关键词: 松墨天牛, ATP合成酶, 基因表达, RT-qPCR, RACE

Abstract:

[Objective] Mitochondrial ATP synthase exists in the plasma membranes of bacteria, thylakoid membranes of chloroplasts and the inner membranes of mitochondria, and is described as a splendid molecular machine, and a key enzyme for energy conservation in mitochondria. The proton motive force generated across the membrane by electron flow is used to drive the ATP synthesis from ADP and inorganic phosphate. The Japanese pine sawyer, Monochamus alternatus Hope (Coleoptera: Cerambycidae), is a longicorn beetle that is notorious as a vector of the pinewood nematode, Bursaphelen chusxylophilus (Steiner etBuhrer) Nickle, which causes pine wilt disease. This beetle is widely distributed in East Asian countries, including Japan, China and South Korea, vectoring the pine wilt disease there. The objective of the present study is to provide molecular information and references for further researches on insect ATP synthase gene, and physiological and toxicological functions of the ATP synthase gene of M. alternatus. [Method] An expressed sequence tag (EST) from Monochamusalternatus cDNA Library was amplified by 3' rapid amplification of cDNA ends. The cloned gene was characterized by splicing with the EST, open reading frame(ORF)research and Blast homologous comparison. ProtParam tool and DNAMAN software were used to analyze the characteristics of deduced protein and construct phylogenetic tree, respectively. The expression characteristics of the cloned gene at different developmental stages and in different parts of adults and larval tissues of M. alternatus were analyzed by real time quantitative PCR. [Result] A cDNA with 1133bp length was cloned, containing a 89 bp at its 5'-UTR, a 294 bp at its 3'-UTR, and an ORF of 750 bp which codes for ATP synthase D subunit designated as MaATPSE (GenBank accession number: KM101044). The MaATPSE encodes 249 amino acid residues, and the deduced molecular weight, isoelectric point and stability coefficient are 28.21 kDa, 9.40 and 31.13, respectively. The MaATPSE contains 3 serine phosphorylation sites, 4 threonine phosphorylation sites, 2 tyrosine phosphorylation sites and 1 glycosylation sites. The MaATPSE has 87% identity with Tribolium castaneum, 83% identity both with Bombyx mori and Drosophila melanogaster, respectively. M. alternatus and T. castaneum are on the same branch in phylogenetic tree, and there is a long genetic distance between M. alternatus and non Coleoptera insects. The relative expression level of MaATPSE in pupae and larvae was 3.70 times and 2.65 times of that in adults, respectively. There was a significant difference in expression among different developmental states (P<0.05). The MaATPSE was expressed in various tissues of larvae, the expression in fat body was highest, and 5.94 times of that in control, and the expression in body wall and hemolymph ranked the second, and was 3.93 times and 2.88 times of that in control, respectively. The expression in midgut and Malpighian tubules was relatively low, and was 0.24 times and 0.28 times of that in control, respectively. There were significant differences in MaATPSE expression among various larval tissues, except that between Malpighian tubules and midgut (P<0.05). The relative expression level in adult head, abdomen, antennae, feet, wings and thorax was 2.24, 3.09, 3.52, 3.39, 4.45 and 0.68 times of that in control, respectively. There were significant differences in MaATPSE expression among various parts in adults (P<0.05). [Conclusion] The Monochamus alternatus ATP synthase D subunit gene containing 5'-UTR, 3'-UTR and a complete ORF was cloned in this study. The MaATPSE and ATP synthase of T. castaneum are the most similar at amino acid level. The MaATPSE is extensively expressed in the larvae, pupae and adults, in various tissues of larvae and parts of adults of M. alternatus.

Key words: Monochamus alternatus, ATP synthase, gene expression, RT-qPCR, RACE