昆虫学报 ›› 2016, Vol. 59 ›› Issue (10): 1050-1057.doi: 10.16380/j.kcxb.2016.10.003

• 研究论文 • 上一篇    下一篇

中华蜜蜂细胞色素CYP9E2基因克隆及其表达分析

江武军, 何旭江, 王子龙, 颜伟玉, 曾志将, 吴小波*   

  1. (江西农业大学蜜蜂研究所, 南昌 330045)
  • 出版日期:2016-10-20 发布日期:2016-10-20

Cloning and expression analysis of cytochrome CYP9E2 gene in the Chinese honeybee, Apis cerana cerana

JIANG Wu-Jun, HE Xu-Jiang, WANG Zi-Long, YAN Wei-Yu, ZENG Zhi-Jiang, WU Xiao-Bo*   

  1. (Honeybee Research Institute, Jiangxi Agricultural University, Nanchang 330045, China)
  • Online:2016-10-20 Published:2016-10-20

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,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】以解剖获得的中华蜜蜂采集蜂中肠组织为材料,提取总RNA。利用RT-PCR技术克隆中华蜜蜂CYP9E2基因的编码区。采用多种生物信息学软件分析该基因的核苷酸和氨基酸序列,利用荧光定量PCR技术(quantitative real-time PCR)分析其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相对表达量及在饲喂氟氯苯菊酯后工蜂中肠组织中的表达变化。【结果】克隆获得中华蜜蜂CYP9E2基因(命名为AcCYP9E2)mRNA序列, 长度为1 600 bp(GenBank登录号:KX394629),编码区长1 494 bp,编码497个氨基酸,其蛋白质分子量为57.026 kD,等电点为8.32。系统发育树显示,中华蜜蜂AcCYP9E2与西方蜜蜂Apis mellifera、小蜜蜂Apis florea CYP9E2基因聚成一支。对中华蜜蜂工蜂成虫期不同阶段头部和中肠组织AcCYP9E2相对表达量测定发现,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差异,其中,采集蜂头部和中肠组织中AcCYP9E2相对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中的AcCYP9E2相对表达量均显著高于其头部(P<0.05)。饲喂氟氯苯菊酯后,工蜂中肠组织中AcCYP9E2的相对表达量显著高于对照组(P<0.05)。【结论】推测AcCYP9E2可能参与了中华蜜蜂机体外源物质的代谢与解毒过程。

关键词: 中华蜜蜂, 工蜂, 细胞色素P450, 基因克隆, 基因表达, 解毒酶

Abstract: 【Aim】 The objective of this study is to clone the coding region sequence of cytochrome CYP9E2 gene of the Chinese honeybee, Apis cerana cerana, and to analyze its expression profiles in workers, which will contribute to our understanding on the biological function of this gene. 【Methods】 Total RNAs were extracted from the dissected midgut tissues of A. cerana cerana foragers, and the coding sequence of CYP9E2 gene of A. cerana cerana was cloned using RT-PCR. The nucleotide and deduced amino acid sequences of the gene were analyzed using bioinformatics software. The relative expression levels of the gene in heads and midguts of the newly emerged workers, nurses, guarders and foragers were compared via quantitative real-time PCR (qRT-PCR). The relative expression levels of the gene between workers fed with flumethrin and the ones fed with 30% sucrose (control) were also compared. 【Results】The full length mRNA sequence of CYP9E2 gene of A. cerana cerana (named AcCYP9E2) is 1 600 bp (GenBank accession number: KX394629). Its coding region is 1 494 bp, which encodes 497 amino acids. The molecular weight and isoelectric point of the encoded protein are 57.026 kD and 8.32, respectively. The phylogenetic tree analysis showed that AcCYP9E2 of A. cerana cerana was firstly clustered with CYP9E2 genes from A. mellifera and A. florea. The qRT-PCR results showed that the relative expression level of AcCYP9E2 was different among the four worker bee groups, while the relative expression levels of AcCYP9E2 in the heads and midguts of foragers were both significantly higher than those of the newly emerged workers, nurses and guarders (P<0.05). The relative expression level of AcCYP9E2 was significantly higher in the midgut than that in the head in all the four worker bee groups (P<0.05), and the relative expression level of AcCYP9E2 in the midgut of the flumethrin-treated groups was significantly higher than that in the control group (P<0.05). 【Conclusion】 These results suggest that AcCYP9E2 may be involved in the metabolism process and detoxification of exogenous substances in the body of A. cerana cerana.

Key words:  Apis cerana cerana, worker bee, cytochrome P450, gene cloning, gene expression, detoxification enzyme