Functional Analysis Of Two Sers Genes And Primary Purification Of Siderophore From Biological Bacterium Act12

Iron is one of the essential elements for almost all living organisms to survive,which plays an important role in metabolic process.However,iron in nature mainly exists in the form of insoluble compounds,most of which are not directly absorbed.With biological evolution,microorganism can use siderophores to absorb and transport insoluble iron.Biocontrol bacteria Act12 was isolated from Qinghai-Tibet plateau,which was identified as Streptomyces pactum by 16 S rDNA sequence analysis.Act12 shows significantly inhibition activity against a variety of soil pathogenic fungi,and good performance of growth-promoting on Cucumis melo L and other crops,but its mechanisms of growth-promoting and inhibition activity are unclear.Its whole genome has been sequenced and annotated.Through annotation analysis,Act12 has two different siderophore gene clusters.In this study,method of homologous double crossover was used to research functions of siderophore synthase gene SerB and regulatory gene SerF.Moreover,siderophore was preliminarily isolated and purified.All of these lay the foundation for revealing mechanisms of growth-promoting and inhibition activity of Act12,and the results are as follows:?1?Kanamycin resistance gene as a marker,upstream and downstream fragments of the target genes as the homologous arms,E.coli-streptomyces shuttle plasmid pKC1132 was used to construct the knock-out vector.Method of conjugational transfer was used to knock out the two target genes,the mutants were identified by PCR and RT-PCR,and 13 SerB mutants and6 SerF mutants were obtained.?2?Several biological characteristics of SerB mutant and SerF mutant were analyzed such as siderophore production,seed germination of Cucumis melo L and inhibition activity of pathogenic fungi compared with wild-type strain Act12.Siderophore production of SerB mutant was 11% less than Act12,SerB mutant inhibits seed germination of Cucumis melo L,and shows less inhibition activity against Macrophoma kawatsukai than Act12.However,Siderophore production of SerF mutant was 16.4% more than Act12,but has no significant effects on seed germination of Cucumis melo L and no difference on inhibition activity against M.kawatsukai compared with Act12.?3?After verification of complement,siderophore production and other phenotypes of the mutants were similar to wild strain Act12,further proofing the differences of siderophore production and other phenotypes of the deletion mutants caused by gene knock-out.?4?Through FeCl₃ experiment,Csaky experiment and Arown experiment,siderophores of Act12 are hydroxamic acid siderophores.Act12 was fermented under condition of iron-restricted,and 3 siderophore samples were isolated and purified by macroporous resin XAD-2 and sephadex LH-20.Sample 100 A has a certain inhibition activity against M.kawatsukai.In this experiment,gene SerB involves in the synthesis of siderophore and gene SerF regulates siderophore synthesis negatively.Siderophore of Act12 plays a role in seed germination of Cucumis melo L and inhibition activity against M.kawatsukai.These lay the foundation for further study of the mechanisms on bio-control and growth-promoting of Act12,and constructed a more effective biocontrol strain to be used in agriculture.