【作者】 乔奇；

【导师】 原国辉；

【作者基本信息】 河南农业大学 ， 作物安全生产， 2008， 博士

【摘要】 嗅觉在昆虫的生存和种族繁衍过程中发挥着重要作用。昆虫对气味的识别过程非常复杂,气味分子与嗅觉神经元树突膜上气味受体的结合,参与了昆虫嗅觉识别的初始过程。昆虫气味受体被普遍认为是G蛋白偶联受体,而一些证据却不支持此观点。针对上述两种不同的观点,本研究对重要农业害虫烟夜蛾的气味受体基因、G蛋白α亚基基因以及两种基因之间的关系进行了研究,以期为昆虫识别气味分子机理的探明提供一些试验依据。主要研究内容包括:烟夜蛾气味受体和G蛋白α亚基基因的克隆、原核表达以及在烟夜蛾不同组织和发育期的表达;利用原位杂交和免疫组化方法研究两基因在烟夜蛾触角内的表达异同,初步分析两基因间的关系。主要研究结果如下:(1)利用RT-PCR和RACE-PCR方法扩增获得了烟夜蛾Or83b-like基因的全长cDNA(1946bp),序列分析结果表明,该基因编码区全长1422bp,5′端非编码区长135bp,3′端非编码区长389bp;编码区推导表达473个氨基酸残基,预测所编码的蛋白质的分子量为53.51kD,等电点为8.73,预测氨基酸序列中含有7个跨膜区;此外,烟夜蛾Or83b类似受体与其他昆虫的Or83b家族受体的氨基酸序列有较高的一致性。证明获得的基因为昆虫的Or83b家族受体基因之一,暂命名为HassOr83b,此基因已在Genbank上登录,登录号为EU057178。(2)从烟夜蛾基因组中克隆出了一个长度为1202bp的HassOr83b基因片段,序列比对结果表明,该片段含有1087bp的内含子序列。此片段已在Genbank上登录,登录号为EU497670。(3)利用跨内含子引物和RT-PCR方法,研究了烟夜蛾HassOr83b基因在烟夜蛾不同发育期和组织内的表达情况。结果表明,HassOr83b基因在幼虫早期、幼虫晚期、蛹期、成虫期皆有表达,而在卵期没有表达;在雌雄成虫触角、下唇须和喙有表达,而在翅、足和去掉足、翅等附肢的躯干部没有表达。(4)利用原位杂交方法研究了HassOr83b基因在雄性烟夜蛾触角内的转录情况。结果表明,阳性标记出现在触角表皮下方的很多感觉细胞中,HassOr83b基因很可能在毛形感器下方的神经元胞体中表达。(5)利用RT-PCR方法,获得了烟夜蛾G蛋白αq亚基基因长度为1103bp的cDNA序列,其中编码区全长1062bp,5′端非编码区长21bp,3′端非编码区长20bp;该基因推导表达353个氨基酸残基,预测蛋白质的分子量为41.5kD,等电点为5.15。序列比对结果表明,烟夜蛾Gαq亚基与其他昆虫Gαq亚基的氨基酸序列有较高的一致性。此基因已在Genbank上登录,登录号为EU057176,该基因暂命名为HassGαq。(6)利用RT-PCR方法,研究了烟夜蛾HassGαq基因在烟夜蛾不同发育期和组织内的表达情况,结果表明,HassGαq基因在卵期、幼虫早期、幼虫晚期、蛹期和成虫期皆有表达,在触角、下唇须、喙、翅、足和躯干部也皆有表达。HassGαq基因在烟夜蛾体内为非组织特异性表达。(7)成功构建了HassGαq基因的原核融合表达载体,经过IPTG诱导以及SDS-PAGE和Western blot分析,结果显示原核表达产物的分子量约为66kD。由于HassGαq蛋白的预测分子量为41.5kD,GST标签的分子量为26kD,因此表达产物的大小与预测一致,试验结果证明融合蛋白得到了有效表达。(8)利用SDS-PAGE分离的目的条带作为抗原,免疫小鼠,获得了HassGαq蛋白的抗血清,Western blot分析结果表明,抗血清的特异性较差,而商品化的anti-Gq/11α抗血清的特异性较好。(9)利用anti-Gq/11α抗血清和免疫组化方法,对烟夜蛾HassGαq蛋白在雄性触角内的定位进行了研究,试验结果表明,HassGαq在毛形感器和锥形感器的基部以及感器腔中皆有表达。(10)通过比较HassOr83b基因和HassGαq蛋白在烟夜蛾触角内的表达情况(二者很可能皆在行使嗅觉功能的毛形感器下方的神经元胞体表达),支持烟夜蛾嗅觉信号传导与G蛋白偶联的观点。更多还原

【Abstract】 Olfaction plays a crucial role in insect survival and reproduction. Insect odorant perception is a complex process. The initial steps in odor detection involve the binding of an odor to the odorant receptor (OR) displayed on dendrites of olfactory sensory neurons (OSNs). There is a widespread assumption that insect ORs belong to the family of G protein–coupled receptor (GPCR). However, there are several challenging evidences against the assumption. In this research, the genes encoding OR and G proteinαsubunit from Helicoverpa assulta, and the relationship between the two proteins were studied. The main results are as follows:(1) The full length cDNA encoding Or83b-like receptor of Helicoverpa assulta was isolated by using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Nucleotide sequence analysis revealed that the cDNA contains a putative coding region of 1422 bp, 5′untranslated region (UTR) of 135bp and 3′UTR of 389bp. Sequencing analysis showed that the open reading frame (ORF) of the cDNA encodes a protein of 473 amino acids with a predicted molecular weights (MW) of 53.51kD and isoelectric point (PI) of 8.73. The predicted protein obtained, containing seven putative transmembrane domains, shares high amino acid identity with other Or83b family receptors. The results suggest that the gene obtained is one member of Or83b family genes and this gene was hence named as HassOr83b. This sequence has been deposited in the GenBank database with accession number EU057178.(2) A fragment of HassOr83b was amplified from genomic DNA. The sequencing of the DNA fragment generated a total of 1202bp nucleotide sequence. The intron position for the fragment was determined on the basis of a comparison of cDNA with the genomic sequence. The intron sequence in the fragment is 1087bp in length. The partial sequence of HassOr83b from genomic DNA has been deposited in the GenBank database with accession number EU497670.(3) The expression of HassOr83b transcript in several adult tissues as well as during preadult stages was described by using intron-spanning primers and RT-PCR. The results showed that the HassOr83b transcript is clearly observed in the antennae, labial palps and proboscises, but not in bodies, wings and legs. In addition, HassOr83b is also expressed in several preadult stages, including early-stage larvae, late-stage larvae and pupae, but not in embryos.(4) To assess HassOr83b are in fact expressed in antennal sensory cells, in situ hybridization experiments were performed. In situ hybridization of antennal sections with the HassOr83b antisense RNA probe provided clusters of labelled cells, most likely representing sensory cells housed in trichodic sensilla.(5) A 1103bp cDNA encoding Gαq protein of Helicoverpa assulta was isolated by RT-PCR. Nucleotide sequence analysis revealed that the cDNA contains a putative coding region of 1062 bp, 5′UTR of 21bp and 3′UTR of 20bp. Sequencing analysis showed that the ORF of the cDNA encodes a protein of 353 amino acids with a predicted MW of 41.5kD and PI of 5.15. The predicted protein obtained shares high amino acid identity with other known Gαq subunits. This sequence has been deposited in the GenBank database with accession number EU057176 and this gene was named as HassGαq.(6) The expression pattern of the HassGαq gene transcript was examined by RT-PCR analysis of RNA isolated from several developmental stages and adult tissues of Helicoverpa assulta. The HassGαq transcript is expressed in all adult tissues tested, as well as throughout preadult development. The expressions of HassGαq are not tissue specific.(7) HassGαq gene was constructed into expression vector pGEX-4T-2 for over expression in prokaryotic cells. The SDS-PAGE and Western blot analysis showed that induced by Isopropyl-β-D-Thiogalactoside (IPTG), the GST-HassGαq fusion protein is expressed in Escherichia coli BL21, and its MW was found to be about 66kD nearly equal to the predicted.(8) The specific band of expression was excised from the gel and used to immunize the mice. The antiserum was collected from the immunized mice. Male antennal homogenate of H. assulta was separated by SDS-PAGE and detected by Western blot using the antiserum prepared, and anti-Gq/11αantiserum purchased respectively. Of the tested sera, anti-Gq/11αantiserum detected a single immunoreactive band with an apparent molecular mass of 40 kD that was consistent with the molecular weight of HassGαq protein, and the antiserum prepared in this research detected several bands. The results indicated that anti-Gq/11αantiserum can recognize HassGαq protein with high specifity.(9) The expression sites of HassGαq in adult male antennae were examined by immunocytochemistry method using anti-Gq/11αantiserum against HassGαq. The results revealed that HassGαq was associated with trichodic sensilla and basiconic sensilla as immunolabeling were typically observed inside and at the base of the two kinds of sensilla.(10) The location of HassOr83b and HassGαq in the male adult antennae of H. assulta supports the view that HassOr83b belongs to the family of GPCR.There is no publication concerned with studies above.更多还原