【作者】 王芳；

【导师】 王磊； 钱岑；

【作者基本信息】 安徽农业大学 ， 生物化学与分子生物学， 2016， 硕士

【摘要】 昆虫在外界不良生存环境下能够有效地抵御外源病菌的入侵,除了机体保护色和物理屏障如鳞毛、甲壳和角质表皮外,昆虫也具有识别和除清体内异原物的高效免疫系统,用来抵御微生物的寄生和感染,因而其先天性免疫应答机制的研究备受广泛关注,这对阐释生物的起源进化、多样性研究和生长发育有重要的意义。本研究以鳞翅目昆虫柳蚕为研究对象,对柳蚕的defense基因和apolipophorin-Ⅲ(ApoLp-Ⅲ)基因进行了克隆、鉴定、比较和进化研究,也初步探讨了它们的诱导调控模式和功能,以期丰富鳞翅目昆虫先天免疫防御机制的理论基础,有利于我们了解新的昆虫防御对策和病原物感染模式。本研究主要内容与结果如下:1.从柳蚕中克隆并鉴定了defense基因,全长包含691个核苷酸预测的开放阅读框498 bp,编码165个氨基酸残基并有一个卷曲结构。defense蛋白的序列与其他鳞翅目昆虫如蓖麻蚕、柞蚕、尺蠖、家蚕、美国白蛾、烟草天蛾等的defense蛋白序列有较高的同源性。在大肠杆菌中defense与表达载体PET-32a(+)连接并成功表达出包涵体和可溶性蛋白,并制备多克隆抗体用于后续实验。实时定量PCR和蛋白印迹检测结果表明defense基因在脂肪体中有特别高的表达水平量,对柳蚕进行免疫刺激后表明,defense基因可以被大肠杆菌、微黄藤球菌和核型多角体病毒刺激上调表达,并表现出对真菌、病毒和革兰氏阳性菌较强的敏感性,而对革兰氏阴性菌的敏感性较弱。抑菌圈实验验证了抑菌圈直径接近相同时,革兰氏阳性菌所需的时间比阴性菌要短。这些数据说明,defense基因在柳蚕的先天免疫应答中起着重要的作用。2.同时从柳蚕中克隆分离出ApoLp-Ⅲ基因,它包含660个核苷酸,预测的开放阅读框561 bp并编码186个氨基酸组成的蛋白,柳蚕ApoLp-Ⅲ蛋白序列与其他物种昆虫的ApoLp-Ⅲ蛋白序列同源性很高,尤其是柞蚕。重组的ApoLp-Ⅲ质粒在大肠杆菌中表达出可溶性的重组蛋白,并制备了多克隆抗体。qRT-PCR检测柳蚕ApoLp-Ⅲ基因在脂肪体和表皮中表达量较高,而在血细胞、性腺、丝腺、马氏管和中肠中很低。在抵御外源物如大肠杆菌、藤黄微球菌、球孢白僵菌和核型多角体病毒入侵时,ApoLp-Ⅲ基因均被上调表达,并表现出对革兰氏阴性菌和真菌比对革兰氏阳性菌和病毒有更高的灵敏度。通过RNA干扰实验,下调的ApoLp-Ⅲ基因直接或间接地影响着超氧化物歧化酶Cu/ZnSOD基因的表达也下调,这些在免疫应答反应中起重要作用的相关基因之间可能存在着一定的相互作用的关系。更多还原

【Abstract】 Under the adverse external living environment insects can effectively resist the invasion of foreign bacteria,in addition to the protection of the body color and physical barriers such as Dryopteris,crustaceans and horny skin,insects also have to identify the body and different original Chuqing efficient immune system,to resist microbial and parasitic infections,thus it attached much attention to study the innate immune response mechanisms,which is to explain the origin of biological evolution,diversity and growth significantly.In this study,wild-type Actias selene(Lepidoptera: Saturniidae)for the study,immune system-related genes were cloned and characterized,as well as comparative and evolutionary studies,and explore their immune regulation induced modes and functions,in order to can We improve the the theoretical basis of lepidopteran innate immune defense mechanism,to provide a solid theoretical basis for subsequent effective use and develop applications.The main research contents and results are as follows:We isolated a novel defense gene(AsDef)cDNAs from Actias selene(Hubner 1806)(Lepidoptera: Saturniidae).It contained 691 nucleotides with a putative open reading frame(ORF)of 498 bp encoding 165 amino acid residues with a reeler domain.It showed significant similarities with defense proteins from several other lepidopteran insect species including Samia ricini(Donovan 1798),Antheraea mylitta(Drury 1773),Bombyx mori(Linnaeus 1758),Lonomia oblique(Walker 1855),Hyphantria cunea(Drury 1773)and Manduca sexta(Linnaeus 1763).The recombinant protein of As Def was expressed successfully in Escherichia coli(Migula 1895)cells,and the anti-AsDef antibody against rabbit was prepared.Results of real-time quantitative PCR(RT-qPCR)and western blot showed that ApDef gene was detected at a particularly high levels in fat body,while ApDef protein can only be detected in hemolymph.For immune functions,AsDef gene can be upregulated by E.coli,Micrococcus luteus(Cohn 1872),Beauveria bassiana(Bals 1912)and nuclear polyhedrosis virus(NPV),and showed a stronger sensitivity for gram positive bacteria than fungi and virus,while the least sensitivity for gram negative bacteria.Zone of inhibition experiments indicate the inhibition zone diameter of approximately the same,the time required shorter for gram-positive bacteria than negative bacteria These data indicated that AsDef plays an important role in innate immune responses of A.selene.Apolipophorin-Ⅲ(ApoLp-Ⅲ)is involved in lipid transport and innate immunity in insects.In this study,we isolated ApoLp-Ⅲ gene(As-ApoLp-Ⅲ)cDNA from Actias selene Hübner(Lepidoptera: Saturniidae).It contained 660 nucleotides with a putative open reading frame(ORF)of 561 bp encoding 186 amino acid residues.It showed similaritieswith ApoLp-Ⅲ proteins from other insect species,especially Antheraea pernyi.The recombinant protein of As-ApoLp-Ⅲ was expressed in Escherichia coli,and anti-As-ApoLp-Ⅲ antibodies were prepared.The As-ApoLp-Ⅲ gene was expressed at high levels in the fat body and integument,and at low levels in the hemolymph,testis,silk gland,malpighian tubule,ovary and midgut.As-ApoLp-Ⅲ gene can be upregulated by E.coli,Micrococcus luteus,Beauveria bassiana and nuclear polyhedrosis virus(NPV),and showed a greater sensitivity for gram negative bacteria and fungi than for gram positive bacteria and virus.These data indicated that As-ApoLp-Ⅲ may play an important role in innate immune responses of A.selene.By RNA interference experiments,ApoLp-Ⅲ gene down-regulated directly or indirectly affect the expression of superoxide dismutase Cu/ZnSOD genes also down,there may be some interactive relationship among these important related genes in immune responses.更多还原