褐飞虱寄主定位的嗅觉感受机制
发布时间：2019-05-17 10:21
【摘要】：对于植食性昆虫而言,通过灵敏的嗅觉系统搜寻并定位寄主植物至关重要。如果能够明确昆虫的嗅觉机制,将为开发新型有效的害虫生物防治和监控技术提供新思路。褐飞虱Nilaparvata lugens(St?l)是水稻上重要的害虫,其暴发性和毁灭性对我国的粮食产量具有严重威胁,以往以化学药剂为主的防治措施,已经引起了一系列的问题,如褐飞虱抗药性的增加,环境的严重污染等。如果能够明确褐飞虱在寄主定位中的嗅觉感受机制,可以为应用昆虫的嗅觉行为调控褐飞虱为害提供理论依据。为此,本课题以水稻害虫褐飞虱为研究对象,在实验室前期的工作基础上,对褐飞虱寄主定位的信息化学物质进行分离鉴定,克隆与嗅觉相关的气味结合蛋白基因(OBPs)Nlug OBPs基因并进行原核表达纯化,分析了与Nlug OBP4有良好结合特性的气味物质对褐飞虱的行为活性,以期为明确褐飞虱的嗅觉感受机制及研发其行为调控技术提供理论基础和技术支持。主要研究结果如下:1褐飞虱寄主定位信息化学物质的分离鉴定利用GC-EAD和GC-MS技术,对褐飞虱触角具有活性的水稻挥发物进行了分离鉴定。结果表明,褐飞虱成虫触角对水稻挥发物有电生理反应的有5种物质:其中反应显著且实验重复性最高的有2种,分别为1,6-Octadien-3-ol,3,7-dimethyl-(芳樟醇)和Undecane,2,6-dimethyl-(2,6-二甲基-十一烷),重复性达到13次以上;其次为Benzene,1,4-dichloro-(1,4-二氯苯)、cis-Linaloloxide(顺式氧化芳樟醇)和Tridecane(正十三烷)等3种化合物,重复性也有10-13次,推测这几种物质对褐飞虱可能有引诱或者趋避作用。2褐飞虱Nlug OBPs基因的克隆及分析依据已经建立的褐飞虱转录组数据,克隆了褐飞虱Nlug OBP8、Nlug OBP9、Nlug OBP10(Gen Bank登录号依次为KC663687、KC663688和KC663689)的3个基因。分析结果表明,3个褐飞虱OBPs都具有完整的开放阅读框,全长分别为414bp、576bp、756bp,分别编码137、191、251个氨基酸,蛋白分子量分别为15.09k Da、20.98k Da、27.88k Da,等电点分别为4.38、6.06、7.97;经过多重序列比对分析序列的保守特征表明,Nlug OBP8基因具有6个保守的Cys位点,与经典OBPs的特征相符合,属于Classic OBPs,而Nlug OBP9和Nlug OBP10都具有8个保守的Cys位点和1个Pro位点,属于Plus-C OBPs;预测3个Nlug OBPs序列的N-末端信号肽表明,仅Nlug OBP8含有21个氨基酸组成的信号肽序列,Nlug OBP9和Nlug OBP10均无信号肽。3褐飞虱Nlug OBPs原核表达载体的构建成功构建了原核表达载体p GEX-6P-1-Nlug OBPs,p ET-17b-Nlug OBPs,p ET-20b(+)-Nlug OBPs,p ET-22b(+)-Nlug OBPs,p ET-30a(+)-Nlug OBPs以及p ET-32a(+)-Nlug OBPs的重组质粒,并将重组质粒导入表达菌株BL21(DE3)/BL21(DE3)Plys S中进行试表达,仅有p GEX-6P-1-Nlug OBP8/Nlug OBP10和p ET-32a(+)-NLug OBP8/NLug OBP10试表达成功。4褐飞虱行为选择试验基于实验室已完成的褐飞虱Nlug OBP4蛋白的荧光竞争结合实验结果,利用H型嗅觉仪,对结合活性高的22种水稻挥发物单体和GC-EAD筛选出的2种活性物质单体(其中芳樟醇是重复存在的)进行了行为测定。结果表明,香叶烯和2-庚酮在稀释50倍的浓度下均对褐飞虱成虫具有引诱作用,但2-庚酮在稀释10倍和100倍浓度时却又对褐飞虱成虫驱避反应显著;不同浓度下均对褐飞虱成虫有明显驱避反应的有芳樟醇和辛烯,而(+)-柠檬烯、蒈烯、反式石竹烯、侧柏烯、二十一烷、正十三烷、庚醇、反-2-己烯醇、苯甲醛、己醛、?-紫罗兰酮以及苯甲酸甲酯等物质仅在一定浓度下对褐飞虱成虫具有驱避反应。
[Abstract]:For phytophagous insects, it is essential to search and locate the host plant by a sensitive olfactory system. If the olfactory mechanism of the insect can be defined, a new idea for the development of new and effective pest control and monitoring technology will be provided. Nilaparvata lugens (St? l), a brown planthopper, is an important pest in rice, and its fulminence and destructive effects are a serious threat to our country's grain output. In the past, it has caused a series of problems, such as the increase of the drug resistance of the brown planthopper, the serious pollution of the environment, etc. If the olfactory sense mechanism of the brown planthopper in the host location can be determined, the theoretical basis for controlling the damage of the brown planthopper can be provided for the application of the olfactory behavior of the insect. in ord to solve that problem, the research object of the brown planthopper of the rice pests is carried out on the basis of the work of the early stage of the laboratory, the information chemical substance of the host position of the brown planthopper is isolated and identified, and the odor-binding protein gene (OBPs) Nlug OBPs gene related to the smell is cloned and purified in a prokaryotic expression, The behavioral activity of the odorous substance with good binding property with Nlug OBP4 to the brown planthopper was analyzed, with a view to providing the theoretical basis and technical support for the identification of the olfactory receptive mechanism of the brown planthopper and the research and development of its behavior regulation and control technology. The main results of this study were as follows:1. The isolation and identification of the host location information chemical of the brown planthopper (BPH) were identified by GC-EAD and GC-MS, and the active rice volatiles were isolated and identified by the method of GC-EAD and GC-MS. The results showed that the antennae of the adult brown planthopper had 5 species of the electrophysiologic response to the volatile matter of the rice: the reaction was significant and the experimental repeatability was the highest in 2 species, namely,1,6-Octdien-3-ol,3,7-dimetyl-(linalool) and Undecane, 2,6-dimetyl-(2,6-dimethyl-undecane), and the repeatability was more than 13 times, followed by Benzene, 1,4-dichloroo-(1,4-dichlorobenzene), cis-Linalooxide (cis-oxidized linalool) and Tridecane (n-tridecane), and the repeatability is also 10-13 times, It is suggested that these substances may induce or prevent the brown planthopper. The cloning and analysis of the Nlug OBPs gene of the brown planthopper is based on the data of the already established brown planthopper transcriptome, and the three genes of the brown planthopper Nlug OBP8, the Nlug OBP9 and the Nlug OBP10 (Gen Bank accession number are KC663687, KC663688 and KC663689) are cloned. The results showed that the three brown planthopper (OBPs) had a complete open reading frame, with the full length of 414bp, 576bp and 756bp, respectively, with the molecular weight of 15.09k Da, 20.98 k Da, 27.88 k Da and the isoelectric point of 4.38, 6.06 and 7.97, respectively. The Nlug OBP8 gene has six conserved Cys sites, which is consistent with the characteristics of the classical OBPs, and belongs to Classic OBPs, while the Nlug OBP9 and Nlug OBP10 have 8 conservative Cys sites and one Pro site, belonging to the Plus-C OBPs; and the N-terminal signal peptide of the three Nlug OBPs sequences is predicted to indicate that, The expression vector pGEX-6P-1-Nlug OBPs, pET-17b-Nlug OBPs, pET-20b (+)-Nlug OBPs, pET-22b (+)-Nlug OBPs, pET-30a (+)-Nlug OBPs and p-ET-32a (+)-Nlug OBPs were successfully constructed. The recombinant plasmid was introduced into the expression strain BL21 (DE3)/ BL21 (DE3) Plys S to be tested and expressed. The expression of pGEX-6P-1-Nlug OBP8/ Nlug OBP10 and pET-32a (+)-NLug OBP8/ NLug OBP10 was successfully expressed. The activity of two active material monomers (which are repeated in the presence of linalool) of 22 rice volatile monomers and GC-EAD with high binding activity were measured. The results showed that the allene and 2-heptanone had an alluring effect on the adults of the brown planthopper in the 50-fold dilution, but the 2-heptanone had a significant effect on the repellent reaction of the adults of the brown planthopper when the concentration of the 2-heptanone was 10-fold and 100-fold. In the different concentrations, the adults of the brown planthopper have an obvious repellent effect of linalool and octene, and (+)-limonene, triene, trans-caryophyllene, arborene,21-alkyl, n-tridecane, heptanol, trans-2-hexenol, benzaldehyde, hexanal, and the like; And the substances such as the ionone and the methyl benzoate only have the repellent effect on the adult brown planthopper adult at a certain concentration.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.39
本文编号：2479024
[Abstract]:For phytophagous insects, it is essential to search and locate the host plant by a sensitive olfactory system. If the olfactory mechanism of the insect can be defined, a new idea for the development of new and effective pest control and monitoring technology will be provided. Nilaparvata lugens (St? l), a brown planthopper, is an important pest in rice, and its fulminence and destructive effects are a serious threat to our country's grain output. In the past, it has caused a series of problems, such as the increase of the drug resistance of the brown planthopper, the serious pollution of the environment, etc. If the olfactory sense mechanism of the brown planthopper in the host location can be determined, the theoretical basis for controlling the damage of the brown planthopper can be provided for the application of the olfactory behavior of the insect. in ord to solve that problem, the research object of the brown planthopper of the rice pests is carried out on the basis of the work of the early stage of the laboratory, the information chemical substance of the host position of the brown planthopper is isolated and identified, and the odor-binding protein gene (OBPs) Nlug OBPs gene related to the smell is cloned and purified in a prokaryotic expression, The behavioral activity of the odorous substance with good binding property with Nlug OBP4 to the brown planthopper was analyzed, with a view to providing the theoretical basis and technical support for the identification of the olfactory receptive mechanism of the brown planthopper and the research and development of its behavior regulation and control technology. The main results of this study were as follows:1. The isolation and identification of the host location information chemical of the brown planthopper (BPH) were identified by GC-EAD and GC-MS, and the active rice volatiles were isolated and identified by the method of GC-EAD and GC-MS. The results showed that the antennae of the adult brown planthopper had 5 species of the electrophysiologic response to the volatile matter of the rice: the reaction was significant and the experimental repeatability was the highest in 2 species, namely,1,6-Octdien-3-ol,3,7-dimetyl-(linalool) and Undecane, 2,6-dimetyl-(2,6-dimethyl-undecane), and the repeatability was more than 13 times, followed by Benzene, 1,4-dichloroo-(1,4-dichlorobenzene), cis-Linalooxide (cis-oxidized linalool) and Tridecane (n-tridecane), and the repeatability is also 10-13 times, It is suggested that these substances may induce or prevent the brown planthopper. The cloning and analysis of the Nlug OBPs gene of the brown planthopper is based on the data of the already established brown planthopper transcriptome, and the three genes of the brown planthopper Nlug OBP8, the Nlug OBP9 and the Nlug OBP10 (Gen Bank accession number are KC663687, KC663688 and KC663689) are cloned. The results showed that the three brown planthopper (OBPs) had a complete open reading frame, with the full length of 414bp, 576bp and 756bp, respectively, with the molecular weight of 15.09k Da, 20.98 k Da, 27.88 k Da and the isoelectric point of 4.38, 6.06 and 7.97, respectively. The Nlug OBP8 gene has six conserved Cys sites, which is consistent with the characteristics of the classical OBPs, and belongs to Classic OBPs, while the Nlug OBP9 and Nlug OBP10 have 8 conservative Cys sites and one Pro site, belonging to the Plus-C OBPs; and the N-terminal signal peptide of the three Nlug OBPs sequences is predicted to indicate that, The expression vector pGEX-6P-1-Nlug OBPs, pET-17b-Nlug OBPs, pET-20b (+)-Nlug OBPs, pET-22b (+)-Nlug OBPs, pET-30a (+)-Nlug OBPs and p-ET-32a (+)-Nlug OBPs were successfully constructed. The recombinant plasmid was introduced into the expression strain BL21 (DE3)/ BL21 (DE3) Plys S to be tested and expressed. The expression of pGEX-6P-1-Nlug OBP8/ Nlug OBP10 and pET-32a (+)-NLug OBP8/ NLug OBP10 was successfully expressed. The activity of two active material monomers (which are repeated in the presence of linalool) of 22 rice volatile monomers and GC-EAD with high binding activity were measured. The results showed that the allene and 2-heptanone had an alluring effect on the adults of the brown planthopper in the 50-fold dilution, but the 2-heptanone had a significant effect on the repellent reaction of the adults of the brown planthopper when the concentration of the 2-heptanone was 10-fold and 100-fold. In the different concentrations, the adults of the brown planthopper have an obvious repellent effect of linalool and octene, and (+)-limonene, triene, trans-caryophyllene, arborene,21-alkyl, n-tridecane, heptanol, trans-2-hexenol, benzaldehyde, hexanal, and the like; And the substances such as the ionone and the methyl benzoate only have the repellent effect on the adult brown planthopper adult at a certain concentration.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.39
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