吡咯伯克霍尔德氏菌JK-SH007吲哚-3-乙酰胺IAA合成功能及其依赖途径鉴定

doi:10.11707/j.1001-7488.20190913

摘要/Abstract

摘要： [目的]探索吡咯伯克霍尔德氏菌JK-SH007（Burkholderia pyrrocinia JK-SH007）合成生长素吲哚-3-乙酸（indole-3-acetic acid，IAA）的能力，并阐明B.pyrrocinia JK-SH007存在色氨酸依赖型的吲哚-3-乙酰胺（indole-3-acetamide，IAM）IAA合成途径。[方法]使用Salkowski比色法和酶联免疫吸附法（ELISA）进行L-色氨酸对B.pyrrocinia JK-SH007合成IAA能力影响的检测；以B.pyrrocinia JK-SH007全基因组为模板，设计特异性引物克隆iaaM和iaaH基因，并使用生物信息学方法对其蛋白质和亲缘关系进行分析；通过实时荧光定量PCR方法分析B.pyrrocinia JK-SH007中的iaaM和iaaH基因在L-色氨酸处理下的差异表达。[结果]B.pyrrocinia JK-SH007菌株能够合成15.663 μg·mL^-1的IAA，在以L-色氨酸为前体的条件下其合成IAA的量显著增加，达到了44.404 μg·mL^-1。iaaM和iaaH基因均含有一个完整的开放阅读框，其中iaaM基因全长1 782 bp，编码593个氨基酸，属于Amino_oxidase super family家族；iaaH基因全长1 368 bp，编码455个氨基酸，属于Amidase super family家族。在L-色氨酸的处理下，B.pyrrocinia JK-SH007中的iaaM和iaaH基因的表达量均显著增加。[结论]B.pyrrocinia JK-SH007具有合成IAA的能力，L-色氨酸对其合成IAA具有显著促进作用，且克隆得到了iaaM和iaaH基因，其在进化上具有一定的保守性，证明B.pyrrocinia JK-SH007中存在吲哚-3-乙酰胺IAA合成途径。

关键词: 吡咯伯克霍尔德氏菌, 吲哚-3-乙酸, 吲哚-3-乙酰胺途径, 色氨酸2-单加氧酶基因, 吲哚乙酰胺水解酶基因

Abstract: [Objective] This study aimed to explore the ability of Burkholderia pyrrocinia JK-SH007 to synthesize the indole-3-acetic acid (IAA) and to elucidate the existence of a tryptophan-dependent indole-3-acetamide(IAM)IAA synthesis pathway in Burkholderia pyrrocinia JK-SH007.[Method] The ability of B. pyrrocinia JK-SH007 to synthesize IAA was detected by Salkowski colorimetry and enzyme-linked immunosorbent assay (ELISA) under the effect of L-tryptophan. The iaaM and iaaH genes were isolated from B. pyrrocinia JK-SH007 by PCR using specific primers, and their protein and phylogenetic relationships were analyzed by bioinformatics method. The differential expression of iaaM and iaaH genes in B. pyrrocinia JK-SH007 under L-tryptophan treatment were analyzed by real-time fluorescence quantitative PCR.[Result] B. pyrrocinia JK-SH007 strain was able to synthesize 15.663 μg·mL^-1 of IAA, and with L-tryptophan as precursor, the yield of IAA synthesized by B. pyrrocinia JK-SH007 increased significantly, reaching 44.404 μg·mL^-1. Both iaaM and iaaH genes contained a complete open reading frame. The full length of iaaM gene was 1 782 bp, encoding 593 amino acids which belongs to the Amino_oxidase super family; the full length of iaaH gene was 1 368 bp, encoding 455 amino acids which belongs to the Amidase super family. The expressions of iaaM and iaaH genes in B. pyrrocinia JK-SH007 were significantly increased under the treatment of L-tryptophan.[Conclusion] B. pyrrocinia JK-SH007 has the ability to synthesize IAA, L-tryptophan has a significant effect on its synthesis of IAA, and the cloned iaaM and iaaH genes have certain evolutionary conservatism, this study showed that B. pyrrocinia JK-SH007 exists indole-3-acetamide pathway.

Key words: Burkholderia pyrrocinia, IAA, IAM pathway, iaaM, iaaH

中图分类号:

S763.1

刘婉慧, 陈飞飞, 叶建仁, 王朝恩, 康熠, 付欢欢. 吡咯伯克霍尔德氏菌JK-SH007吲哚-3-乙酰胺IAA合成功能及其依赖途径鉴定[J]. 林业科学, 2019, 55(9): 121-129.

Liu Wanhui, Chen Feifei, Ye Jianren, Wang Chaoen, Kang Yi, Fu Huanhuan. Identification of Indole-3-Acetamide IAA Synthesis Function and Its Dependent Pathway in Burkholderia pyrrocinia JK-SH007[J]. Scientia Silvae Sinicae, 2019, 55(9): 121-129.

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