小菜蛾RyR调控蛋白FKBP基因克隆及其时空表达谱  

Cloning and spatiotemporal expression analysis of RyR regula⁃tory protein FKBP gene in Plutella xylostella(L.)

作　　者：张万里 葛天成 陈琦椿 彭争科 肖勇 李振宇[2] 尹飞[2] ZHANG Wan-li;GE Tian-cheng;CHEN Qi-chun;PENG Zheng-ke;XIAO Yong;LI Zhen-yu;YIN Fei(Guangdong Tianhe Agricultural Resources Co.,Ltd.,Guangzhou,Guangdong 510080,China;Institute of Plant Protection,Guangdong Academy of Agricultural Sciences/Guangdong Provincial Key Laboratory of High Technology for Plant Protection,Guangzhou,Guangdong 510640,China)

机构地区：[1]广东天禾农资股份有限公司,广东广州510080 [2]广东省农业科学院植物保护研究所/广东省植物保护新技术重点实验室,广东广州510640

出　　处：《南方农业学报》2024年第10期3023-3032,共10页Journal of Southern Agriculture

基　　金：广东省基础与应用基础研究基金项目(2022A1515012395);广东省农业科学院协同创新中心项目(XTXM202209)。

摘　　要：【目的】明确FK506结合蛋白(FKBP)基因在小菜蛾对氯虫苯甲酰胺抗性形成中的作用,筛选出起主要作用的关键FKBP基因,为阐明小菜蛾抗双酰胺类杀虫剂的分子机理提供新思路。【方法】以抗氯虫苯甲酰胺种群和敏感种群小菜蛾为研究对象,采用浸叶法测定不同种群小菜蛾对氯虫苯甲酰胺的敏感性差异;利用cDNA末端快速扩增(RACE)方法克隆3个FKBP基因(FKBP8、FKBP12和FKBP52)的全长序列,并通过GSDS 2.0、CDD和MEME在线分析软件分析其基因结构与蛋白保守结构域和Motif;采用实时荧光定量PCR分析抗性种群不同发育阶段和敏感、抗性种群4龄幼虫不同组织FKBP基因的时空表达特性。【结果】生物测定结果显示,抗性种群小菜蛾对氯虫苯甲酰胺的抗性倍数为122.67倍,属于高水平抗性。实时荧光定量PCR分析发现,抗性种群小菜蛾体内3个FKBP基因的相对表达量均显著高于敏感种群(P<0.05,下同),其中FKBP8基因的表达差异最显著,是敏感种群的968倍。通过RACE方法克隆得到FKBP52、FKBP8和FKBP12基因的全长序列,长度分别为1473、1525和649 bp,分别编码423、307和108个氨基酸残基,其蛋白均为亲水蛋白。FKBP52、FKBP8和FKBP12基因具有相同的基因结构,FKBP52和FKBP12蛋白具有相同的保守结构域FkpA,而FKBP8蛋白具有2个不同的保守结构域。抗性种群小菜蛾4龄幼虫体内3个FKBP基因表达量均高于蛹期,其中FKBP12和FKBP8基因在4龄幼虫中的表达量最高,显著高于其他龄期和蛹期。FKBP8基因在抗性种群小菜蛾中肠和表皮中的表达量显著高于敏感种群,分别是敏感种群的3.2和3.7倍。【结论】FKBP52、FKBP8和FKBP12基因在氯虫苯甲酰胺抗性种群小菜蛾中均过量表达,其中FKBP8基因在高抗氯虫苯甲酰胺种群小菜蛾的中肠高表达,表明其可能通过过量表达参与小菜蛾对氯虫苯甲酰胺的抗药性。【Objective】The role of FK506-binding protein(FKBP)gene in the development of resistance of Plutella xylostella(L.)to chlorantraniliprole was determined,and key FKBP genes involved in this process was identified,provi-ding new insights into the molecular mechanism underlying diamide insecticide resistance in P.xylostella.【Method】Chlorantraniliprole resistant and susceptible populations of P.xylostella were used to determine chlorantraniliprole susceptibility difference of P.xylostella by leaf-dip bioassay.The full-length sequence of 3 FKBP genes(FKBP8,FKBP12 and FKBP52)were cloned using rapid amplification of cDNA end(RACE),and its gene structure,protein conserved domains and Motifs were analyzed using online softwares including MEME,CDD and GSDS 2.0.Real-time fluorescence quantitative PCR(RT-qPCR)was used to analyze spatiotemporal expression patterns of FKBP genes in different developmental stages of the resistant population,and in different tissues of fourth-instar larvae of both resistant and susceptible populations.【Result】The bioassay revealed a 122.67-time resistance to chlorantraniliprole in the resistant population of P.xylostella,indicating high-level resistance.RT-qPCR analysis showed that the relative expression levels of all 3 FKBP genes of P.xylostella in the resistant population were significantly higher than those in susceptible population(P<0.05,the same below).The full-length sequence of FKBP52,FKBP8 and FKBP12 genes were cloned by the RACE method,with lengths of 1473,1525 and 649 bp,encoding 423,307 and 108 amino acids residues respectively.All 3 proteins were predicted to be hydrophilic.They shared similar gene structures.FKBP52 and FKBP12 protein had the same conserved domain FkpA,while FKBP8 protein contained 2 different conserved domains.The difference of FKBP8 gene expression was the most significant,which was as 968 times as that in the resistant population.The expression levels of 3 FKBP genes in fourth-instar larvae in the resistant population were higher than those in pupae.T

关 键 词：FK506结合蛋白 FKBP基因 小菜蛾 氯虫苯甲酰胺 时空表达特性 抗药性 

分 类 号：S433.4[农业科学—农业昆虫与害虫防治]