Sequencing And Analysing The Genes Of Stenotrophomonas Maltophilia D2Genaral Secretory Pathway Proteins And Knocking Out Partial Gsp2E Gene

Objctive:We isolated Stenotrophomonas maltophilia strain D2from digestive tract of Perinereis aibuhitensis Grube in2003.Research finding described that D2secrete a great quantity protein in extracellular.Based on the sequenced genes, we found the gene of this protein was clustered by genes of general secretory pathway proteins. So we speculated the type Ⅱ secretion system may be responsible for the the hyperproducion of SMP in D2. We expect to know more about the high level protein secretion mechanism of D2according to exploring the type Ⅱ machinery. Methods:According to the sequence of S. maltophilia D2obtained previously and the sequences of K279a、R551-3、JV3、D457（Genbank log number:NC₀1094、NC₀15947、NC₀17671、NC₀11071） from Genbank, the reward primers were designed based on the sequenced genes by the methord of gene moving. Then the PCR amplified products were connected to pMD18-T-vector and transformed into Escherichia coli JM109.The recombinant colonies identified by enzymes cutting.After stitched those sequencing fragments together,we find its ORF which analyzed by bio informatics techniques.2. We designed up and down homologous arms and take the tet place of partial gspE,these fragments were fusion by over lap PCR technology. After enzyme cutting the PCR product and the suicide vector then linged them together.after that transformed the suicide vector△Gsp2E-pEX-18TC into Ecol. SM10λpir.the suicide vector△Gsp2E-pEX-18TC were converyed into S. maltophilia D2Via the first Homologous recombination and choosed by the chloramphenicol and streptomycin.we filtrated the△Gsp2E and using polyacrylamide gel electrophoresis to detect protein D2.Ressult:We finded11ORFs （Gsp2E%Gsp2F、 Gsp2G、Gsp2H、Gsp2I、Gsp2J、Gsp2K、Gsp2L、Gsp2M、Gsp2N、Gsp2D） in T2SSs of Stenotrophomonas maltophilia D2and applied GenBank accession for each （KF234409、KF234410、KF234411、KF234412、KF2344113、KF234414、KF234415、 KF234416、KF234417、KF234418、KF234419）.After blasted with Stenotrophomonas maltophilia K279A and R551-3, we finded gene homo logy of D2T2SS1with K279A and R551-3up to80%, the sequence of correspond amino acid are all more than97%, homo logy of gene sequences and amino acid sequences of T2SS1were higher than T2SS2. the gene homology of GspE and Gsp2E、GspF and Gsp2F、Gspl and Gsp2I are over50%, other gene homology between35-68%,correspond amino acid sequence range15-61%.The homology of Stenotrophomonas maltophilia D2T2SSs with P.aeruginosa PAO1wre higher than Yersinia enterocolitica.there were not very significant difference between phenotypes and wild type.2. The polyacrylamide gel electrophoresis displayed that the protein D2was not very significant difference between phenotypes and wild type.Through medicine sensitive experiment we found the△Gsp2E was more resistance to chloramphenicol compar to the wild D2,it reminder that the gene of chloramphenicol replaced the partial△Gsp2E successfully.Biochemistry detection finding that L-aminocaproic acid-AMC was positive,however L-tryptophan-AMC changed for negative.Conclusion:The complete genetic sequence of T2SSs Stenotrophomonas mahophilia D2were successfully cloned. we found higher homology of11gene sequence with the same species,inconstract lower homology with distinct species though blast and phylogenetic tree.Knocking out Gsp2E provided a basis for further exploring of the mechanism of D2protein secretion.