Initial Screening Of Genes Regulating The Acid Tolerance Two-component Regulatory System

Lactobacillus delbrueckii subsp. bulgaricus is currently one of the mostvaluable bacteria and is widely used in global dairy industry. The acid-toleranceability of lactic acid bacteria is the key point of its survival and proliferation duringfermentation process and in gastrointestinal tract of human body. It is also the mainconstraint for bacteria cell density change in the fermentation environment usinglactic acid bacteria as starter. The study of acid-tolerance ability and mechanism ofL. delbrueckii subsp. bulgaricus can provide a theoretical basis for future industryproduction.This research plans to explore the genes activated/inactivated bytwo-component system HPK1/RR1which is involved in acid tolerance regulation,provide theory basis to build the acid-tolerance regulation network and lay afoundation to clarify and improve the system. The research in this dissertation firstlyextracted total RNA from L. delbrueckii subsp. bulgaricus wild type CH3, RR1mutant type CH3-R and HPK1mutant type CH3-H and chose genes with certainfunctions, such as two-component system and adaption to atypical condition, toamplify using RT-qPCR. The differences of gene expression level were then studiedamong the three types above. Among the63genes in two mutants,22of126havedifferences in gene expression level. Mutant CH3-H has6genes up-regulated and2genes down-regulated, mutant CH3-R has3genes up-regulated and11genesdown-regulated. The expression level of other genes has no obvious changes. Thisresearch implies that several genes’ expressions are induced or suppressed after themutation of HPK1/RR1, which shows that the regulation of acid-tolerance ability ofL. delbrueckii subsp. bulgaricus is a comprehensive regulation realized throughmultiple paths.The research then used16pairs of random primers and cDNA-AFLP to exploreunknown genes with expression level differences which may be regulated by thetwo-component system HPK1/RR1. To our knowledge, this is the first reportdemonstrating the effect of two-component system HPK1/RR1on important relatedgenes. The present study will offer experimental basis for clarifying the acidtolerance regulation mechanism.