褐飛虱寄主定位的嗅覺感受機(jī)制
發(fā)布時(shí)間：2019-05-17 10:21
【摘要】：對(duì)于植食性昆蟲而言,通過靈敏的嗅覺系統(tǒng)搜尋并定位寄主植物至關(guān)重要。如果能夠明確昆蟲的嗅覺機(jī)制,將為開發(fā)新型有效的害蟲生物防治和監(jiān)控技術(shù)提供新思路。褐飛虱Nilaparvata lugens(St?l)是水稻上重要的害蟲,其暴發(fā)性和毀滅性對(duì)我國(guó)的糧食產(chǎn)量具有嚴(yán)重威脅,以往以化學(xué)藥劑為主的防治措施,已經(jīng)引起了一系列的問題,如褐飛虱抗藥性的增加,環(huán)境的嚴(yán)重污染等。如果能夠明確褐飛虱在寄主定位中的嗅覺感受機(jī)制,可以為應(yīng)用昆蟲的嗅覺行為調(diào)控褐飛虱為害提供理論依據(jù)。為此,本課題以水稻害蟲褐飛虱為研究對(duì)象,在實(shí)驗(yàn)室前期的工作基礎(chǔ)上,對(duì)褐飛虱寄主定位的信息化學(xué)物質(zhì)進(jìn)行分離鑒定,克隆與嗅覺相關(guān)的氣味結(jié)合蛋白基因(OBPs)Nlug OBPs基因并進(jìn)行原核表達(dá)純化,分析了與Nlug OBP4有良好結(jié)合特性的氣味物質(zhì)對(duì)褐飛虱的行為活性,以期為明確褐飛虱的嗅覺感受機(jī)制及研發(fā)其行為調(diào)控技術(shù)提供理論基礎(chǔ)和技術(shù)支持。主要研究結(jié)果如下:1褐飛虱寄主定位信息化學(xué)物質(zhì)的分離鑒定利用GC-EAD和GC-MS技術(shù),對(duì)褐飛虱觸角具有活性的水稻揮發(fā)物進(jìn)行了分離鑒定。結(jié)果表明,褐飛虱成蟲觸角對(duì)水稻揮發(fā)物有電生理反應(yīng)的有5種物質(zhì):其中反應(yīng)顯著且實(shí)驗(yàn)重復(fù)性最高的有2種,分別為1,6-Octadien-3-ol,3,7-dimethyl-(芳樟醇)和Undecane,2,6-dimethyl-(2,6-二甲基-十一烷),重復(fù)性達(dá)到13次以上;其次為Benzene,1,4-dichloro-(1,4-二氯苯)、cis-Linaloloxide(順式氧化芳樟醇)和Tridecane(正十三烷)等3種化合物,重復(fù)性也有10-13次,推測(cè)這幾種物質(zhì)對(duì)褐飛虱可能有引誘或者趨避作用。2褐飛虱Nlug OBPs基因的克隆及分析依據(jù)已經(jīng)建立的褐飛虱轉(zhuǎn)錄組數(shù)據(jù),克隆了褐飛虱Nlug OBP8、Nlug OBP9、Nlug OBP10(Gen Bank登錄號(hào)依次為KC663687、KC663688和KC663689)的3個(gè)基因。分析結(jié)果表明,3個(gè)褐飛虱OBPs都具有完整的開放閱讀框,全長(zhǎng)分別為414bp、576bp、756bp,分別編碼137、191、251個(gè)氨基酸,蛋白分子量分別為15.09k Da、20.98k Da、27.88k Da,等電點(diǎn)分別為4.38、6.06、7.97;經(jīng)過多重序列比對(duì)分析序列的保守特征表明,Nlug OBP8基因具有6個(gè)保守的Cys位點(diǎn),與經(jīng)典OBPs的特征相符合,屬于Classic OBPs,而Nlug OBP9和Nlug OBP10都具有8個(gè)保守的Cys位點(diǎn)和1個(gè)Pro位點(diǎn),屬于Plus-C OBPs;預(yù)測(cè)3個(gè)Nlug OBPs序列的N-末端信號(hào)肽表明,僅Nlug OBP8含有21個(gè)氨基酸組成的信號(hào)肽序列,Nlug OBP9和Nlug OBP10均無信號(hào)肽。3褐飛虱Nlug OBPs原核表達(dá)載體的構(gòu)建成功構(gòu)建了原核表達(dá)載體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的重組質(zhì)粒,并將重組質(zhì)粒導(dǎo)入表達(dá)菌株BL21(DE3)/BL21(DE3)Plys S中進(jìn)行試表達(dá),僅有p GEX-6P-1-Nlug OBP8/Nlug OBP10和p ET-32a(+)-NLug OBP8/NLug OBP10試表達(dá)成功。4褐飛虱行為選擇試驗(yàn)基于實(shí)驗(yàn)室已完成的褐飛虱Nlug OBP4蛋白的熒光競(jìng)爭(zhēng)結(jié)合實(shí)驗(yàn)結(jié)果,利用H型嗅覺儀,對(duì)結(jié)合活性高的22種水稻揮發(fā)物單體和GC-EAD篩選出的2種活性物質(zhì)單體(其中芳樟醇是重復(fù)存在的)進(jìn)行了行為測(cè)定。結(jié)果表明,香葉烯和2-庚酮在稀釋50倍的濃度下均對(duì)褐飛虱成蟲具有引誘作用,但2-庚酮在稀釋10倍和100倍濃度時(shí)卻又對(duì)褐飛虱成蟲驅(qū)避反應(yīng)顯著;不同濃度下均對(duì)褐飛虱成蟲有明顯驅(qū)避反應(yīng)的有芳樟醇和辛烯,而(+)-檸檬烯、蒈烯、反式石竹烯、側(cè)柏烯、二十一烷、正十三烷、庚醇、反-2-己烯醇、苯甲醛、己醛、?-紫羅蘭酮以及苯甲酸甲酯等物質(zhì)僅在一定濃度下對(duì)褐飛虱成蟲具有驅(qū)避反應(yīng)。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S433.39
本文編號(hào)：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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S433.39
【參考文獻(xiàn)】
相關(guān)期刊論文 前6條
1 王明勇;;褐飛虱發(fā)生情況·防治現(xiàn)狀及可持續(xù)治理技術(shù)[J];安徽農(nóng)業(yè)科學(xué);2007年10期
2 余海忠;;昆蟲觸角感受器研究進(jìn)展[J];安徽農(nóng)業(yè)科學(xué);2007年14期
3 蔡海林;柏連陽;周小毛;黃燦;;褐飛虱抗藥性研究進(jìn)展[J];江西農(nóng)業(yè)學(xué)報(bào);2007年04期
4 周強(qiáng),徐濤,張古忍,古德祥,張文慶;蟲害誘導(dǎo)的水稻揮發(fā)物對(duì)褐飛虱的驅(qū)避作用[J];昆蟲學(xué)報(bào);2003年06期
5 鄧飛;倪深;朱旭東;;水稻抗褐飛虱育種研究的現(xiàn)狀與展望[J];中國(guó)農(nóng)學(xué)通報(bào);2011年24期
6 劉佳敏;徐華潮;;星天牛成蟲對(duì)楝揮發(fā)性物質(zhì)的GC-EAD反應(yīng)[J];浙江林業(yè)科技;2014年02期
，本文編號(hào)：2479024
本文鏈接：http://sikaile.net/kejilunwen/nykj/2479024.html
最近更新
教材專著
