拟黑多刺蚁tailless基因的克隆及其在个体发育的mRNA水平表达的定量研究

【作者】 刘晓明；

【导师】 奚耕思；

【作者基本信息】 陕西师范大学 ， 动物学， 2010， 硕士

【摘要】 Tailless隶属于核受体家族第二亚家族,已经在多种无脊椎动物及脊椎动物如果蝇、家蝇、意大利蜜蜂、鼠类、人类中发现,该蛋白在胚胎发育早期调控分节作用及神经系统的生成。由于该受体的生理配基至今仍未被识别,tailless为孤儿核受体。拟黑多刺蚁(Polyrhachis vicina Roger),隶属于昆虫纲(Insecta)、膜翅目(Hymenoptera),蚁科(Formicidae),多刺蚁属(Polyrhachis),是一类典型的营群居性生活的社会性昆虫,同时,也是国家卫生部指定的药食兼用型资源昆虫。拟黑多刺蚁具有社会性昆虫的典型特征,具有广泛的多态现象,品级分化为工蚁、雌蚁和雄蚁,此外,此类昆虫还具有高度复杂的神经系统。综上所述,拟黑多刺蚁是研究昆虫发育的优良动物实验材料。本论文以拟黑多刺蚁为实验材料,主要内容分为三个部分：首先,利用RT-PCR与RACE等技术获得拟黑多刺蚁tailless基因的全长cDNA序列；其次,利用生物信息学等相关方法对拟黑多刺蚁tailless的核苷酸序列和蛋白序列及其相关信息进行预测、分析；最后,提取拟黑多刺蚁不同发育阶段的个体和成虫脑部等样品的总RNA,通过荧光实时定量RT-PCR方法研究tailless基因在拟黑多刺蚁不同发育阶段的个体、不同品级成虫整体及成虫脑部的mRNA表达水平是否存在差异。主要研究结果为：1.本实验材料中所克隆出来的拟黑多刺蚁tailless基因的全长cDNA序列为1643 bp。通过开放阅读框分析,发现其包含一个长度为1260bp的最大开放阅读框,所编码的蛋白含有419个氨基酸残基。该基因5’末端非编码区域序列长度为198 bp,3’末端存在一个序列长度为177bp的非编码区域,此外,3’末端非编码区域存在典型的AATAAA加尾信号及序列长度为18bp的PolyA尾。2.运用生物信息学等相关方法对拟黑多刺蚁tailless氨基酸序列进行分析后,得出tailless基因编码的蛋白质分子量大小为47.8 kDa,等电点pI为6.36。通过对该蛋白质进行同源性分析,显示该蛋白与意大利蜜蜂、赤拟谷盗、库蚊和果蝇的氨基酸序列相似性分别为88%、71%、70%和69%。生物信息学分析表明,该蛋白为非分泌型蛋白,这一结论与报道一致。因此,可以确定本次实验所获得的序列就是拟黑多刺蚁tailless基因的全长cDNA序列,将该序列命名为Pvtailless,并上传至GenBank,获得的基因序列号为HM064499。3.提取拟黑多刺蚁不同发育阶段的个体及成虫脑部等样品的总RNA,采用荧光实时定量RT-PCR方法相对定量研究tailless在拟黑多刺蚁不同发育阶段虫体、不同品级成虫及成虫脑部的mRNA表达水平。结果表明,拟黑多刺蚁tailless基因在全部待分析的样本内均有表达。具体表现为,在拟黑多刺蚁的发育过程中,tailless在拟黑多刺蚁胚胎阶段表达量较高,随着幼虫的发育,tailless的表达量逐渐降低,待幼虫发育至第4龄,其表达量又缓慢升高。成虫的表达量相对于幼虫阶段具有明显增加,但是仍然次于胚胎时期的表达量。从拟黑多刺蚁的三个品级成虫整体比较来看,三个品级之间的表达量有明显的差异,雄蚁的表达量最高。在比较不同品级脑部表达量时,雄蚁脑部的表达量最高,其表达量为工蚁脑部的1.35倍,雌蚁脑部的表达量为工蚁脑部的0.66倍。在拟黑多刺蚁的胚胎和不同品级成虫中,tailless基因的表达水平相对较高,表明该基因在蚂蚁的生长发育中可能扮演重要的角色。本次试验成功的获得了社会性昆虫拟黑多刺蚁的tailless基因的全长cDNA序列,并将该序列上传至GenBank;利用分子生物信息学相关方法研究了该基因的核苷酸序列和蛋白序列；并采用相对实时定量方法研究了tailless在拟黑多刺蚁中mRNA水平上的表达差异。实验所获得的研究结果可作为进一步探讨昆虫tailless的相关研究的理论基础。更多还原

【Abstract】 Polyrhachis vicina Roger belongs to the genus Polyrhachis (Hymenoptera:Formicidae). As a typical kind of eusocial insects, P.vicina possesses the characteristic of castes differentiation, sophisticated behaviors, behavioral plasticity and highly complex nervous system. Also, p.vicina is a very important resource insect. P.vicina is a good material to study the mechanisms of insect development.Tailless is a member of nuclear receptor family, presents in both invertebrates and vertebrates, such as human, Musculus, Drosophila melanogaster Meigen, Musca domestica, Apis mellifera. Tailless initially expresses in the embryonic termini, which is critical gene for normal pattern formation including development of the brain and formation of the nervous system. As an orphan nuclear receptor, the ligands of tailless have not been found up to now.In this study, the full-length cDNA of P.vicina tailless gene is coloned by RT-PCR and RACE methods. The bioinformatics method is used to analyze characteristics of full-length cDNA and predict functional motifs in the ORF. The mRNA expression levels of tailless in the ants were investigated by real-time RT-PCR. The major experiment results are as follow:1. The full-length cDNA of tailless in P.vicina is 1643 bp, containing an open reading frame of 1260 bp, which encodes a deduced 419-amino acid peptide with a predicted molecular mass of 47.8 kDa and with the theoretical pI of 6.36, which contains a 5’-untraslated region (5’-UTR) of 198 bp and a 3’-UTR of 177 bp, and a putative polyadenylation signal AATAA was found at 18 bp upstream from the 18-nucleotide poly(A) tail in 3’-UTR.2. The results of homologous analysis showed that the full length cDNA of tailless in P.vicina is similar to Apis mellifera, Tribolium castaneum, Culex quinquefasciatus and Drosophila melanogaster at 88%、71%、70% and 69%. Bioinformatics analysis showed that tailless protein in P.vicina belongs to a non-secreted member. The nucleotide sequence of tailless in P.vicina, named Pvtailless, is submitted to GenBank and assigned the number HM064499.3. In this study, a fluorescent real-time quantitative RT-PCR is used to analyze the relative quantification expression of the mRNA level of tailless gene in different developmental periods, different castes adults and different adults’brain. The results showed that tailless is expressed in each tested sample. During the development stage, the highest expression level is found in embryo, and declined gradually until the fourth instar. Then the expression of pupa increased again. Among three caste adults, the highest expresse of tailless is male ants. The tailless mRNA expression analysis among the heads of the three casts showed that the expression level from height to low was male, worker and female ants in turn. From these results,we speculate that the high mRNA levels in both the embryo and adults suggest the essential role of tailless in ant development.The full-length cDNA of tailless gene have been cloned from the eusocial insect, P. vicina for the first time, and submitted the full-length cDNA sequences to GenBank. Real-time quantitative RT-PCR analysis showed that the tailless mRNA was differentially expressed in ants’ developmental stages, different castes and heads. These results may provide the theory foundation for further researching on the concrete function of tailless in insects.更多还原