Black Spiny Ant PAH And TPH CDNA Sequencing MRNA Gene Expression And Developmental Relevance Of The Proposed Gene

Aromatic amino acid hydroxylase (AAAH) plays a critical role in physiological action of aromatic amino acid metabolism. Aromatic amino acid hydroxlase include phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH). Phenylalanine hydroxylase widely exist in animals, plants and microorganism, which is a key and rate-limiting enzyme in Phe metabolism. Phenylalanine hydroxylase catalyzes the hydroxylation of phenylalanine to produce tyrosine, which is the only rate-limiting step in Phe normal catabolism in mammals. Phenylalanine hydroxylase gene is closely related to Phenylketonuria in human. In insects, phenylalanine hydroxylase gene is related to skin hardening, eye pigment synthesis and nervous activity in Drosophila and is related to immune response melanism in Aedes too. It is very important significance to make clear the physiological role and biological function of PvPAH gene in Polyrhachis vicina which as the model organisms representative of social insects. It is further important significance to research the economic value and medicinal value of Polyrhachis vicina. Meanwhile, it is of important reference value for PKU treatment in human.Tryptophan hydroxylase (TPH) is a key and rate-limiting enzyme in serotonin (5-HT) synthesis, its activity determines the content of serotonin, and becomes a good marker for5-HT synthesis in neurons. Serotonin distributes widely in nervous system of vertebrates and invertebrates, as well as participates in of modulators a series behaviors and development. Clinieally, polymorphism of tryptophan hydroxylase gene is associated with many mental disorders. In insets, the reports about tryptophan hydroxylase are a very little, but only in Drosophila. It is not only necessary that to understand the5-HT neurotransmitter systems and the mode of action in Polyrhachis vicina which as the model organisms representative of social insects, but also has an important value to research the5-HT neurotransmitter systems.Polyrhachis vicina Roger belongs to the genus Polyrhachis (Hymenoptera: Formicidae). It is a species of typical eusocial insects characterized by castes differentiation, sophisticated behaviors and behavioral plasticity. Consequently, P. vicina is a good material for the studies of the mechanisms of insect development and behavior. Based on the above, the clone, expression and development relevance of phenylalanine hydroxylase and tryptophan hydroxylase in Polyrhachis vicina had been done. The main results are as follows:1. The full-length cDNA of P.vicina phenylalanine hydroxylase gene (PvPAH) is1670bp, which contains an open reading frame of1344bp and encodes447amino-acid peptide. The5’-UTR is87bp and the3’-UTR is239bp. The cDNA sequence of PvPAH was submitted to GenBank and get the accession number is JQ782583.2. The full-length cDNA of P.vicina tryptophan hydroxylase gene (PvTPH) is1956bp, which contains an open reading frame of1620bp and encodes539amino-avid peptide. The5’-UTR is108bp and the3’-UTR is228bp. The cDNA sequence of the PvTPH was submitted to GenBank and gain the accession number is KC600559.3. Through the bioinformatics analsis, the result showed that the PvPAH’s relative molecular weight is51237.0Da and pI is6.12. There is no signal peptide of this protein, which belongs to the hydrophilic nonsecreted protein. The PvPAH include a4a-peroxyterahydropterin site that belongs to the aromatic amino avid family. The secondary structure of PvPAH was determined by bioinformatics software and the result is a kind of mix protein. Three-dimensional structure analysis displayed that the best match template is DIMER (2.00A). The PvPAH shares87%with Apis mellifer, about60%with vertebrate’s PAH. These results indicated that many functional information of PAH primary structure and the predicted functional areas of the conserved domain are highly conserved with other species.4. Through the bioinformatics analsis, the result showed that the PvTPH’s relative molecular weight is61590.3Da and pI is5.33. There are no signal peptide of the PvTPH, which belongs to the hydrophilic nonsecreted protein. The PvTPH includes a4a-peroxyterahydropterin site and belongs to the ATC structure domain of heme iron depend on the aromatic amino acid hydroxylase family. The secondary structure of PvTPH was determined by bioinformatics software and the result is a kind of mix protein. Three-dimensional structure analysis displayed that the best match template is MONOMER (1.71A). The PvPAH shares88%with Apis mellifer, about60%with vertebrate’s TPH. These results indicated that many functional information of TPH primary structure and the predicted functional areas of the conserved domain are highly conserved with other species.5. Using the RQ-PCR technology research the expression of PvPAH mRNA, the result showed as follows:the PvPAH mRNA expresses in every developmental stages of ant. but there are many differences in them. We guess that phenylalanine hydroxylase is the key enzyme for the synthesis of melanin, which plays an important role in insect immune defense mechanism. From egg to adult, the more increase of individual volume, the more quantity of melanin, and the PvPAH mRNA expression is increase. In adult, because of the workers have miscellaneous social division of labor and defense mechanism, so its expression is the highest.6. Tryptophan hydroxylase is the key enzyme for the synthesis of5-TH, which is an important neurotransmitter and participate in the animal’s learning and memory behavior. Using the RQ-PCR technology research the expression of PvTPH mRNA, the result showed as follows:from egg to the third instar, the PvTPH mRNA expression is a trend of increasing, so we guess the PvTPH mRNA expression is associated with the development of mercous tissue. But the PvTPH’s expression is decrease in the fourth instar, may be due to the neural tissue development completed during this period. In the pupal stage, this proptein’s expression is increase sharply, presumably because of complete metamorphosis insects’ the central nervous system is reconstruct in the pupal stage. In audlt, the highest expression is worker, which is associated with the labor of workers. The result shows that the females’ PvTPH mRNA expression is more than males’ may be due to the males need higher visual information and spatial discrimination in the mating process.