SMT1负调控狗牙根抗旱性的分子机制

Bermudagrass is one of the most widely used turfgrass species with drought tolerance. It is important to understand the mechanism of drought tolerance in bermudagrass, while little is known at molecular levels. Sterol methyltransferase 1 (SMT1) is the first key enzyme in phytosterol synthesis. Down-regulation of SMT1 gene expression leads to accumulation of cholesterol. However, the physiological role of cholesterol in plants is unknown. In our previous studies supported by NSFC, it has been revealed that dwarfism phenotype with elevated drought tolerance in a bermudagrass mutant was associated with down-regulated SMT1 expression, as a result, cholesterol was accumulated, which in turn induced expression of lots of genes including MYB59 transcription factor and S-adenosylmethionine decarboxylase gene. On the basis of the above progress, this project continues investigations on regulation of SMT1 and cholesterol on drought tolerance in bermudagrass through analysis of MYB59 function, for understanding of the physiological role of cholesterol and how SMT1 negatively regulate drought tolerance. Transgenic bermudagrass plants overexpressing and down-regulating MYB59 expression by RNAi will be generated, followed by analysis of drought tolerance, transcript profiles, and response to cholesterol in transgenic plants in comparison to the wild type. In addition, interactions of MYB59 with the promoters of its downstream gene(s) will be tested by using yeast one hybrid system and electrophoretic mobility shift assay. Transcriptome and metabolites in the above transgenic plants as compared with the wild type will also be examined in the project to reveal the processes regulated by SMT1, cholesterol, and MYB59.

狗牙根是全球应用最广、抗旱性最强的暖季型草种之一，揭示狗牙根抗旱机制，有重要科学意义和潜在应用价值，但目前对狗牙根抗旱分子机理所知很少。环类固醇甲基转移酶1（SMT1）是植物甾醇合成途径的第一个关键酶，其基因表达下调后导致胆固醇积累，但胆固醇的植物生理作用并不清楚。前一个基金课题取得了突破性进展，初步揭示SMT1下调导致胆固醇积累，从而调节MYB59、SAMDC等系列基因表达，是狗牙根突变体表现出矮化和抗旱的原因。在此基础上，本项目拟重点围绕狗牙根MYB59开展深入研究，获得上调和下调MYB59的转基因植物，阐明MYB59及其调控的下游基因与抗旱性的关系、MYB59是否为胆固醇提高抗旱性所必需；以酵母单杂实验证明MYB59与其调控的下游基因启动子的互作关系，比较SMT1、胆固醇和MYB59调控的基因表达谱及代谢途径的异同，从而揭示SMT1负调控狗牙根抗旱性的的分子机制和胆固醇的生理功能。

本项目研究了SMT1及胆固醇调控的基因表达谱及其生化生理反应、受胆固醇调控的狗牙根CdMYB59及水稻同源基因OsMYB48-1的功能，以揭示胆固醇调控的分子机理。获得的主要结果和结论包括：（1）胆固醇处理提高狗牙根和水稻抗旱性，抑制植物生长，导致植株矮化；OsSMT1的RNAi植株积累胆固醇，植株表现出矮化和抗旱性，证明了胆固醇的重要作用。（2）胆固醇处理的狗牙根和水稻OsSMT1的RNAi水稻植株种多胺合成关键酶基因SAMDC相对表达量提高，导致亚精胺和精胺积累；亚精胺和精胺具有提高植物抗旱性的作用，外源精胺处理引起植株矮化，亚精胺处理未降低株高；结果证明胆固醇通过调控多胺合成和积累进而调控株高和抗旱性。研究结果揭示胆固醇具有生理功能，创新性较强，发表在Plant Cell and Environment（2018）上。（3）克隆了受胆固醇诱导表达的狗牙根CdMYB59基因，其表达受低温诱导，但不受干旱和盐胁迫诱导；过量表达CdMYB59转基因水稻株高正常，耐冷性提高，但耐旱、耐盐性与野生型差异不大；结果表明虽然胆固醇诱导CdMYB59的表达，但CdMYB59表达与株高和耐旱性无关。（4）克隆了水稻中CdMYB59的同源基因OsMYB48-1，但其不响应逆境；过量表达或基因编辑的突变体抗逆性也不发生改变，表明其功能与抗逆性无关；过量表达植株种子萌发和根系生长受到促进，但基因编辑植株与野生型无差异，表明OsMYB48-1与CdMYB59的功能不同。