Sequencing Of GspF And GspE Gene Of Type Ⅱ Serection System In Stenotrophomonas Maltophilia D2 And Constructing Of The Recombinant Suicide Plasmid Of Smp Gene

Objective We isolated a Stenotrophomonas maltophilia strain from the digestive tract of Perinereis aibuhitensis Grube, which mass secreted a extracellular protease (SMP) and in high purity; The previous study had gotten the complete coding genetic sequences of smp gene and found that it was related to typeⅡsecretion system. To study the secretory mechanism and lay foundation for exploitation and utilization, we made the smp gene be replaced by the foreign gene with the method of homologous recombination technology. We constructed the recombinant suicide plasmid(pDS132NotI-A-tet-B) to knock out the smp gene in D2. And we got the two complete coding genetic sequences GspF and GspE of the transmenbrane protein gene cluster in typeⅡsecretion system in Stenotrophomonas maltophilia D2.Methods 1.Through the drug sensitive test (microbe and drug analysis system and dilution method), we detected the sensitive drugs and the situation of the drug resistance about D2 so that we could select the suitable suicide plasmid and marker gene.2. According to the sequence of smpgene and the sequences of K279a and R551 recorded in Genbank, we designed the primes. The up and down fragments of smp gene was amplified by polymerase chain reaction with Genome walking tecnology.3.We analysed the sequence and biology information of the GspF and GspE of typeⅡsecretion system in the up fragment of smp gene.4. According to the up and down sequence of smp gene, the primers was designed and added suitable restriction enzyme cutting site. We got the fragments of homologous arm A and B; According to the sequence of the tetracyclin resistant sequences of plasmid pBR322 recorded in Genbank, we designed the primers and got the complete Open Reading Frames(ORFS) of the selective marker gene; We added the complementary sequences to the primers when they were designed, and it would be used to splice overlap extension PCR.5. The fragment of A-tet-B was gotten by splicing overlap extension PCR technique.6. The suicide plasmid pDS132NotI and the plasmid pMD18T-A-tet-B were digested by NotI, the tow products were ligated, the recombinant suicide plasmid pDS132NotI-A-tet-B were constructed, and analyzed after sequencing.Results 1. The result of two methods were that the sensitive drugs of D2 were chloramphenicol and tetracycline, and there were several resistant drugs, so we selected pDS132NotI (catr) and DH5 aλpir(cat-) to be the tool in homologous recombination, the gene of tet was the selected marker.2. Successively we got the 5'sequencing of smp gene, found two complete Open Reading Frames (ORFS). They were 1200bp GspF gene (GenBank accession number:GU377212.1) and 1434bp GspE gene (GenBank accession number: HM151387). According to the blasting result in GenBank, the homology of GspF gene and amino acids between D2 and S. maltophilia R551-3 are 93 percent and 98 percent, GspE gene and amino acids are 92 percent and 99 percent.3. The recombinaton suicide plasmid was constructed successfully by enzyme digestion analysis. In the sequencing of A-tet-B in the suicide plasmid pDS132NotI, the sequence of A is the same to the original sequencing, the sequence of tet is the same to the sequencing of the tetracyclin resistant in plasmid pBR322 recorded in Genbank, and there were five bps which were different to the original sequencing in part B, the fidelity was 98 percent.Conclusion We obtained the MIC of chloramphenicol and tetracycline are 25μg/ml and 12.5μg/ml about D2. We cloned complete coding gene sequence GspF and GspE of the transmenbrane protein gene cluster in typeⅡsecrete system in S. maltophilia D2. The sequences are highly conserved in S. maltophilia, and are significantly different from other bacteria, which show that the type II secrete system in S. maltophilia D2 may be different from those in other Gram-negative bacteria. We constructed the recombinant plasmid pDS132NotI-A-tet-B. Successfully three gene fragments were spliced and amplified by the technology of SOE-PCR. The recombinant plasmid pDS132NotI-A-tet-B which successfully was constructed can be used in the next experiment.Postgraduate:XIN Xiao-Ni (Clinical Laboratory Diagnostics)Advised by Prof. YAN Zhi-Yong...