【作者】 康桂娟；

【导师】 祝增荣；

【作者基本信息】 浙江大学 ， 农业昆虫与害虫防治， 2010， 博士

【摘要】 稻水象甲(Lissorhoptrus oryzophilus kuschil)原产于北美,20世纪70年代入侵亚洲以来,范围不断扩大,是一种重要的水稻害虫。昆虫嗅觉和味觉在感受气味物质、寻找寄主、交配等方面起着重要的作用。G蛋白偶联信号传导系统是生物体内一类重要的细胞信号传导途径,参与了昆虫的嗅觉、视觉和内分泌激素等多种信号的传导过程。开展昆虫G蛋白的研究,不仅可以明确昆虫对信号分子的感受机理；还可以了解G蛋白对昆虫的行为和生理信号传导的调节作用,为今后深入研究病虫害信号传递机制和调控机理奠定理论基础。本研究克隆了稻水象甲G蛋白α亚基基因的读码框(ORF),并对序列特征及进化关系进行了分析,构建了该基因的原核表达载体pET30a/Gαo,用纯化后的蛋白免疫兔子制备了多克隆抗体,同时用荧光定量PCR研究了该基因转录水平的表达情况,并利用制备的抗体将G蛋白α亚基在稻水象甲触角进行了免疫定位。本文构建了稻水象甲头部cDNA文库,并通过酵母双杂交的方法筛选与G蛋白α亚基相互作用过的蛋白。研究结果如下：1.稻水象甲G蛋白α亚基基因的克隆和序列分析。利用RT-PCR和RACE方法从稻水象甲头部cDNA中克隆了一个G蛋白α亚基基因,Lo Gαo。该基因的ORF为1065个核苷酸,编码354个氨基酸残基,预测的分子量为40.416kDa,等电点为5.34。通过多序列比对、系统进化及相似性分析,发现这个G蛋白α亚基应属于Gαo家族,主要功能域和结合位点的氨基酸序列在与其他Go蛋白基本相同。我们推测Lo Gαo的功能可能与其他已报道的Gao蛋白相类似,可能结合相似的受体以及调控相同的下游效应器。2.稻水象甲Gαo基因的原核表达、多克隆抗体制备及其组织分布的Western blot检测。我们构建了Lo Gαo基因的原核表达载体pET30a/Gαo, IPTG诱导原核表达。用原核表达的蛋白制备了兔源多克隆抗体,并用Elisa和Western blot检测了该抗体的效价和特异性。Anti-Lo Gαo抗血清的效价为2.048×106,并且Western blot结果显示该抗体在稻水象甲各个组织的粗提液中只检测到一条40kDa的特异性条带,与其他G蛋白亚基没有交叉反应。我们制备的抗体的特异性和效价均符合下一步实验的要求,对于Lo Gαo在稻水象甲的免疫定位提供了必要的前提。3 Real-time PCR鉴定稻水象甲Gao基因的转录表达,用相对定量的方法,我们研究了稻水象甲各器官Lo Gαo转录水平上的表达谱,发现Lo Gao mRNA没有组织特异性,在各个组织都有表达,但是其在触角的表达显著高于其他部位。触角是昆虫感觉系统的重要组成部分,行使着嗅觉、味觉等功能,该基因在触角的高丰度表达,使我们推测其可能参与嗅觉或味觉的信号转导。4稻水象甲触角感器的扫描电镜观察,利用扫描电镜技术,我们观察了孤雌生殖和两性生殖稻水象甲触角感器的类型和分布；比较了两种生殖方式和雌雄之间的差异,并探讨了各类型感器的功能,为研究稻水象甲G蛋白α亚基在嗅觉感器上的定位奠定了基础。5稻水象甲Gαo亚基在触角感器中的免疫定位,利用本研究制备的anti-LoGαo多克隆抗体,用免疫胶体金标记的方法研究了稻水象甲G蛋白αo亚基在触角的定位情况,发现该亚基主要定位在毛型感器的树突神经中,根据本文第五章对触角感器功能的探讨,毛型感器可能是稻水象甲的味觉/嗅觉感器,该结果表明Lo Gαo可能参与稻水象甲味觉/嗅觉信号的转导过程。6酵母双杂交方法筛选与稻水象甲Gαo相互作用的蛋白,本文构建了稻水象甲头部的酵母cDNA文库,文库的滴度为6.8×109个细胞/ml文库(>2.0×107个细胞/ml),符合文库构建的要求,可用于进行下一步的酵母双杂交实验。用Gαo诱饵蛋白对稻水象甲头部cDNA文库的筛选之后,我们得到一些与α亚基相互作用的克隆,如肌球蛋白调节轻链激酶(myosin light chain kinase)、载脂蛋白D(apolipoprotein D)、肌球蛋白Ⅵ(mysinⅥ)、铁蛋白(ferritin)、山梨醇脱氢酶(sorbitol dehydrogenase)、锌指蛋白(zinc finger protein)等。这些蛋白与稻水象甲Gαo的相互作用还需要进一步的验证。更多还原

【Abstract】 Rice water weevil Lissorhoptrus oryzophilus Kuschel (Coleoptera: Curculionidae), native to North America, is the key insect pest of rice. In China, since its first detection in 1988 from Tangshan city of Hubei province, it had been reported in 15 provinces. Olfactory and gustatory of insects play important roles in the detection of food, hosts, or partners and therefore determine the spreading process of the alien weevil in a new environment. G protein-coupled signaling system is an important signaling pathway involved in insect olfactory, visual and endocrine hormones and other signal transductions. The research on the G protein of insect is helpful to understand not only the molecular mechanism of how insect feel outside signals, but also the functions of G protein on the behavioral and physiological regulation of signal transduction of insect. This will offer an theoretical basis to study signal transduction and to explore new approach pest control in the future.In this dissertation, I used reverse transcription (RT)-PCR and RACE method to clone the ORF of a G protein a subunit from L. oryzophilus, and analysis the sequence and evolution relationship. The expression profile of transcript of G protein a subunit was conducted by real-time quantitative PCR. Meanwhile, we expressed it in prokaryotic system and prepared for polyclonal antisera, with the anti-Lo Gαo antiserum we immunolocalized the G proteinαsubunit in the antennae of the weevil. We constructed a L. oryzophilus head’s cDNA library, and screened for the related proteins of Lo Gαo by Yeast Two-Hybrid system. The results are presented as follows:1. A cDNA encoding a G proteinαsubunit was cloned from the head of the rice water weevil L. oryzophilus Kuschel (Coleoptera:Curculionidae) by the combination of reverse transcription-PCR (RT-PCR) and RACE-PCR. The cDNA contains a 1065 bp open reading frame (ORF) that encodes a 354 amino acid protein. The predicted molecular weight (MW) and pI was 18.192 kDa and 5.08, respectively. Through multiple sequence alignment, phylogenetic and similarity analysis, we found that this G protein a subunit belongs to the Gαo family, the amino acid sequences of main functional domain and binding sites are essentially the same with the other Go protein. From the results we speculated that the functions of Lo Gαo might be similar with other Gαo proteins had been reported, and might be combined with similar receptors and regulated the same downstream effector.2. Prokaryotic expression, polyclonal antibody preparation and tissue distribution of the Gαo. Prokaryotic expression vector of pET30a/Lo Gαo was constructed and successfully expressed in BL21. The recombinant protein was purified and prepared the polyclonal antibody. With ELISA and Western blot, the antibody titer and specificity was studied. The titer of anti-Lo Gao antiserum was 2.048×106, and the result of Western blot showed that there was only one specific band of 40 kDa was detected from crude extracts of various organs of the weevil, there was no cross-reaction with other G protein subunits. The titer and specificity of the antibody were consistent with the requirements of next experiments, it provided a precondition for the immunolocalization of Lo Gao in the weevil3. Spatial expression profile of Lo Gao transcript was analyzed by Real-time quantitative PCR. With the relative quantitative method, we studied the expression profiles of the transcription level of Lo Gao and found that the Lo Gao mRNA expressed in various organs, without tissue-specific, but the expression in the antennae was significantly higher than in other parts of weevil. Antenna is an important part of insect sensory systems, performed the sense of smell, taste and other functions, highly expressed of the gene in the antennae, lead we speculated that the protein may be involved in the sense of olfactory/gustatory signal transduction.4. SEM observation of the antennae of the rice water weevil. Using scanning electron microscopy, we observed types and distribution of antennal sensilla of parthenogenesis and bisexual rice water weevil; observed the differences between two reproductive modes and male and female, and explored the functions of various types of sensilla, as a basis to study the immunolocalization of Lo Gao in the olfactory sensilla of the rice water weevil.5 Immunolocalization of Lo Gao in the antennal sensilla of the rice water weevil. With anti-Lo Gao polyclonal antibody prepared in this study, using immune colloid labeling method to study the localization of G protein ao subunit in the antennal of rice water weevil, and found that Lo Gao was mainly expressed in the dendrites of the trichoid sensilla. According to the general functions of antennal sensilla proposed by previous reports and our fore-mentioned observation, trichoid sensilla were such kinds of olfactory/gustatory sensilla. We speculate that the Lo Gαo protein may be involved in olfaction or gustatory signal transduction of the weevil.6. Interaction proteins were screened from the cDNA library of L. oryzophilus head with Lo Gao as bait protein by Yeast Two-Hybrid System. We built a yeast cDNA library of the head of rice water weevil, the titer of the library was 6.8×109 cell/ml (>2.0×107cell/ml), can be used for the yeast two-hybrid experiments. The rice water weevil head cDNA library was screened with Gao bait protein, get some interaction clones, e.g. myosin light chain kinase, apolipoprotein D, mysin VI, ferritin, sorbitol dehydrogenase, and zinc finger protein etc. The interaction of these proteins with Gao need further experimental proof.更多还原