橡胶树衰老相关HbWRKY3基因的克隆与鉴定及其调控割面干涸的分子机制

Natural rubber (cis-1,4-polyisoprene) is an important industrial raw material with unique physical properties superior to petroleum-derived synthetic rubber. Among over 2,000 polyisoprene-producing plant species, brazilian rubber tree (Hevea brasiliensis Muell. Arg.) is the only commercial source of natural rubber due to its high production and quality of rubber. Over the past decades, the rubber yield has been significantly increased due to the wide cultivation of high-yielding clones and the extensive utilization of ethephon (an ethylene generator). Meanwhile, steadily increased occurence of a complex physiological syndrome called Tapping Panel Dryness (TPD), which is characterized as tapping incision blocked partly or entirely during latex exploiting, caused great losses of latex production and has become the most important limiting factor on the rise of yield per unit area in rubber tree. Although a great deal of effort has been made on TPD, the molecular mechanism underlying remains poorly understood due to its complex causes. Up-regulated expression of a large number of senescence-associated genes and down-regulated expression of many anti-apoptosis-associated genes in TPD-affected trees suggests that TPD may be a type of programmed cell death (PCD) due to overproduction of reactive oxygen species (ROS) resulted from high-strength tapping and ethylene over-stimulating. WRKY is a plant-specific transcription factor family encoded by multiple genes. In Arabidopsis, at least 13 of 74 family members play a positive or negative role in leaf senesing. Our previous studies suggested the senescence-associated HbWRKY3 gene is a key factor involved in TPD occurence of rubber tree. To elucidate the main network of HbWRKY3 gene in TPD occurrence, in this project, biochemistry and molecular biology techniques are carrying out to isolate and characterize this gene and its targets, which promises to lay a basis for TPD-related molecular breeding and for developping new technologies on prevention and treatment of TPD in Hevea.

天然橡胶是重要的工业原料和战略物资，其主要来源为巴西橡胶树。生产上，天然橡胶通过用胶刀周期性地切割树皮收集胶乳而获得。割面干涸(TPD)是影响天然橡胶产量的关键因素。目前我国橡胶树TPD的发生率高达25%，每年因此而造成的经济损失近40亿元，然而，至今对TPD的发生机制知之甚少。项目组已有的研究表明，转录因子HbWRKY3是调控TPD发生和叶片衰老的关键因子。本项目拟在前期克隆HbWRKY3基因的基础上，综合应用qRT-PCR、RNA-seq和酵母双杂交等研究技术，鉴定HbWRKY3基因的功能，研究基因的表达模式及其与TPD发生的相关性，筛选并鉴定HbWRKY3调控的下游基因。本研究可初步揭示HbWRKY3调控TPD发生的分子机制及其代谢网络，为开展TPD相关分子育种和研发TPD综合防控新技术奠定基础。

天然橡胶是一种不可替代的重要战略资源和工业原料，主要来自于巴西橡胶树的胶乳。我国橡胶树种植区域主要分布在海南、云南和广东三省，属于非传统植胶区。目前，包括我国在内的橡胶树种植业面临的重要难题之一就是引起橡胶树胶乳产量大幅下降的死皮病害。橡胶树死皮病又称为割面干涸（TPD），是指橡胶树割线发生局部或全部不排胶的现象。据调查，我国胶园橡胶树死皮病发生率高达30%，局部地区发病率可能更高，严重影响了橡胶树的产胶量，是世界各植胶国都非常重视、也是天然橡胶产业亟待解决的重大研究课题。.前期研究表明，WRKY家族转录因子可能在橡胶树叶片衰老和TPD发生过程中发挥重要调控作用。本项目在此基础上重点研究了如下4个方面的内容：①橡胶树叶片衰老和TPD相关WRKY家族转录因子HbWRKY3基因的克隆与表达特性；②HbWRKY3基因表达与橡胶树叶片衰老及TPD形成的相关性分析；③HbWRKY3转基因功能分析及其下游调控基因的筛选与鉴定；④橡胶树TPD发生的基因表达特性及其分子调控网络。.橡胶树HbWRKY3属于第II类WRKY家族转录因子基因，在TPD橡胶树的胶乳和树皮组织中具有不同的表达模式，在胶乳中呈现下调表达趋势，这与该基因在外源乙烯利诱导橡胶树叶片衰老进程中呈现的上调表达趋势明显不同。转基因拟南芥研究表明，HbWRKY3过表达可诱导许多抗逆相关下游基因的表达，从而提高转基因拟南芥植株的抗逆性，因此，橡胶树HbWRKY3可能是1个可提高植物抗逆性的转录因子。结合本项目转录组和蛋白质组研究结果，HbWRKY3基因在TPD橡胶树胶乳中的下调表达，一方面可能导致了橡胶树乳管细胞中活性氧（ROS）清除能力的下降，使乳管细胞生理紊乱，从而引起TPD的发生；另一方面还可能导致了橡胶生物合成途径中FPS等重要功能基因表达的下降，从而抑制了橡胶树的橡胶生物合成活性，导致产胶能力下降或丧失。.本项目研究结果对深入研究橡胶树TPD发生的分子机制及调控网络奠定了重要基础，并为通过转基因技术提高橡胶树乳管细胞清除ROS的能力及建立橡胶树TPD防控技术提供了一定的理论依据。