基于转录组的阳春砂挥发性萜类代谢关键功能基因的系统发掘

Volatile terpenoids have significant pharmacological and biological activities, therefore, it is important to study their metabolic regulation. Amomum villosum is the main variety of Fructus Amomi, one of the famous "Four South China Herbals". Volatile oil is the main compounds of A. villosum which contribute the medicinal functions of aromaticity and dissipating dampness. However, volatile terpenoid metabolic pathway of A. villosum remains unclear, therefore, its key functional gene research is very necessary. Methyl jasmonate (MeJA) is an important signal molecule regulating plant terpenoid metabolism. The applicant found that MeJA induced the key enzyme genes expression and regulated the volatile terpenoids metabolism in A. villosum. This project will use MeJA-treated A. villosum and its different developing stage fruits as research materials, ① transcriptome sequencing and expression analysis of the materials will be carried out using high throughput sequencing technology, and their volatile terpenoid components will be determined and analyzed by GC-MS; ② the transcriptome and metabolome informations will be associated, and candidate functional genes will be screened by data analysis; ③ then quantitative PCR for candidate genes and GC-MS analysis for targeted terpenes will be carried out to verify the correlation between the gene expression and terpenes accumulation; ④ two or three critical functional genes will be selected to clone and verify their functions. This project assembles JA regulatory pathway and fruit maturity transcriptomes analysis to massively excavate the critical functional genes involved in volatile terpenoid metabolism of A. villosum, and reveal its molecular mechanisms, which will help to establish the foundation for in-depth understanding the pattern and regulation of volatile terpenoid metabolism of medicinal plant.

挥发性萜类具有显著的药理和生物活性，研究其代谢调控有重要价值。阳春砂是"四大南药"砂仁的主流品种，挥发油是其芳香化湿的主要物质，然而阳春砂挥发性萜类的代谢途径仍不清楚，对其关键功能基因的研究十分必要。茉莉酸甲酯（MeJA）是调控植物萜类代谢的重要信号分子，申请人研究发现MeJA诱导阳春砂关键酶基因的表达，并调控挥发性萜类的代谢。本项目将以MeJA处理的阳春砂和不同成熟期的果实为材料，①用高通量测序技术进行转录组测序和表达谱分析，用GC-MS测定挥发性萜类成分；②关联转录组和代谢组信息，批量筛选功能基因；③对候选功能基因进行定量PCR和目标萜类的GC-MS分析，验证两者的相关性；④克隆2-3个代表性关键基因并验证其功能。本项目联合JA调控通路和果实成熟期转录组分析，系统发掘阳春砂挥发性萜类代谢的功能基因，揭示阳春砂挥发性萜类代谢的分子机制，为深入认识药用植物挥发性萜类代谢调控规律奠定基础。

挥发性萜类具有显著的药理和生物活性，研究其代谢调控有重要价值。阳春砂Amomum villosum是“四大南药”砂仁的主流品种，挥发油是其芳香化湿的主要物质，然而阳春砂挥发性萜类的合成途径仍不清楚。茉莉酸甲酯（MeJA）是调控植物萜类代谢的重要信号分子。为了认识阳春砂的挥发性萜类合成途径，本项目主要以MeJA处理的阳春砂和不同成熟时期的果实及其根茎叶为材料，分别构建了2套转录组数据AvM和AvD，从2套转录组数据中分别获得70,323条和72,002条被注释的unigene；分别获得萜类上游合成途径的unigene有137和141条。.基于MeJA诱导的转录组和11个表达谱数据，初步构建了JA信号转导与部分转录因子和萜类合酶基因的调控网络；筛选出与萜类合酶基因表达和挥发性萜类含量均相关的6个WRKY、3个MYC和6个MYB转录因子；克隆了其中的AvMYC2、AvMYC4b和AvWRKY40。.从转录组中筛选出了多个候选萜类合酶基因，克隆了其中7个挥发性萜类合酶基因，并鉴定了其中AvTPS1和AvTPS3的功能，分别鉴定为蒎烯合酶基因（pinene synthase, PS）和龙脑基二磷酸合酶（bornyl diphosphate synthase，BPPS）基因。其中阳春砂蒎烯合酶（AvPS）主要负责阳春砂叶片和果皮中α-蒎烯和β-蒎烯的合成，α-蒎烯和β-蒎烯是阳春砂叶片和果皮中的主要单萜，并克隆了AvPS的启动子；而阳春砂龙脑基二磷酸合酶（AvBPPS）的直接产物莰烯、月桂烯和柠檬烯以及间接产物龙脑、樟脑和乙酸龙脑酯是阳春砂果实中的主要单萜，因此，AvBPPS负责了阳春砂药用部位果实中主要单萜的合成。AvPS和AvBPPS的发现为阳春砂挥发性萜类的代谢调控提供了重要的分子调控元件，其中AvBPPS更是有重要的研究前景。AvBPPS是一种特殊的多产物单萜合酶，以龙脑和乙酸龙脑酯的前体龙脑基二磷酸为主要产物，据目前国内外报道仅从3种植物中发现了BPPS基因，对该基因的深入研究有可能发现阳春砂的主要药效物质乙酸龙脑酯在种子团中富集的分子机制，对于阳春砂药材的品质调控具有重要价值。本项目为后续获得完整的乙酸龙脑酯生物合成途径奠定了基础，对于阳春砂的挥发性萜类调控有重要意义。.