Molecular Cloning And Expression Analysis Of Adipokinetic Hormone Gene In The Adult Oriental Armyworm, Mythimna Separata(Walker)

The oriental armyworm, Mythimna separata(Walker), is one kind of typical, seasonal and long-distance migratory pests which may seriously threaten the food security in China. It is widely distributed in many countries of Asia and Oceania, and can migrate between the north and south of China several times every year. There has been one aggravating trend of the occurrence and damage of M. separata recently, which is due to the change of the agro-ecosystem and the enhanced adaptive capacity of the changeable environment in view of M. separata, especially the nationwide outbreak in 2012 and 2013. The difficulty on the control of M. separata is mainly on account of the long-distance migration habit, the dynamics of which stems from the energy metabolism. Adipokinetic hormone(AKH) is one neuropeptide, which is primarily synthesized, stored and released from the corpora cardiaca(CC) into the hemolymph. AKH peptide plays an important role in the aspect of energy metabolism, flight, reproduction, stress physiology, immune response, and so on. The flight muscle of M. separata is well-developed, which can mobilize the carbohydrate and lipid efficiently. However, the molecular mechanism about energy metabolism regulation of the oriental armyworm during migration has not yet been reported.The female adults of M. separata were served as research object in this study. The full-length cDNA sequences of AKH gene from M. separata were cloned by the reverse transcription PCR(RT-PCR) and rapid-amplification of cDNA ends(RACE), and the corresponding expression pattern was analyzed then by the real time quantitative PCR(qPCR) method. The main results are as follows:1. Two novel full-length cDNA sequences of AKH were obtained by RT-PCR and RACE, which were named as MsepAKH1(GenBank accession No: KP979739.1) and MsepAKH2(GenBank accession No: KX096711.1), respectively. The results showed that the cDNA of MsepAKH1 was 449 bp, which included an open reading frame(ORF) of 207 bp, encoding 68 amino acid residues, with the predicted molecular mass of 7.61 kD and theoretical isoelectric point of 8.46. MsepAKH2 was 668 bp in full-length, and the ORF was 216 bp, encoding 71 amino acid residues, with the predicted molecular mass of 7.78 kD and theoretical isoelectric point of 6.13. It was found that both MsepAKH1 and MsepAKH2 contained the typical structural characteristics of the known insectile AKH gene superfamily by the conserved domain search, and there were phenylalanine, tryptophan and glycine residues at position 4, 8 and 9 of the mature peptide, respectively.2. Amino acid sequences alignment and phylogenetic tree of MsepAKH1 and MsepAKH2 with AKHs from other insects were analyzed using bioinformatics software. The results indicated that MsepAKH1 shared a high amino acid identity above 53% with MsexAKH, HarmAKH1, CsupAKH1, PxylAKH1 from Manduca sexta, Helicoverpa armigera, Chilo suppressalis and Plutella xylostella, respectively. Furthermore, the maximum similarity was 56.9% between MsepAKH1 and MsexAKH. With regard to MsepAKH2, it had a high consistency of amino acid sequence above 45% with HarmAKH2, PxylAKH2, CsupAKH2, BmorAKH2 from H. armigera, P. xylostella, C. suppressalis, Bombyx mori, respectively. It could not be ignored that the supreme consistency between MsepAKH2 and HarmAKH2 was more than 83%. The phylogenetic analysis based on the amino acid sequences confirmed the obtained results of the sequences alignment, which was likely to reveal the evolutionary relationship to some extent.3. The temporal and spatial expression patterns of MsepAKH1 and MsepAKH2 were analyzed by qPCR method. The result of qPCR analysis showed that the relative expression levels of MsepAKH1 were significantly different in various tissues of the 1 day-old female adults. MsepAKH1 was mostly expressed in the head and flight muscle, while it was very lowly expressed in fat body, ovary and midgut of the newly emerged female adults. Compared with that in the different developmental stages, the relative expression levels of MsepAKH1 in the head of the 7 day-old female adults and the flight muscle of the 3 day-old female adults were significantly higher than the same tissues of other day-old female adults. The spatial expression pattern of MsepAKH2 was nearly identifical with MsepAKH1 in various tissues of the 1 day-old female adults. However, the highest peak of MsepAKH2 mRNA expression in head emerged at 7 day-old, and the 5 day-old flight muscle of female adults had significant difference in the relative expression levels with that in the same tissues of female adults during the other development stages.The full-length cDNA sequences of MsepAKH1 and MsepAKH2 from M. separata were cloned for the first time. Moreover, the temporal and spatial expression patterns of these two genes at the level of mRNA were made clear in the female adults. The results in this study may be beneficial for us to clarify the molecular mechanism of the flight energy metabolism of M. separata, such as the carbohydrate and lipid. This study can not only enrich our knowledge on the occurrence and regulation of migration in M. separata, but also lay the foundation for the further function study of MsepAKH1 and MsepAKH2. Besides that, this study can also provide a theory reference for the establishment of the genetic control system, aiming at realization the goal of the sustainable pest management for M. separata.