| 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. |
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