electrophoresis, last glue reclaims, obtain the promoter Pxyl sequence of upstream belt BamHI restriction enzyme site, its base sequence is such as shown in SEQIDNO.4, and the base sequence of described Pxyl-F is such as shown in SEQIDNO.2, and the base sequence of described Pxyl-R is such as shown in SEQIDNO.3;
Buy the plasmid pSG1170 containing gfp sequence, and with plasmid pSG1170 for template, with Gfp-F and Gfp-R for primer, carrying out pcr amplification, then pcr amplification product carries out sepharose electrophoresis, last glue reclaims, obtain gfp sequence, its base sequence is such as shown in SEQIDNO.7, and the base sequence of described Gfp-F is such as shown in SEQIDNO.5, and the base sequence of described Gfp-R is such as shown in SEQIDNO.6;
With the genomic DNA of described bacillus cereus for template, with Teminator-F and Teminator-R for primer, carry out pcr amplification, then pcr amplification product is carried out sepharose electrophoresis, last glue reclaims, it is thus achieved that the terminator terminator sequence in downstream belt XbaI enzyme cutting site, and its base sequence is such as shown in SEQIDNO.10, the base sequence of described Teminator-F is such as shown in SEQIDNO.8, and the base sequence of described Teminator-R is such as shown in SEQIDNO.9;
Step 2, builds Pxyl-gfp sequence, including,
Step 2.1, the promoter Pxyl sequence obtained in step 1 and gfp sequence are used XhoI single endonuclease digestion respectively, then single endonuclease digestion product carries out sepharose electrophoresis, and last glue reclaims, it is thus achieved that the XhoI single endonuclease digestion product of promoter Pxyl sequence and the XhoI single endonuclease digestion product of gfp sequence;
Step 2.2, connects the XhoI single endonuclease digestion product of promoter Pxyl sequence obtained in step 2.1 and the XhoI single endonuclease digestion product of gfp sequence through T4DNA ligase, then reclaims through sepharose electrophoresis, it is thus achieved that connect product;
Step 2.3, with the connection product of step 2.2 acquisition for template, with Pxyl-F and Gfp-R for primer, carries out pcr amplification, then pcr amplification product carries out sepharose electrophoresis, and last glue reclaims, it is thus achieved that Pxyl-gfp sequence;
Step 3, builds Pxyl-gfp-teminator sequence of modules
Step 3.1, the terminator terminator sequence obtained in the Pxyl-gfp sequence obtained in step 2.3 and step 1 is used BglII single endonuclease digestion respectively, then single endonuclease digestion product is carried out sepharose electrophoresis, last glue reclaims, it is thus achieved that the BglII single endonuclease digestion product of Pxyl-gfp sequence and the BglII single endonuclease digestion product of terminator terminator sequence;
Step 3.2, connects the BglII single endonuclease digestion product of the BglII single endonuclease digestion product of Pxyl-gfp sequence and terminator terminator sequence through T4DNA ligase, then reclaims through sepharose electrophoresis, it is thus achieved that connect product;
Step 3.3, the connection product obtained with step 3.2, for template, with Pxyl-F and Teminator-R for primer, carries out pcr amplification, and glue reclaims after pcr amplification product carries out sepharose electrophoresis, it is thus achieved that Pxyl-gfp-teminator sequence of modules;
Step 4, builds shuttle plasmid pPG
Step 4.1, Pxyl-gfp-terminator sequence of modules bacillus cereus expression vector pHY300PLK and step 3.3 obtained respectively is through BamHI and XbaI double digestion, then double digestion product is carried out sepharose electrophoresis, last glue reclaims, it is thus achieved that the double digestion product of bacillus cereus expression vector pHY300PLK and the double digestion product of Pxyl-gfp-terminator sequence of modules;
Step 4.2, the double digestion product of bacillus cereus expression vector pHY300PLK step 4.1 obtained and the double digestion product of Pxyl-gfp-terminator sequence of modules connect through T4DNA ligase, then reclaim through sepharose electrophoresis, it is thus achieved that shuttle plasmid pPG.
4. the preparation method of shuttle plasmid pPG according to claim 3, it is characterised in that the bacterial strain adopted when extracting the genomic DNA of described bacillus cereus is bacillus subtilis 168 (Bacillussubtilis168).
5. the preparation method of the shuttle plasmid pPG according to any one of claim 3-4, it is characterized in that, described with Pxyl-F and Pxyl-R for primer, the reaction system carrying out pcr amplification includes: 10 �� PCR buffer of 5 �� L, the primer Pxyl-F of 1 �� L, the primer Pxyl-R of 1 �� L, the genomic DNA of bacillus cereus of 5 �� L, the dNTP of 8 �� L, the archaeal dna polymerase of 1 �� L, 29 �� L ddH2O, reacts cumulative volume 50 �� L;
Described with Gfp-F and Gfp-R for primer, the reaction system carrying out pcr amplification includes: 10 �� PCR buffer of 5 �� L, the primer Gfp-F of 1 �� L, the primer Gfp-R of 1 �� L, the plasmid pSG1170 of 5 �� L, the dNTP of 8 �� L, the archaeal dna polymerase of 1 �� L, 29 �� L ddH2O, reacts cumulative volume 50 �� L;
Described with Teminator-F and Teminator-R for primer, the reaction system carrying out pcr amplification includes: 10 �� PCR buffer of 5 �� L, the primer Teminator-F of 1 �� L, the primer Teminator-R of 1 �� L, the genomic DNA of bacillus cereus of 5 �� L, the dNTP of 8 �� L, the archaeal dna polymerase of 1 �� L, 29 �� L ddH2O, reacts cumulative volume 50 �� L;
The program of described pcr amplification is: the first step, and 94 DEG C maintain 4min; Second step, including 30 circulations, the condition of each of which circulation be 95 DEG C maintain 30s, afterwards 55 DEG C maintain 45s, afterwards 72 DEG C maintain 1min; 3rd step, 72 DEG C maintain 10min; 4th step, temperature reduces to 4 DEG C, and PCR program stopped.
6. the preparation method of the shuttle plasmid pPG according to any one of claim 3-4, it is characterized in that, the reaction system of the promoter Pxyl sequence XhoI single endonuclease digestion of step 2.1 includes: the promoter Pxyl sequence of 39 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the XhoI restriction endonuclease of 2 �� L, 4 �� L ddH2O, reacts cumulative volume 50 �� L; Reaction temperature 37 DEG C, response time 2h;
The reaction system of the gfp sequence XhoI single endonuclease digestion of step 2.1 includes: the gfp sequence of 39 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the XhoI restriction endonuclease of 2 �� L, 4 �� L ddH2O, reacts cumulative volume 50 �� L;Reaction temperature 37 DEG C, response time 2h;
The reaction system of the Pxyl-gfp sequence BglII single endonuclease digestion of step 3.1 includes: the Pxyl-gfp sequence of 39 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the BglII restriction endonuclease of 2 �� L, 4 �� L ddH2O, reacts cumulative volume 50 �� L; Reaction temperature 37 DEG C, response time 2h;
The reaction system of the terminator sequence BglII single endonuclease digestion of step 3.1 includes: the terminator sequence of 39 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the BglII restriction endonuclease of 2 �� L, 4 �� L ddH2O, reacts cumulative volume 50 �� L; Reaction temperature 37 DEG C, response time 2h.
7. the preparation method of the shuttle plasmid pPG according to any one of claim 3-4, it is characterized in that, the bacillus cereus expression vector pHY300PLK of step 4.1 includes through the reaction system of BamHI and XbaI double digestion: the bacillus cereus expression vector pHY300PLK of 37 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the BamHI restriction endonuclease of 2 �� L, the XbaI restriction endonuclease of 2 �� L, 4 �� L ddH2O, cumulative volume 50 �� L; Reaction temperature 37 DEG C, response time 2h;
The Pxyl-gfp-terminator sequence of modules of step 4.1 includes through the reaction system of BamHI and XbaI double digestion: the Pxyl-gfp-terminator sequence of modules of 37 �� L, 10 �� NEB enzyme cutting buffering liquid of 5 �� L, the BamHI restriction endonuclease of 2 �� L, the XbaI restriction endonuclease of 2 �� L, 4 �� L ddH2O, cumulative volume 50 �� L; Reaction temperature 37 DEG C, response time 2h.
8. the preparation method of the shuttle plasmid pPG according to any one of claim 3-4, it is characterized in that, in described step 2.2, the coupled reaction system that the XhoI single endonuclease digestion product of promoter Pxyl sequence and the XhoI single endonuclease digestion product of gfp sequence connect through T4DNA ligase includes: the XhoI single endonuclease digestion product of the promoter Pxyl sequence of 5 �� L, the XhoI single endonuclease digestion product of gfp sequence of 5 �� L, 10 �� T4DNA ligase buffer of 2 �� L, the T4DNA ligase of 1 �� L, 7 �� L ddH2O, reacts cumulative volume 20 �� L; Reaction temperature 22 DEG C, the response time is 1h;
In described step 3.2, the coupled reaction system that the BglII single endonuclease digestion product of Pxyl-gfp sequence and the BglII single endonuclease digestion product of terminator terminator sequence connect through T4DNA ligase includes: the BglII single endonuclease digestion product of the Pxyl-gfp sequence of 5 �� L, the BglII single endonuclease digestion product of terminator terminator sequence of 5 �� L, 10 �� T4DNA ligase buffer of 2 �� L, the T4DNA ligase of 1 �� L, 7 �� L ddH2O, reacts cumulative volume 20 �� L; Reaction temperature 22 DEG C, the response time is 1h;
In described step 4.2, the coupled reaction system that the double digestion product of bacillus cereus expression vector pHY300PLK and the double digestion product of Pxyl-gfp-terminator sequence of modules connect through T4DNA ligase includes: the double digestion product of bacillus cereus expression vector pHY300PLK of 5 �� L, the double digestion product of Pxyl-gfp-terminator sequence of modules of 5 �� L, 10 �� T4DNA ligase buffer of 2 �� L, the T4DNA ligase of l �� L, 7 �� L ddH2O, reacts cumulative volume 20 �� L; Reaction temperature 22 DEG C, the response time is 1h.
9. the shuttle plasmid pPG application of expressing green fluorescent protein in bacillus cereus based on bacillus cereus as claimed in claim 1.
CN201610066098.XA 2016-01-25 2016-01-25 Shuttle plasmid pPG based on bacillus and preparation method and application thereof Pending CN105647955A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1782080A (en) * 2005-07-21 2006-06-07 复旦大学 Bacillus pumilus expression system

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1782080A (en) * 2005-07-21 2006-06-07 复旦大学 Bacillus pumilus expression system

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
余小霞等: "枯草芽孢杆菌表达系统及其启动子研究进展", 《生物技术通报》 *
殷幼平等: "生防菌枯草芽孢杆菌 CQBS03 的绿色荧光蛋白基因标记及其在柑橘叶片上的定殖", 《中国农业科学》 *

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