Cloning And Functional Analysis Of Voltage-gated Calcium Channel α1 Subunit Gene Of Ditylenchus Destructor

Voltage-gated calcium channel(VGCC)is a kind of functional channel,which widely distributed on biological cell membranes to control the flow of calcium ions.And it is a macromolecular compound protein composed of the main functions of α1 subunit and several auxiliary subunits,α1,α2/δ,β and γ subunits.The VGCC has the function of controlling the internal flow of calcium ions,stabilizing the internal and external voltage difference of the cells,and plays an important role in regulating the organism.As the main subunit of VGCC,α1 subunit plays a leading role in the operation and regulation of VGCC.However,so far there were so few studies on the VGCC of plant parasitic nematodes,and their structure and function have not been determined.The purpose of this study was to cloning VGCC α1 subunit gene of Ditylenchus destructor(D.destructor),and analyzing its protein structure and function.In this study,the α1 subunits mRNA sequence of VGCC gene of D.destructor was amplified by RT-PCR and RACE technique.Built on the sequencing results,the bioinformatics analysis of nucleic acid and putative amino acid was conducted.Then,the amino acid sequence and functional column analysis of each α1 subunit were carried out by reference to the relevant research results of the VGCC of Caenorhabditis elegans(C.elegans).The expression position of various types VGCC in D.destructors was preliminarily determined by RNA-DIG in situ hybridization experiment.Using RNAi technology,the corresponding genes were silenced by the method of immersion treated with both dsRNA and fluorescent dyes FITC reagent,so that it could presume the function of those three types VGCC in D.destructors.Finally,three types of VGCC alpha-1 subunit were acquired,which were L-,P/Q-and Ttype VGCC.L-type VGCC alpha-1 D subunit(DdCα1D,GenBank: MG585272)was 6236 basic pair(bp)long and included a 5208 bp ORF encoding 1736 amino-acid protein.P/Q-type VGCC alpha-1 A subunit(DdCα1A,GenBank: MG585271)was 6676 bp long and included a 6276 bp ORF encoding 2091 amino-acid protein.T-type VGCC alpha-1 subunit(DdCα1G,GenBank: MG585273)was 6457 bp long and included a 5988 bp ORF encoding 1996 aminoacid protein.RNA-DIG in situ hybridization experiment results show that DdCα1D were presented within the pharynx and body-wall muscles.DdCα1A were expressed in the esophageal gland,vulva of female and vas deferens of male.And DdCα1G were expressed in the esophagus and medium bulb.After 48 h L-dsRNA soaking,the relative gene expression of DdCα1D was 33.54%.And most of L-dsRNA treated worm was rigid and curled after silence of DdCα1D.And after 48 h P/Q-dsRNA soaking,the relative gene expression of DdCα1A was 26.36%.And the P/QdsRNA treated worms had less progeny compared to the control test.And after 48 h T-dsRNA soaking,the relative gene expression of DdCα1G was 14.64%.And the medium bulb muscle of T-dsRNA treated worms was explicit contraction,which restrained the feeding amount of DdCα1G silence worm compared to the control test.