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

Polyrhachis vicina Roger belongs to the genus Polyrhachis (Hymenoptera:Formicidae). As a typical kind of eusocial insects, P.vicina possesses the characteristic of castes differentiation, sophisticated behaviors, behavioral plasticity and highly complex nervous system. Also, p.vicina is a very important resource insect. P.vicina is a good material to study the mechanisms of insect development.Tailless is a member of nuclear receptor family, presents in both invertebrates and vertebrates, such as human, Musculus, Drosophila melanogaster Meigen, Musca domestica, Apis mellifera. Tailless initially expresses in the embryonic termini, which is critical gene for normal pattern formation including development of the brain and formation of the nervous system. As an orphan nuclear receptor, the ligands of tailless have not been found up to now.In this study, the full-length cDNA of P.vicina tailless gene is coloned by RT-PCR and RACE methods. The bioinformatics method is used to analyze characteristics of full-length cDNA and predict functional motifs in the ORF. The mRNA expression levels of tailless in the ants were investigated by real-time RT-PCR. The major experiment results are as follow:1. The full-length cDNA of tailless in P.vicina is 1643 bp, containing an open reading frame of 1260 bp, which encodes a deduced 419-amino acid peptide with a predicted molecular mass of 47.8 kDa and with the theoretical pI of 6.36, which contains a 5'-untraslated region (5'-UTR) of 198 bp and a 3'-UTR of 177 bp, and a putative polyadenylation signal AATAA was found at 18 bp upstream from the 18-nucleotide poly(A) tail in 3'-UTR.2. The results of homologous analysis showed that the full length cDNA of tailless in P.vicina is similar to Apis mellifera, Tribolium castaneum, Culex quinquefasciatus and Drosophila melanogaster at 88%,71%,70% and 69%. Bioinformatics analysis showed that tailless protein in P.vicina belongs to a non-secreted member. The nucleotide sequence of tailless in P.vicina, named Pvtailless, is submitted to GenBank and assigned the number HM064499.3. In this study, a fluorescent real-time quantitative RT-PCR is used to analyze the relative quantification expression of the mRNA level of tailless gene in different developmental periods, different castes adults and different adults'brain. The results showed that tailless is expressed in each tested sample. During the development stage, the highest expression level is found in embryo, and declined gradually until the fourth instar. Then the expression of pupa increased again. Among three caste adults, the highest expresse of tailless is male ants. The tailless mRNA expression analysis among the heads of the three casts showed that the expression level from height to low was male, worker and female ants in turn. From these results,we speculate that the high mRNA levels in both the embryo and adults suggest the essential role of tailless in ant development.The full-length cDNA of tailless gene have been cloned from the eusocial insect, P. vicina for the first time, and submitted the full-length cDNA sequences to GenBank. Real-time quantitative RT-PCR analysis showed that the tailless mRNA was differentially expressed in ants' developmental stages, different castes and heads. These results may provide the theory foundation for further researching on the concrete function of tailless in insects.