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

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。

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.