橡胶树乳管质膜H+-ATPase基因在乳管产排胶中的功能研究

Both of latex regeneration and flow are considered as two main factors in the yield control of Hevea brasiliensis. The laticifer plasma membrane H+-ATPases(PMA), as the powerhouses for transport processes across the plasma membrane of the laticifer cell, involved in multiple physiological process related to latex regeneration and flow, such as nutrients uptake, latex pH and turgor pressure. And it also might participate in the stimulating production of H. brasiliensis by ethylene. It is reasonable to assume that the HbPMA expressed in laticifer cell played critical roles in the regeneration and flow of latex as well as the regulation of stimulating production by ethylene, which would be a key point for the yield formation of H. brasiliensis. Based on cloning of all members of HbPMA gene families from H. brasiliensis, and identification of four HbPMA genes expressed in laticifer cell, the changes in the level of genes expression and proteins as well as the p-Thr phosphorylation respondings to ethylene stimulation were explored in this work, to confirm ethylene activated the laticifer HbPMA, thereby promoting the latex regeneration and flow in H. brasiliensis. In Taraxacum brevicorniculatum, we will test the effects to laticifer on suger transport, latex pH regulation, rubber biosynthesis and latex flow after the HbPMA were activated artificially in laticifer cell. This works will provide an insight into the important function for regeneration and flow of latex as well as the regulation of latex yield by ethylene, and it will also provide the target gene and theoretical basis for the genetic improvement of the high yield traits in H. brasiliensis.

胶乳再生(产胶)与排胶是橡胶树产量形成的关键。橡胶树乳管H+-ATPase (PMA)作为其物质跨膜运输的动力来源，与乳管营养吸收、胶乳pH调节、膨压形成等产排胶相关的生理过程密切相关，并可能参与了橡胶树的乙烯调控增产。这些均表明橡胶树乳管PMA在其产胶和排胶过程中发挥了重要作用，是其产量形成的关键节点。基于此，本项目在全面克隆橡胶树PMA家族基因，并分离鉴定出4个乳管HbPMA基因的基础上，在橡胶树中研究乳管HbPMA的基因表达、蛋白水平及其活性位点磷酸化对乙烯的响应，明确乳管HbPMA作为乙烯信号的靶标调控橡胶树产排胶，并确定其调控的方式。在橡胶草乳管中过表达高酶活HbPMA，研究人为激活乳管PMA对橡胶草乳管的糖转运、胶乳pH、橡胶合成以及排胶等产量形成相关性状的调控。最终揭示橡胶树乳管PMA在其产排胶和乙烯调控增产中的生理功能，为橡胶树高产分子育种提供可靠的靶标基因和理论指导。

胶乳再生(产胶)与排胶是橡胶树产量形成的关键。橡胶树乳管H+-ATPase (PMA)是乳管质膜的关键酶，作为其物质跨膜运输的动力来源，与乳管营养吸收、胶乳pH调节、膨压形成等产排胶相关的生理过程密切相关，并可能参与了橡胶树的乙烯调控增产。为了解乳管PMA在乙烯调控增产过程中的作用，本研究经两相体系纯化质膜的优化，构建了橡胶树树皮高质量质膜的提取方法。通过体外酶活的测定明确了PMA活性受乙烯的上调。同时运用Nano LC-MS对乙烯处理不同时间的树皮质膜蛋白进行了定性定量分析，共鉴定到2157个蛋白，并1450个蛋白进行了定量，其中上调大于2倍的蛋白184个，如物质运输相关的ABC转运蛋白、ATP酶、蔗糖转运蛋白、离子转运蛋白，水通道蛋白，受体激酶以及核酸结合蛋白等；下调大于2倍的蛋白217个，包括信号转导相关的受体激酶、细胞氧化还原相酶、硫氧化还原蛋白、细胞壁合成相关的微管蛋白等。该研究在质膜蛋白质组水平上揭示乙烯促进橡胶树增产的相关机制，同时明确了乙烯对PMA蛋白的上调作用；采用qPCR分析了乙烯处理后橡胶树树皮和胶乳中PMA基因表达变化，确定了乙烯可上调质膜PMA基因表达；从而明确了乙烯可通过转录翻译，上调橡胶树乳管PMA的基因表达和蛋白水平，是乳管乙烯信号的重要靶标。为研究乳管PMA 激活对乳管产排胶的影响，本研究利用YAK2酵母突变体筛选与鉴定了截取自抑制区PMA1（Truncated PMA1）的高酶活特性，构建了该基因片段的乳管特异表达载体，导入了橡胶草中，并对其胶乳可溶糖含量、干胶含量及排胶量进行了测定。结果显示，3个转高酶活PMA基因载体的植物中胶乳可溶性糖和干胶含量均显著提高，明确了通过转基因提高乳管PMA活性，可促进乳管可溶性糖运输和橡胶的合成。本研究证实橡胶树乳管PMA是橡胶树乙烯调控增产的靶标，而乳管PMA的人为调控可作为橡胶树产量调控手段。这为橡胶树高产分子育种提供可靠的靶标基因和理论指导。