松墨天牛中性肽链内切酶2基因的鉴定与表达
Identification and Expression of Neprilysin 2 Gene in Monochamus alternatus (Coleoptera:Cerambycidae)

为了探讨M13家族中的中性肽链内切酶2基因在昆虫中的表达特性，以松墨天牛为研究对象，从cDNA文库中筛选到松墨天牛中性肽链内切酶2基因，命名为 [QX(Y15] MaNep2[QX)] (GenBank:KU245762)。该序列长为2 471 bp，其中编码序列为2 280 bp，共编码759个氨基酸。由此预测的蛋白质二级结构主要由α-螺旋与规则卷曲组成，其次是延伸链与β-转角。 MaNep2基因编码蛋白定位于细胞质，为跨膜蛋白，跨膜区长度为23个氨基酸(C25LTLSTAVAFVIGISLVVALATV47)，并存在一个低复杂度区域：I423SAGSLSIASGAL435。通过DNAMAN软件比对发现MaNep2与山松甲虫和赤拟谷盗同源性最高，为82%,且存在4个保守结构域序列：NAFY（553-556）、HEITH（594-598）、ENIAD（656-660）、CXXW（756-759）；用MEGA4.0构建系统发育树，显示松墨天牛与山松甲虫处在同一分支。qPCR分析表明： MaNep2的表达与松墨天牛的变态发育相关，各虫态不间断表达，幼虫期表达表现先上升后下降，化蛹期间表达表现为先下降后上升，羽化期间表达表现为先上升后下降，并在初羽化的成虫中表达量达到最大值；幼虫和成虫头部表达量最高，且在马氏管中均有表达；成虫的足、翅、触角和卵巢中也都有表达。
To explore the expression characteristics of Neprilysin 2 gene from M13 family in insects，Neprilysin 2 gene named MaNep2 (GenBank:KU245762) was filtrated from cDNA library of Monochamus alternatus.The sequence of MaNep2 is 2 471 bp in length,containing a 2 280 bp open reading frame (ORF) encoding 759 amino acids.The predicted secondary structure of MaNep2 is mainly composed of alpha helix and random curl,followed by a beta turn and extension chain.Subcellular localization analysis showed Neprilysin 2 is in cytoplasm.The transmembrane of Neprilysin 2 (C25LTLSTAVAFVIGISLVVALATV47) was 23 amino acids in length.A low-complexity region（LCR ,I423SAGSLSIASGAL435）was found. MaNep2 showed the highest homology with the Neprilysin 2 gene from Tribolium castaneum and Dendroctonus ponderosae,respectively (82%) through amino acids sequence multiple alignment.There are four conserved domains,i.e.NAFY(553-556),HEITH(594-598),ENIAD（656-660）and CXXW（756-759）in MaNep2.Phylogenetic tree built by MEGA4 indicated M.alternatus and D.ponderosae are in the same branch.RT-qPCR analysis showed that MaNep2 is expressed in each insect stage continuously,which is associated with development of M.alternatus.Expression level declined after rising in larval stage,rising after falling during the period of pupation,falling after rising during the period of eclosion,and reached the maximum amount in 0d-1d pharate adults.The expression level of MaNep2 is the highest in heads of larvae and adults. MaNep2 is also expressed in malpighian tubes of larvae and is in feet,wings,antennae and ovaries of adults.