Analysis Of Indigenous Plasmid In Xanthomonas Oryzae Pv.Oryzicola And Construction Of Shuttle Plasmid Vectors

Bacterial plasmid is a kind of extra-chromosomal hereditary material that is able to replicate autonomously,and most of which is circular double stranded deoxyribonucleic acid(DNA).The plasmid often code functional genes involved in antibiotic resistance,heavy metal ion resistance,protein secretion system and so on that help the host bacteria adapt the environment better.What’s more,the ability of replicating and transferring autonomously equips plasmid with enormously potential of serving as genetic engineering vector.Genetic engineering vector has been playing a key role in the study of plant pathogenesis and production of xanthan and extracellular protease.Xanthomonas oryzae pv.oryzicola(Xoc)was taken as research subject in this study.Based on the sequence information of pXOCgx01-the first discovered indigenous plasmid in Xoc,general survey and analysis of plasmid and construction of geneticengineering vector were conducted in this study.1.Among the 257 isolates of Xanthomonas oryzae pv.oryzicola that came from different regions of China,9 kinds of plasmid were identified:pXOCgx02,pXOCB116,pXOCB209,pXOCB216,pXOCB220,pXOCB409,pXOC709,pXOC820 and pXOC905.Some of the indigenous plasmids were sequenced and analyzed.At the same time,three important functional genes from plasmid pXOCgx01 were selected to make an investigation on the nine plasmids.The result showed that pXOCB209 carried all of the three genes and pXOCgx02,pXOCB116,pXOCB216,pXOCB220,pXOC905 carried one of the functional genes respectively,while the rest four plasmid pXOCB409,pXOC709,pXOC820 seems to have none of the functional genes selected.2.Currently,none of the genetic engineering plasmids commonly utilized in Xanthomonas is modified based on plasmids from Xanthomonas,which may not be able to fully meet the requirements of functional genomics of Xanthomonas.Based on the sequence analysis,pXOCgx01 was modified to construct genetic engineering plasmids that are applicable to Xanthomonas in this research.The modification including deletion of genes that should be no contribution to the plasmid replication and introduction of critical functional genes such as resistance screening marker,multiple cloning sites and replication origin.The original plasmid of(53 kb)was constructed to two shuttle plasmidvectors pXUG and pXUK,which are around 11.6 kb.Plasmid vector pXUG holding resistance against Gentamycin while pXUG holding resistance against Kanamycin.After the construction being finished,pXUG was used to complement the extracellular polysaccharide deficient strain of Xoc GX01 and pXUK was used to complement the extracellular protease deficient strain of Xoc GX01.The result indicated that both phenotypes of the two deficient strains were complemented to the level of wild type.At the same time,the stabilities of pXUG and pXUK were tested,the investigation showed that both of the two shuttle plasmid vectors were able to achieve stable replication and efficient foreign gene expression without influence the phenotype of strain itself simultaneously in Escherichia coli and Xanthomonas.In summary,the research provides abundant information for study on indigenous plasmid of Xoc through the general survey,sequence analysis,function investigation as well as plasmid modification of plasmid from Xoc Chinese isolates.Meanwhile,the genetic.engineering vectors of pXUG and pXUK constructed in this research are the first two plasmid vectors modified on the basis of indigenous plasmid from Xoc,which can be applied in both Escherichia coli and Xanthomonas.Application of pXUG and pXUK is able to simplify the traditional procedure of gene function study on Xanthomonas significantly.The two plasmid vectors,therefore,laid foundation for the development of efficient molecular tools.