【作者】 吴敏；

【导师】 韩召军；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2008， 博士

【摘要】 piggyBac转座子是典型的DNA转座子,最初的序列在粉纹夜蛾(Trichoplusia ni)细胞系中分离(IFP2)。该转座子目前已经开发成为转基因昆虫中应用最为广泛的载体之一。虽然目前的研究工作已经表明,piggyBac广泛分布于各类生物的基因组中,但由于发生移码突变或者缺失而使绝大多数序列结构不完整,缺乏转座活性。此外,与mariner等其他转座子相比,piggyBac家族转座子的生物学以及分子进化研究仍然比较滞后,这种现状与piggyBac类转座子在转基因昆虫和基因分离、功能分析研究中的重要地位不相称。因此,本文对鳞翅目昆虫的13个科的41种昆虫展开了piggyBac类转座子分布的调查,对夜蛾中发现的转座子进行全长基因克隆,并对克隆获得的全长转座子进行了活性测定和互作亲和性分析,发现piggyBac转座子末端和亚末端反向重复序列都是活性自主转座子必需的元件。1鳞翅目昆虫piggyBac转座子的调查采用简并引物PCR和Southern点杂交技术筛查了鳞翅目的13个科,41种昆虫中piggyBac因子的分布情况。结果显示,夜蛾科的五种昆虫被检测到内源性piggyBac因子的存在。这五种昆虫分别是小地老虎(Agrotis ypsilon),银纹夜蛾(Argyrogrammaagnata),银锭夜蛾(或称为连纹夜蛾Macdunnoughia crassisigna),斜纹夜蛾(Spodoptera litura),瘦银锭夜蛾(Macdunoughia confusa)。从以上五种昆虫基因组中克隆获得的片断长350bp～380bp,经Blast比对和序列分析,它们彼此之间以及与IFP2序列的相似性均在57%～97%不等。2 piggyBac转座子全序列的克隆采用反向PCR和ITR PCR技术,从银锭夜蛾,小地老虎和银纹夜蛾三种昆虫中成功克隆到相应的piggyBac转座子全长,分别命名为McrPLE,AyPLE和AaPLE。McrPLE全长2472bp,编码595个氨基酸,与IFP2序列高度相似,一致性达99.5%。AyPLE1.1和AaPLE1.1分别编码完整的598和600个氨基酸的转座酶,两者之间的相似性为79%,它们与IFP2转座酶的相似性分别为59%和62%,属于IFP2中度相似序列。而AyPLE1.2和AaPLE1.2两个转座子序列均积累了较大的变异,不能编码完整转座酶蛋白。此外,AyPLE和AaPLE两类因子含有完全一致的反向末端重复序列,但都不存在亚末端反向重复。3 piggyBac转座子基因的系统进化分析收集了14种生物的25个piggyBac类似因子的氢基酸序列,利用CLUSTALX1.8和MEGA3.1构建亲缘关系树,进行系统进化分析。结果显示,进化树中piggyBac转座酶大致分为三大支,即CladeⅠ,CladeⅡ和CladeⅢ。这三大支中都既包含昆虫又包括哺乳动物的piggyBac因子。本文所克隆的McrPLE,AyPLE1.1和AaPLE1.1都出现在CladeⅠ中:McrPLE毗邻IFP2;AyPLE1.1,AaPLE1.1以及HaPLE1所在小分支与IFP2序列亲缘关系最为接近,靴值高达100%。根据以上的进化分析结果以及转座子序列结构特征推断,AyPLE1.1,AaPLE1.1以及HaPLE1因子很可能是同一类新发现的piggyBac亚家族成员。此外,进化树信息还显示不但在不同生物中可含有高度相似的piggyBac因子,而且转座子序列与宿主进化关系并不一致。以上结果揭示:piggyBac转座子的进化不仅仅遵循垂直遗传的规律,而且存在着生殖隔离物种间的水平转移机制。4三个piggyBac类似因子转座活性分析在果蝇S2细胞中建立起转座子活性检测系统,研究了piggyBac类似因子McrPLE,AaPLE1.1和AyPLE1.1的功能。结果表明,McrPLE不但能够在细胞中精确的剪切,而且能够准确地将KOα片段插入Target载体上的TTAA特异性位点。McrPLE因子的28个转座事件分布于Target载体上可插入位点的85位,101位,209位,363位,491位,603位,945位,992位和1863位,表现出插入位点的非特异性。AyPLE1.1的转座酶不能识别缺少亚末端重复序列的pHa[KOα]Donor载体,但可以准确剪切和转座另一个加入IFP2的亚末端反向重复的pHB[KOα]Donor载体。结果表明,AyPLE1.1转座酶是一个有功能的蛋白,并且亚末端反向重复序列在转座酶识别以及剪切反应中起着重要作用,一旦缺失就会严重影响转座子的功能。AaPLE1.1因子利用本研究采用的方法没有检测到活性检,还有待进一步研究。5 piggyBac因子相互作用初探利用果蝇S2细胞转化系统,研究了两类活性因子IFP2,McrPLE和AyPLE1.1之间互相识别和作用的关系。结果显示,两类转座酶表现出很强的专一性,均只能识别各自的反向末端重复序列。根据该研究结果推测,反向末端重复序列很可能是决定转座酶识别和作用专一性的重要序列,而亚末端反向重复则似乎没有种类的特异性。在剪切实验阴性对照中,偶然地发现pHB[KOα]Donor载体单独转染果蝇细胞48小时后能检测到不精确地剪切活动,该结果暗示着果蝇S2细胞中的某个未知因子能够与piggyBac转座子载体互相作用,并有待进一步的实验验证分析,对piggyBac转座子的转基因安全性做出评估和鉴定。综上所述,本研究调查了鳞翅目中41种昆虫内源性piggyBac转座子的分布情况,分离到一个IFP2高度相似的自主活性因子McrPLE,和一个编码活性转座酶的非自主因子AyPLE1.1。转座子互作研究表明,piggyBac转座子的末端和亚末端反向重复序列都是构成自主活性转座子的必要元件,其中末端反向重复很可能决定着转座酶识别和作用的专一性,而亚末端反向重复序列似乎没有种类的特异性。本文探索了在未知基因组生物中分离piggyBac转座子的方法,为其他生物中克隆转座子提供了可借鉴的手段;本研究深入了解了鳞翅目昆虫内源piggyBac转座子的分布,分子进化并探讨了内源性piggyBac转座子和外源IFP2载体的相互作用。本文研究成果为更好地设计piggyBac转基因载体以及为piggyBac载体的生物风险评估提供了理论依据,也为构建具有自主知识产权的昆虫转基因系统奠定了坚实的基础。更多还原

【Abstract】 PiggyBac is a kind of classⅡtransposable elements,and its first sequence(IFP2) was originally isolated from a cell line of Trichoplusia ni.This piggyBac transposon has been modified and widely used as an effective gene-transfer vector to transform the germ-line of more than a dozen species of insects spanning five different orders.Recently,it was reported that piggyBac also efficiently transposes in vertebrate embryos and cell lines. Actually,previous studies had revealed that piggyBac-like elements(PLEs) were widespread in a variety of organisms.However,among all of these hundreds of PLEs recovered,few were found active or even with intact structure.Furthermore,knowledge of the biological characteristics and evolutionary history of piggyBac elements is still limited.This work focused on cloning new intact and potential functional PLEs sequences from lepidopteran insects where the active piggyBac was originally found.And a naturally automobile McrPLE element was isolated,which belongs to the highly conserved IFP2 class.Another moderate related IFP2 sequence amplified was AyPLE1.1,which encoding a functional transposase.Cross-mobility assay showed that the sub-inverted terminal repeat sequence was essential for the transposase recognition,while the inverted terminal repeat determines the speciality of a functional transposase.1 Investigation piggyBac-like element in lepidopteran insectsWith nested degenerate PCR,41 lepidopteran insect species spanning thirteen different families have been surveyed for the presence of endogenous PLEs.Only in five noctuid species,Argyrogramma agnate,Agrotis ypsilon,Macdunnoughia confuse,M.crassisigna and Spodoptera litura,the degenerate primers successfully amplified target fragments about 350bp～380bp in length.Sequence analysis revealed these partial fragments sharing 57-97%identities with each other and with IFP2. The southern dot blot analysis using degenerate PCR fragments as probes showed clear hybridization signals,confirming the presence of endogenous piggyBac-like element in the genome in five noctuid species.2 Cloning of full-length PLEsWith inverse PCR and ITR PCR,the full length of piggyBac-like element in M. crassisigna,A.ypsilon and A.agnate were cloned and designated as McrPLE,AyPLE and AaPLE respectively.McrPLE is 2472 bp in length with a single open reading frame(ORF) encoding for a transposase of 595 amino acid residues.It shares 99.5%identity with active IFP2 element,and belongs to highly conserved IFP2 class.AyPLE1.1 and AaPLE1.1 encode transposases of 598 and 600 amino acid residues respectively.Sequence alignment showed that AaPLE1.1 shares 79%similarity with AyPLE1.1.These two elements are moderated related IFP2 sequences,and share the same 16bp inverted terminal repeats.3 Phylogenetic analysisPhylogenetic tree was made with a total 25 transposase sequences from 14 species, including Daphnia pulicaria,Homo sapiens,Mus musculus and insect species from Lepidoptera,Diptera and Coleoptera.The phylogenetic tree reveals three main clades(Ⅰ,ⅡandⅢ),these three clades contain mixture of elements from both insect and vertebrate. Obviously,the evolutionary pattern within the piggyBac family deviates from the phylogeny of their host species,and the existence of nearly identical piggyBac sequence in reproductively isolated species indicate that horizontal transfer were probably involved in the evolution of PLEs.According to the phylogenetic tree,McrPLE formed the common clade with IFP2 sequence,which was strongly supported by 100%bootstrapping value.AaPLE1.1,HaPLE1 and AyPLE1.1 clustered in the other same branch,considering these elements sharing the same ITR sequence,it is likely that they might be members of a new sub-family of piggyBac transposon.4 Mobility assay of McrPLE,AyPLE1.1 and AaPLE1.1Excision and transposition assays were performed in Drosophila S2 cell culture to verify the activity of PLEs.McrPLE was proved to be an active automobile transposable element and transposition in the characteristic cut-and-paste and TTAA target-site specific manner.The transposase of AyPLE1.1 can’t recognize the donor pHa[KOα]which had no sub-terminal repeats,while it successfully mediated the excision and transposition of pHB[KOα]donor plasmid into the target pGDV1,which indicated AyPLE1.1 encoding a functional transposase,and sub-terminal repeat sequences were essential for the functional transposon.Although the element AaPLE1.1 shows no active signal in our mobility assay,it is worthy of further study.5 Cross-mobility assay of IFP2,McrPLE and AyPLE1.1Cross-mobility assay showed that functional IFP2 transposase couldn’t recognized the donor pHB[KOα],and the active AyPLE1.1 couldn’t mobilize the pB[KOα]either.The difference between two donor plasmids were the inverted terminal repeat sequences,the ITR of pB[KOα]was 13bp and the ITR of pHB[KOα]was 16bp.It seems the inverted terminal repeats determine the speciality of a functional transposase.In the negative control of excision assay,the excision of donor pHB[KOα]in the absence of piggyBac transposase in S2 cell culture led us to propose that S2 cells contains related factors that could cross-mobilized with piggyBac vector.This phenomenon is worthy of further study.更多还原