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

【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（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. 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】 FKBP52，FKBP8 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.