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A method for establishing a Lactobacillus reuteri non-resistance marker gene integration system and its application

A technology of Lactobacillus reuteri without resistance markers, which is applied in the field of genetic engineering to achieve the effect of avoiding mutations

Inactive Publication Date: 2019-02-01
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Using the pheS point mutation gene as the reverse screening gene for gene insertion in Lactobacillus reuteri and the method of constructing the integration system have not been reported in China

Method used

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  • A method for establishing a Lactobacillus reuteri non-resistance marker gene integration system and its application
  • A method for establishing a Lactobacillus reuteri non-resistance marker gene integration system and its application
  • A method for establishing a Lactobacillus reuteri non-resistance marker gene integration system and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0045] Example 1 Cloning of pheS gene and construction of its mutant gene

[0046] Design specific primers according to the Lactobacillus reuteri pheS gene sequence published in Genebank, then use the Lactobacillus reuteri XNY genomic DNA as a template to amplify, connect to the cloning vector pMD19-T for sequencing, and obtain the correct L. reuteri XNY pheS gene; then carry out pheS gene amino acid sequence alignment ( figure 1 ), combined with the published literature to determine the mutation site as the second base (C mutation to G) of the 312th amino acid (Ala), and then use the overlapping PCR technique to construct the point mutation gene pheSM of the pheS gene ( figure 2 ).

Embodiment 2

[0047] Example 2 Construction of pheSM gene recombinant L. reuteri XNYM strain

[0048] Select the insertion site of the target gene, and use Lactobacillus reuteri XNY genomic DNA as a template for PCR amplification to obtain 700bp homology arm fragments (LR, RR) upstream and downstream of the insertion site; then amplify the promoter fragment (P), The erythromycin resistance gene (EM) fragment was connected with the pheSM gene and the homology arm fragment by overlapping PCR technology to obtain the LR-P-pheSM-EM-RR integration frame, which was connected to the cloning vector pMD19-T for sequencing, After obtaining the correct transformant, double enzyme digestion to obtain the LR-P-pheSM-EM-RR integration box, electrotransform into L. reuteri XNY competent cells, spread the MRS plate containing erythromycin, and extract the genomic DNA of the positive transformant And carry out PCR detection, obtain pheSM gene recombinant Lactobacillus L.reuteri XNYM ( image 3 ).

Embodiment 3

[0049] Example 3 Construction of L.reuteri recombinant cellulase genetically engineered bacteria

[0050] The cellulase gene cel15 (including the promoter) was connected with the homology arm sequences at both ends of the insertion site by overlapping PCR technology to obtain the cel15 gene integration framework LR-cel15-RR, which was connected to the cloning vector pMD19-T for sequencing to obtain the correct After the transformant, the integrated frame was obtained by double enzyme digestion, electrotransformed into pheSM gene recombinant Lactobacillus L. reuteri XNYM competent cells, spread on the MRS plate containing p-Cl-Phe, extracted positive transformant genomic DNA, and detected by PCR The L. reuteri recombinant bacteria ( Figure 4 ).

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Abstract

The invention discloses a building method and application of a lactobacillus reuteri resistance-marker-free gene integration system. By means of the point mutation technology, a point mutation gene pheSM of a lactobacillus reuteri pheS gene is obtained, the point mutation gene pheSM and a erythromycin resistance gene are used for building an integrated framework LR-pheSM-em-RR, the integrated framework LR-pheSM-em-RR is electrically converted into L.renteri XNY competent cells, and pheSM genetic recombination lactobacillus L.renteri XNYM is obtained through erythromycin screening; then, the gene integration framework is constructed and inserted, then the gene integration framework is electrically converted into pheSM gene recombination lactobacillus reuteri competent cells, and the lactobacillus reuteri without resistance markers can be obtained through para-chlorophenylalanine acid resistance screening. According to the method, resistance genes are not introduced into host bacteria genomes, so that biological safety hidden hazards of the resistance genes and the polarity effect of the resistance genes on upstream genes and downstream genes are avoided. By means of the system, gene targeted integration can be carried out on the host bacteria for multiple times.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for establishing a Lactobacillus reuteri resistance-free marker gene integration system and its application. Background technique [0002] Lactic acid bacteria are generally recognized as safe (Generally Regarded As Safe Organisms, GRAS) microorganisms, and are the dominant probiotics in the digestive tract of animals; Lactobacillus is beneficial to animals, and can produce active substances such as lactobacillus Lactocidin, Lactacin, Acidophilucin and Acidocin, and pass Mechanisms such as biological antagonism, improvement of the ecological environment in the intestinal tract of animals and improvement of animal immunity play its role. At the same time, lactic acid bacteria have many characteristics, such as the ability to secrete hydrophobic substances on the cell surface and extracellular matrix molecules, convenient gene manipulation, and the ability of l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12R1/225
Inventor 陈玉林曹平华王小龙杨雨鑫
Owner NORTHWEST A & F UNIV
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