唾液蛋白在白背飞虱刺吸危害水稻中的作用和机理研究

The white-backed planthopper (WBPH), Sogatella furcifera, is one of the most destructive rice pests. In this project, by combining analysis three sets of data, salivary transcriptome, proteome of salivary gland tissue and proteome of watery saliva, we select the candidate genes which might play important roles in WBPH feeding and WBPH-rice interactions. The genes highly expressed in the salivary gland will be selected by quantitative real-time PCR technique (RT-qPCR), and are further screened by using RNAi and Western blot techniques. The full length of the salivary protein gene is obtained by RACE-PCR, and the sequence and expression patterns are analyzed. After silencing target genes of WBPH and transient overexpression of the corresponding proteins in rice, the biological indicators of WBPH are screened such as feeding amount, feeding behavior, survival rate of nymphs and the fecundity of female adults. On the other hand, the concentrations of jasmonic acid (JA), jasmonoyl-isoleucine (JA-Ile), salicylic acid (SA) and hydrogen peroxide (H2O2) contents in rice plants are detected after infestation by WBPH with knockdown of target genes, transient overexpression of the corresponding proteins in rice with and without infestation by WBPH. This project will provide a theoretical basis for investigating the interaction between WBPH and rice, and give aid to controlling strategies of WBPH.

白背飞虱是水稻上一种重要的刺吸式害虫，本项目拟通过综合分析白背飞虱唾液腺转录组、唾液腺组织蛋白组和唾液源蛋白组三套数据，筛选出具有重要注释功能的唾液蛋白。经RT-qPCR分析，筛选白背飞虱唾液腺特异表达的基因，结合RNAi和Western blot技术鉴定出重要的唾液分泌蛋白。采用RACE-PCR获得唾液蛋白基因的全长，对序列和表达模式进行分析。在沉默白背飞虱的目标基因以及在水稻中瞬时表达相应唾液蛋白后，检测对白背飞虱的取食量、取食行为、存活率和单雌产卵量生物学指标的影响。另一方面，沉默目标基因的白背飞虱危害水稻后、在水稻中瞬时表达单个唾液蛋白以及白背飞虱取食瞬时表达唾液蛋白的水稻后，检测对水稻防御反应信号分子茉莉酸（JA）、茉莉酸-异亮氨酸复合物（JA-Ile）、水杨酸（SA）和过氧化氢（H2O2）含量的影响。本研究结果将有助于白背飞虱-水稻互作调控作用的研究。

白背飞虱是水稻上一种重要的刺吸式害虫。本项目综合分析了白背飞虱唾液腺转录组、唾液腺组织蛋白组和唾液源蛋白组三套数据，通过蛋白质组学分析、唾液分泌蛋白基因的克隆和时空表达、鉴定出了具有重要注释功能的唾液蛋白，丰富了白背飞虱唾液蛋白的数据库。筛选出了在白背飞虱唾液腺中高表达的三种基因，分别是多铜氧化酶4基因（multicopper oxidase 4，MCO4）、黏液样蛋白基因（Mucin）和内切-β-1,4-葡聚糖酶基因（endo-β-1,4-glucanase，SfEG1）。MCO4是白背飞虱取食过程的重要基因，对于克服水稻防御反应，完成取食过程具有重要的作用。在沉默SfEG1和Mucin后白背飞虱取食行为受到了明显的干扰，无法取食到水稻的韧皮部汁液，从而降低了在水稻上的存活率。SfEG1和Mucin对于诱导植物防御反应具有重要的作用。Mucin参与了白背飞虱唾液腺和唾液鞘结构的形成，是白背飞虱成功取食的关键基因。三种唾液基因对白背飞虱取食具有关键作用。以上研究结果将有助于白背飞虱-水稻互作调控作用的研究，为白背飞虱的防治提供新的思路和理论基础。