Quasi Black Prickly Ant Tyrosine Hydroxylase (th) And Dopa Decarboxylase (ddc), Clone And Express Mrna Level In Development

As an important neurotransmitter in hypothalamus and pituitary gland, dopamine is widely expressed in vertebrates and invertebrates. Lacking dopamine will cause people lose the ability to control muscles, even to get Parkinson when serious. Dopaminergic neurons can ingest L-tyrosine in the blood. In the cytoplasm, normally L-tyrosine can be catalyzed to L-dihydroxyphenylalanine(L-dopa) by tyrosine hydroxylase(TH), and then L-dopa will be catalyzed to dopamine by dopa decarboxylase(DDC).Polyrhachis vicina Roger, which belongs to genus Polyrhachis(Hymenoptera:Formicidae), is a species of typical eusocial insect with the characteristic of castes differentiation, sophisticated behaviors and behavioral plasticity. It is a good material for studying the mechanisms of insect development and behavior.In this thesis, the full-length cDNA of TH and DDC gene are cloned from P. vicina by RT-PCR and RACE methods for the first time; the mRNA expression levels of PvTH and PvDDC are investigated by real-time RT-PCR and in situ hybridization. Y maze trained and untrained workers are also investigated by the two methods. The results of investigations are as follows:1. The full-length cDNA of PvTH is2429bp, which contains an open reading frame of1713bp. The5’and3’-untranslated regions are357and359bp long, respectively. The open reading frame encodes a deduced570amino-acid peptide with a calculated molecular mass of65.09kilodaltons and an isoelectric point of5.26. The results of sequence alignments analysis showed that this protein shares70%identity with Nasonia vitripennis, Apis mellifera, Tribolium castaneum and Bombyx mori, about50%identity with the vertebrate THs. By bioinformatics technology, many functional information of TH primary structure and the predicted functional areas of the conserved domain are highly conserved with other species. These results indicate that the newly isolated cDNA sequence encodes the ant TH protein. The full-length cDNA sequence of the PvTH was submitted to GenBank. The accession number is JF499654.2. Real-time quantitative reverse transcription polymerase chain reaction is used for comparing the expression during P. vicina development, the whole bodies of different castes, and the heads of different castes and for the trained and untrained workers’heads. The result shows that PvTH mRNA is differentially expressed in the ant development, in whole bodies and the heads of different castes. During the development, the highest expression level is in pupa. Among castes, the highest level is in workers. In the heads, the heads of females and males are much higher than in workers. The expression of the heads of trained workers is higher than untrained. The result may be related to the complex physiological function of dopamine in different body location.3. The prominent positive reactions are found in the optic lobes, the olfactory lobe and the Keyron cells of mushroom body. The relatively higher expression levels are found in the optic lobes of males brain, the central complex body of females and males brain, the olfactory lobe and mushroom body of worker’brain. The trained worker brain of the optic lobes, olfactory lobe and central complex body show more prominent positive reactions than the untrained worker brain. The results show that TH is related with the behavior of different castes and in learning and memory.4. The full length cDNA of PvDDC is1893base pairs and contains a5’-untranslated region71nucleotides and3’-UTR379bp long. The open reading frame of PvDDC encodes a deduced480-amino acid peptide. The protein shares70%identity with Apis mellifera, Nasonia vitripennis and Tribolium castaneum. The functional motifs of the DDC primary structure and the predicted functional areas of the conserved domain are highly conserved with other species. The cDNA sequence was submitted to the GenBank and named PvDDC. The accession number is JN979785.5. By real-time RT-PCR, the result shows that the highest expression level is in pupa. The levels also vary among castes; the highest level is in males. In the heads, the heads of males are also highest. The expression is higher in trained workers’heads than in untrained. It maybe related with the expression of dopamine and5-hydroxy tryptamine(5-HT) in different castes and developments.6. The distribution of PvDDC mRNA is analyzed by in situ hybridization:the prominent positive reactions appear in the Keyron cells, the optic lobes and central complex body. The relatively higher expression levels are in the olfactory lobe and in the optic lobes of males’brain. The higher prominent positive reaction is in the coronula and pedunculus of mushroom body of trained workers’heads than untrained. We have cloned the full-length cDNAs of TH and DDC genes from P. vicina for the first time, and submitted the sequences to GenBank. The results of real-time quantitative RT-PCR and in situ hybridization methods revel that TH and DDC are related with insects’development, nervous system and behavior. The results may be useful for further investigation on the functions of TH and DDC in insects.