铁皮石斛多糖合成途径相关基因的克隆与功能分析

Dendrobium officinale is a precious traditional chinese medicinal material and had been used as a medicinal herb for 2000 years. D. officinale is used extensively base on effective composition of Dendrobium polysaccharide, and content of polysaccharide and drug effects is directly relationship. Polysaccharide is a polymeric compound of monosaccharide molecules that was formed by sucrose through transportation, conversion and polymerization. At present, research on polysaccharide metabolism of Dendrobium officinale mainly concentrated in the polysaccharide structure, biological activity and so on, but related gene of polysaccharide synthesis is poorly understood. This study is based on a normalized stems cDNA library, and further constructed a stem, leaf and growing tips normalized cDNA library. Then gene related of polysaccharide synthesis are cloned, and functional, expression analysis are conducted. The results will offer basic information for functional genome of Dendrobium officinale, genetics and breeding and regulation mechanism of polysaccharide synthesis. Moreover, the relationship between sucrose synthesis and degrading enzymes and sucrose and polysaccharide were elucidated. It sheds light on molecular mechanism of polysaccharides synthesis and accumulation.

铁皮石斛（Dendrobium officinale Kimura et Migo）是传统名贵的中药材，具有2000多年的药用历史，可溶性多糖是铁皮石斛的主要活性成分，其含量与药效直接相关。多糖是光合产物蔗糖经过运输、转化、聚合等复杂过程形成的许多单糖分子的聚合物。 目前，对铁皮石斛多糖代谢的研究主要集中在多糖的结构、生物活性等方面。对多糖的合成相关基因的认识还知之甚少，本研究拟在课题组已有的cDNA文库构建和基因克隆的基础上，以铁皮石斛茎、叶和生长点为材料，通过构建均一化cDNA文库，结合从genebank数据库中查询并收集相近石斛属的兰科等植物多糖代谢候选功能基因的序列信息，克隆与石斛多糖合成相关的基因，并进行功能和表达分析，探讨铁皮石斛多糖合成相关基因与糖含量间的可能关系，为阐明铁皮石斛多糖的合成与积累分子生物学机制奠定基础。也为铁皮石斛功能基因组学研究、遗传育种和调控机制积累资料。

铁皮石斛是传统名贵的中药材，可溶性多糖是铁皮石斛的主要活性成分，其含量与药效直接相关。项目通过cDNA文库构建，克隆7个与与石斛多糖合成相关的基因，并进行功能和表达分析；筛选了铁皮石斛qRT-PCR内参基因，选择了7个候选基因，根据序列比对分析、稳定性分析、NormFinder软件和GeNorm软件的分析结果，鉴定到最适内参基因为UBC和GAPDH。完成了铁皮石斛的转录组测序分析，鉴定出9个蔗糖合酶基因，并与拟南芥、水稻和小兰屿蝴蝶兰的蔗糖合酶基因家族做系统进化树分析，发现铁皮石斛SUS基因和小兰屿蝴蝶兰的有更近的聚类关系。另外选育高产、优质铁皮石斛新品种光明1号、光明2号、红鑫5号和红鑫6号4个新品种、开展铁皮石斛与近缘种的遗传多样性研究及分子鉴定。项目的完成初步阐明了铁皮石斛多糖的合成与积累分子生物学机制，为铁皮石斛遗传育种和调控机制积累资料。