根际促生解淀粉芽孢杆菌SQR9中ysnE参与合成吲哚乙酸的代谢途径研究

Plant growth-promoting rhizobacteria (PGPR) has been widely applied in agricultural production because it is friendly and safe to environment. The PGPR strain Bacillus amyloliqueficiens SQR9, which was isolated by this lab and has been widely used in bio-organic fertilizer, could synthesis phytohormone indole-3-acid (IAA) as an important plant growth-promoting factor. However, the details of biosynthesis pathways utilized by Bacillus remain less clear. YsnE was supposed to be the key gene of IAA synthesis in SQR9 and the gene-knockout mutant ΔysnE was got in previous study. In this project, the difference of indolic compounds produced by the wild type strain SQR9 and the IAA synthesis mutant ΔysnE will be studied. Enzymes and genes participated in the ysnE involved IAA synthesis pathway will be classified and verified. Finally, the ysnE involved IAA synthesis pathway will be determined and cloned. This research will deepen our knowledge in the mechanism of IAA synthesis in SQR9, and provide molecular basis for the agricultural application of PGPR strain Bacillus sp.

植物根际促生细菌（PGPR）因其环境友好、安全无毒等优点在农业生产中逐渐得到广泛的应用。解淀粉芽孢杆菌SQR9是本实验室筛选并已得到广泛应用的PGPR菌株，其合成的吲哚乙酸（IAA）是促进植物生长的重要物质。芽孢杆菌中IAA的合成途径尚不清楚。本申请在已经获得SQR9中IAA合成关键基因ysnE缺失突变体（ΔysnE）的基础上，主要分析比较野生型SQR9与ΔysnE IAA合成中间产物吲哚类物质的差异，鉴定SQR9中基于基因ysnE的IAA合成途径的其他基因并分析相关基因功能，预测并克隆验证ysnE基因在SQR9中参与IAA合成的完整代谢途径。本研究将使我们深入理解SQR9中合成IAA的机制，并且为促生芽孢杆菌在农业上的应用提供理论基础。

解淀粉芽孢杆菌SQR9合成的吲哚乙酸（IAA）对其促生效果具有重要作用。基因ysnE预测为编码色氨酸酰基转移酶，是菌株SQR9中IAA合成代谢途径中的关键基因。本实验以野生型SQR9和突变体ΔysnE为研究材料，通过高效液相色谱（HPLC）和液相色谱-质谱联用（LC-MS）技术分析SQR9和突变体ΔysnE发酵液中差异性组分，研究基因ysnE在SQR9中所参与的IAA的代谢途径。分析发现突变体ΔysnE的吲哚乙醇（TOL）含量降低了约56%，而吲哚乳酸（ILA）和色胺(TAM)显著升高（是野生型菌株的1.8倍和1.5倍）。通过对添加不同吲哚衍生物处理的SQR9和ΔysnE发酵液中IAA和各种IAA代谢途径关键中间产物的分析，发现在添加吲哚丙酮酸（IPA）的处理中，突变体ILA合成量显著高于野生型菌株，但是IAA和TOL合成显著低于野生菌株。在TAM处理中，突变体ΔysnE的TOL和IAA合成都低于野生型SQR9。当ILA为底物时，突变子TOL和IAA的产量明显低于野生型菌株。由以上研究结果，推测基因ysnE参加吲哚丙酮酸途径和色胺途径，并在吲哚丙酮酸到吲哚乙醛之间或者色胺到吲哚乙醛之间发挥功能。