甜菜夜蛾PBAN-PBANR关键结合区域鉴定及功能分析

The beet armyworm, Spodoptera exigua is a serious agricultural pest across the world. The project will identify gene function and the critical binding domains between the pheromone biosynthesis activating neuropeptide (PBAN) and its receptor (PBANR) . Organic connections among PBAN-PBANR binding activity, sex pheromone biosynthesis and release and adult reproduction will be investigated. Mutation of amino acids will be introduced in different site of PBANR by sit-directed mutagenesis technique, then the mutants of PBANR will be cloned into the expression plasmid, pCDNA5-FRT, transfected into CHO or HEK cells, and will be allowed to bind with synthesized PBAN peptide in vitro. Binding function expression and critical binding domains of PBAN-PBANR will be identified according to the binding activity in Ga2+ or cAMP level of the mutants as compared with the wild-type PBANR. Furthermore, mimetic peptides with high binding or inhibiting activity which were designed according to the character of PBAN gene sequence will be selected based on their binding experiment with PBANR. Finally, mimetic peptides with physiological activity and biological function will be chosen by using the models in vitro or in vivo to screen sex pheromone quantification which can be determined by GC-MS in the treatment of mimetic peptides and sex gland, and by analyzing adult coupling, mating and egg laying behavior. Meanwhile, RNAi system of PBANR gene will be established by detecting the transcript expression after microinjecting the dsRNA, and the PBANR gene express function will be further identified by measurement of the sex pheromone and female mating and egg laying. This project will provide theoretical and experimental support for disclosing sex pheromone biosynthetic pathway in S. exigua, develop a novel pest management technique using mimetic peptides and RNAi to disturb adult reproduction, and accumulate experience for research on the neuropeptides function of other insect species.

以重大农业害虫甜菜夜蛾为对象，围绕调控性信息素合成与释放的神经肽PBAN及其受体PBANR的结合活性、性信息素释放以及成虫生殖之间的有机联系，不仅采用点突变技术构建不同位置氨基酸突变型PBANR，转染细胞株后与PBAN结合引起的Ca2+或cAMP浓度效应鉴定PBANR结合功能及PBAN-PBANR关键结合区域；而且根据甜菜夜蛾PBAN基因结构设计合成模拟肽，通过与PBANR结合活性测定筛选激动剂或拮抗剂，并建立性腺体外和体内模型，利用GC-MS鉴定其对性信息素释放的促进或抑制作用，结合成虫求偶、交配与产卵行为变化，最终筛选出具有生理活性和生物学功能的模拟肽；同时，建立PBANR基因RNAi体系，测定基因沉默后性信息素含量和生殖参数变化揭示PBANR基因功能。为阐明甜菜夜蛾性信息素合成传导途径，发展模拟肽和RNAi干扰生殖的新型防治技术提供理论和实验依据，并为其它昆虫神经肽功能研究提供借鉴。

甜菜夜蛾是世界性一种重要的农业害虫，生产上防治困难。本项目围绕调控甜菜夜蛾求偶与交配行为的神经肽PBAN及其受体PBANR的结合活性、关键结合区域、以及PBAN-PBANR基因功能方面开展研究,以期揭示PBAN-PBANR在防治上作用。首先，通过体外表达体系的构建，测定分析了甜菜夜蛾PBAN-PBANR的结合功能，鉴定了甜菜夜蛾PBAN神经肽是PBAN受体的良好结合配体；进一步根据其PBAN的C端特征序列，人工设计、合成并筛选出了与PBANR结合良好的模拟肽，结合试验表明，FSPRL关键氨基酸对与PBAN受体的结合活性影响顺序为R> L> P>去酰胺化> F> S。同时，在分析PBANR胞外结构域和7个跨膜区特征的基础上，鉴定出了TM3和TM6为甜菜夜蛾PBANR的关键结合结构域；根据上述结果以及PBAN-PBANR基因的结构特征，建立了甜菜夜蛾PBANR的RNAi干涉体系，获得了5个对甜菜夜蛾PBANR基因有干涉效果的RNAi片段，其中R002蛹期注射72h的干扰效果显著，抑制率为22.6%。R003在蛹期注射后72h的干扰效果显著，抑制率为33%。R004注射初羽化雌蛾腹部8-9节后48h及72h的干扰效果显著，抑制率分别为29.02% 和30.57%。R005和R006雌蛾腹部注射后72h抑制率分别为34.87%和44.3%。筛选出1个对甜菜夜蛾PBAN基因有干涉效果的siRNA，并揭示了基因干涉后对甜菜夜蛾交配与产卵的影响。羽化前一天雌蛹前胸注射siRNA（N001） 24h后PBAN基因表达量抑制率为66.3%，N001 在注射后1d显著降低了成虫交配率和交配次数，随着注射后时间的延后这种抑制效果逐渐消失。这些结果为阐明甜菜夜蛾性信息素合成传导途径，发展模拟肽和RNAi干扰生殖的新型防治技术提供了理论和实验依据。