过表达SNAT基因增强内生菌褪黑素合成能力和葡萄抗逆性的研究

Grape (Vitis vinifera) is one of the most important fruit trees in China, however, the domestic grape producing region is affected by the stress of adversity, and the yield and quality of grapes face serious challenges. Therefore, researches on the resistance of vine is significant to the development of the domestic grape industry. Recently, it has been reported that melatonin can improve the resistance of plants in many ways. The applicant had reported a biological phenomenon that the endophytes isolated from grapevine roots has a capacity of secreting melatonin, and acquired a high melatonin-producing strain B. amyloliquefaciens SB-9, which enhanced the endogenous melatonin levels and stress-resistance of grapevine via interaction between endophytes and its host. Unfortunately, the melatonin productivity of endophytes is generally low, and the responsible genes for melatonin synthesis pathway are still unknown. The genome of SB-9 strain had been de novo sequenced, and found several gene sequences which may have the function of the SNAT protein in melatonin biosynthesis. This project aims to make clear the key SNAT gene sequence of melatonin synthesis in SB-9 strain with prokaryotic expression and enzyme activity analysis, construct mutant strain by gene knockout and analyze its melatonin synthesis pathway using isotopic tracer. We will boost the melatonin-producing ability of this strain by homologous over-expression of SNAT gene, and promote the endogenous melatonin levels and the stress resistance of grape roots by infecting grapevine roots with SNAT over-expressed strain. This project will provide theoretical basis for the development of high melatonin-producing endogenous bacteria, as well as enhancing the endogenous melatonin levels and stress resistance via communication between beneficial endophytes and host plants.

葡萄是我国重要果树树种之一，然而国内葡萄产区常年遭受逆境胁迫影响，产量和品质面临严峻挑战，提高葡萄抗逆性对国内葡萄产业发展具有重要意义。近年来，研究发现褪黑素能通过多途径增强植物抗逆性。申请人曾报道葡萄根系内生菌产褪黑素的生物现象，其中解淀粉芽孢杆菌SB-9能与葡萄互作提高根系内源褪黑素含量和抗逆性。然而，目前内生菌产褪黑素能力普遍较低，并且其合成关键基因仍未知。申请人已对此菌株进行了全基因组测序，并找到多个可能具有褪黑素合成关键酶SNAT功能的基因序列。本项目拟采用原核表达和酶活分析，明确SB-9菌株SNAT基因序列，通过基因敲除构建褪黑素合成突变体，并采用同位素示踪法检测其褪黑素合成路径；进而过表达SNAT基因增强菌株褪黑素合成，通过定殖侵染提高葡萄根系内源褪黑素含量和抗逆性。申请项目的实施将为高产褪黑素内生菌种的开发，以及通过内生菌-根系互作提高宿主褪黑素含量和抗逆性的研究提供依据。

我国85%以上的葡萄产区常年遭受低温、干旱、盐碱、重金属等逆境胁迫的影响，逆境胁迫对葡萄生产的影响已成为当前我国葡萄产业上一个不可回避的突出问题。褪黑素属于吲哚类化合物，可以作为自由基清除剂结合活性氧、活性氮类物质，现在普遍认为，外源施用褪黑素够显著缓解植物逆境胁迫下过氧化自由基的伤害，提高植物抗氧化酶的活性以及系统抗逆性。. 本研究前期在葡萄根系中筛选出一株高产褪黑素的内生细菌，它能与葡萄根系互作有效提高宿主内源褪黑素含量和抗逆性。然而，目前根系内生菌产褪黑素能力较低，并且细菌褪黑素合成关键基因仍不清楚。在此基础上，本研究首先拟明确参与细菌褪黑素合成的相关基因，并通过过表达增强其褪黑素合成能力，最终侵染葡萄根系，验证过表达菌株是否能更有效的提高葡萄根系内源褪黑素含量以及抗逆性。主要取得以下进展：. （1）通过序列比对和基因克隆，发现细菌苯丙氨酸4-羟化酶可能是5-羟色氨的生物合成，并进一步影响细菌的褪黑素合成，可能是细菌褪黑素合成的关键基因之一。. （2）利用原核表达和蛋白酶活分析，纯化的苯丙氨酸4-羟化酶不仅能够羟基化苯丙氨酸，而且还能够将色氨酸转化为五羟色胺酸，尽管活性相关较低。. （3）通过基因敲除试验，表明苯丙氨酸4-羟化酶缺失，显著降低了细菌褪黑素的合成以及微生物的抗逆性。. 综上所述，本研究揭示了细菌苯丙氨酸4-羟化酶在控制5-羟基色氨酸和褪黑素生物合成中的可能作用，研究结果对微生物的褪黑素合成以及高产褪黑素菌株的开发利用具有重要意义。