拟黑多刺蚁semaphorin 2a基因的克隆与mRNA水平表达的研究

【作者】 罗晶；

【导师】 奚耕思；

【作者基本信息】 陕西师范大学 ， 发育生物学， 2009， 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),是一类营群居的典型的社会性昆虫,同时也是一类重要的资源昆虫。该种昆虫具有独特的品级分化、行为复杂、分工明确等社会性特征以及高度复杂的神经系统,因此是研究昆虫发育、行为调控机制及神经功能方面的很好材料。Semaphoring 2a(sema2a)是semaphorin家族中的第二亚家族中的的分泌蛋白,曾经在果蝇(Drosophila melanogaster Meigen)、沙漠蝗虫(Schistocerca gregaria Forskal)、蟋蟀(Gryllusbimaculatus De Geer)中发现,该蛋白可以作为选择性靶标抑制突触的形成。本论文以拟黑多刺蚁为研究材料。通过RT-PCR及RACE技术从拟黑多刺蚁体内克隆sema2a基因的全长cDNA序列,并对其核苷酸序列利蛋白序列进行生物信息学分析;通过实时定量RT-PCR方法对sema2a基因在拟黑多刺蚁体内的mRNA表达水平进行相对定量研究;研究结果如下:1.拟黑多刺蚁sema2a基因cDNA序列全长2763 bp,包含一个长度为2151 bp的开放阅读框,编码716个氨基酸残基。5’端非编码区(5’-UTR)长度为92 bp,3’-UTR为521 bp,3’-UTR区可见典型的加尾信号AATAAA及18bp长度的PolyA尾。编码的蛋白质分子量为81.1 kDa,等电点pI=7.05。同源性分析表明该蛋白与意大利蜜蜂mAchR的氨基酸序列相似性达98%,与金小蜂、拟谷盗、沙漠蝗虫和果蝇的氨基酸序列相似性分别为98%、95%、83%、71%和69%。生物信息学分析表明,该蛋白为分泌型蛋白,这一结论与报道一致。因此,可以确定本次克隆所获得序列为拟黑多刺蚁sema2a基因的全长cDNA序列,命名为Pv-sema-2a,上传GenBank后,获得的基因序列号为EU836737。2.采用荧光实时定量RT-PCR方法对拟黑多刺蚁各发育阶段虫体、不同品级成虫及不同品级成虫头部的Pv-sema-2a mRNA表达水平进行相对定量分析。结果表明,Pv-sema-2a基因在所检测的各个样本内均有表达,从整个发育阶段来说,从胚胎到第一龄幼虫,Pv-sema-2a的表达量有所增加,但随着幼虫的发育,Pv-sema-2a的表达量逐渐降低,直到蛹期,其表达量又再次升高。在三个品级成虫比较中,各品级之间的表达量有明显的不同,工蚁的表达量最高,雌蚁次之,在雄蚁成体中的表达量最低。在三个品级成虫头部比较中,雌蚁头中的表达量最高,其表达量为工蚁头的1.25倍,雄蚁头的表达量为工蚁头的0.26倍。Pv-sema-2a基因在胚胎和蛹中的高表达显示了该基因在蚂蚁发育中的作用。3.采用荧光实时定量RT-PCR方法对拟黑多刺蚁行为学训练后,已训练的拟黑多刺蚁和未训练的拟黑多刺蚁之间头部Pv-sema-2a mRNA表达水平进行相对定量分析。发现已训练的拟黑多刺蚁和未训练的拟黑多刺蚁之间头部Pv-sema-2a mRNA表达差异显著,未训练的拟黑多刺蚁头部的表达要比训练过的拟黑多刺蚁头部表达量高。由此可以推出Pv-sema-2a mRNA在经过训练的拟黑多刺蚁头部低表达可能有助于新的突触连接的建立,与增强学习和记忆有关。本项研究首次从社会性昆虫拟黑多刺蚁中克隆sema基因的全长cDNA序列,并将全长cDNA序列上传GenBank;采用相对实时定量方法研究发现,Pv-sema-2a mRNA在拟黑多刺蚁不通不同发育阶段虫体、不同品级成虫及不同品级成虫头部差异表达:并对成年拟黑多刺蚁工蚁进行行为训练,用相对实时定量的方法发现,未训练的拟黑多刺蚁头部的表达要比训练过的拟黑多刺蚁头部表达量高。这些研究结果将为深入探讨昆虫sema2a的相关研究奠定基础。更多还原

【Abstract】 Polyrhachis vicina Roger as eusocial insects belongs to the genus Polyrhachis(Hymenoptera: Formicidae).This species possess the characteristic of castes differentiation,sophisticated behaviors, behavioral plasticity and highly complex nervous system.At the same time,p.vicina is also a very important resource insect.Consequently,P.vicina as a good material is used for study the mechanisms of insect development,behavior,and the function of insects’nerve system.Semaphorin 2a belongs to the secreted members of the semaphorin family identified as class 2 and only present in invertebrates,which has been identified in the fruit fly(Drosophila melanogaster Meigen),grasshopper(Schistocerca gregaria Forskal),and Cricket(Gryllus bimaculatus De Geer). Semaphorin 2a may be acting the selective target to inhibiting synaptic arborization formation.In this paper,the full-length cDNA of sema2a 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 sema2a in the ant were investigated by real-time RT-PCR.The major experiment results are as follow:1.The full-length cDNA of Pv-sema-2a is 2763 bp,including an open reading frame(ORF) of 2151 bp,which encodes a deduced 599-amino acid peptide with a predicted molecular mass of 81.1 kDa and with the theoretical pI of 7.05,which contains a 5’-untraslated region(5’-UTR) of 92 bp and a 3’-UTR of 521 bp,and a putative polyadenylation signal AATAAA was found at 18 bp upstream from the 16-nucleotide poly(A) tail in 3’-UTR.The results of sequence alignments analysis showed that full length cDNA of Pv-sema-2a is similar to Apis mellifera,Nasonia vitripennis,Tribolium castaneum,Schistocerca gregaria and Drosophila melanogaster at 98%,95%,83%,71%and 69%. From the results of bioinformation analysis show that this protein is a secreted member,this is identical with previous research.The nucleotide sequence of Pv-sema-2a gene is submitted to GenBank and assigned the number EU836737.2.The relative quantification expression of the mRNA level of Pv-sema-2a genes in different developmental periods and the different castes were analyzed using real-time quantitative RT-PCR. The results showed that Pv-sema-2a mRNA is expressed in all samples tested.During development, the expression of Pv-sema-2a in first instar higher than embryos,and then declined gradually from the second instar to the fourth instar,but in pupa expression increased again,In three castes,the Pv-sema-2a expression was higest in worker ants,then female ants,lowest in male.The Pv-sema-2a mRNA expression in the heads of different castes from height to low was female,worker and male ants.The high mRNA levels in both the embryo and pupa suggest the essential role of Pv-sema-2a in ant development.3.To obtain information on whether sema2a associated with learning and memory,we analyzed Pv-sema-2a expression levels from the trained ants and untrained ants.We found Pv-sema-2a was expressed at a greatly lower in the trained ants than that in untrained ants.Consequently,the low expression of Pv-sema-2a may be beneficial in establishing these new connections.For the first time,we have cloned the full-length cDNAs of Pv-sema-2a genes from the eusocial insect,P.vicina,and submitted the cDNA sequences to GenBank.Real-time quantitative RT-PCR analysis revealed that Pv-sema-2a express at distinct developmental stages,different castes,heads and between the trained and untrained ants.These results may provide the theory foundation for further researching on the concrete function of Pv-sema-2a in insects.更多还原