[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.%【目的】研究松墨天牛 ATP合成酶 D亚基基因的鉴定及其表达特性，为开展昆虫 ATP 合成酶基因研究提供分子信息和参考，为深入研究 ATP合成酶基因在松墨天牛中的生理、毒理作用奠定基础。【方法】对从松墨天牛 cDNA文库中克隆的一条表达序列标签(EST)进行3'cDNA 末端快速扩增，将获得的扩增序列与 cDNA文库中的序列进行拼接，经开放阅读框( ORF)检索和 Blast同源比对鉴定基因；用 ProtParam tool软件分析基因编码的蛋白质性质，用 DNAMAN软件构建系统发育树，用实时荧光定量 PCR 分析基因在不同虫态、成虫各部位和幼虫组织中的表达特性。【结果】获得了1条长度为1133 bp的 cDNA，其5'端非编码区长89 bp，3'端非编码区长294 bp，ORF 长750 bp，编码 ATP 合成酶 D 亚基，命名为 MaATPSE ( GenBank 登录号：KM101044)。MaATPSE编码249个氨基酸残基，预测分子质量为28.21 kDa，等电点为9.40，稳定系数为31.13。MaATPSE 含有3个丝氨酸磷酸化位点、4个苏氨酸磷酸化位点、2个酪氨酸磷酸化位点和1个糖基化位点。松墨天牛与黑腹果蝇和家蚕的 MaATPSE相似性均为83%；松墨天牛与赤拟谷盗在系统发育树上聚成一支，相似性为87%，与其他非鞘翅目昆虫的遗传距离较远。MaATPSE在蛹和幼虫的相对表达量分别是成虫的3.70和2.65倍，各虫态之间表达有显著差异(P＜0.05)； MaATPSE在幼虫各组织中均有表达，在脂肪体中的表达量最高，为对照的5.94倍，其次是体壁和血淋巴，分别为对照的3.93和2.88倍；中肠和马氏管的表达量较低，分别为对照的0.24和0.28倍；除马氏管和中肠的相对表达量无显著差异外，各组织均有显著差异(P ＜0.05)。MaATPSE 在成虫头、腹、触角、足和翅的相对表达量分别为对照的2.24，3.09，3.52，3.39和4.45倍，胸部的表达量较低，为对照的0.68倍；成虫各部位之间相对表达量有显著差异( P＜0.05)。【结论】克隆到松墨天牛 ATP合成酶 D亚基基因MaATPSE，包括5'和3'非编码和完整的 ORF。MaATPSE 与赤拟谷盗 ATP 合成酶最为相似，MaATPSE 在松墨天牛幼虫、蛹和成虫中都有表达，在幼虫各组织和成虫各部位中广泛分布。
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