铁皮石斛是国家重点保护的濒危药用植物，其生物碱具有抗肿瘤等药理活性。AP2/ERF转录因子是植物中广泛存在的一类转录因子，相关研究表明AP2/ERF转录因子参与萜类吲哚生物碱的生物合成调控。申请者前期通过对铁皮石斛茎的转录组进行高通量测序，获得22条编码AP2/ERF转录因子的基因。本研究在此基础上进一步利用MeJA诱导筛选得到候选基因，分别构建候选基因的过表达载体和RNAi表达载体转化铁皮石斛，通过检测目的基因表达量、生物碱含量变化及转录因子蛋白表达量，快速筛选与铁皮石斛生物碱合成调控相关的编码AP2/ERF转录因子的基因。采用实时荧光定量PCR技术确定其时空表达模式，并进行亚细胞定位分析；凝胶阻滞实验明确与其结合的顺式作用元件，酵母双杂交寻找其互作蛋白，阐明AP2/ERF转录因子在调控铁皮石斛生物碱合成中的功能和机制，为铁皮石斛的品种选育和遗传改良奠定基础。

Dendrobium officinale is an endangered medicinal plant under the national key protection. Alkaloids are one of its main components, which exhibit antitumor and other activities. AP2/ERF transcription factor is one of the widespread transcription factors in plants. Previous studies showed that AP2/ERF transcription factor are involved in the regulation of terpenoid indole alkaloids biosynthesis. Applicant has obtained 22 genes which possible encoded AP2/ERF transcription factor using high-throughput sequencing of the transcriptome of the stem of Dendrobium officinale. On the basis of these results, the candidate genes will be screened by induced with MeJA. Then the over-expression vector and RNA interference of expression vector of the candidate genes will be built and transformed to Dendrobium officinale, respectively. By detecting the expression quantity of the target genes, the contents of alkaloid and protein expression of the transcription factors, AP2/ERF transcription factor involved in the regulation of Dendrobium officinale alkaloids biosynthesis will be rapidly screened. The spatio-temporal expression profile of the AP2/ERF transcription factor will be determined by real-time fluorescent quantitative PCR method, and will be located by transient expression analysis. The cis-acting element of the AP2/ERF transcription factor will be determined by gel retardation assay and the interaction protein will be located using yeast two-hybrid method. In a word, the function and mechanism of the AP2/ERF transcription factor involved in regulation of alkaloids biosynthesis of Dendrobium officinale will be clarified, which will lay the foundation for the variety breeding and genetic improvement of Dendrobium officinale.

铁皮石斛是国家重点保护的濒危药用植物，其生物碱具有抗肿瘤等药理活性，AP2/ERF转录因子是植物中广泛存在的一类转录因子，相关研究表明AP2/ERF转录因子参与萜类吲哚生物碱的生物合成调控。本项目基于铁皮石斛的全基因组数据，联合二代和三代测序技术，构建铁皮石斛的全长转录组数据库，鉴定铁皮石斛的可变剪切事件，分析铁皮石斛茎和叶转录组的差异表达基因。此外，检测铁皮石斛药效活性成分多糖及生物碱在其不同组织部位的含量差异，结合不同组织部位的转录组学数据，筛选和克隆了6个可能参与铁皮石斛生物碱合成调控相关的AP2/ERF转录因子，其中有5个AP2/ERF转录因子编码基因在叶片中的表达要高于在茎中的表达。本项目研究为铁皮石斛生物碱生物合成途径的研究提供了理论基础，为铁皮石斛品种选育和遗传改良提供基础。