【作者】 马艳；

【导师】 鲁成；

【作者基本信息】 西南大学 ， 特种经济动物饲养， 2009， 硕士

【摘要】 ZP是zona pellucida的缩写,ZP基因是指包含ZP功能域的一群基因。ZP功能域包含约260个氨基酸,其中有8-10个保守的半胱氨酸(Cys)残基。ZP蛋白大多数是糖基化的蛋白,其功能多种多样,从精子受体到结构原件的动力传导器,从卵壳的主要成分到参与信号传导,以及哺乳动物和非哺乳动物的各种受体。ZP功能域的突变会引起蛋白质功能的缺失以及很多的疾病。因此,许多ZP蛋白都引起了人们的高度关注,因为它们在特定组织大量表达,并且和很多人类疾病相关。翅的发生使昆虫在觅食、寻偶、扩大分布和避敌等多方面获得了优越的竞争能力,是昆虫纲成为最繁荣的生物类群的重要原因。家蚕作为鳞翅目昆虫的模式代表,又是目前完成基因组测序的唯一的一种鳞翅目昆虫,其ZP基因,尤其是在翅中的研究还未见相关报道。本研究在家蚕基因组中鉴定出了一群ZP基因,并对它们进行了生物信息学分析。同时为了研究ZP基因在家蚕翅中的作用,克隆了其中一个dusky基因,对其在家蚕不同发育时期的表达进行了检测,并采用RNAi技术对其功能进行了初步研究,获得结果如下:1、家蚕ZP基因家族的生物信息学分析已报道在果蝇中有16个ZP基因,其中部分基因的功能在果蝇中有相关的研究。通过同源检索,我们发现这16个基因在家蚕基因组中均存在同源基因。其中部分基因的功能在果蝇中有相关的研究。通过分析家蚕ZP基因的结构域特点发现其和果蝇ZP基因的结构域非常相似。通过进化分析还发现它们的进化关系也比较对应。这一结果表明,ZP基因在鳞翅目和双翅目昆虫中还是比较保守的。另外,还基于家蚕基因组数据对家蚕ZP基因在染色体上的位置特点进行了分析,通过芯片数据对其表达特点进行了分析。2、家蚕dusky基因的克隆及序列分析在对家蚕基因组数据进行生物信息学分析的基础上,设计引物克隆了家蚕dusky基因的全长CDS序列。结果表明,家蚕dusky基因是单外显子基因,ORF长1398bp,编码465个氨基酸。物种间同源蛋白质序列的比对结果表明,家蚕同其他不同物种间的dusky基因的ZP domain序列保守,且都能找剑10个半胱氨酸残基。3、家蚕dusky基因的mRNA表达模式家蚕clusky基因在在家蚕不同组织中的表达检测结果表明,其基因在家蚕5龄第6天幼虫的头部、体壁、卵巢、精巢、马氏管、丝腺、血液、脂肪体和中肠中都没有表达,而在翅原基中却有大量表达,这一结果说明dusky基因在家蚕翅发育过程中特异表达,推测dusky基因对家蚕翅的发育有重要作用。家蚕dusky基因在在家蚕翅发育中的表达检测结果说明,dusky基因在幼虫和蛹前期的家蚕翅中都有表达,而在蛹后期和化蛾后没有表达。4、家蚕dusky基因在翅发育中的RNAi研究本实验对dusky基因在家蚕翅中进行了RNAi干涉实验,结果表明,家蚕dusky基因被干涉后,翅表现为小翅的表型。同时,对干涉后翅的毛孔进行的扫描电镜观察发现,毛孔有变密的趋势。最后,通过定量PCR的方法对干涉后dusky基因的表达作了分子水平的检测,结果表明dusky基因被干涉后表达量明显降低。更多还原

【Abstract】 The designation ZP stands for zona pellucida,while ZP genes mean that genes contain ZP domain which consists of～260 amino acids(aa) and has 8 -10 conserved cysteine(Cys) residues. Most ZP domain proteins are glycosylated.The functions of ZP domain proteins,from sperm receptors and mechanotransducers to structural components,mutations in genes encoding ZP domain proteins can result in loss of functions of ZP proteins and severe human pathologies.Many ZP proteins attracted attention because of their abundant expression in certain organs and their relation to various diseases.The wings of insects make them taking many advantages in predation,mate choice,expanding distribution and safe-keeping,and so on.This is the most important reason for them to become the most prosperous organism.Silkworm is the typical model insects in Lepidoptera,is also the only one lepidopterous insect which has been completed the genome sequencing at present.The study of ZP genes,especially in the wing,has not been reported yet.We identified a family of genes encoding zona pellucida(ZP) domain proteins in silkworm,and analysis them by bioinformatics.Also,we cloned the silkworm dusky homologue,detected its expression of different stages during silkworm development and preliminarily studied on its function by RNA interference technique.The main reserch contents and results are as follows.1.Bioinformatics Analysis of Silkworm ZP GenesIn Drosophila,there are 18 genes.Through the homology search,we found 16 genes have the homologous in the Bombyx mori genome.Some of them also have been reported in Drosophila.The gene sequence analysis indicated that the similarities of ZP domain of ZP genes are very high between Bombyx mori and Drosophila.Phylogenetic analysis showed that their phylogenetic relationship are also corresponding.These results showed that ZP genes are conserved in Lepidoptera and Diptera.We also analysised the location of ZP genes in the genome based on the genome data,and the expression profile based on the gene microarray data during the course of mature silkworm to adult.2.Clonging of silkworm dusky geneDepending on the results of bioinformatics analysis,we designed primers for silkworm dusky gene and then cloned this gene.The cloned mRNA length of the dusky gene comprised an open reading frame.Results showed that silkworm dusky gene has only one exon which length is 1398bp, coding for 466 amino acids.Protein sequence alignment shown silkworm ZP domain of different DUSKY protein are highly conversed,all of which have 10 conserved cysteine(Cys) residues.3.Expression profile analysis of silkworm dusky gene mRNART-PCR detection revealed that dusky gene expressed in all detected tissues from Silkworm 5th instar larva,such as head,epidermis,ovary,tetise,malpighian tube,silk gland,hemolymph,fat body, midgut and wing.The result showed that dusky gene participates developmental regulation only in the wing in silkworm.And then,we detected the expression profile of dusky gene during silkworm wing development.Results of RT-PCR showed the expression of dusky gene express in larve stage and early days of pupa,while no expression in late pupa and Moth period.4.RNAi of silkworm dusky gene in the wing of silkwormRNAi experiment on dusky gene was performed in the wing development.Results showed that after RNAi the wing reduced Seriously.And then observed the pores of the wing by using Scanning electron microscope(SEM),it showed that the pores turn dense.At last we used Quantitative PCR to detect the expression level of dusky gene after RNAi,the result showed that its expression level is lower.更多还原