松褐天牛气味结合蛋白基因克隆及功能分析

Because of the larvae cryptic feeding habit of longhorned beetles, control strategies of such wood-boring pests have to be based on controlling or killing longhorned beetle adults by using insect semiochemicals. It also has been suggested that the perception of volitiles by ordant binding protein (OBPs) is essential for odorant recognition, transportation, and signal transduction of insect, although the precise functions of OBPs have not been completely elucidated. Studying the precise functions of OBPs will help in developing more efficient attractants of the beetle. In this study, based on the anaysis of the antenna transcriptome of a serious pest Monochamus alternatus Hope, the major vector that transmit the pine wood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle which causes a devastating pine wilt disease, all of the OBP genes will be colned. And in order to elucidate the functions of OBPs in olfacotory recognition of M. alternatus, the correlation between OBP genes expression patterns and the behavior of M. alternatus, will be analyzed. Based on the results of the three-dimensional models and in situ hybridisation of co-expressed pattern of OBPs in some antennal sensilla, the purified recombinant OBPs and their ligand-binding properties were investigated one by one or by combination. And also the effect to expression level of OBP genes and the olfacotory recoganition will be analyzed by knockdown of OBP genes one by one or by combination. The results of the project not only can elucidate the regulating olfactory by OBPs, but also will be helpful for designing and developing the more effective methods of monitoring and management of M. alternatus.

利用信息化学物质监测和防治天牛种群，已成为探索天牛防治新途径的重要方面；气味结合蛋白（OBPs）和气味分子之间的作用是昆虫嗅觉神经传递的关键步骤，如果能够明确OBPs在嗅觉反应中的功能，则可为高效诱剂的研制提供分子基础。但目前OBPs在昆虫嗅觉反应中的功能还不是很清楚。为此，本项目以松材线虫传播媒介松褐天牛Monochamus alternatus为研究对象，在分析前期触角转录组数据的基础上，克隆其触角OBPs基因，分析OBPs基因表达模式与天牛行为选择的相关性，结合OBPs三维结构预测及组织定位检测，测定重组的单个OBP或OBPs组合与气味的结合特性，探明单个或多个OBPs基因沉默对天牛嗅觉反应的影响，以期明确OBPs在松褐天牛嗅觉感受中的功能。本研究结果对于揭示松褐天牛嗅觉感受的分子机制，探索针对OBPs设计和开发更为高效的化学通讯调控技术，最终实现松褐天牛的可持续治理具有重要的意义。

气味结合蛋白（OBPs）和气味分子之间的作用是昆虫嗅觉神经传递的关键步骤，如果能够明确OBPs在嗅觉反应中的具体功能，则可为高效诱剂的研制提供分子基础。本项目以林业毁灭性病害松材线虫的传播媒介松褐天牛Monochamus alternatus Hope为研究对象，对其OBPs基因在其嗅觉反应中的功能进行研究。取得以下主要的研究结果：.对松褐天牛触角转录组信息进一步筛选比对，通过PCR技术克隆得到了松褐天牛转录组中获得的29个OBPs基因和8个化学感受蛋白CSPs基因的全长；利用比较转录组学系统分析了松褐天牛OBPs及CSPs基因与其天敌花绒寄甲在进化中的关系，有些聚类族包含上述两个物种的OBPs，而有些聚类族仅包含松褐天牛或其天敌花绒寄甲。通过qPCR检测松褐天牛MaltOBPs基因的时空表达情况。结果表明，这MaltOBPs基因在雌雄虫下唇须和下颚须的表达量普遍较高，在触角、头、足、腹和翅也有一定的表达；且MaltOBPs基因在触角中的表达量因成虫龄期、性别和交配状态的不同而呈现差异。成功构建了重组质粒pET20b-MaltOBPs或pET17b-MaltOBPs或pGEX-6P-1-MaltOBPs，在大肠杆菌中表达成功后并获得纯蛋白。选择1-NPN为荧光探针，利用荧光竞争结合技术测定了MaltOBPs在中性和酸性pH环境下与18种寄主植物挥发物单体的结合特性。结果表明，MaltOBPs在中性pH环境下与挥发物的结合能力普遍高于酸性pH环境，但不同MaltOBPs与气味物质结合特性方面表现不同。利用免疫胶体金技术对MaltOBP9和MaltOBP10在松褐天牛触角感器中进行定位，结果表明MaltOBP9主要雌雄触角的刺形感器、毛形感器、耳形感器中表达，在锥形感器中相对较少；MaltOBP10则主要在雌触角的锥形感器、耳形感器、刺形感器和毛形感器。利用RNAi对MaltOBPs的功能进行了进一步分析，但结果显示，当注射dsRNA-MaltOBPs时，相关MaltOBPs基因的表达量下调并不明显。对于产生这种效应的原因还需要进一步的研究和分析。.本研究结果对于明确松褐天牛嗅觉反应的分子机制，探索针对OBPs设计和开发更为高效的化学通讯调控技术，进而最终实现松褐天牛的可持续治理具有重要的意义。