Coloning And Function Analysis Of Pigment Genes FabG And AroE In Acidovorax Citrulli

Bacterial fruit blotch of watermelon(BFBW)is a world-spared disease that causes seriousdamages on the plants of cucurbits worldwide. BFBW is a typical seed borne bacterial diseaseand caused by a Gram-negative bacterium Acdovorax citrulli (Schaad et al.,2008). It was firstreported in1965and is a worldwide quarantine disease. Those infected are mainly plants ofcucurbits, such as watermelon, melon and cucumber, etc., among which the watermelon andmelon got the worst damage. Therefore, the development of cucurbits industry is severelyrestricted. Researches of this disease mainly focus on the pathogen identification, occurrenceregularity, flagellum and fimbriae. Among researches of bacteriology, the effect of pigment onbacteria is always one of the important contents. However, the focus is mainly the study of itseffect in the bacteria classification and its physicochemical property, while the pathogenicityof the pathogenic bacteria is rarely involved.In this study, the mutant library of Aac-5Transposon (Mini-Tn5) was developedsuccessfully with the method of triparental mating, and15,000cloned children were obtained.Then, two pigment mutant strains were screened out of the6000cloned children: pigmentoverexpression mutant strain Aac-c64and pigment deletion mutant strain Aac-d17. The bandof591bp fragment was amplified from the mutant strains with kanamycin resistance genespecial primers Aac-c64and Aac-d17, while no band was amplified from the wild type strains.It indicated that Transposon Mini-Tn5was inserted in the Aac-5chromosome. The Southernblot hybridization results revealed that transposon Mini-Tn5generated the random andsingle-copy insertions in the chromosome of strain. The Mini-Tn5flanking sequences werecloned with shotgun, and the disruption genes were confirmed to be fabG and aroE afteranalysis by sequencing and comparison. Currently, researches on these two genes are less, andit has not been reported in the research of bacterial fruit blotch. The pathogenicity,allergenicity, growth capacity, moveability and other factors of the mutant strains,complementary strains and wild strain were measured. The results showed that, thepathogenicity of pigment deletion mutant strain Aac-d17on the host plant decreased obviouslyand moveability were both reduced, and the growth capacity was diminished; thepathogenicity of pigment overexpression mutant Aac-c64was significantly reduced, but itsallergenicity, growth capacity and moveability had no significant changes compared to thewild strain.