石斛属植物CAM型PEPC基因的鉴定及功能分析

Dendrobium, one of the three largest genera in the Orchidaceae, is famous for their higher ornamental and medicinal value. In our previous research, we found not only obligate CAM and C3/CAM intermediate but obligate C3 plant all exist in the genus Dendrobium. CAM represents higher carbon-concentrating mechanisms than C3 photosynthesis for a warmer and drier environmental. CAM type PEPC gene (one member of PEPC gene family) play a key role in the switch from C3-photosynthesis to CAM, but the molecular mechanism required for the C3/CAM switches remain unclear on. In this proposal, based on our cloning and cDNA sequence analysis of three type PEPC isoforms, obligate C3, C3/CAM intermediate (or facultative) and obligate CAM plant of Dendrobium will be selected as materials, CAM-PEPC isoforms will be identified by comparing spatiotemporal expression patterns under natural and stressed conditions. Also, functional characterization of CAM-PEPC is used to confirm the function of CAM-PEPC in vitro, mutation sites, enzyme kinetics and combined with genetic engineering by overexpression of CAM-PEPC in Arabidopsis thaliana and silencing of CAM-PEPC in Dendrobium species under control of the EβFS promoter. We expect the results not only explore the molecular mechanisms of CAM-PEPC in regulating the photosynthesis C3/CAM shift of Dendrobium species, and contribute to lay the foundation for CAM-into- C3 engineering in the future.

石斛属（Dendrobium）乃兰科三大属之一，大都具有较高的观赏价值或药用价值。在前期工作中，我们发现石斛属同时含有专性C3、C3/CAM中间型和专性CAM三类植物。干旱环境下，CAM途径具有比C3途径更有竞争力的碳浓缩机制，在C3/CAM转换过程中，CAM型PEPC基因起到关键调节作用，然而相关分子机理并不明确。本研究拟在前期对三类PEPC基因亚型克隆和序列分析基础上，以C3、C3/CAM中间型和专性CAM石斛兰为材料，通过研究自然和胁迫状态下时空表达模式，鉴定调控CAM途径的CAM型PEPC基因；通过原核表达、氨基酸位点突变、酶动力学并结合拟南芥转基因超表达和石斛兰基因沉默后表达，明确基因功能。研究结果不仅对揭示石斛属植物中CAM型PEPC基因参与光合碳同化C3/CAM代谢转换的分子机理提供依据，同时也为今后把CAM途径转移到C3植物而提高植物在干旱胁迫下光合能力而奠定基础。

石斛属（Dendrobium）乃兰科三大属之一，大都具有较高的观赏价值或药用价值。在前期工作中，我们发现石斛属同时含有专性C3、C3/CAM中间型和专性CAM三类植物。干旱环境下，CAM途径具有比C3途径更有竞争力的碳浓缩机制，在C3/CAM转换过程中，CAM型PEPC基因起到关键调节作用，然而相关分子机理并不明确。本研究拟在前期对三类PEPC基因亚型克隆和序列分析基础上，以C3、C3/CAM中间型和专性CAM石斛兰为材料，分析自然和胁迫状态下三类PEPC基因异构体的表达差异，分析序列特征及功能，鉴定调控CAM途径的CAM型PEPC基因；结合烟草转基因超表达和铁皮石斛基因沉默后表达，分析基因功能。结果表明，（1）不同类型植物三类PEPC基因的表达模式不同：对照和干旱胁迫后，专性C3类流苏石斛的PpcA、PpcB和PpcC三类PEPC基因相对表达量均较低；专性CAM类美花石斛PpcA和PpcC三类PEPC基因相对表达量均远高于专性C3类流苏石斛的表达量；干旱胁迫能诱导C3/CAM中间型铁皮石斛两类CAM型PEPC基因的表达量明显升高。（2）结合基因表达、序列分析，以及与已明确具有CAM功能的香荚兰PEPC基因等比较，认为石斛属植物至少PPCA类CAM型PEPC基因具有诱导C3/CAM转换的功能，而PPCB也可能具有调节作用。（3）分别获得烟草转PPCA类CAM型PEPC基因超表达和该基因位点突变表达载体的抗性植株；C3/CAM中间型铁皮石斛转沉默载体和专性CAM植物秋石斛转超表达载体未获得抗性植株。（4）建立了铁皮石斛等多种石斛的再生体系；探索了铁皮石斛和秋石斛的转化CAM型PEPC基因的体系。研究结果不仅对揭示石斛属植物中CAM型PEPC基因参与光合碳同化C3/CAM代谢转换的分子机理提供依据，同时也为今后把CAM途径转移到C3植物而提高植物在干旱胁迫下光合能力而奠定基础。