Application of sucrose lethal gene SacB in gene deleted reverse screening marker, and marker-free deleted suicide vector of sucrose lethal gene SacB

A suicide carrier and reverse screening technology, which is applied in the direction of introducing foreign genetic material, carrier, nucleic acid carrier, etc., can solve the problems of limited range of resistance gene selection, trace deletion, non-transcription of downstream genes, etc., and achieve biological Universality, effects of avoiding polar effects

Inactive Publication Date: 2018-09-07
SICHUAN AGRI UNIV
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

The current method for gene deletion of Riemerella anatipestifer is mainly scar deletion, and scar deletion will leave a resistance "scar" in the genome and cause non-transcription of downstream genes, that is, "polar effect"
In addition, due to the multidrug resistance of R. anatipestifer, the selection of resistance genes for the scarred deletion of R. anatipestifer is limited

Method used

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  • Application of sucrose lethal gene SacB in gene deleted reverse screening marker, and marker-free deleted suicide vector of sucrose lethal gene SacB
  • Application of sucrose lethal gene SacB in gene deleted reverse screening marker, and marker-free deleted suicide vector of sucrose lethal gene SacB
  • Application of sucrose lethal gene SacB in gene deleted reverse screening marker, and marker-free deleted suicide vector of sucrose lethal gene SacB

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Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1, the amplification of SacB gene

[0031] The SacB gene was amplified using the plasmid pEX18GM (Gene.1998; 212(1):77–86) as a template, and the primers were SacBP1: 5'-catgccatggcaatgaacatcaaaaagtttgc-3' (SEQ ID NO.1), SacBP2: 5'-ccgctcgagttatttgttaactgttaattgtcc- 3' (SEQ ID NO.2), the reaction program is: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 55°C for 30s, extension at 72°C for 1min and 30s, cycled 30 times; final extension at 72°C for 7min, and storage at 16°C. The amplified products were subjected to agarose gel electrophoresis, and the results were as follows: figure 1 shown.

Embodiment 2

[0032] Embodiment 2, test SacB gene as the reverse screening marker experiment of scarless deletion

[0033] The amplified SacB fragment was cloned into the NcoI and XhoI restriction sites of the shuttle plasmid pLMF02 (Sci Rep.2016, 6:37159), and then transformed into R. anatipestife ATCC by conjugative transfer to obtain strain R. anatipestife ATCC pLMF02::SacB; at the same time, the plasmid pLMF02 was transferred into R.anatipestife ATCC to obtain R.anatipestife ATCC pLMF02, and the recombinant plasmid identification results were as follows figure 2 shown. The strains R.anatipestifeATCC pLMF02 and R.anatipestife ATCC pLMF02::SacB were cultured on the blood plate containing sucrose, and R.anatipestife ATCC pLMF02::SacB showed sensitivity to sucrose, while the control R.anatipestifeATCC pLMF02 was not sensitive to sucrose sensitive. This result indicated that the SacB gene can be used for subsequent studies on scarless deletions.

Embodiment 3

[0034] Embodiment 3, sucrose sensitivity test

[0035] will contain 10 7 CFU of R.anatipestifer ATCC pLMF02::SacB and R.anatipestiferATCC pLMF02 bacterial solution were applied to the blood plate containing different concentrations of sucrose (0%, 2.5%, 5%), cultivated in a 37°C incubator for 18h, and the growth conditions were as follows: image 3 shown. The results showed that the strain carrying the SacB gene did not grow when the sucrose concentration was 5%.

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Abstract

The invention relates to an application of a sucrose lethal gene SacB in a gene deleted reverse screening marker, and a marker-free deleted suicide vector of the sucrose lethal gene SacB. A sacB geneis used to encode sucrose fructanase which can catalyze the hydrolysis of sucrose into glucose and fructose, the fructose is polymerized to form high molecular weight fructan, but the accumulation ofhigh molecular weight fructan achieves a potential toxic effect on cells and can cause cell death, so the suicide vector is constructed by using the sucrose lethal gene SacB without replacing a targetgene with a resistant gene, so any possible polar action of the resistant marker on a downstream gene is avoided, and the sucrose lethal gene SacB is of great significance to study the functions of aRiemerella anatipestifer gene.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the application of the sucrose lethal gene SacB in gene deletion reverse screening markers, and also relates to a traceless deletion suicide vector containing the sucrose lethal gene SacB. Background technique [0002] Gene knockout is an important method to study the function of bacterial genes, including the research on the gene function of Riemerella anatipestifer. The method of gene knockout mainly includes scar deletion mediated by resistance gene, and scarless deletion mediated by reverse selection gene. The current method for gene deletion of Riemerella anatipestifer is mainly scar deletion, and scar deletion will leave a resistance "scar" in the genome and cause non-transcription of downstream genes, that is, "polarity effect". In addition, due to the multi-drug resistance of R. anatipestifer, the range of selection of resistance genes for the scarred deletion of R. anatipesti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74
CPCC12N15/74C12N2800/101
Inventor 刘马峰黄月罗睿心程安春汪铭书朱德康
Owner SICHUAN AGRI UNIV
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