小热激蛋白在橡胶树死皮发生中的分子机制研究

Tapping panel dryness (TPD) of rubber tree is one of the major constrains in natural rubber production. The elucidation of molecular mechanism of TPD is prerequisite for taking effective measures to prevent TPD, but the molecular mechanisms underlying TPD remain poorly understood. Our previous works suggested that the programmed cell death (PCD) caused by overdose of reactive oxygen species (ROS) was the key pathway, meanwhile small heat shock protein (sHSP) interacting with HbTCTP, a key gene associated with TPD, may play important roles in scavenging ROS. Based on the detailed transcriptional data of several varieties of rubber trees, 13 sHSPs family members were cloned. In this work, we propose to evaluate the roles of every sHSP member in the TPD induced by ROS through comparative analysis the relationship between the expression of sHSP and enzymatic activity related to ROS production or scavenging. Additionally, we plan to detailed screen the interaction between HbTCTP and each sHSP family members using yeast two-hybrid and rubber protoplast transient expression system. Finally, we will investigate the function of sHSP through the transgene system of E.coli and tobacco.The outcomes of this project will help to elucidate the molecular mechanism and build up new prevention and control strategies on TPD.

死皮是限制橡胶树单产提高的主要因素，阐明死皮发生机制是防治死皮的前提，但对其分子机理的了解十分有限。我们的前期工作表明：过量活性氧导致的细胞程序化死亡 (PCD)是死皮发生的关键途径，而与死皮发生关建因子HbTCTP互作的小热激蛋白 (sHSP)可能在ROS清除过程中发挥重要作用。我们已经完成多个橡胶树转录组测序，并成功获得了13个橡胶树sHSP成员。本项目拟通过比较分析乙烯处理前后和不同死皮等级橡胶树中各sHSP表达量与ROS含量、ROS产生-清除相关酶的活性，评估各sHSP成员在ROS导致死皮成因途径中的作用；利用酵母双杂交，橡胶树叶片原生质体瞬时表达体系进一步明确与HbTCTP互作的sHSP成员；鉴于橡胶树遗传转化体系尚不成熟，利用大肠杆菌和烟草转基因体系鉴定sHSP的生物学功能｡项目结果有望揭示ROS导致死皮成因途径的分子机制，为研制安全高效的新型死皮防治制剂提供理论依据｡

死皮是限制橡胶树单产提高的主要因素，阐明死皮发生机制是防治死皮的前提，项目申请之初，对其分子机理的了解十分有限。项目组前期工作表明：过量活性氧导致的细胞程序化死亡 (PCD)是死皮发生的关键途径，并推测与死皮发生关建因子HbTCTP互作的小热激蛋白 (sHSP)可能在ROS清除过程中发挥重要作用。本项目进一步利用酵母双杂交揭示HbTCTP互作的sHSP成员只有HbsHSP17.5kDa，且该基因与HbTCTP基因在乙烯处理前后和不同死皮等级橡胶树中协同表达；此外，HbsHSP17.5kDa 转基因大肠杆菌和烟草均可以显著提高抵御H2O2的胁迫能力；上述证据提示HbsHSP17.5kDa 通过清除ROS负调控死皮病的发生。. 本项目进一步丰富了橡胶树死皮病的发生机制，同时将为研制安全高效的新型死皮防治制剂提供理论依据