Coloning And Functional Analysis Of GacA Gene In Pseudomonas Syringae Pv. Actinidiae

Bacterial canker caused by Pseudomonas syringae pv.actinidiae(Psa)is a devastating disease on kiwi fruit production.This disease was discovered for the first time in California and Japan's shizuoka on 1980,subsequently spreading to Italy,New Zealand,China,etc.Kiwi fruit industry has been caused great economic losses by this disease.gacA gene widely exists in pseudomonas,which can control the bacteria toxicity and secondary metabolites.However,This disease is defferent from other pseudomonas on host selection type and disease epidemic conditions.whether gacA gene relates to the pathogenicity of bacteria or not in the Psa,has not been reported.According to the regulation of gene sequence of gacA in Pseudomonas syringae pv.tomato,a pare of primers was designed to clone gacA gene from the Psa.Using the method of homologous recombination,we constructed the mutant strain and function complementary strain which we analyzed its preliminary function,to lay the theory foundation of revealing gacA gene in the regulation pathway and seeking the pathogenic mechanism of Psa.The research content is as follows:1.Cloning and bioinformatics analysis of gacA gene in Pseudomonas syringae pv.actinidiae gacA gene is clustering analysis among Pseudomonas?Xanthomonas and Erwinia.The results show that gacA gene is highly homologous in pseudomonas,similarity of 98%.With wild type strains of AHP-18,according to the conserved sequence of gacA gene in Pseudomonas,we designed a pair of specific primers gacA3/gacA4 to clone gacA gene,then sequencing validation.Meanwhile,we used NCBI to analyze the sequence.Using website SMART to predict protein domain of Gac A.The results show that the N end of Gac A possess a Signal receiver domain,accepting the phosphate groups transferred by signal protein.The C end possess a HTH structure,responsible for multi-polarization and combined with the promoter DNA.2.Construction and screening the mutant of gacA gene in Pseudomonas syringae pv.actinidiae Using the method of homologous recombination,we constructed the mutant strain.First of all Designing specific primers(gac-1U/gac-1R?gac-2U/gac-2R)amplify upstream and downstream homologous segments of gacA gene.In order to reduce the difficulty of screening the mutant strains,at the same time the introduction of chloramphenicol resistance fragment.Then three fragments used fusion PCR to merge into a long fragment,sequencing,when verifying correct,linked with suicide carrier plasmid pK18 mobsac B,building a recombinant plasmid pK18 gacA.This study used three close combination method putting recombinant plasmid into wild strainAHP-18,After homologous exchange,this study choose 20 strains of double exchanging from 260 strains of single exchanging,which are gacA mutants.3.Construction and screening function complementary strain of ?gacA Design specific primers(gac-F/gac-R)to amply complementary gene fragments,and the fragments introducing restriction sites(Pst I/Bam HI),and extraneous fragment and broad host range plasmid pLAFR3 proceed enzyme digestion,respectively,when verifying correct,constructing complementary recombinant plasmid and verified.using then using the three close combination putting recombinant plasmid into ?gacA,the complementary strains the was validited by colony PCR.4.Functional analysis of gacA gene in Pseudomonas syringae pv.actinidiae The wild type strains,mutant strains and reply mutant strains were inoculated into living branches and detached leaves of Kiwi Fruit,and inoculated into tobacco leaf in vitro,studying the pathogenicity of gacA mutation,the test results show that gacA gene participated in the regulation of bacteria toxins and extracellular enzyme synthesis,swimming the ability of bacteria and pathogenic,etc.After mutation of gacA gene,Pathogenic bacteria lossed secretion capacity of phaseotoxion,and lower ability of the extracellular enzyme synthesis and flagella swimming.