Quasi Black Spiny Ant Muscarinic Acetylcholine Receptor Gene (machr) And Tumor Suppressor Genes (qm) Cloning And Expression Study

Polyrhachis vicina Roger belongs to the genus Polyrhachis(Hymenoptera:Formicidae).It is a species of typical eusocial insects characterized by castes differentiation,sophisticated behaviors and behavioral plasticity.Consequently,P.vicina is a good material for the studies of the mechanisms of insect development and behavior.This thesis contains two parts.1.The cloning and expression analysis of the muscarine acetylcholine receptor(mAchR) from P.vicina:The full-length cDNA of P.vicina mAchR (PvmAchR) was cloned using RT-PCR and RACE methods;the mRNA expression levels of PvmAchR in the ant were investigated by real-time RT-PCR and in situ hybridization methods.2. The cloning and expression analysis of a QM homologue from P.vicina:The full-length cDNA of P. vicina QM(PvQM) was cloned by RT-PCR and RACE methods;the mRNA expression levels of PvQM in the ant were investigated by real-time RT-PCR;and the PvQM prokaryotic expression vector was constructed by the prokaryotic expression vector pGEX-5X-1,and the specific expression of the GST-fused protein was detected by SDS-PAGE.The results of investigations are offered as follows:1.The full-length cDNA of PvmAchR is 2699 bp,containing a 5′-untraslated region(5′-UTR) of 189 bp and a 3′-UTR of 711 bp.The open reading frame(ORF) of PvmAchR is 1800 bp,which encodes a deduced 599-amino acid peptide with a predicted molecular mass of 67.3 kDa and with the theoretical pI of 9.11.The results of sequence alignments analysis showed that this protein shares 76.9%identity with Apis mellifera(Hymenoptera:Apidae) mAchR,and an overall identity of 45.0%-48.3%with the vertebrate mAchRs.Sequence analysis by bioinformatics showed that,there are a structure domain of seven-transmembrane helix and a G-protein coupled receptors signature in the primary structure of the animo acid sequence of the PvmAchR.These results indicate that the newly isolated cDNA sequence indeed encodes the ant mAchR protein.The full-length cDNA sequence of the PvmAchR was submitted to GenBank and assigned the accession number EU573239.2.Real-time quantitative RT-PCR analysis was performed to quantify the relative expression levels of PvmAchR transcripts at different developmental stages,in adults as well as in the heads of three castes.The results showed that PvmAchR mRNA is expressed in all samples tested at different levels.The expression of PvmAchR is higher in eggs than in larval stages,and the highest level is found in the adults of three castes.In the adults,the PvmAchR expressed highest in workers,and there is no significant difference between the male and female ants.The expression analysis among the heads of the three castes showed that the expression levels are higher in the heads of workers and females than in that of males.From these results,we speculated that the main physiological functions of PvmAchR in the ant are involved in regulating the behaviors or other physiological activities in adults.The variant expression levels of PvmAchR mRNA in different castes and in the heads of different castes may reflect the functions of PvmAchR in behaviors of different caste.3.The distribution of PvmAchR mRNA was analyzed by in situ hybridization to cryosections of the brain of adult castes.The results showed that the hybridization signals are present extensively in the brains of three castes.The calyx and collaret of the mushroom bodies are weakly stained.And the prominent positive reactions appeared in the Keyron cells,the optic lobes and deutocerebrum, which may reflect the functions of PvmAchR involved in obtaining and integrating the visual and olfaction information in the nervous system.In the three castes,the relatively higher expression levels are found in the deutocerebrum of workers'brain as well as in the optic lobes of males'brain, which may be relevant with their functions in social community.4.The full-length cDNA of PvQM is 827 bp,containing a 5′-UTR of 91 bp and a 3′-UTR of 77 bp.The ORF of PvQM is 660 bp,encodes a deduced 219-amino acid peptide with a predicted molecular mass of 25.1 kDa,and the theoretical pI is 9.93.The result of sequence alignments indicate that PvQM protein shares 98.2%identity with M.mellifera QM,and the lowest identity with the vertebrate QM homologues is more than 77.2%.The phylogenetic tree showed that PvQM is clustered with QMs from other Hymenoptera,and is most closely related to the A.mellifera QM. Sequence analysis by bioinformatics showed that,there are a series of predicted functional motifs in the PvQM protein sequence,and multiple functional motifs are well conserved in all QM homolgues. The full-length cDNA sequence of PvQM was submitted to GenBank and assigned the accession number EU360768.5.The PvQM prokaryotic expression vector was constructed by the prokaryotic expression vector pGEX-5X-1,and the recombined plasmid was transfected into E.coli BL21.After induction by IPTG,the specific expression of the GST-fused protein was detected by SDS-PAGE.The results of SDS-PAGE showed that the molecular weight of the recombined protein is approximately 51 kDa, which is consistent with its theory molecular weight.And there is no interest protein expression in the negative control.Results analysis indicated that the expression products exist mainly in a dissolvability manner and a small quantity of them is in inclusion body.6.Expression levels of PvQM mRNA at different developmental stages,in adults as well as in the heads of three castes were detected by real-time quantitative RT-PCR.PvQM mRNA is expressed in all samples at different levels.The expression level is highest in embryos and lowest in late larvae and pupae.During larval growth,PvQM mRNA levels are gradually increased from the first instar till third instar,while decreased significantly in the fourth instar.The PvQM gene is found to be expressed at higher levels in workers as well as in heads of workers,compared to both in the male and female ants.The highest expression levels in embryos suggest that PvQM may be related with cell proliferation and differentiation during the ant development.And in adults,the higher level of PvQM mRNA expression in the workers may highlight the important physiology function of PvQM in that caste.For the first time,we have cloned the full-length cDNAs of mAchR and QM genes from the eusocial insect,P.vicina,and submitted the cDNA sequences to GenBank.The results of real-time quantitative RT-PCR and in situ hybridization methods revealed that the PvmAchR mRNA was found in all developmental stages and all castes.The higher expression levels in adults and the differential expression in heads of different castes revealed the main physiological functions of PvmAchR in the ant are involved in regulating the behaviors in adults.PvQM was also expressed in all developmental stages and all castes,the highest expression levels in eggs and the differential expression in different developmental stages revealed that PvQM may be related with the ant development.The PvQM prokaryotic expression vector was successfully constructed,which contributed to the further analysis of PvQM protein.These results may provide the theory foundation for further researching on the concrete function of mAchR and QM in insects.