Quasi Black Spiny Ant Myocyte Enhancer Factor 2 And Troponin I Subunit Gene Cloning And Expression In The Developing

The ant Polyrhachis vicina Roger is one of the important economic insects in China, which belongs to the genus of Polyrhachis (Hymenoptera, Formicidae). There are four main stages in the ontogenesis, described as embryo, larva, pupa and adult. Also because of the characteristic of caste differentiation, the ant is gradually becoming a special experimental material for the research of development and differentiation.Methods of RT-PCR, molecular cloning, bioinformatics and real-time quantitative PCR are first used to study the Mef2 and TnI of Polyrhachis vicina to understand the development or the physiological function of muscles. The main results are as follows:1) Two isoforms (isoform A and B) of PvMef2 cDNA sequence were obtained which are 2074bp and 1793bp respectively. The two sequences which are only different in 5'-UTR possess a same ORF of 1419bp encoding a 472-amino acid protein. All of the two sequences were submitted to the GenBank and assigned the accession numbers:GQ386849 and GQ386850.2) The deduced protein of PvMef2 holds highly conserved MADS and MEF2 domains which share about 100% identity with the corresponding regions of some other insects. Results of phylogenetic analysis show that PvMEF2 is most closely related to MEF2 of Apis mellifera and Nasonia vitripennis and the tree illustrates the evolutionary relationship of different selected animals.3) The theoretical isoelectric point (PI) and molecular weight of MEF2protein were 6.83 and 50191.5 Da respectively. Its chemical formula is C2168H3392N628O714S16, comprising 6918 atoms. Analysis of post-translational modification shows that there are 40 possible O-glycosylation sites,2 possible N-glycosylation sites and 42 phosphorylation sites including 30 Serine sites,6 threonine sites and 6 Tyrosine sites, and no signal peptide and transmembrane domain in the sequence.4) Prediction of protein structure shows that N-terminal of PvMEF2 contains helix, sheet and coil,which is related to the physiological function of PvMEF2 by mean of combining other factors.5) Results of real-time quantitative RT-PCR show expression levels of different stages change constantly with distinctly high levels in first instars relative to the following stages, while in the adult ants the PvMef2 gene was highly expressed in males compared with workers and females. Different expression levels will affect the regulation to different Mef2-regulated genes.The difference in expression levels of two PvMef2 mRNA isoforms can also be detected according to the results. Those results suggest that PvMef2 is developmentally and caste-specifically regulated at the level of transcription.6) Two isoforms (isoform A and B) of PvTnl cDNA sequence were obtained which are different in 3'-UTR. The two sequences of 2074bp and 1793bp in length contain 660bp and 615bp ORFs respectively, encoding 219 and 204 amino acid protein. All the sequences were submitted to the GenBank and assigned the accession numbers:HM484304 and HQ230044.7) The obtained Tnl protein sequences share high identity with some insects such as Apis mellifera. Phylogenetic tree shows that Polyrhachis vicina is grouped with Apis mellifera, and then they were grouped with other insects, showing the relationship among different groups. But the relationship among the protein subtypes of different species cannot be learned from the tree.8) There is closed physicochemical property between different TNI subtypes which is also similar to that of Apis mellifera. Few posttranslational modification sites could be found besides 1 O-glycosylation site and 11 phosphorylation sites, and no transmembrane domain was found.9) The secondary structure of the two TNI subtypes contains helix and coil, but no distinct sheet. And 3 helix regions can be found respectively in their tertiary structures.10) Expression of Tnl gene can be detected but different in developmental stages of Polyrhachis vicina by means of real-time quantitative RT-PCR. The expression level may be related to the muscular structure and their physiological function of different stages. But all need farther studies.The regulation of muscle differentiation and function may be related to Mef2 and Tnl in different developmental stages and different castes. Some theoretical foundation may be learned from our reports, but more researches are needed to investigate their specific mechanism and function.