Cloning And Functional Analysis Of Voltage-gated Calcium Channel α₂δ Subunit Gene Of Ditylenchus Destructor

Theα₂δsubunit is one of the auxiliary subunits of voltage-gated calcium channel（VGCC）,which plays an important role in the transport and function of high-voltage activated（HVA）calcium channel.Currently,studies on theα₂δsubunit have focused on mammals,while no reports have been made on theα₂δsubunit of plant parasitic nematodes.Therefore,it is important to explore the function of the plant parasitic nematode VGCC by cloning theα₂δsubunit of Ditylenchus destructor and conducting preliminary analysis of its expression and function.In this study,we cloned the DdCa_vα₂δsubunit from D.destructor and analyzed its gene sequence and expression levels at different developmental stages.By RNA interference,the HVA VGCCα₁ andα₂δsubunits of the D.destructor were silenced separately,and the biological functions played by the DdCa_vα₂δgene in the D.destructor were investigated.The biological functions of DdCa_vα₂δgene were investigated by RNA interference.The main results obtained are as follows.1.We cloned the DdCa_vα₂δgene by RT-PCR and RACE using D.destructor c DNA as template.the open reading frame of DdCa_vα₂δgene was 3825 bp,and its sequence analysis showed that the amino acid sequence of DdCa_vα₂δcontained a VWA structural domain,and a MIDAS motif was included in the structural domain.2.The results of quantitative real-time PCR showed that the expression of DdCa_vα₂δgene was stage-specific,and although it was expressed in all developmental stages of D.destructor,the expression was highest in the egg stage,followed by the J3 and J4 stages,and lowest in the J2 stage.3.RNA-DIG in situ hybridization experiments showed that the DdCa_vα₂δsubunit was expressed in the body wall muscle,oesphageal gland,uterus,post uterine and spicules of D.destructor.4.The biological functions of DdCa_vα₂δsubunit were initially analyzed by silencing of D.destructorα₂δsubunit（DdCa_vα₂δ）,L-typeα₁ subunit（DdCa1D）and Non-L-typeα₁subunit（DdCa1A）by ds RNA soaking method,respectively,and the two genes were silenced simultaneously using a multi-target gene co-silencing method.The results showed that the expression of each target gene was significantly down-regulated by ds RNA soaking for 24 h.The sand column passage rate of D.destructor decreased to 25.33%and 44.67%at 6 h and24 h after silencing the DdCa_vα₂δsubunit gene,respectively,the attraction rate of sweet potato to D.destructor decreased to 12.00%and the reproduction coefficient decreased to35.20%.The sand column passage rate of D.destructor at 6 h and 24 h after silencing the DdCa1D subunit gene decreased to 23.67%and 42.00%,the attraction rate decreased to 7.33%and the number of stylet thrusting decreased to 33.00.When co-silencing the DdCa_vα₂δand DdCa1D subunit genes,the sand column passage rate at 6 h and 24 h decreased further to19.67%and 41.00%,respectively;the attraction rate decreased to 5.67%and the number of stylet thrusting decreased to 33.00.The protein content of D.destructor decreased to19740.00μg/m L and the reproduction coefficient decreased to 22.23 after the silencing of DdCa1A subunit gene.Moreover,the protein content of D.destructor further decreased to19664.44μg/m L and the reproduction coefficient decreased to 17.20 after the co-silencing of DdCa1A subunit gene with DdCa_vα₂δ.From the results of RNA interference experiments,it was concluded that L-type VGCC mediates the motility,chemotaxis,stylet thrusting of D.destructor,Non-L-type VGCC mediates protein secretion and reproduction of D.destructor,and the DdCa_vα₂δsubunit has some auxiliary effects on these biological functions of L-type and Non-L-type VGCC.Meanwhile,the silencing of DdCa_vα₂δsubunit gene alone also affected the motility,chemotaxis and reproduction coefficient of D.destructor.