拟黑多刺蚁homothorax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】 吴静；

【导师】 奚耕思；

【作者基本信息】 陕西师范大学 ， 发育生物学， 2010， 硕士

【摘要】 拟黑多刺蚁(Polyrhachis vicina Roger)隶属于膜翅目(Hymenoptera),蚁科(Formicidae),多刺蚁属(Polyrhachis),属于典型的营群居真社会性昆虫。具有广泛的多态现象,品级分化为工蚁、雌蚁(蚁后)和雄蚁，具有行为复杂和分工明确的特征。是研究昆虫发育机制方面较好的材料。Homothorax (Hth)是Meis家族中的成员,是Hox基因的辅助因子,主要存在于节肢动物中,对胚胎和成虫的发育起着重要的作用,它与附肢、中枢和外周神经系统、眼、唾液腺等发育密切相关。本论文以拟黑多刺蚁为研究材料,提取总RNA,采用RT-PCR及RACE技术从拟黑多刺蚁体内克隆出Hth基因的全长cDNA序列,并利用生物信息学方法对其核苷酸序列和预测的蛋白序列进行分析；通过荧光实时定量PCR方法对Hth基因在拟黑多刺蚁体内的mRNA水平的相对定量表达情况进行了研究。研究结果如下：1.基因克隆：拟黑多刺蚁Hth基因cDNA序列全长为2111bp,包含一个长度为1257 bp的开放阅读框。5’端非编码区(5’-UTR)和3’端非编码区(3’-UTR)长度分别为290 bp和567bp,3’-UTR区可见典型的加尾信号AATAAA以及长度为17 bp的PolyA尾。编码含有418个氨基酸残基的蛋白质,该蛋白质分子量和等电点分别为45.2 kDa和5.65。将该基因命名为Pv-Hth,所获序列上传到GenBank,获得的序列号为GU324093。2.序列分析：同源性分析表明Pv-Hth与意大利蜜蜂Hth的氨基酸序列相似性达98.2%,与脊椎动物Meis家族成员的相似性在62.7%-79.4%之间。生物信息学分析表明,该蛋白具有同源异形结构域,含有1个N-糖基化位点、4个蛋白激酶C磷酸化位点、9个酪蛋白激酶Ⅱ磷酸化位点、2个酪氨酸激酶磷酸化位点、1个酰胺位点和8个N-豆蔻酰化位点,这些位点为研究该蛋白质的功能提供了信息。3.相对定量研究：采用荧光实时定量PCR方法对拟黑多刺蚁发育的不同阶段虫体、不同品级成虫整体及不同品级成虫头部的Pv-Hth在mRNA表达水平上进行相对定量分析。结果显示,Pv-Hth在除了蛹期的发育阶段均有表达,胚胎期表达量最高,幼虫期开始表达量显著下降,发育到第二龄幼虫时,表达量有小幅上升,但之后下降,三龄和四龄处于同一表达水平,蛹期停止表达。Pv-Hth基因从胚胎到蛹期表达的变化趋势与拟黑多刺蚁全变态发育的过程和特征密切相关,但是在蛹期停止表达的具体原因还有待于进一步的研究。Pv-Hth基因mRNA在拟黑多刺蚁三个品级成虫全身及头部中的表达情况是一致的,在雄蚁中表达量最高,工蚁次之,雌蚁最低。雄蚁全身及头部的表达量分别比工蚁全身及头部高2.6倍和5倍,比雌蚁全身及头部高6.5倍和9倍。Pv-Hth基因雄蚁中的高表达,一方面与其形态特征有关,另一方面可能与品级分化及相应的功能有关。本项研究首次从社会性昆虫拟黑多刺蚁中克隆Hth基因的全长cDNA序列,并将序列上传到GenBank;序列比对分析表明,Pv-Hth与脊椎动物的Meis家族成员、秀丽线虫Unc-62、节肢动物的Hth有很高的相似性,表明Meis家族基因在动物中的进化是很保守的；采用相对荧光实时定量方法研究发现,Pv-Hth mRNA在拟黑多刺蚁不同发育阶段虫体、不同品级成虫及不同品级成虫头部的表达情况有很大差异,提示Hth基因与拟黑多刺蚁的发育密切相关。这些结果将为深入探讨Hth在昆虫中的相关功能研究奠定基础。更多还原

【Abstract】 Plyhachis vicina Roger belongs to the genus Polyrhachis (Hymenoptera Formicida). It is a typical eusocial insects characterized by common polymorphism. The group mostly consists of three castes:worker, female (queen) and male. This species possess the characteristic of sophisticated behaviors and behavioral plasticity. Consequently, P. vicina is a good material for study the mechanisms of insect development.Homothorax (Hth), which belongs to the Meis family that present in invertebrates and is the Hox genes cofactor, plays important role in the development of appendage, central nervous system, peripheral nervous system, eyes and salivary gland during the embryo and adult development.In this paper, the full-length cDNA of Hth gene is coloned from the P. vicina by RT-PCR and RACE methods. The bioinformatics method is used to analyse characteristics of full-length cDNA and predict functional motifs in the ORF. The mRNA expression levels of Hth in P. vicina were investigated by real-time RT-PCR. The major experiment results are as follow:1. Cloning:The full-length cDNA of P. vicina homothorax, termed Pv-Hth, is 2111 bp, which contains an ORF of 1257 bp that encodes a 418-amino acid protein with a predicted molecular mass of 45.2 kDa and the theoretical pI of 5.65. The length of the 5’and 3’UTRs are 290 bp and 567 bp, respectively. A putative polyadenylation signal AATAAA was found at 19 bp upstream from the 17-nucleotide poly (A) tail in 3’-UTR. The nucleotide sequence of Pv-Hth gene is submitted to GenBank and assigned the accession number GU324093.2. Sequence analysis:The results of sequence alignments analysis showed that Pv-Hth shares 98.2% identity with Apis mellifera, and an overall identity of 62.7%-79.4% with the vertebrate Meis. Sequence analysis by bioinformaties showed that, there is a Homeodomain in the the Primary structure of the animo acid sequence of the Pv-Hth. A series of predicted function motifs are found in the Pv-Hth protein sequence, including one N-glycosylation site, four Protein kinase C phosphorylation sites, nine Casein kinaseⅡphosphorylation sites, eight N-myristoylation sites, and one Amidation site, these sites provide functional information of Pv-Hth.3. Relative quantification expression:The relative quantification expression of the mRNA level of Pv-Hth genes in different developmental periods and the different castes were analyzed using real-time quantitative RT-PCR. The results showed that Pv-Hth was expressed in all templates at different levels expect pupae stage. During development, the expression of Pv-Hth was highest in embryos and was zero in pupae. In the course of larval growth, the levels of expression of Pv-Hth transcripts gradually increased from the first to the second instar and then decreased noticeably in the third instar,then remained the same level in fourth instar.The distinct expression of Pv-Hth from embroy to pupa is related to the Metamorphosis of holometabolous insects, herefore,the result, that the undetectable level of Pv-Hth expression in the pupa, needs more research to determine the precise role of Pv-Hth in the pupa. In the three castes, Pv-Hth gene was found to be expressed at significant higher levels in males than in either workers or females no matter in the whole bodies or heads. The levels of Pv-Hth mRNA expression in males whole body and heads is 2.6 and 5 times more than workers,6.5 and 9 times more than females, respectively. In spite of the feature in the males, the high level of Pv-Hth transcript in the males may shed light on the important physiological function of Pv-Hth mRNA in that caste.The full-length cDNAs of Pv-Hth genes have been cloned from the eusocial insect, P. vicina for the first time, and submitted the cDNA sequences to GenBank. Alignment analysis manifests that Pv-Hth has a high similarity to the vertebrate MEIS family and Caenorhadits elegans Unc-62, as well as arthropods Hth. It is indicated that the evolutionary pathways of MEIS genes in animals is similar. The result from real-time quantitative RT-PCR analysis revealed that Pv-Hth express at distinct developmental stages, whole body and heads of different castes, which revealed that Pv-Hth maybe related with the ant development. These results may provide the theory foundation for further researching on the concrete function of Pv-Hth in insects.更多还原