醇腈酶基因在石蒜抗旱性中的作用和分子机制研究

Lycoris radiata is one of the important ornamental and medical plants cultivated widely in our country. It comes into flower at a time when few other bulbous plants are active. The flowers are characterized by their pastel and plentiful colors as well as by beautiful and varied shapes. Additionally, L. radiata is usually regarded as a type of xerophytes because of its own characteristics and merits. But its mechanism is largely unknown. Hydrocyanic acid (HCN), as a gaseous molecule, is well known for its strong toxicity to all living cells including those of plants. It has been demonstrated that the sources of HCN in plants are diversified, while hydroxynitrile lyases (EC.4.1.2.x, HNLs) is the main enzymes which catalyzes the cyanohydrins to form it. Recently, some attentions have focused on the potential signal roles of HCN in plants under biotic/abiotic stress, while the potential roles of HNL are rarely characterized. In this study, based on our former transcriptome database of Lycoris, the full length cDNA and its promoter sequences of Lycoris radiata HNL (LaHNL) gene would be cloned. The expression model of LrHNL and the content of HCN release under drought stress would be tested. By using bioinformatics analysis, identification and prediction of drought stress responsive transcription factors involved in abiotic stress signaling would also be analyzed. Meanwhile, the significant differences between wild-type and LaHNL-overexpressing transgenic Arabidopsis would be observed, with the parameters of root elongation and chlorophyll contents under drought stress. Additionally, combining with RNA-Seq analysis of transcriptomes in HCN-treated and control Lycoris radiata plants, the significant induction of genes involved in drought stress signal transduction would also be identified. These results would clarify the positive roles of HNL/HCN under drought stress conditions and provide some new theoretic basis for the abiotic stress study in Lycoris.

石蒜是一类抗旱性较强的特色观赏花卉，对其抗旱性研究具有重要的理论和应用价值。氢氰酸(HCN) 是由醇腈酶（HNL）催化产生的极性小分子气体，在植物体内有着广泛的来源。目前已知，HCN在响应胁迫应答过程中具有潜在的信号转导作用。为了进一步分析HNL及其催化的HCN在石蒜抗旱性中的作用，本项目拟以先前构建的石蒜转录组数据库为基础，克隆石蒜HNL基因及其启动子序列；分析干旱胁迫处理下，石蒜HNL基因表达模式及其酶活性的变化；构建石蒜HNL基因过表达及其启动子序列截短的转基因拟南芥，比较它们在干旱胁迫下的表型差异；结合RNA-Seq等技术，分析和鉴定HCN参与石蒜干旱胁迫信号转导途径中关键基因和调控元件；探查HNL及其催化的HCN在石蒜抗旱性中的作用及分子调控机制；研究结果不仅为石蒜在干旱胁迫适应性中的研究提供一定的理论基础，同时也为丰富其他观赏植物的抗性品种培育起到指导作用。

石蒜是一种重要的传统药用植物，同时也是一类抗旱性较强的特色观赏植物。本项目通过开展干旱处理下的石蒜根和叶差异转录组测序，全面分析了可能受干旱胁迫诱导的醇腈酶（HNL）基因的转录本。在此基础上，结合基因克隆、构建原核/真核表达体系、启动子克隆、亚细胞定位和拟南芥转基因功能验证等，重点对石蒜HNL1和HNL2基因进行研究。研究结果表明：HNL1和HNL2体外表达的蛋白具备催化扁桃腈和邻氯扁桃腈降解的活性；HNL1和HNL2不同程度的受干旱、冷害等胁迫诱导，同时响应激素（MeJA和ABA）的应答；利用SEFA-PCR的方法克隆得到了HNL1基因上游约1,500 bp的片段，该基因启动子区含有受干旱、冷害和热胁迫等诱导的重要顺式作用元件，同时还包括一些响应植物激素（MeJA和ABA）和光应答等相关的作用元件。通过构建HNL1过表达载体，并进行拟南芥转基因，对HNL1基因在干旱胁迫中的作用进行了分析。HNL1基因过表达提高了拟南芥NaCl胁迫的耐性，并降低了对ABA的敏感性，从而降低了干旱耐受性。此外，结合拟南芥AtHNL基因受非生物胁迫诱导表达模式、启动子受诱导模式分析和转AtHNL基因拟南芥在抗旱性种的作用研究，进一步验证了HNL基因可能参与植物体干旱胁迫应答，并在其中起到重要作用。本项目研究内容的完成，明确了HNL基因在石蒜抗旱性中的作用，初步解析了HNL1基因的受诱导和作用机制。为深入研究石蒜的逆境生理提供了理论基础。