【作者】 张升祥；

【导师】 崔为正； 徐世清；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 家蚕是和人们的生产、生活联系最密切的昆虫,是目前唯一完成全基因组测序的鳞翅目昆虫,是国际鳞翅目学会公认的鳞翅目模式生物;数十年来,蚕蛾还一直是研究蛾类嗅觉接受和传导的模式昆虫,其中气味结合蛋白(以性信息素结合蛋白为主)是研究的热点之一。气味结合蛋白(Odorant binding proteins,OBPs)在昆虫和外界环境化学信息交流过程中起着重要作用,对昆虫觅食、求偶、繁殖具有重要意义。因此,开展家蚕气味结合蛋白的研究对进一步认识其功能,阐明家蚕的嗅觉机制,以及利用昆虫嗅觉进行害虫治理和益虫饲养具有重要理论和实践意义。为此,本文利用生物信息学、基因定位、基因表达谱分析、基因克隆、RNAi、分子进化分析和分子标记等方法研究了家蚕气味结合蛋白基因家族的基因结构、定位、表达谱、功能,以及家蚕对人工饲料摄食性相关的分子标记。主要结果如下:1对NCBI公布的家蚕OBPs家族生物信息学分析发现,该家族基因分布于7条染色体上,形成4个基因簇;基因的结构多态性明显,核苷酸序列相似性、遗传距离差距较大;家蚕OBP基因的电子表达谱广,多个基因在多种组织中具有表达。对OBP家族蛋白质分析发现,该家族的生化特性和昆虫OBP蛋白基本类似,其中可能有2个蛋白为跨膜蛋白,5个膜相关蛋白,蛋白质的疏水性区域类似;序列一致性较低,遗传距离差异大,但半胱氨酸和色氨酸非常保守;家蚕的OBP家族同样存在Minus-C类群,但未发现Plus-C类群基因。结果表明,OBP家族可能具有多种功能。2性信息素结合蛋白/普通气味结合蛋白(Pheromone binding protein/general binding protein)是鳞翅目昆虫OBP家族的一个重要单系群。染色体上定位发现这些基因以基因簇的形式存在于第19染色体的nscaf3052上,基因结构相似,转录方向一致,表明这些基因可能由同源基因复制产生,并具有类似功能;对其潜成虫和成虫阶段多个发育时期的雌、雄虫多种组织中进行表达分析,发现这些基因在不同发育时期的不同组织间差异明显,相对表达量均以触角中为最高,其它非嗅觉组织中也多有表达,性别间差异不大,说明了该基因簇基因除了具有嗅觉相关的功能外,很可能具有其它尚未被发现的功能;而蚕蛾趋性反应结果说明,蚕蛾的嗅觉反应以雄蛾对性引诱的应答为主,而对幼虫期敏感的引诱物或忌避物没有反应,成虫期OBPs的主要功能可能和感受性信息素刺激有关。3对家蚕幼虫期ABP(Antennal binding protein)和ABPX(Antennal binding protein X)基因的蛋白质结构、功能位点、基因表达和功能分析发现,这2个蛋白差异较大,分属不同的OBP群;且这2个基因在嗅觉组织(器官)中均有较高的表达量,但并非仅在嗅觉器官中表达,基本上在所检测的组织中均有表达。摘除实验结果显示,二者可能和幼虫对食物的趋性相关;但5龄期注射dsRNA和口服细菌表达的dsRNA实验表明,RNAi能够显著降低目的基因的表达,但未引起家蚕的嗅觉失灵,而对家蚕幼虫5龄期的发育经过有明显的延缓作用。这些结果表明,这2个基因在幼虫期可能和幼虫的对食物的趋性相关,并具有其他重要的、和发育相关的生理功能。4克隆得到了野桑蚕性信息素感受过程中的重要基因PBP1、PBP2、PBP3、OR1和OR3。序列分析发现,家蚕和野桑蚕基因间的遗传距离很近,基因变异较小,且以转换为主,核苷酸变异未引起蛋白质二级结构的变化,其重要的功能位点也未发生变异,推测其基因的功能未发生变化,故二者能够相互感受、识别。为通过杂交开发、利用野桑蚕资源提供了理论依据;同时,这也是家蚕起源于野桑蚕的分子证据。5普遍认为,GOBP(general OBP)具有结合食物和环境中各种普通的气味分子的能力。对13种昆虫GOBP亚家族分子进化分析结果表明,GOBP1和GOBP2氨基酸和核苷酸的一致性很高,序列差异性很小,其亲缘关系非常近,其中半胱氨酸和色氨酸非常保守,但是它们来自不同的祖先基因,并在物种分化之前已经分化完成。基于氨基酸序列的ω分析表明,Ka/Ks率一般都很低,仅在烟夜蛾和棉铃虫GOBP1的分化进化过程中受到正向选择作用,而在其他分化过程中均未检测出正向选择作用。表明GOBP基因在昆虫间可能具有相似的功能。6家蚕的食性相关分子标记研究表明,家蚕对人工饲料育摄食性相关性状可能主要受不食基因控制;RAPD标记结果具有明显的多态性,SSR标记的多态性较少,其对应的特异引物主要为染色体第10、11、17、19、20、27、28号上的标记,其中以第20号染色体的标记最多,且多为高摄食性亲本的特异性条带,说明家蚕对人工饲料摄食性的基因很可能位于其上;而位于17号染色体的标记扩增出的特异性条带多来自于低摄食性亲本,表明家蚕对人工饲料不食基因可能位于其上。这为进一步确定食性相关性状的分子标记,利用分子标记辅助育种,培育对人工饲料育高摄食性品种奠定了基础,有助于产业进步,也有利于害虫的防治和其他益虫的饲养。更多还原

【Abstract】 Domestic silkworm Bombyx mori is the closest insect with the production and living of homo-sapiens. Bombyx mori is the only Lepidopteran insect whole genome sequence available. And it has been considered as the lepidopteran model insect by The International Lepidoptera Society, TILS. In the past decades years, silk moths were also as the model insect utilized in moth olfactory reception conduction research, the odorant binding proteins （OBPs） were on the focus, especially the pheromone-binding proteins （PBPs）. OBPs in insects play a primordial role in foraging, courtshiping, reproduction, and chemical communication with the environments. Accordingly, researches on the OBPs of silkworm would further recognize the functions, and illuminate the mechanism of silkworm olfactory, and be utilized in pests governing and in beneficial insects rearing. For those reasons, we investigated the genetic structure, chromosome location, expression profiles, gene function of OBPs, and the molecular markers related to feeding habit to artificial diet of silkworm utilized bioinformatics, gene localization, expression profile analysis, gene cloning, RNA interference （RNAi）, molecular evolution analysis, and molecular markers analysis. The results as follows:1 We analyzed the OBP family members of Bombyx mori released in NCBI with bioinformatics methods and softwares, and found the family members located on the 7 disparity chromosomes, and emerged 4 gene clusters. The genetic structure showed obviously polymorphism. The disparities were fairly larger of nucleotide sequence similarity and genetic distance. The electronic expression profiles were extensive, and many genes expressed in different tissues. The family had similar biochemistry character to insect OBPs, and two were transmembrane proteins, 5 embrane-associated proteins. The amino acids had low level sequence identities and remote genetic distances, but cysteine and tryptophan were very conservative. There possessed Minus-C group like Drosophila melanogaster and other insects, and no Plus-C group in the family. The results indicated OBP probably functioned in many aspects in silkworm.2 Pheromone binding proteins/general binding proteins（PBPs/GOBPs） were an important monophyletic group of Lepidoptera. We mapped the genes in the genome of silkworm. The results exhibited those genes organized in the nscaf3052 of Chromosome 19^th in form of gene cluster, with similar gene structure and identical transcript direction, indicated them originated from homologous gene duplication and possessed functional similarity probably. Analysis the expression profiles in different tissues in different female and male tissues during pupal and adult stages, the results revealed that those genes possessed conspicuous temporal and special differences, the maximum relative expression level occured in the antennae, and lower in other most no-olfactory organization without significant sex differences. The results indicated the gene cluster may function as a olfactory organism, and others undiscovered. Otherwise, taxis experiment of silkworm adult males showed olfactory reaction between adults was the responses to sexual attraction and independence to attractant or repellent, the major function of OBPs were related to receipt sexual pheromone irritation.3 We analyzed the predicted tertiary structures and functional sites, gene expression profiles of the whole stages, and function of ABP and ABPX by RNAi in the 5th larva stage. The two proteins were significantly differences, and belonged to different groups. They were transcribed highly in olfactory tissues （organism）, and by no means merely in those tissues, but expressed in all most tissues detected. Removal olfactory organisms experiment showed the two genes possibly correlated to the taxis to ingestas in larva stage. But the transcript levels of targeted genes significantly decreased after injected with dsRNA or oral application of bacterial expressed dsRNA. RNAi did not cause olfactory malfunction of silkworm, but postponed the developmental process of the 5th larva significantly. Those results indicated the two genes probably concerned with the taxis to ingestas and shouldered other important physiological functions related to development in larva stage.4 Sex pheromone signal transduction in moths was focused on PBP subfamily member and OR （olfactory receptor） genes at present. It formed a paradigm for the study of insect chemical communication. PBPs, OR1 and OR3 played an important role in the process of the male silkmoths perception and location of the females. For the purpose of finding out the molecular mechanism of difficult hybridization,and understanding the evolution of related genes to sexual recognition between the demostic and wild silkworm. We cloned the 3 PBP and 2 OR genes from wild silkmoths Bombyx mandarina. The results, of sequence analysis of coding regions, revealed that the genetic distances were very intimate and the base transversion was the predominant form of basic mutations. Secondary structure predicted suggested mutation sites had no influence on the adjoining regions, and nor on the functional sites. They are presumably no functional differences in the two related moths,So the two related species can receipt, recognize each other, and it founded the theoretical evidence for the exploitation of wild silkworm through mutual cross. Meanwhile, it was the molecular evidences of domestic silkworm originated from wild silkworm. 5 General OBPs （GOBPs） were considered as the binding protein of variety of general odorant molecules associated with food and the environment. The molecular evolution based on GOBP subfamily sequences of 13 insects released by NCBI indicated there presented high identity in the GOBP1 and GOBP2 amino acid and nucleotide sequences, and low level sequence divergence. The genetic relationship was very close between the two genes. Also, cysteine and tryptophan were the most conservative amino acids. But the two genes originated from two different ancestral gene, and had been duplicated prior to the divergence of those 13 species.Theω（ka/ks） analysis based on the nucleotide and amino sequences indicated Ka/Ks ratios were generally small. Only one positive selection was detected during the evolutionary process of GOBP1 between Helicoverpa assulta and Helicoverpa armigera. Those results demonstrated GOBPs were probably functional similarity among insects.6 Correlated characters of feeding habit on artificial diet of silkworm perhaps regulated by no-feeding genes according to the molecular markers experiments. Polymorphism of RAPD （Random Amplified Polymorphic DNA） markers exceeded those of SSR （Simple Sequence Repeat）. And special marks of SSR mainly came from the chromosome 10th, 11th, 17th,19th, 20th, 27th, and 28th. The major were on the chromosome 20th, and many special bands amplified from the parents with high feeding ability. It indicated the genes related foraging artificial diet located on the chromosome probably. And special bands amplified by the SSR marks of chromosome 17th mainly came from the parents with low feeding ability. The no-feeding genes perhaps located on it. Those results founded the base of determination molecular markers of feeding habit related characters and assisting molecular seed breeding. It would promote sericulture,and benefit in pests control and useful insects breeding.更多还原