Analysis Of YsnE Involved In IAA Synthesis Pathways In Plant Growth-Promoting Rhizobacteria Bacillus Amyloliqueficiens SQR9

Plant growth promoting rhizobacteria(PGPR),which can promote plant growth,control plant diseases,increase crop yield,has been widely used in agricultural production.Bacillus genus,one of PGPR community,can produce spores with special resistance under adverse conditions,is now extensively studied.B.amyloliquefaciens SQR9 is a strain of Bacillus amyloliquefaciens,isolated from the rhizosphere of healthy cucumber,which can produce indole-3-acetic acid(IAA)and other plant growthpromoting factors.Gene ysnE,predicted to be tryptophan acyltransferase,is a key gene in the IAA anabolic pathway of SQR9 strain.In this study,we used wild-type SQR9 and its mutant ?ysnE as the research materials,determined the content of IAA in fermentation broth by HPLC,and then found the difference components treated with tryptophan,for finding out the function of ysnE in IAA metabolic pathway.The main results are as follows:1.IAA biosynthesis of SQR9 is mainly dependent on tryptophan pathway,and gene ysnE plays an important role in IAA metabolic pathway.IAA quantification of SQR9 and mutant ?ysnE fermentation broth with and without adding tryptophan was performed by High performance liquid chromatography(HPLC).We found that IAA yield of SQR9 and?ysnE treated without 3 mM tryptophan was significantly less than the other treatments.And there was no significant difference in IAA production without adding tryptophan between SQR9 and AysnE;the IAA production of SQR9 and ?ysnE treated with 3 mM tryptophan increased significantly,with production of mutant AysnE only 38.78 ?g/mL,accounting for about 33.6%of wild-type SQR9 production.2.The gene ysnE was highly likely to be involved in the indole pyruvate metabolic pathway or tryptamine metabolic pathway in the IAA synthesis pathway.SQR9 and ?ysnE fermented broth treated with 3 mM tryptophan were analyzed by High performance liquid chromatography(HPLC)and Liquid chromatography-Mass Spectrum(LC-MS).Four kinds of related substances were identified:indole ethanol?indole pyruvate?indole lactic acid and tryptamine.In the quantitative determination of indole ethanol,the content in the mutant?ysnE was only 53.63%of the content in SQR9.Indole lactic acid and tryptamine was also significantly higher than that of SQR9.IAA was found in both SQR9 and ?ysnE fermentation broth by using LC-MS analysis,but indole pyruvate was detected only in the mutant ?ysnE.3.We treated SQR9 and ?ysnE fermentation broth with different indole derivatives,analyzed contents of IAA and the key intermediates in IAA metabolic pathways.Then we found that in the treatment of indole pyruvate and tryptamine,IAA synthesis was increased in both SQR9 and mutant ?ysnE.And IAA content of SQR9 was also significantly higher than that of mutant ?ysnE,especially in indole pyruvate pathway.However when they were treated with indole acetamide(IAM)and indole acetonitrile(IAN),IAA synthesized much less,and there's no significant difference between the two.In the treatment with tryptophan(TRP)and indole pyruvate acid(IPA),the ILA content in the ?ysnE fermentation broth was much higher than that of the wild-type,while the indole acetaldehyde was significantly lower than that of the wild-type.In the treatments with tryptamine(TAM)and tryptophan(TRP),the TAM content in the AysnE fermentation broth was much higher than that of the wild-type,the thetryptophol(TOL)content of the mutant ?ysnE was lower than that of the wild-type.We speculated that the gene ysnE involved in the indole pyruvate pathway and tryptamine pathway,and also formal the function between indole pyruvate and indole acetaldehyde or tryptamine to indole acetaldehyde.