Expression And Alternative Splicing Analysis Of A Large Conductance Calcium-activated Potassium Channel Gene In Plutella Xylostella

The large-conductance calcium-activated potassium channel(BK_Ca),which are regulated by intracellular Ca²⁺concentration and membrane depolarization signals,plays an important role in the regulation of insect neural circuits and locomotion.In this study,we injected iberiotoxin(IBTX,an inhibitor of BK_Ca)into fourth instar Plutella xylostella larvae,and obeserved the effect on Plutella xylostella.To further investigate the possible functions of the BK channel in P.xylostella,we cloned the coding sequence of slowpoke gene(pxslo,the alpha subunit of a BK_Ca)from the P.xylostella,and characterized the gene expression profile and alternative splicing analysis.The main results are as follows:（1）The results showed that after the 4th instar larvae were anesthetized with ethyl acetate at 25°C for 3 minutes and the 5min（95%confidence interval:4.85-6.35 min）could be recovered after injection of normal saline.After injection 50 n L of 1 n M or 1μM IBTX,the anesthetic time of ethyl acetate was 10 min（95%CI:9.0-11.47 min for 1 n M,9.33-11.19min for 1μM）.It is suggested that injection of IBTX can significantly prolong the anesthetic time of ethyl acetate.There was no significant difference between the two doses of IBTX,indicating that IBTX could effectively inhibit the BK_Ca of Plutella xylostella.（2）Four full-length coding sequences were cloned from the 4th instar larvae.The sequencing results showed that there was alternative splicing,and the coding sequence（CDS）of pxslo with the highest frequency of splicing（Gen Bank ID:MN938456）was 3,405 bp.Genomic sequence analysis showed that the full length of pxslo gene was 19.9 kb,which was composed of 25 exons and 24 introns.The deduced protein pxslo contained 1135 amino acids with a molecular weight of 126769.30 Da,and an isoelectric point of 5.45.NCBI conservative domain search analysis showed that pxslo was a calcium-activated BK_Capotassium channelαsubunit.（3）To detect the expression level of pxslo gene in different growth stages and different tissues of Plutella xylostella.It was found that pxslo gene was expressed in all developmental stages of P.xylostella,and the expression level of P.xylostella was higher in adults.In the larval stage,pxslo gene is mainly expressed in the head and epidermis,but rarely in the midgut.In the adult stage,pxslo gene was highly expressed in the head,followed by ovarian tubules,almost no expression in the testis and wings,and the expression level in the head was 93.4 times higher than that in the testis.It is suggested that BK_Camay play a regulatory role in neural circuit,movement and ovulation.（4）In order to determine the specific way of splicing tissue,the alternative splicing region was amplified by PCR technique and four alternative splicing sites of pxslo gene were found,named pxslo-ASP-（A C E G）.Selective splicing analysis showed that variable exon ASP（A1,C1,E1）was preferred in both tested tissue samples and single adult samples.In this study,a total of 18 combinations were found through a large number of cloning and sequencing for the first time,among which A1/C1/E1/G1 appeared most frequently in the head and ovarian tube.In this study,we cloned the slo gene of P.xylostella by homologous sequence alignment.The temporal and spatial expression of slo gene was detected by q RT-PCR.At the same time,the alternative splicing of Slo in adult head and reproductive organs was studied.In order to further analyze the function of Slo subunit,we injected BK_Ca inhibitor（IBTX）into P.xylostella.The results showed that IBTX could significantly prolong the anesthetic time of ethyl acetate on Plutella xylostella.This study provides a basis for further study of the physiological function of BK_Ca channels.