Intended Black Spiny Ant Homothorax Gene Cloning And Its Mrna Levels In The Ontogeny Of Expression Quantitative Research

Plyhachis vicina Roger belongs to the genus Polyrhachis (Hymenoptera Formicida). It is a typical eusocial insects characterized by common polymorphism. The group mostly consists of three castes:worker, female (queen) and male. This species possess the characteristic of sophisticated behaviors and behavioral plasticity. Consequently, P. vicina is a good material for study the mechanisms of insect development.Homothorax (Hth), which belongs to the Meis family that present in invertebrates and is the Hox genes cofactor, plays important role in the development of appendage, central nervous system, peripheral nervous system, eyes and salivary gland during the embryo and adult development.In this paper, the full-length cDNA of Hth 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 Hth in P. vicina were investigated by real-time RT-PCR. The major experiment results are as follow:1. Cloning:The full-length cDNA of P. vicina homothorax, termed Pv-Hth, is 2111 bp, which contains an ORF of 1257 bp that encodes a 418-amino acid protein with a predicted molecular mass of 45.2 kDa and the theoretical pI of 5.65. The length of the 5'and 3'UTRs are 290 bp and 567 bp, respectively. A putative polyadenylation signal AATAAA was found at 19 bp upstream from the 17-nucleotide poly (A) tail in 3'-UTR. The nucleotide sequence of Pv-Hth gene is submitted to GenBank and assigned the accession number GU324093.2. Sequence analysis:The results of sequence alignments analysis showed that Pv-Hth shares 98.2% identity with Apis mellifera, and an overall identity of 62.7%-79.4% with the vertebrate Meis. Sequence analysis by bioinformaties showed that, there is a Homeodomain in the the Primary structure of the animo acid sequence of the Pv-Hth. A series of predicted function motifs are found in the Pv-Hth protein sequence, including one N-glycosylation site, four Protein kinase C phosphorylation sites, nine Casein kinaseⅡphosphorylation sites, eight N-myristoylation sites, and one Amidation site, these sites provide functional information of Pv-Hth.3. Relative quantification expression:The relative quantification expression of the mRNA level of Pv-Hth genes in different developmental periods and the different castes were analyzed using real-time quantitative RT-PCR. The results showed that Pv-Hth was expressed in all templates at different levels expect pupae stage. During development, the expression of Pv-Hth was highest in embryos and was zero in pupae. In the course of larval growth, the levels of expression of Pv-Hth transcripts gradually increased from the first to the second instar and then decreased noticeably in the third instar,then remained the same level in fourth instar.The distinct expression of Pv-Hth from embroy to pupa is related to the Metamorphosis of holometabolous insects, herefore,the result, that the undetectable level of Pv-Hth expression in the pupa, needs more research to determine the precise role of Pv-Hth in the pupa. In the three castes, Pv-Hth gene was found to be expressed at significant higher levels in males than in either workers or females no matter in the whole bodies or heads. The levels of Pv-Hth mRNA expression in males whole body and heads is 2.6 and 5 times more than workers,6.5 and 9 times more than females, respectively. In spite of the feature in the males, the high level of Pv-Hth transcript in the males may shed light on the important physiological function of Pv-Hth mRNA in that caste.The full-length cDNAs of Pv-Hth genes have been cloned from the eusocial insect, P. vicina for the first time, and submitted the cDNA sequences to GenBank. Alignment analysis manifests that Pv-Hth has a high similarity to the vertebrate MEIS family and Caenorhadits elegans Unc-62, as well as arthropods Hth. It is indicated that the evolutionary pathways of MEIS genes in animals is similar. The result from real-time quantitative RT-PCR analysis revealed that Pv-Hth express at distinct developmental stages, whole body and heads of different castes, which revealed that Pv-Hth maybe related with the ant development. These results may provide the theory foundation for further researching on the concrete function of Pv-Hth in insects.