橡胶树排胶相关ADF基因参与乳管堵塞的机制研究

The duration of latex flow is a key factor that limits the latex yield, which is mainly determined by the plugging formation at the end of severed laticifers. A protein-network mainly consists of actin cytoskeleton at the end of severed laticifers, and it plays a critical role in laticifer plugging. As an import member of actin binding protein, the actin depolymerizing factor (ADF) regulates the dynamic assembly process of actin cytoskeleton by controlling actin assembly and disassembly. In rubber production, ethephon is utilized to decrease the plugging index of laticifer vessels and postpone the duration of latex flow. Four ADFs in rubber tree were cloned, and their expression levels were up-regulated by ethephon. It was speculated that ADFs in rubber tree might be involved in laticifer plugging by regulating actin cytoskeleton, but the molecular mechanism is still unclear. Based on the database of de novo transcriptome developed by our research group and other gene databases, the ADFs related to latex flow will be screened and identified in rubber tree. With the different rubber tree clones indicating specific latex flow as materials, the ADFs expression patterns, the dynamic transformation of actin and the influence on actin cytoskeleton are further analyzed under the treatments regulating latex flow. These results will contribute to not only elucidate the mechanism how ADFs regulat actin cytoskeleton and reveal ADFs function on latex flow, but also provide the theoretical basis on developing safe and highly efficient stimulus of latex flow in rubber tree.

排胶时间是限制天然橡胶产量的关键因子，而乳管伤口堵塞物的形成直接决定排胶时间长短。乳管伤口末端蛋白质网主要由肌动蛋白细胞骨架构成，它在乳管堵塞过程中起关键作用。肌动蛋白解聚因子（actin depolymerizing factor, ADF）调节肌动蛋白解聚与聚合，进而调控肌动蛋白细胞骨架的动态装配过程。在橡胶生产中,乙烯可解除乳管堵塞延长排胶时间。我们已获得4个受乙烯上调表达的橡胶树ADF基因，据此推测ADF通过调控蛋白质网中肌动蛋白细胞骨架参与乳管堵塞过程，但其调控机制仍不清楚。本项目拟利用课题组构建的橡胶树转录组等数据，全面筛选和鉴定橡胶树排胶相关ADF，研究排胶相关处理对不同排胶特性品系橡胶树中ADF表达模式、肌动蛋白动态变化和细胞骨架的影响。研究结果有望阐明橡胶树ADF调控肌动蛋白细胞骨架的机制，揭示ADF在乳管堵塞中的作用，为研制安全、高效的新型橡胶树排胶刺激剂提供理论依据。

排胶时间是限制天然橡胶产量的关键因子，而乳管伤口堵塞物的形成决定着排胶时间的长短。乳管伤口末端的蛋白质网在乳管堵塞过程中起关键作用，该蛋白质网主要由肌动蛋白细胞骨架构成。肌动蛋白解聚因子（actin depolymerizing factor, ADF）通过解聚与聚合肌动蛋白来调节肌动蛋白细胞骨架的动态装配过程。本项目全面筛选和鉴定了橡胶树排胶相关ADF基因，对各种处理条件下橡胶树排胶相关ADF基因表达与肌动蛋白动态变化进行研究，并鉴定橡胶树ADF基因的生物学功能，取得的研究结果包括1）克隆了8个橡胶树胶乳ADF基因全长cDNA序列，获得其中6个基因的基因组序列和4个基因的启动子序列，序列分析表明6个ADF基因均由3个外显子和2个内含子组成，启动子中含有多个激素或胁迫响应元件；2）利用qRT-PCR技术对橡胶树ADF基因在不同组织表达模式进行分析，其中6个ADF基因均在胶乳中表达量最高，另外2个分别在叶片或树皮中高表达；3）研究乙烯、碘化钾、不同死皮程度和排胶时间处理条件下橡胶树ADF基因的表达模式和G/F-actin的动态变化，结果发现橡胶树ADF基因表达受乙烯和碘化钾调控，但两个橡胶树品系间ADF基因表达模式不同。乙烯和碘化钾处理后G/F-actin比例都呈先升高后降低的趋势，两个处理的最大比值均出现在24 h。橡胶树ADF基因表达和G/F-actin的动态变化与死皮和排胶相关，随着死皮严重程度的加剧G/F-actin比例逐渐增加。随着割胶后排胶时间的进程，G/F-actin比例逐渐降低，直至停止排胶，但复割后5 min比值又有所增加；4）构建原核表达载体，在大肠杆菌中表达和鉴定橡胶树ADF重组融合蛋白，制备抗体；5）构建过表达载体，在裂殖酵母中异源表达橡胶树ADF基因，结果发现过表达橡胶树ADF的酵母细胞形态变长，双核和多核比例增加。以上结果表明橡胶树ADF通过参与调控肌动蛋白动态变化在乳管堵塞中发挥重要作用，研究结果有助于阐明橡胶树排胶机制，并为安全高效的排胶刺激剂的研发奠定了理论基础。发表相关论文6篇，授权专利1项，登记成果1项。