The Polyrhachis Barbed Ants Dopamine Transporter Gene Cloning And Expression Of Mrna Levels Associated With The Development Of Research

Dopamine belongs to a neurotransmitter which plays an important role in regulating human and animal's physiological, biological activities, development and behavior. The investigation had shown that there is a certain relationship between dopamine and dopamine transporter. Usually, physiological functions of dopamine are regulated by dopamine receptors which including postsynaptic membrane receptor and presynaptic membrane receptor. Especially, the function of DA is controlled by dopamine transporter, that can not be substituted by other ways.In this study, the dopamine transporter gene of P. vicina is cloned by the technology of RT-PCR and RACE. And the mRNA levels of Pvdat gene was performed by the quantitative real-time PCR in different developmental stages and grades. The results are as follows:(1) Pvdat is cloned by use the technology of RT-PCR and RACE. Full-length cDNA sequence of dopamine transporter of P. vicina is the sequence of 2520bp which encodes 634 amino acid residues including the length of 1905bp coding region,5 'untranslated region of 365bp,3'non-coding region of 250bp. Homology search shown that PvDAT is high similar to insects. Especially the Amino acids squence of PvDAT is 87% identical with Apis mellifera. The full-length cDNA sequence of P. vicina is named Pvdat, and was submitted to GenBank and we assigned the accession number: HQ898856.(2) Predicted amino acid sequence indicated that Pvdat gene encodes 634 amino acid residues of the protein. Homology analysis showed that predicted protein posses highly homologous with other insects, and PvDAT is more than 80% similarity to insects that have been found. Phylogenetic analysis of PvDAT revealed that this protein has high conservation and belongs to Na/Cl-ion-dependent transporter family. Phylogenetic tree indicated that P. vicina and Apis mellifera obviously belong to same cluster. We found that PvDAT has no O-glycosylation sites but two N-glycosylation sites and multiple phosphorylation sites. Molecular weight of PvDAT is 71650.3Da, and theoretical isoelectric point is 6.06. PvDAT as a lipophilic polarity protein has 14 transmembrane regions and signal peptide of N terminus. Secondary structure prediction showed that PvDAT belong to a multi-helix structure, folding and curling hybrid protein. Third structure prediction revealed that this protein contains the structure of bound cell membrane and function of molecular recognition.(3) Pvdat expression levels are detected by use the real-time quantitative RT-PCR in different developmental stages and in all grades. The results showed that Pvdat is expressed in all developmental stages, and it is significantly higher during embryo than larva and pupa. The mRNA level of third instar larvae is the lowest. These differences suggest that the Pvdat may be higher associated with embryo development than developments of the larvae. Expression levels in different grades are higher than embryo, and especially 2.5 times higher in workers. In ant queens, Pvdat expression levels significant decreased after amating flight. mRNA expression levels has significant difference between workers and queens (P<0.05). According to developmental and behavioral differences of different grades in P. vicina, our data suggest that dopamine transporter gene is associated with embryo development and social behavior, particularly worker behavior.