Cloning And Functional Analysis Of Voltage-Gated Calcium Channel β Subunit Gene Of Ditylenchus Destructor

Voltage-gated calcium channels（VGCCs）are a kind of macroprotein molecule,which is composed of a porousα₁subunit andα₂/δ,β,γand other auxiliary subunits.It participates in many excitatory processes in cells and plays an important role in organisms.As the main auxiliary subunit of VGCCs,βsubunit plays an important role in regulating the surface expression and gating characteristics of high voltage activated calcium channel.At present,the study of voltage-gated calcium channelβsubunit（Ca_vβ）in plant parasitic nematodes have not been reported,and its structure and function have not been determined.In this paper,using Ditylenchus destructor as material,the full c DNA sequence of Ca_vβsubunit was cloned by RT-PCR and RACE techniques.The analysis showed that its full length was 2036bp,and the maximum ORF was 1530bp,encoding 509 amino acids.The accession number of Gen Bank is:MN867027;has SL1 shearing at the 5’end of the sequence and Poly A at the 3’end.Through amino acid sequence alignment analysis,it was found that the obtainedβsubunit had high homology with Ca_vβsubunit（ccb-1）,and had a typical voltage gated calcium channelβsubunit domain,and there were residues interacting with AID（α₁interaction domain）in the GK domain.The relative expression of Ca_vβsubunit in different ages of D.destructor was determined by RT-q PCR.The results showed that compared with the expression of J2,the expression of this gene increased in egg,J2 and J3,decreased significantly in J4,and was the highest in J3 and the lowest in J4.Through the original hybridization experiment of RNA-DIG,it was confirmed that theβsubunit gene was mainly expressed in the parietal cells,esophageal gland,female vulva and male vas deferens of D.destructor.The genes ofβsubunit,L-type and non-L-typeα₁subunit were interfered with RNA by ds RNA and FITC co-bath method.The results showed that after soaking for 12 hours,the expression of target genes decreased significantly,the movement of nematodes was slower,the sand column passage rate of nematodes decreased significantly compared with the water control,especially in the treatment of co-silencing ofβand L-typeα₁ subunits.Through inoculation of sweet potato,it was found that the bran heart phenomenon of the treatment group was slighter than that of the water control.The reproduction coefficient of nematodes treated with gene silencing showed that the reproduction coefficient of nematodes treated with each treatment was significantly lower than that of the control,and the reproduction coefficient of nematodes treated withβsubunit and non-L-typeα₁ subunit decreased most obviously.From the above results,it is speculated that the silencing of Ca_vβsubunit may affect the function of L-type and non-L-type voltage-gated calcium channelα₁ subunit,and further affect the function of VGCCs.This leads to the decrease of locomotor abilities,feeding behavior and reproduction rate of D.destructor.