转录因子Nrf2的克隆及其对橡胶六点始叶螨抗氧化酶的调控功能研究

Natural rubber is important and irreplaceable strategic material for national economy and defense. Eotetranychus sexmaculatus (Riley) is the most dangerous mite to rubber tree in China. However, the tall rubber tree make pesticide difficult to target the mite, therefore, effective control becomes very challenging. To now, breeding of mite-resistant cultivars is considered as the most persistent, economic and convenient strategy for mite control. Several studies indicated that the activities of antioxidant enzymes in pests were closely related to host adaptability. In previous study we also found that the development, reproduction and activities of antioxidant enzymes in E. sexmaculatus feeding on mite-resistant rubber cultivars were significantly inhibited. In vertebrates transcription factor Nrf2 plays a key role in regulating antioxidant enzymes, but in pest there is a lack of evidence about this function. In present study we aim to clone the transcription factors Nrf2 from E. sexmaculatus, and interfere Nrf2 expression via gene induction, inhibition and silence respectively. After that, the changes of antioxidant enzyme activities, development and reproduction of E. sexmaculatus feeding on mite-resistant and mite-susceptible rubber cultivars were analyzed respectively. This study can demonstrate the function of transcription factors in regulating antioxidant enzymes in E. sexmaculatus, moreover, provide theoretical basis for the studies of mite-resistant rubber molecular breeding and mite resistant mechanism.

天然橡胶是关系到国计民生的重要战略物质，在国防和国民经济建设中具有不可替代的作用。六点始叶螨是危害我国橡胶树最严重的一种世界危险性害螨，但橡胶树高大，药剂难以靶标，防治难度很大。目前，培育抗性品种被国内外确定为能从根本上持久防控害螨的最有效、经济、简便的防治途径。相关研究表明，农业害虫（螨）抗氧化酶的活性与其寄主适应性关系密切。我们前期研究也发现，抗螨性橡胶树种质能显著抑制六点始叶螨发育、繁殖及其抗氧化酶活性。转录因子Nrf2对脊椎动物抗氧化酶的调控起关键作用，但迄今为止尚未见Nrf2具有对害螨抗氧化酶调控功能相关报道。因此，本项目首次从六点始叶螨体内克隆Nrf2，研究分别诱导、抑制和沉默Nrf2对六点始叶螨取食抗、感螨橡胶树种质时抗氧化酶活性、发育和繁殖的影响，从分子水平解析Nrf2对六点始叶螨抗氧化酶的调控功能，并为橡胶抗螨分子设计育种及分子机理研究提供理论依据。

农业害螨抗氧化酶的活性与其寄主适应性关系密切，转录因子Nrf2对脊椎动物抗氧化酶的调控起关键作用，但迄今为止尚未见Nrf2具有对害虫（螨）抗氧化酶调控功能相关报道。本项目首次从六点始叶螨体内克隆获得长度为1719 bp的EsNrf2 编码区全长序列，并经生物信息学分析确定为六点始叶螨抗氧化酶转录组因子。采用geNorm，Bestkeeper，Normfinder和RefFinder分析软件从9个候选内参基因RPL13, RP49, β-actin, α-TUB, β-TUB, SDHA, 18SrRNA, 5.8SrRNA 和 GAPDH中筛选出在六点始叶螨不同龄期表达稳定性最佳的两个内参基因β-actin和β-tub用于抗氧化酶基因表达量的RT-qPCR分析。进一步采用筛选出的EsNrf2抑制剂鸦胆子苦醇（BA）、两种EsNrf2激活剂特丁基对苯二酚（TBHQ）和二噻环戊二烯硫酮 （D3T）处理，以及RNA干扰手段研究分别诱导、抑制和沉默EsNrf2对六点始叶螨取食抗、感螨橡胶树种质时抗氧化酶基因表达和酶活性的影响，结果表明当转录因子EsNrf2的基因表达受到诱导、抑制和沉默后，相应地会导致六点始叶螨抗氧化酶基因EsCu/ZnSOD、EsCAT1、EsPOD1、EsPPO的表达和SOD、CAT、POD和PPO的酶活表现出协同的变化趋势。本研究从分子水平证实Nrf2对六点始叶螨抗氧化酶的调控功能，为橡胶抗螨分子设计育种及分子机理研究提供理论依据。