Subcellular Localization And Expression Analysis Of OMP Genes Of Candidatus Liberibacter Asiaticus

Huanglongbing(HLB)is one of the most devastating diseases of citrus,prevalenting throughout the citrus grown areas worldwide.The causal agent is phloem-limited unculturable Gram-negative bacteria,and given genus name Candidatus Liberibacter.Outer membrane proteins(OMPs)play a significant important role in the pathogenicity of gram-negative bacteria.In this issue,we focused on the subcellular localization and expression analysis of MotB gene of Candidatus Liberibacter asiaticus(CaLas),which works as elicitor and can trigure HR response in Nicotiana benthamian based on the previouse study in our laboratory.The main results obtained in the present study are summarized as follows:1.Subcellular localization of MotB and ?tMotB(mutant without transmembrane domain)in Nicotiana betamian:MotB and ?tMotB genes were constructed to entry vector pDONRTM/Zeo separately and then subcloned to plant expresseion vector pMW390 following the protocol of gateway technology.The recombinant plasmid containing MotB and ?tAMotB were obtained and transformed into GV3101 and transient expressed in Nicotiana benthamian.Subcellular localization of MotB and ? tMotB were visuralized by laser confocal microscope at 48-72h after infiltration.The result indicated strong fluorescent signals were produced in cell cytoplasmic and nucleus by both MotB construct and ?tMotB mutant.2.The analysis of expression level of ten HR marker genes in Nicotiana benthaian after infiltration of MotB and ?tMotB respectively:qRT-PCR analysis results show that SIPK,WIPK,BAK1,RbohB,TOBPAL and ACRE31 genes' has similar expression level induced by MotB-103 and ?tMotB-103,appearing normal distribution.The highest expression leval was at 2dpi.Compared to MotB gene,the expression of the above genes induced by?tMotB gene trending a lower level.In addition,the expression level of Plastocyanin gene in N.benthamian induced by ?tMotB-103 was up-regulated,while it appeared down-regulated by MotB-103.3.The construction of prokaryotic expression vector,fusion protein expression and preparation of polyclonal antibody:the above two genes were constructed into three different bacterial expression vectors,which were pET22b(+),pET28a(+)and pET28b(?).SDS-PAGE analysis revealed that the recombinant plasmids ?tMotB-22b and ?tMotB-28b can express target proteins successfully under the condition of 16 centugrade and supplied with 0.5mmol/L IPTG.However,MotB construct failed to express target proteins.After purification with Ni-NTA column,target protein was sent out to the company to acquire polyclonal antibody.4.The localization and secretion character of MotB gene in E.coli:MotB and ?tMotB genes were constructed into the bacterial expression vector 1GFP by Ligation Independent Cloning(LIC)method.Under the condition of 28 centugrade and 0.1mmol/L IPTG supplied,the recombinant plasmids MotB-1GFP and ?tMotB-1GFP could express target proteins successfully.MotB was localized to the bacterial poles,when fused to the GFP gene and expressed in E.coli,meanwhile the 1GFP empty vector and ?tMotB were distributed throughout the whole E.coli's cellobserved under fuorescent microscope.SDS-PAGE and Western blot analysis revealed that target fusion protein was not secreted to the outside of the cell.