小茴香中聚炔合成第一步关键酶基因的克隆和功能分析

Polyacetylenes are a class of natural products with conjugated triple bonds on carbon chains. They have only been identified in a number of Apiales plants such as fennel, and have potent antifungal, antibacteria and anticancer activities. To regulate polyacetylene levels in plants through molecular bredding or genetic engineering, it is prerequisite to study their biosynthesis pathways and regulation means. Our project intends to clone and functionally analyze genes encoding key enzyme regulating the first key step of polyacetylene biosynthesis using fennel as plant material. Based on the finding that polyacetylene accumulation is induced by fungi infection, we will compare high-throughput sequencing results between cDNAs of fungi-induced fennel seeds and those of non-induced seeds, and thus search for genes with RNA expression significantly induced. Based on hypothesis that unsaturated fatty acids are precursors of polyacetylene biosynthesis, we will identify homologs of FAD2 in induced gene pool, and clone full-length sequence of this gene; to confirm that the gene we clone is a key gene responsible for triple bond formation in polyacetylene biosynthesis, we will characterize biological activities and physiological roles of this gene through multiple means such as complementation tests in Arabidopsis mutants and fungi infection tests. In addition, we will search for genes possibly involved in later steps of polyacetylene biosynthesis. Results of this study will set foundation for the fuuther studies on polyacetylene biosynthesis pathways, and provide gene targets for future endeavors of improvement of pharmaceutical and food values of fennel through genetic engineering and molecular breeding of polyacetylene levels.

聚炔类化合物是一类在碳链上含共轭三键的天然化合物。此类化合物存在于小茴香等伞形目植物中,具有高度的抗菌和抗癌活性。通过分子育种和基因工程调控植物中聚炔的含量，必须以了解其生物合成途径为基础。本项目拟以小茴香为植物材料，克隆负责聚炔生物合成第一个关键步骤的基因。基于聚炔合成受真菌诱导这一事实，我们将比较真菌诱导与非诱导的小茴香种子cDNA库的高通量测序结果，寻找受显著诱导表达的基因。基于聚炔生物合成的前体是不饱和脂肪酸这一假设，将在受显著诱导表达的基因中进一步鉴定FAD2基因的同源基因，并克隆其全长序列；通过在拟南芥突变种子中的互补试验、真菌侵染试验等方式，阐明此基因的生物活性，从而确认其为聚炔类生物合成中负责三键形成的关键基因。此外，我们将寻找可能参与聚炔合成后续步骤的基因。本项目的开展，将为进一步研究聚炔生物合成途径建立基础，并为通过基因工程和分子育种调控小茴香中聚炔含量，提供基因靶标。

聚炔化合物是一类在碳链上含共轭三键的特殊天然化合物。此化合物存在于小茴香等伞形目植物、以及三七等五加科植物中,具有高度的抗菌和抗癌活性。如何大量获取聚炔化合物，是利用其造福人类的基础，而通过分子育种和基因工程调控植物中聚炔的含量，必须以了解其生物合成途径为前提。本项目通过组学与分子生物学方式的结合，从小茴香中克隆得到了两条聚炔合成酶基因。首先，本项目确立了flg22能成功诱导小茴香伞形组织中相关基因表达，激活植物天然免疫机制。同时，本项目发展了一套高通量基因挖掘的生物信息学方法。将flg22诱导的组织取样，进行转录组测序，在拼装结果中获得6条FAD2同源基因序列。根据这些序列设计引物，成功克隆得到了3条全长基因，其中确认2条属于聚炔合成酶基因。Flg22能成功诱导这两条聚炔合成酶基因表达，在诱导后2-6h其表达量达到峰值。该基因是FAD2基因的同源基因，并含有一个保守结构域。在系统发育树中，该基因与其它已知聚炔合成酶基因一起，形成一个显著的独立枝。此外，我们通过转录组测序，系统研究了小茴香在flg22诱导后不同时间点的基因表达模式，发现了一系列差异表达基因。本项目为更深入地研究聚炔化合物的生物合成通路提供了靶标，并为今后通过基因工程生产聚炔化合物提供了基础。