中国农业科学Issue(15)：3058-3068,11.DOI:10.3864/j.issn.0578-1752.2014.15.015
N-乙酰胞壁质酶缺失对保加利亚乳杆菌LJJ自溶及形态的影响
Effect of N-acetylmuramidase Knockout onAutolysis and Morphology of Lactobacillus bulgaricus LJJ
摘要
Abstract
[Objective] Autolysis of lactic acid bacteria exists widely in the production of fermented dairy products. The rate and extent of autolysis not only play an important role in the whole fermentation process, but also have a vital effect on the quality, flavor and production cycle of the product. As main component of peptidoglycan hydrolase, N- acetylmuramidase can destroy the cell wall integrity in lactic acid bacteria autolysis process. It plays a crucial role in the autolysis of lactic acid bacteria. This paper mainly studied the influence of N-acetylmuramidase deletion on autolysis and morphology ofLactobacillus bulgaricus LJJ.[Method] The primers of upstream homologous arm m-up, downstream homologous arm m-down of N-acetylmuramidase and erythromycin resistance genes were designed based on the sequences ofL. bulgaricus genome and pMG36e vector. Then m-up, m-down and erythromycin resistance gene were amplified by PCR and verified by gene sequence analysis. Then erythromycin resistance gene and pUC19 were degested withKpnΙ,XbaΙ and connected together. The recombinant vector pUC:Emr was verified by double-enzyme cleavage method. Next, m-down and recombinant vector pUC: Emr were digested withKpnΙ,SacΙ and connected together. The recombinant vector pUC:Emr:m-down was confirmed by double-enzyme cleavage method. Finally, the m-up and recombinant plasmid pUC:Emr:m-down were digested withPstΙ,XbaΙ and connected together. The recombinant vector pUC:m-up:Emr:m-down was verified by double-enzyme cleavage method and gene sequence analysis. The recombinant vector pUC:m-up:Emr:m-down as the homologous recombinant N-acetylmuramidase knockout component, was transformed intoL. bulgaricus under the electroporation conditions of electric voltage, resistence and capacity of 1.5kV, 400 Ω and 25 μF, respectively. N-acetylmuramidase gene knockout mutant strains were screened on MRS agar containing erythromycin and verified by PCR. Then the autolysis rate of the gene deletion mutant and the wild type strain was detected and the morphological changes of gene deletion mutant and wild-type strain were observed by scanning electron microscope.[Result] N-acetylmuramidase knockout components with erythromycin resistance as selection marker was constructed and used to knock N-acetylmuramidase gene ofL. bulgaricus out. N-acetylmuramidase knockoutL. bulgaricus mutant strain with erythromycin resistance gene was constructed successfully. Compared with the wild-type strain, the characteristics and morphology of the mutant strain changed significantly. The autolysis rate also decreased significantly. The autolysis rate of the mutant strain was about 35% while the autolysis rate of the wild type strain was about 73% after 24 hours incubation at 37℃. The single cell length of thewild type strain was about 10 μm while that of the mutant strain was approximately 30-40 μm. Compared with the wild-type strain, the single cell length of the mutant strain increased by 3-4 times.[Conclusion]N-acetylmuramidase ofL. bulgaricus plays an important role in autolysis and cell division. The deletion of N- acetylmuramidase fromL. bulgaricus will result in loss of cell autolysis and the cell length will be increased by about 3-4 times.关键词
乳酸菌/基因敲除/自溶/N-乙酰胞壁质酶Key words
lactic acid bacteria/gene knockout/autolysis/N- acetylmuramidase引用本文复制引用
崔文明,逄晓阳,张书文,刘鹭,李红娟,吕加平..N-乙酰胞壁质酶缺失对保加利亚乳杆菌LJJ自溶及形态的影响[J].中国农业科学,2014,(15):3058-3068,11.基金项目
“十二五”国家科技支撑计划 （）
