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 regulatory protein FKBP gene in Plutella xylostella(L.)

  • 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(FKBP8FKBP12FKBP52)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FKBP52FKBP8FKBP12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。FKBP52FKBP8FKBP12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FKBP12FKBP8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】 FKBP52FKBP8FKBP12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。

     

    Abstract: 【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,providing 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(FKBP8FKBP12 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 FKBP52FKBP8 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. The expression levels of FKBP12 and FKBP8 genes were the highest in fourth-instar larvae,significantly higher than in other larval stages and pupae. The expression levels of FKBP8 gene in the midgut and epidermis of the resistant population were significantly higher than those in the susceptible population,being 3.2 and 3.7 times as those in susceptible population respectively. 【Conclusion】 FKBP52FKBP8 and FKBP12 genes of P. xylostella in the chlorantraniliprole-resistant population are all over-expressed. The high expression of FKBP8 gene in the midgut of the highly resistant population suggests its potential involvement in chlorantraniliprole resistance through over-expression.

     

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