Expression Of Inward-Rectifying Potassium Channel Subunits In Plutella Xylostella

Inward rectifier potassium channels(Kir)consisted of channel structure of alpha subunit(Kir subunit)and essential subunits,which controlled insects lay eggs,silk gland secretion and Malpighian tube drainage and other important physiological functions.The abnormal structure and function of Kir subunit would affect the normal physiological activities of insects,which was a potential pesticide target.In this study,the conserved domain sequences of mosquito and melanogaster were used as bait to identify five Kir subunit genes from transcriptional databases of Plutella xylostella and Danaus plexippus and six Kir subunit genes from Bombyx mori and Manduca sexta.According to phylogenetic analysis,it was found that the four lepidoptera insects were similar mosquito,melanogaster and Nilaparvata lugens.There were three different evolutionary branches(Kir1-3),of which Kir3 included two subtypes Kir3A and Kir3B.In addition,a new branch of evolution(Kir4)would have emerged in lepidoptera compared with other insects.Among them,Kir4 of B.mori and M.sexta contained two subtypes of Kir4A and Kir4B,while only one subtype of Kir4 was found in P.xylostella and D.plexippus.Kir subunit gene full-length cDNA sequence of P.xylostella was successfully obtained by using specific primers:PxKir1:MT274448;Pxkir2:MN894569;Pxkir3A:MN894570;Pxkir3B:MN894571 and PxKir4:XM＿011554721.The protein prediction sequences encoded by five genes were analyzed and found that all PxKir subunits had typical Kir subunit structural characteristics:K~+selection filter existed between two transmembrane helices(TM1 and TM2)and G-loop gate formed by C/N-terminal in the cytoplasmic domain(CTD).Further sequence alignment revealed that the sequence similarities of PxKir1 and the direct homologous proteins of fruit flies,Culicidae,drosophilid and other insects were 52.3-67.3%,55.0-76.8%,31.8-38.9%and 36.1-38.5%,respectively.The sequence similarity between PxKir4and lineal homologous proteins was 47.2-61.3%.qRT-PCR was used to analyze the expression pattern of pxkir1-pxkir4 in different developmental stages and tissues of P.xylostella.The dynamic expression of pxkir1 genes was higher in each developmental stage of P.xylostella than in the other four genes and the expression level of pxkir1 was significantly higher in male adults than in other developmental stages.The expression level of pxkir2 increased sharply in the adult stage and the expression level of pxkir3A was high throughout the growth and development cycle.The expression level of pxkir3B gene increased gradually in the larval stage,reached the highest expression level in the 3rd instar and decreased gradually after entering the 4th age,while the expression level was the lowest in the pupal stage and increased rapidly after entering the adult stage.The expression level of pxkir4 in the egg stage was significantly lower than that in other developmental stages.The expression levels of pxkir1 and pxkir2 were found to be higher in the chest,feet and wings;the expression levels of pxkir3 and pxkir4 were higher in the Malpighian tubes of larvae and adults than in other tissues.When comparing the expression differences between males and females,it was found that pxkir1,pxkir3A and pxkir4 were highly expressed in the testis but only pxkir3B was highly expressed in the ovarian tube and pxkir2 was low expressed in the testis and ovary tubes.The spatiotemporal expression patterns lay foundation for the in-depth study of the pharmacology and electrophysiology of Kir subunits in lepidoptera.