【作者】 江武军； 何旭江； 王子龙； 颜伟玉； 曾志将； 吴小波；

【Author】 JIANG Wu-jun;HE Xu-jiang;WANG Zi-long;YAN Wei-yu;ZENG Zhi-jiang;WU Xiao-bo;Honeybee Research Institute,Jiangxi Agricultural University;

【机构】 江西农业大学蜜蜂研究所；

【摘要】 [目的]克隆中华蜜蜂Apis cerana cerana细胞色素CYP9E2基因的完整编码区序列,并分析CYP9E2基因在工蜂体内的表达特征,为研究该基因的生物学功能提供理论基础。[方法]以中华蜜蜂为实验材料,解剖其中肠获得中肠组织,提取中肠组织的总RNA。利用RT-PCR技术克隆中华蜜蜂CYP9E2(AcCYP9E2)基因的编码区。采用多种生物信息学软件分析AcCYP9E2基因序列,利用荧光定量PCR技术(Real-time Quantitative PCR)分析AcCYP9E2在中华蜜蜂工蜂成虫期不同阶段(初生蜂、哺育蜂、守卫蜂以及采集蜂)头部和中肠组织中的表达量及饲喂氟氯苯菊酯后工蜂中肠组织AcCYP9E2基因的表达变化。[结果]克隆获得中华蜜蜂CYP9E2基因mRNA序列长度为1600 bp(GenBank登录号:KX394629),编码区长1494bp,编码497个氨基酸,其蛋白质分子量为57.026kD,等电点为8.32。系统发育树显示中华蜜蜂CYP9E2与西方蜜蜂Apis mellifera CYP9E2、小蜜蜂Apis florea CYP9E2基因聚成一支。经过对中华蜜蜂工蜂成虫期不同阶段头部和中肠组织AcCYP9E2基因相对表达量进行测定发现:该基因在中华蜜蜂工蜂成虫期不同阶段的表达量存在一定的差异,其中,采集蜂头部和中肠组织AcCYP9E2基因表达量均显著高于初生蜂、哺育蜂以及守卫蜂(P<0.05),而且4个阶段工蜂中肠组织的AcCYP9E2表达量均显著高于其头部(P<0.05)。饲喂农药处理后,工蜂中肠组织AcCYP9E2基因的表达量显著高于对照组(P<0.05)。[结论]推测中华蜜蜂CYP9E2可能参与了机体外源物质的代谢与解毒过程。更多还原

【Abstract】 [Aim] The objective of this study is to clone the full-length cDNA sequence of CYP9 E2 gene of Apis cerana cerana and then to analyze its expression,which will contribute our understanding to the biological function of AcCYP9 E2 gene in Apis cerana cerana.[Methods] Total RNA was extracted from the dissected midgut tissues of Apis cerana cerana,and the coding sequence of AcCYP9 E2 was cloned using RTPCR.The sequence of AcCYP9 E2 of AcCYP9 E2 was analyzed using a variety of bioinformatics software.Newly emerged worker bees,nursing worker bees,guard worker bees and foragers were captured,and the expression of AcCYP9 E2 in heads and midguts of these four worker bee groups was compared via quantitative real-time PCR(RT-qPCR).We also compared the mRNA expression level of AcCYP9 E2 between workers fed with flumethrin and the ones fed with sucrose(control).[Results] The full length of AcCYP9 E2 mRNA sequence is 1600 bp(Accession number KX394629).The coding region is 1494 bp,which encodes 497 amino acids.The molecular weight and an isoelectric point of AcCYP9 E2 protein are 57.026 kD and 8.32,respectively.Further,the phylogenetic tree analysis of gene AcCYP9 E2 showed that Apis cerana cerana was firstly clustered with Apis mellifera,and Apis florea.Moreover,the results of qRT-PCR showed that the gene expression of AcCYP9 E2 was different among these four worker bee groups while.the expression levels of AcCYP9 E2 in the heads and midguts of foragers were both significantly higher than that of newly emerged worker bees,guard worker bees and nursing worker bees.It was significantly higher in midguts compared to heads in all four groups(P<0.05).In addition,the expression level of AcCYP9 E2 in the midguts of the pesticide-treated group was significantly higher than that in the control group(P<0.05).[Conclusion] These results suggest that the AcCYP9 E2 may be involved in honeybee metabolism process and detoxification of exogenous substances in vivo.更多还原