利用“组学”方法解析岩溶高钙环境下华南忍冬叶片腺体储钙和气孔泌钙的代谢网络

High level of calcium in karst ecosystem was formed in the process of weathering pedogenesis of numerous carbonate rocks. Research on the adaptation mechanism for plant that growth in karst area could provide the theoretical and practical basis for vegetation recovery. Take the Lonicera confusa which can suitable for grown in karst area as research object, the gland storing and stomata secreting calcium are the two main adative modes for this plant to high level of calcium content in karst area were observed previous. However, the molecular mechanism of these two adative modes was remained unclear. In this project, the Lonicera confusa that cultivated in non-karst area with lower calcium was used as control, the calcium content in glands in different developmental stages and stomata in different calcium secreting stages were analyzed that cultivated in karst area by using energy dispersive X-ray spectroscopy(EDX) and laser scanning confocal microscope(LSCM), the gene and protein expression profiles were analyzed by using RNA sequencing combined with two dimensional electrophoresis(2-DE) of protein techniques in order to found the differentially expressed genes and proteins. The function of these differentially expressed genes and proteins were verified by using fluorescence quantitative PCR and transgenic plants. The results can provide us a basis for construction the metabolic network for gland storing and stomata secreting calcium process and cultivate the new germplasm resources of Lonicera confusa.

碳酸盐岩风化成土过程造就了岩溶生态系统土壤的富钙性，对岩溶区植物适应高钙机制进行研究，可为岩溶区植被恢复提供理论和实践依据。前期研究表明：腺体储钙和气孔泌钙是岩溶高钙适生植物华南忍冬适应高钙环境的两种主要方式，但腺体储钙和气孔泌钙的分子机制尚不明确。本研究以非岩溶低钙土壤栽培的华南忍冬为对照，以岩溶高钙土壤栽培华南忍冬腺体储钙和气孔泌钙不同阶段的叶片为材料，在能谱和激光共聚焦显微镜分析腺体不同储钙阶段腺体内钙含量和气孔泌钙不同阶段气孔及周围细胞钙含量的基础上，利用RNA-Seq技术结合蛋白质组学技术对比分析腺体储钙和气孔泌钙不同阶段的基因表达谱和蛋白质表达谱，找到差异表达的关键酶基因和蛋白质，并对差异基因和差异蛋白质对应基因进行转基因功能验证，为解析华南忍冬腺体储钙和气孔泌钙的代谢网络及培育新的华南忍冬种质资源打下基础。

碳酸盐岩风化成土过程造就了岩溶生态系统土壤的富钙性，对岩溶区植物适应高钙机制进行研究，可为岩溶区植被恢复提供理论和实践依据。前期研究表明：腺体储钙和气孔泌钙是岩溶高钙适生植物华南忍冬适应高钙环境的两种主要方式，但腺体储钙和气孔泌钙的分子机制尚不明确。本研究以非岩溶低钙土壤栽培的华南忍冬为对照，以岩溶高钙土壤栽培华南忍冬腺体储钙和气孔泌钙不同阶段的叶片为材料，利用能谱和激光共聚焦显微镜分析了腺体不同储钙阶段腺体内钙含量和气孔泌钙不同阶段气孔及周围细胞钙含量；同时利用RNA-Seq技术结合蛋白质组学技术对比分析腺体储钙和气孔泌钙不同阶段的基因表达谱和蛋白质表达谱，找到差异表达的关键酶基因和蛋白质，并对差异基因和差异蛋白质对应基因进行转基因功能验证，为解析华南忍冬腺体储钙和气孔泌钙的代谢网络及培育新的华南忍冬种质资源打下基础。.研究结果表明：大量Ca2+被根系吸收后，通过Ca2+转运系统运输到叶片表面腺体和气孔周边细胞，并最终通过叶片表面腺体和部分气孔分泌到植物体外，该过程势必涉及了钙离子的吸收、转运以及腺体储存和气孔分泌等多个过程；针对钙离子诱导后的金银花转录组和蛋白质组学分析研究发现，Carbohydrate metabolism，Amino acid metabolism，Energy metabolism，Lipid metabolism在钙离子短期诱导和长期诱导后均有较大变化，同时与Excretory system相关的代谢以及钙离子诱导的转运蛋白等均被发现；根据这些基因表达变化构建了华南忍冬吸收转运钙离子的代谢网络，并对部分基因进行了转基因功能验证。对于华南忍冬有关腺体储钙和气孔泌钙可能的分子机理以及特异的钙转运蛋白（通道）及其调控途径还未见相关报道。