中华蜜蜂感觉神经元膜蛋白基因克隆、组织表达分析及原核表达

为明确中华蜜蜂Apis cerana cerana嗅觉形成中重要功能因子的信号转导通路, 本研究利用RT-PCR方法, 克隆了中华蜜蜂感觉神经元膜蛋白 (sensory neuron membrane protein, SNMP) 基因编码区, GenBank登录号为KC012595, 命名为AccSNMP1。序列分析表明, 该编码区开放阅读框长1 563 bp, 编码520个氨基酸, 推测的编码蛋白的相对分子量和等电点分别为58.02 kD和5.83。同源性比较发现, 中华蜜蜂AccSNMP1与其他昆虫感觉神经元膜蛋白基因同源性差异很大, 在氨基酸水平上与西方蜜蜂Apis mellifera SNMP基因一致性达99.2%, 与熊蜂Bombus impatiens SNMP基因一致性达90.9%, 而与赤拟谷盗Tribolium castaneum SNMP基因一致性仅为22.7%。系统发育树显示中华蜜蜂与西方蜜蜂遗传距离最近。实时荧光定量PCR结果分析表明, AccSNMP1在触角中表达量最高, 在足中表达量较高, 与胸、 腹、 头（去除触角和喙）、 喙中表达量相比差异显著（P<0.05）。构建原核表达载体pEASY-E1-AccSNMP1, 经IPTG诱导, 中华蜜蜂感觉神经元膜蛋白在大肠杆菌Escherichia coli BL21 （DE3）中高效表达。结果为进一步研究AccSNMP1在中华蜜蜂体内的作用机理奠定了基础。

Cloning, tissue expression profiling and prokaryotic expression of a sensory neuron membrane protein gene from Apis cerana cerana (Hymenoptera: Apidae)

To explore the signal transduction pathway of important factors in olfactory formation in Apis cerana cerana, the cDNA sequence encoding a sensory neuron membrane protein (SNMP) (GenBank accession no. KC012595), named as AccSNMP1, was cloned by RT-PCR from the Chinese honey bee, Apis cerana cerana. Sequence analysis results showed that the open reading frame (ORF) is 1 563 bp in length, encoding 520 amino acids with the predicted molecular weight of 58.02 kD and the theoretical isoelectric point of 5.83. Multiple sequence alignment indicated that AccSNMP1 from A. cerana cerana shares different identities with those from other nine insects at the amino acid level. The AccSNMP1 gene from A. cerana cerana has high amino acid sequence identity with that of Apis mellifera (99.2%) and Bombus impatiens (90.9%), while has the lowest amino acid sequence identity with that of Tribolium castaneum (22.7%). The phylogenetic analysis indicated that A. cerana cerana species has a close relationship with A. mellifera and Bombus impatiens. Tissue expression profiling of AccSNMP1 quantified by real time RT-PCR demonstrated that it was highly expressed in antennae and legs of A. cerana cerana, showing a significant difference with that in thorax, abdomen, proboscis and head without antennae and proboscis (P<0.05). A recombined plasmid pEASY-E1-AccSNMP1, containing the coding sequence of AccSNMP1, was constructed using pEASY-E1 as the fused expression vector, and AccSNMP1 was expressed successfully after induced with IPTG in BL21 (DE3) strain of Escherichia coli. The results provide the basis for further studying the functions of sensory neuron membrane protein gene in A. cerana cerana to better understand its action mechanisms.