茉莉酸甲酯高效调控石蒜加兰他敏生物合成的分子机制

Lycoris radiata is a popular ornamental and medicinal species of Amaryllidaceae plant widely distributed in our country. The natural alkaloids galantamine extracted from amaryllidaceae, has been used clinically for the treatment of Alzeheimer disease. Our former studies have proved that the galanthamine biosynthesis was efficiently up-regulated by methyl jasmonate (MeJA) in Lycoris radiate. By RNA-seq and miRNA/degradation sequencing, we further identified some genes involved in above galantamine biosynthesis or participate in JA signal transduction pathway, and some miRNA induced by MeJA were also confirmed in above process. But, the molecular mechanisms of this regulation are still largely unknown.. For further elucidating of the molecular mechanisms of MeJA in efficient up-regulation of galantamine biosynthesis in lycoris radiata, in this project, the following studies will be carried out. Firstly, we plan to clone the promoter of genes involved in galanthamine biosynthesis, and then find out its cis-acting elements combined with jasmonate by bioinformatics analyzing. Secondly, using the yeast one-hybrid and PCR methods, we can clone the transcription factor genes and miRNAs which may participate in the regulation pathway of galanthamine biosynthesis induced by MeJA and genes of protein combined with the core components of jasmonate signaling casade. Afterwards, the lines of Lycoris radiata which overexpress or inhibit the target genes or miRNAs, by transgenic approaches, were obtained. Finally, we identify the key genes and cis-acting elements involved in the regulation pathway by determination of galantamine content and RNA-Seq sequencing, and elucidated the mechanisms of MeJA in efficient up-regulation of galantamine biosynthesis in lycoris radiate, in the post-transcriptional or transcriptional level. Together, these results would not only provide the theoretical and technological basis for increasing the content of galanthamine in Lycoris radiata, but also enrich the signal transduction theoretical foundation of secondary metabolism regulated by jasmonate in the horticultural crops.

来源于石蒜科植物的生物碱加兰他敏已被临床应用。前期，申请人的研究发现茉莉酸甲酯（MeJA）能高效诱导石蒜体内加兰他敏合成；差异转录组、小RNA/降解组测序分析了其中可能涉及的合成酶和转录因子基因、miRNA、JA信号转导核心组分基因。为进一步明确MeJA诱导加兰他敏合成的分子机制，本项目拟克隆参与加兰他敏生物合成的酶基因启动子并分析其响应茉莉素的作用元件，以钓取受MeJA诱导参与加兰他敏合成调控的转录因子；克隆与JA信号转导核心组分互作的蛋白基因，并鉴定调控加兰他敏合成的miRNA；进而转基因构建目标基因/miRNA过表达或抑制的石蒜株系，结合加兰他敏含量测定RNA-Seq分析和鉴定参与该合成途径调控的关键基因和调控元件；以从转录/转录后调控水平阐释MeJA诱导加兰他敏合成的作用机制。本研究结果为培育高加兰他敏含量石蒜新品种提供理论依据，也将丰富茉莉素调控园艺作物次生代谢信号转导的理论。

石蒜属植物体内含有加兰他敏等石蒜科生物碱，具有重要的药用价值。本项目利用茉莉酸甲酯（MeJA）处理下忽地笑差异转录组和小RNA测序，发掘和鉴定加兰他敏生物合成及调控基因，并对其调控机制进行研究。主要结果如下：（1）完成MeJA处理下转录组测序，筛选到可能参与加兰他敏生物合成的转录因子和茉莉素信号通路核心组分基因；筛选并验证了忽地笑合适的内参基因。（2）筛选到加兰他敏生物合成途径中的氧甲基转移酶（OMT）基因，并对LaOMT1基因进行功能验证，发现其编码蛋白催化降孤挺花啶生成目标产物4’ 氧甲基降孤挺花啶。（3）克隆并鉴定加兰他敏生物合成途径中的酪氨酸脱羧酶基因LaTYDC1，其编码蛋白可以体外催化酪氨酸转化为酪胺。（4）完成忽地笑MeJA处理下小RNA和降解组测序，鉴定到可能调控加兰他敏生物合成途径关键酶基因的miRNAs。通过RLM-RACE实验，验证了LaTYDC1基因有miR396的识别切割位点。（5）克隆并分析了7茉莉酸ZIM结构域家族蛋白基因（LaJAZ1-LaJAZ7），利用酵母双杂交系统筛选到LaJAZ的互作蛋白；克隆到2个MYC类转录因子基因（LaMYC2-1和LaMYC2-2），并完成其转录本变化分析。本项目的实施，初步解析了MeJA信号通路可能调控加兰他敏生物合成的作用机制，为加兰他敏的合成生物学研究奠定了一定的理论基础。

内容获取失败，请点击重试