| Indole-3-acetic acid(IAA)is a common auxin,which plays an important role in plant development.Many microorganisms related to plant growth and development or disease processes also have the ability to produce IAA,so that these microorganisms affect and change the physiological processes of plants.To clarify the IAA synthesis of plant growth promoting bacteria(PGPB)and the IAA synthesis mechanism are helpful to understand the growth promoting mechanism of PGPB.Burkholderia pyrrocinia JK-SH007 is a good PGPB,which can promote the growth of poplars and other trees.It is known that its ability to synthesize the plant hormone IAA is one of the reasons for its promotion effect.This study will further analyze the effects of external environmental conditions on the synthesis of IAA by B.pyrrocinia JK-SH007,and perform a functional analysis of its synthetic pathway.The main findings are as follows:(1)The ability of B.pyrrocinia JK-SH007 to secrete IAA was identified by Salkowski colorimetry,enzyme-linked immunosorbent assay(ELISA),and high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).B.pyrrocinia JK-SH007 was found can synthesize IAA,as well as the intermediates related to IAA synthesis pathway,such as indole-3-pyruvic acid(IPyA),indole-3-acetonitrile(IAN),indole-3-acetamide(IAM)and tryptamine(TAM).The relationship between the growth and IAA synthesis of B.pyrocinia JK-SH007 was studied.The results showed that the IAA synthesis of B.pyrocinia JK-SH007 was the highest in the stable growth period.The effects of L-tryptophan(L-Trp),temperature and pH on IAA synthesis of B.pyrocinia JK-SH007 were studied.The results showed that L-Trp had a significant role in IAA synthesis.The optimal temperature for the synthesis of IAA by B.pyrrocinia JK-SH007 was 37 ?,and the optimum pH was 7.(2)Through the IAA production test of B.pyrocinia JK-SH007 cell-free extract and the comparative annotation analysis of B.pyrocinia JK-SH007 genome and NCBI and KEGG database,it is indicated that B.pyrocinia JK-SH007 contains all the genes required for IAM and TAM pathways,which indicates that B.pyrocinia JK-SH007 is likely to synthesize IAA through the complete IAM and TAM pathways.(3)The iaaM(tryptophan 2-monooxygenase)and iaaH(indoleacetamide hydrolase),the key genes of IAM pathway in B.pyrocinia JK-SH007 were cloned and bioinformatics analyzed.The results showed that the total length of iaaM gene was 1 782 bp,encoding 593 amino acids,protein molecular weight was 64.81 kD,without signal peptide;the total length of iaaH gene was 1 368 bp,encoding 455 amino acids,protein molecular weight was 47.28 k D,without signal peptide.The analysis of secondary structure and phylogeny showed that both of them were conservative in evolution.RT-qPCR analysis of the differential expression of iaaM and iaaH genes under L-Trp treatment showed that the expression of both genes increased significantly,indicating that the IAM pathway played a role in the synthesis of IAA by B.pyrocinia JK-SH007.(4)The activities of tryptophan 2-monooxygenase and indoleacetamide hydrolase,two key enzymes of IAM pathway,were verified by prokaryotic protein expression and substrate feeding experiments.The results showed that both of them were active,and Iam pathway was active in B.pyrocinia JK-SH007.This study shows that B.pyrocinia JK-SH007 has the ability to synthesize IAA and its intermediates,and L-Trp can significantly promote the synthesis of IAA.The temperature of 37? and pH 7 are the best conditions for B.pyrocinia JK-SH007 to synthesize IAA.The whole genome analysis of B.pyrocinia JK-SH007 shows that it may have complete synthesis pathways of IAM and TAM,and respectively from the molecular and protein levels for B.pyrocinia JK-SH007 is based on IAM to verify the function of IAA synthesis pathway,revealing the molecular functional mechanism of IAM pathway in the synthesis of IAA of B.pyrocinia JK-SH007,laying a foundation for exploring the promotion mechanism of B.pyrocinia JK-SH007. |
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