The Construction Of Genes FurA And KatA Mutants Of Clavibacter Michiganensis Subsp.michiganensis

Bacterial canker of tomato is one of the most serious and devastating diseases in tomato production.. It is now widely distributed in the tomato-producing areas of the United States and gradually become a global disease from it was first found in Michigan in 1909. Now, the bacterial canker of tomato has occurred in Beijing, Heilongjiang, Jilin, Inner Mongolia, Xinjiang, Henan, Hebei, Shanxi, Shandong, Shanghai, Hainan and other provinces in China,, and it affects the tomato production in varying degrees in most regions. Previous study showed that gene furA katA may be associated with the pathogenicity of Cmm. In order to study the relationship between gene furA and katA, speculate its pathogenesis and provide a theoretical basis for the resistance breeding,we constructed the different gene furA and katA deletion mutants of Cmm, and transferred them into Cmm wild-type strains by electroporation method, and carried out molecular biological and physiological testing.Use Cmm genome of wild strains as the template DNA, according to furA and katA gene sequence specific primers, the PCR were performed. After electrophoresis, target PCR product was recovered, then linked with pMD18-T vector, named Cmm 1, 2, 3, 4 clips and 123-T. Digestion by BamHâ… , the correct sequencing of the target fragments were gotten from the pMD18-T vector. And then 1 and 2 fragment,3 and 4 fragments were linked by T₄ DNA lingase. Followed by PCR to add Kpnâ… to different fragments, target bands were recycled, then were linked with pMD18-T or pEASY-T1 vector. After digested by Kpnâ… , the target sequencing fragments were recovered from the sequencing vector,then linked with each other by T4 DNA ligase, acquired 1234,12 '3, 12'34;add the EcoRâ… sites to each fragment through PCR,linked with pMD18-T or pEASY-T1 vector,then digested by EcoRâ… ; while pHN216 vector was linearized by EcoRâ… ,Then the recovery of 1234,12 '3, 12'34, 123 fragment and pHN216 vector fragment were linked by T4 DNA lingase. Next,the linked product was transformed into Escherichia coli competent cell(DH5α), the positive clones were picked out and identified. Finally, with the method of electroporation,the correct recombinant were transformed into the wild-type strains of Cmm. The results showed that,when the concentration of Fe²⁺ in the medium was 0 mM and 0.5mM, the consumption of hydrogen peroxide for the wild strain and the mutant strains of Cmm, and the control of vector, showed various differences. When the concentration of Fe²⁺ was 0mM, the various treatments had no significant differences; While the concentration of Fe²⁺ was 0.5mM, the treatments showed significant differences: 12'3-2 (furA-katA-) and 12'34-2 (furA-katA+) is 1.5 times of the wild type (furA+katA+); 123-2 (furA+katA-) is 0.5 times of the wild-type (furA+katA+); the vector control compared with the wild type, the difference was not significant.