Molecular Characterization Of PBAN And JHAMT And Their Effect On Pheromone Biosynthesis From The Seabuckthorn Carpenterworm, Holcocerus Hippophaecolus(Lepidoptera:Cossidae)

In many moth species, females emit a kind of species-specific pheromones to attract conspecific males for mating. Whether successful mating in insect or not is greatly determined by pheromones. Because pheromones are species specific, they become good material, which are used to study evolution of species, reproduction isolation and ecological stability. The synthesis of pheromone is regulated by the series of internal mechanism, especially pheromone biosynthesis activating nuropeptide (PBAN) and juvenile hormone (JH). Juvenile hormone acid methyltransferase (JHMAT) is the key regulatory enzyme for JH synthesis. We could understand the physiological role of JH by the research of JHAMT gene. The article mainly researched the molecular characterization of pest Holcocerus hippophaecolus DH-PBAN and JHAMT by the molecular biology methods of gene sequencing and quantitative PCR. We reported the full-lenth of these two genes for the first time. Therefore, the aim of study is to elucidate the role of JH and PBAN in the regulation of sex pheromone production. The results of these studies were as follows:(i) The full-length cDNA of Hh-DH-PBAN was790bp and contained five peptides:diapause hormone (DH) homolog, a-neuropeptide (NP),β-NP, PBAN, and y-NP. All of the peptides were amidated at their C-terminus and shared a conserved motif, FXPR (or K) L. Moreover, multiple alignments and phylogenetic analysis confimed that Hh-DH-PBAN was closely related to PBANs from Noctuidae.(ii) Real-time quantitative PCR (qRT-PCR) analysis showed that Hh-DH-PBAN mRNA expression peaked in the brain-subesophageal ganglion (Br-SOG) complex, and was also detected at high levels during larval and adult stages. The expression decreased significantly after pupation. These expression profiles suggested that the Hh-DH-PBAN gene might be correlated with larval development and sex pheromone biosynthesis in females of the H. hippophaecolus.(iii) The full-length cDNA of Hh-JHAMT was1659bp. This sequence contained a highly conserved Motif I, which was SAM well-conserved motif I. The result suggested Hh-JHAMT was a member of SAM-dependent MTases. Moreover, multiple alignments and phylogenetic analysis revealed that Hh-JHAMT was closely related to JHAMT from Noctuidae.(iiii) Real-time quantitative PCR (qRT-PCR) analysis showed that JHAMT level was gradually declining during larval, JHAMT mRNA level was not detected during the last instar and the whole pupation. However, JHAMT mRNA was increased to a higher level during adult stages. This mode of JHAMT expression was similar to the mode of JH biosynthesis expression. The result suggested JHAMT was the key regulatory enzyme for JH synthesis. These results of molecular characterization and phylogenetic analysis were provided the molecular basis for effect of JH on insect larvae growth, metamorphosis and adult reproduction.