灰飞虱水通道蛋白基因克隆及其与杀虫剂代谢的关系

Aquaporin gene cloning and its possible contribution to insecticide metabolism in Laodelphax striatellus

[目的]水通道蛋白是细胞膜内嵌蛋白超家族的主要成员,具有传输水分子以及小分子化合物进出细胞膜的功能,本研究目的是分析重要农业害虫灰飞虱体内水通道蛋白功能及其与杀虫剂代谢之间的关系。[方法]根据转录组数据,采用PCR和RACE技术对灰飞虱体内的水通道蛋白基因进行了克隆,并利用进化树对获得的基因进行亲缘关系的分析验证,利用实时荧光定量PCR技术分析这些基因在灰飞虱抗、感品系间的表达量差异,利用体外表达技术分析其转运活性。[结果]从灰飞虱体内克隆获得了6个灰飞虱水通道蛋白基因全长序列,分别命名为LsAQP1、LsAQP2、LsAQP3、LsAQP4、LsAQP5和LsAQP6,其中LsAQP6在灰飞虱的不同抗性品系里均高表达。体外功能表达试验显示:LsAQP6对水具有很强的转运能力,对灭多威也具有显著的转运能力,但对甘油和杀虫双的转运能力不显著。[结论]LsAQP6在灰飞虱体内对水和某些小分子农药具有转运作用。

[ Objectives] Aquaporin, a member of the major intrinsic protein superfamily, functions mainly for transporting water and small molecule compounds across cell membrane, and the aim of this study was to analyze the function of the aquaporin and its possible contribution to insecticide metabolism in an important agricultural pest Laodelphax striatellus. [ Methods ] In this study, aquaporin genes in L. striatellus were cloned based on the transcriptome data and with PCR and RACE cloning techniques. The cloned genes were subjected to phylogenetic relationship analysis by constructing phylogenetie tree with previously reported insect aquaporin genes, tests of the expression level in the susceptible and resistant strains by real-time quantitative PCR, and functional verification for their transporting capability by eombinant expression in vitro. [ Results ] Six full length aquaporin genes were cloned from L.striatellus and designated as LsAQP1 , LsAQP2 , LsAQP3 , LsAQP4 , LsAQP5 and LsAQP6 , respectively. Among them, LsAQP6 was found expressed with higher level in all three resistant strains tested as compared to the susceptible strain. Furthermore, functional expression showed that LsAQP6 had strong transport capacity for water and significant transport capacity for methomyl, but not for glycerol and bisultap. [ Conclusions ] Thus, it is speculated that LsAQP6 plays a significant role in transporting water and some small molecule insecticides in L. striatellus.