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

Identification of Indole-3-Acetamide IAA Synthesis Function and Its Dependent Pathway in Burkholderia pyrrocinia JK-SH007

【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15.663μg·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44.404μg·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Aminooxidase 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。

【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 Aminooxidase 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.