The Counstruction And Utilization Of Shuttle Plasmid In Riemerella Anatipestifer

RA is a gram-negative bacterium belonging to the family Flavobacteriaceae.RA can infect most types of poultry,including ducks,chicken,geese and turkeys especially the waterfowls.At present,the disease is prevalent in the world and the duck industry has suffered huge economic losses.Due to there are few reported studies about gene manipulation techniques of Riemerella anatipestifer,the research of physiological and pathogenicmechanisms of Riemerella anatipestifer infection makes slow progress.A series of experiments have been carried out on the above problems,the results are as follows:1.Construction of shuttle plasmids in E.coli and RAWe generated shuttle vectors by amplifying replication regionof plasmid from R.anatipestifer,and using it to replace the replication region of plasmid pMM47.A which can exist stably in E.coli.We named the recombinant plasmid pLMF01.The plasmid stability test showed that this shuttle plasmid can exist stably in R.anatipestifer.The copy number of the plasmid is 33.Then we amplified the promoters of the high,medium,low transcriptional gene and used them to replace the erm F promoter in plasmid pLMF01,giving the plasmids pLMF03,pLMF04 and pLMF05,respectively.2.Validation of tonB1 gene's functionality in RA ATCCThe tonB1 gene was amplified and cloned into all three vectors to construct high-medium-low complementary strains respectively.The result of the real time-PCR and the western-blot showed that the expression level of tonB1 gene was highest in pLMF03,lowest in pLMF05.Secondly we checked for the ability of heme utilization,the result revealed that all the complementary strains could enhance the ability compared to the mutation accordingly.3.Function investigation of tbf A gene in RA ATCCIn previously study,the tbf A gene couldn't be deleted after several attempts.We use the shuttle plasmid based tbf A gene knockdown and over expression methods to investigate its function.The heme utilization experiment showed that the utilization ability was greatly redeced after the over expression of tbf A gene no matter in the wildtype or the tonB1 and tonB2 double mutation but the heme utilization didn't show any disadvantages in the tbf A-antisense strain.According to the results above,the tbf A gene didn't participate in the heme utilization.The tbf A gene may compet with Ton B1 and Ton B2 to use the Exb B-Exb D complex,and then it could decrease the utilization of heme.4.The utilization of shuttle plasmids in RA CH-1In order to prove shuttle plasmids could be used in different kinds of serotypes in RA,this research transferred pLMF03::B739?1208 into the mutant strain RA CH-1?B739?1208 to construct the complementary strain.The result showed that this complementary strain could recover the iron utilization ability.So this result proved that these shuttle plasmids could be used in different kinds of serotypes in RA.