韭菜迟眼蕈蚊普通气味结合蛋白GOBP鉴定与功能分析

The Chinese chive maggot, Bradysia odoriphaga (Diptera: Sciaridae), is a serous pest on Chinese chive. The current control methods rely heavily on chemical pesticides, which results in environmental pollution and excessive pesticide residues, and the control effect is no satisfactory. Previous studies found that the pest has strong chemotaxis to newly-cut Chinese chive. Subsequently we determine regulating insect behavior by developing and applying attractants from the host plant to trap this pest will be the effective control strategy. Insect general odorant binding protein (GOBP) can combine with fat-soluble of Host plant volatiles, which plays an important role in the interaction between insects and host plant. In order to clarify the mechanism of Bradysia odoriphaga identification and feel to Chinese chive volatiles, this study will clone and identify the general odorant binding proteins with RACE technology, measure the olfactory response of Bradysia odoriphaga to Chinese chive volatiles standards by the electroantennogram recording assay, test affinities of recombinant GOBP with Chinese chive volatiles standards by fluorescence compretitive binding assay, preparation dsRNA of receptor gene, and use RNAi technology, verify the reaction characteristics of Bradysia odoriphaga to Chinese chive volatiles after the receptor gene silenced. The results will contribute to illuminate the mechanism of Bradysia odoriphaga odor recognition and provide a scientific basis for the development of Bradysia odoriphaga attractants and promotion of the sustainable prevention and control.

韭菜迟眼蕈蚊是为害韭菜等百合科蔬菜的重要害虫。因其发生量大且为害严重，生产中一直依赖化学杀虫剂，造成环境污染、农残超标等问题，且防治效果并不理想。申请者前期研究发现韭菜迟眼蕈蚊对刚割过的韭菜有明显趋性，推测利用行为调控技术如植物源引诱剂进行诱杀将是该虫有效防控的重要组成。昆虫普通气味结合蛋白（GOBP）可以和脂溶性的寄主植物挥发物结合，在昆虫与寄主植物互作中起着重要作用。为明确韭菜迟眼蕈蚊对韭菜挥发物的识别及感受机制，本研究将利用RACE技术克隆并鉴定GOBP，利用触角电位仪测定韭菜迟眼蕈蚊对韭菜挥发物标样的嗅觉反应，利用荧光竞争结合技术测定GOBP与寄主挥发物标样的结合能力，同时制备GOBP目的基因dsRNA，利用RNAi技术，将目的基因沉默后，验证韭菜迟眼蕈蚊对韭菜挥发物的反应特性。研究结果有助于明确韭菜迟眼蕈蚊的嗅觉识别机制，为开发韭菜迟眼蕈蚊嗅觉引诱剂，促进可持续防控提供科学依据。

本项目对韭菜迟眼蕈蚊普通气味结合蛋白GOBP基因进行了克隆及功能分析，明确了其嗅觉识别机制，揭示了该虫寻找寄主并产卵的行为机理。首先测定韭菜迟眼蕈蚊成虫触角转录组，从中鉴定到6条OBP基因。经比对测定，所得OBP序列均包含完整的OBF阅读框。通过设计全长引物PCR扩增、胶回收、连接转化、蓝白斑筛选、测序分析获得正确的质粒。利用荧光竞争结合试验对重组蛋白Bodo-OBP6与18种化合物结合特性测定试验表明，只有β-紫罗兰酮能与之结合。为进一步验证OBP基因的功能，采用气相色谱-质谱联用技术测定了韭菜的挥发物八种，并利用触角电图分析引起反应的主要物质。结果表明韭菜挥发物释放量最高的是甲基二烯丙基二硫和二烯丙基二硫。触角电位分析结果表明：韭菜迟眼蕈蚊雌虫对这两种硫化物及其混合物具有强烈的反应。为明确韭菜挥发物对该虫产卵趋性行为的影响，测定了甲基烯丙基二硫、二烯丙基二硫醚单独或者以1:5的比例的混合后喷施韭菜。结果表明，与对照相比，韭菜迟眼蕈蚊雌虫产卵量显著增加。利用Y型嗅觉仪研究了未交配或已交配的韭菜迟眼蕈蚊雌成虫对不同虫态的挥发物的趋性，并利用固相微萃取和气-质联用仪对有引诱活性虫态挥发物进行定性和定量分析，并采用Y型嗅觉仪测定不同化学成分及比例对雌虫的引诱活性。结果表明，未交配或者已交配韭菜迟眼蕈蚊雌成虫明显嗜好4龄幼虫，而对2龄幼虫、蛹和卵块无明显趋性。韭菜迟眼蕈蚊4龄幼虫体表挥发物主要成分是二丙基二硫醚和2,2-二甲基-1,3-噻烷等二硫化物，前者含量是后者的10倍。50 mg二丙基二硫醚单体对韭菜迟眼蕈蚊交配雌成虫具有明显吸引作用；二丙基二硫醚与2,2-二甲基-1,3-噻烷以质量比1：1和10：1混合对韭菜迟眼蕈蚊交配雌成虫具有明显吸引作用；二者10：1混合后与50 mg二丙基二硫醚单体比较，前者对交配雌成虫吸引作用更明显。