关键词: 菜青虫, Pain, 瞬时感受器电位通道, 基因克隆, 系统进化分析, 钙流检测

Abstract: The purpose of this study is to clone and express the Painless (Pain) gene which belongs to transient receptor potential channels in the cabbage white butterfly, Pieris rapae regarded as one of the important pests on cruciferous vegetables, so as to provide an important basis for the further study of its physiological function in Lepidoptera. Based on the transcriptome data of P. rapae, RT-PCR was applied to clone the complete open reading frame (ORF) of Pain gene, and mammalian expression system was employed to express Pain channel. The Pain gene in P. rapae we identified as the sequence for the GenBank accession number as NW_019093434.1. It's ORF included of 2850 nucleotides and encoded 949 amino acids. The calculated molecular mass and theoretical isoelectric point (pI) of Pain were 108.34 kDa and 5.37, respectively. Bioinformatic analysis showed that Pain contained two conserved regions, the ankyrin repeat-containing domain (ANK) and transmembrane domains. ANK domains contained eight ankyrin repeats (ANK1-8), and transmembrane domains contained six segments (TM1-6). Multiple amino acid sequence alignment revealed that PrPain of P. rapae shared over 70% amino acid identities with BmPain of silkworm Bombyx mori, DpPain of monarch butterfly Danaus plexippus, PxPain of diamondback moth Plutella xylostella and MsPain of tobacco moth Manduca sexta, but only 31% amino acid identities with DmPain of fruit fly Drosophila melanogaster. Phylogenetic analysis showed that PrPain had the closest relationship with PxPain and DpPain. The calcium flux assay in vitro revealed that PrPain channel could be activated by 43 ℃ buffer, and this indicated that PrPain channel could sense to the heat.

Key words: Pieris rapae, Pain, transient receptor potential channel, gene clone, phylogenetic analysis, calcium flux assay