拟黑多刺蚁Semaphorin 2a基因mRNA水平的定位和Cathepsin-D基因的克隆研究

【作者】 梁开丹；

【导师】 奚耕思；

【作者基本信息】 陕西师范大学 ， 动物学， 2011， 硕士

【摘要】 Semaphorin 2a (sema)是Semaphorin家族的第二亚群,是一种分泌型蛋白,广泛存在于无脊椎动物中,此基因作为一个神经抑制因子在神经连接以及中枢神经轴突导向中发挥着重要作用。该基因的长度为2763 bp,最长阅读框为2151 bp,编码716个氨基酸,5’端非编码区(5’-UTR)长为92 bp,3’端非编码区(3’-UTR)长为521 bp。该基因编码的蛋白质相对分子量是81.1 kDa,理论等电点为7.05,通过分析发现该蛋白含有信号肽,是一种分泌型蛋白。组织蛋白酶D(Cathepsin-D)是一种天冬氨类溶酶体的肽链内切酶,在酸性pH中可由雌激素诱导产生。在哺乳动物中,Cathepsin-D与肿瘤的发生、发展密切相关,因而被当作是肿瘤侵袭性指标,倍受关注。在昆虫中,Cathepsin-D作为蛋白水解酶参与生物胚胎发育过程,特别对昆虫的变态发育有十分重要的作用。拟黑多刺蚁(Polyrhachis vicina Roger)隶属于膜翅目(Hymenoptera),蚁科(Formicidae),是一类典型的营群居生活的社会性昆虫,同时也是一种重要的资源昆虫。该昆虫具有品级分化、分工明确、社会行为复杂、神经系统高度复杂等特点,因此是研究昆虫生长发育、行为调控机制及神经功能等方面的很好材料。本研究以拟黑多刺蚁为实验材料,应用原位杂交技术对Semaphorin 2a基因的mRNA表达水平在拟黑多刺蚁不同品级的头部进行定位；对拟黑多刺蚁工蚁行为训练后头部进行该基因：nRNA表达水平原位杂交定位研究,并与训练前的结果进行比较；采用RT-PCR,5’与3’RACE技术,克隆Cathepsin-D基因的全长cDNA序列；利用生物信息学方法对Cathepsin-D基因的核苷酸序列和蛋白质序列进行了分析和预测。研究结果如下：1.采用原位杂交组织化学方法对拟黑多刺蚁不同品级的头部Semaphorin 2a mRNA的表达进行定位研究。结果表明Semaphorin 2a在拟黑多刺蚁的脑部广泛表达,在蕈形体、视叶以及中央复合体都具有阳性反应,说明此基因参与了昆虫的学习记忆、嗅觉、视觉和运动行为,此基因作为一个神经抑制因子对昆虫的神经系统起着重要的作用。在不同品级的蕈形体中雌蚁表达量相对较高,视叶中工蚁表达量最高,推测Semaphorin 2a基因在蚂蚁不同品级同一脑部区域表达量的差异与不同品级的蚂蚁在社会中的行为和职责有关。2.利用Y型迷宫对拟黑多刺蚁的工蚁进行训练,训练后采用原位杂交组织化学法对拟黑多刺蚁工蚁头部进行Semaphorin 2a基因mRNA水平的定位研究。结果表明经过训练的蚂蚁Semaphorin 2a的表达量明显低于训练之前的,推测可能是由于Semaphorin 2a是一种神经抑制因子,而学习记忆与突触元的可塑性有关,在对拟黑多刺蚁训练的过程中,突触结构以及数目发生了改变,Semaphorin 2a对突触的抑制作用减弱,因此经训练后工蚁头部的表达量降低。3.采用RT-PCR和RACE技术获得了拟黑多刺蚁的Cathepsin-D基因,全长1655bp,最长阅读框为1155 bp,编码384个氨基酸,5’端非编码区(5’-UTR)长为153bp,3’端非编码区(3’-UTR)长为341 bp。该基因编码的蛋白质相对分子量是41.6kDa,理论等电点为6.25,属于疏水性蛋白质,并且分析发现该蛋白含有信号肽,是一种分泌型蛋白。同源性分析显示该蛋白与预测的意大利蜜蜂相似性为87%,与桑天牛相似性为73%,与拟谷盗相似性为72%,与果蝇相似性为69%,均大于30%。因此可确定本次实验获得的序列即为拟黑多刺蚁Cathepsin-D基因,命名为Pv-cath-D,并将序列上传至GenBank,获得该基因的序列号为JF759824。本项研究首次采用原位杂交组织化学法对拟黑多刺蚁不同品级头部Semaphorin 2a基因进行mRNA表达水平定位研究,并对工蚁经过行为训练后采用原位杂交对头部进行mRNA表达水平定位研究,结果发现训练后的拟黑多刺蚁头部的表达量明显低于未训练蚂蚁头部的表达量；从拟黑多刺蚁中首次克隆出组织蛋白酶D(Cathepsin-D)基因的全长cDNA序列,应用生物信息学技术对拟黑多刺蚁中的Cathepsin-D基因进行生物学分析。这些研究结果将为更加深入研究昆虫Semaphorin 2a基因和Cathepsin-D基因功能奠定一定的基础。更多还原

【Abstract】 Polyrhachis vicina Roger as eusocial insects belongs to the genus Polyrhachis (Hymenoptera;Formicidae),which is an important resource insect. This species has the different castes, highly complex nervous system and social behavior. P. vicina is a good material which is used for study the insect development, behavior and neurological function.Semaphorin 2a (sema) belong to the second sub-group of Semaphorin family which is a a secreted protein exists in invertebrates. This gene as a neural inhibitory factor plays an important role in the neural connections and axon guidance in the central nervous system.Cathepsin-D is a aspartic endopeptidase,which is produced by estrogen in the acidic pH. Cathepsin-D is related with tumor development in mammals, which has been regarded as indicators of tumor aggressiveness. In insects, Cathepsin-D as a proteolytic enzyme is involved in the biological process of embryonic development, especially posesses a very important functionin in the metamorphosis of insect.In this paper, the mRNA expression of Semaphorin 2a were investigated by in situ hybridization in the untrained ant and the trained worker of P. vicina; Cathepsin-D full-length cDNA sequence was cloned through the ways of RT-PCR and 5’and 3’RACE technology, and the analysis and forcast of the nucleotide sequence and protein Sequences were completed by used the bioinformatics methods. These results are as follows:l.The localization expression.of Semaphorin 2a mRNA is studied in the head of different castes by in situ hybridization. The results show that Semaphorin 2a are widely expressed in the brain of P. vicina, indicating that this gene is involved in the learning and memory, olfactory, visual and behavior. This gene as a neural inhibitory factor play an important role on the nervous system of insects.The expression have different level in different castes,so we suggested that the difference expression of semaphorin 2a in the same brain region of different castes was contacted with the behavior and duties of the three castes.2.The expression of Semaphorin 2a was investigated by in situ hybridization in the trained workers of Polyrhachis vicina which were trained by Y-maze. The results show that the expression of Semaphorin 2a in the trained ants was significantly reduced. We analyzed the number and structure of synaptic had changed when the workers was trained and the inhibition of synaptic was weaken, so the expression in the workers’ head was reduced.3.The Cathepsin-D full-length of P. vicina was obtained by RT-PCR and RACE techniques.The full-length is 1655bp,and the longest reading frame is 1155bp, encoding 384 amino acids,also,5’untranslated region (5’-UTR) is 153bp and 3’untranslated region (3’-UTR) is 341bp.The corresponding protein containing a signal peptide is a secreted protein with a predicted molecular mass of 41.6kDa and with the theoretical pI of 6.25.The analysis of homology showed that the full length cDNA of Cathepsin-D has similarity of Apis 87%, Apriona 73%, castaneum 72% and Drosophila 69%, all were more than 30%. Consequently, the sequence of this gene named Pv-cath-D indeed encodes the ant CTSD protein, submitted to GenBank and assigned the accession number JF759824.We have investigated the mRNA expression of level of Semaphorin 2a by in situ hybridization in different castes and the trained worker of P. vicina for the first time, and cloned the full-length cDNA of Cathepsin-D from P. vicina, also analysed the nucleotide sequence and protein sequences through the bioinformatics methods. These results will provide the basis for further research of Semaphorin 2a and Cathepsin-D in insects.更多还原