Cloning And Functional Analysis Of Voltage-gated Calcium Channel α1 Subunit Gene Of Heterodera Elachista And Meloidogyne Graminicola

Voltage-gated calcium channel is a kind of functional channel widely distributed on biological cell membrane to control calcium influx.It is a macromolecular complex protein composed ofα1 main functional subunits,α₂δ,βandγand other auxiliary subunits.As the main subunit of voltage-gated calcium channel,α1 subunit plays a leading role in the operation and regulation of voltage-gated calcium channel.α₂δ,βand other auxiliary subunits play an important role in the regulation of voltage-gated calcium channels.At present,there are relatively few studies on voltage-gated calcium channels of plant parasitic nematodes,and their structure and function have not been determined.In this paper,two important pathogen nematodes of Heterodera elachista and Meloidogyne graminicola,were used to amplify the cDNA sequences of different types of calcium channelα1 subunits of upland rice cyst nematodes by RT-PCR and RACE techniques,which were HeCα1D,HeCα1G and HeCα1A,respectively.The cDNA sequences of different types of calcium channelα1 subunit,βsubunit andα₂δgene from M.graminicola,are MgCα1D,MgCα1A,MgCa_vβand MgCa_vα₂δ,respectively.The results showed that the partial cDNA sequence of L-type voltage-gated calcium channelα1D subunit gene（HeCα1D）was 5309bp in Heterodera elachista,and the full cDNA sequence of non-L-type voltage-gated calcium channelα1A subunit gene（HeCα1A）was 6420 basic pair（bp）long and included a 6057bp ORF encoding 2019 amino-acid protein,and the accession number of GenBank was MT218397.The full cDNA sequence of T-type voltage-gated calcium channelα1 subunit gene（HeCα1G）was 6558 basic pair（bp）long and included a 6162bp ORF encoding 2054 amino-acid protein,The accession number of GenBank is MT218398.Meloidogyne graminicola:the full cDNA sequence of L-type voltage-gated calcium channelα1A subunit gene（MgCα1D）was 6501 basic pair（bp）long and included a 6236bp ORF encoding 2166 amino-acid protein.The accession number of GenBank is MT259202;and the full cDNA sequence of non-L-type voltage-gated calcium channelα1 subunit gene（MgCα1A）was 6432 basic pair（bp）long and included a 6288bp ORF encoding 2095 amino-acid protein,and the accession number of GenBank is MT259203.The full cDNA sequence of voltage-gated calcium channelβsubunit（MgCavβ）was 2060 basic pair（bp）long and included a 1740bp ORF encoding 580 amino-acid protein.The accession number of GenBank is MT218396.The full cDNA sequence of voltage-gated calcium channelα₂δsubunit gene（MgCa_vα₂δ）was 3781 basic pair（bp）long and included a3600bp ORF encoding 1200 amino-acid protein.The landing number of GenBank is MT218395.The amino acid sequences of eachα1 subunit protein were compared,and it was found that the coding amino acid sequence of each calcium channelα1 subunit of H.elachista and M.graminicola had high homology with that of C.elegans and Ditylenchus destructor（D.destructor）.Therefore,the transmembrane structures and functional domains of the obtained calcium channelα1 subunits were determined with reference to C.elegans.The results showed that the proteins encoded by HeCα1D,HeCα1A,HeCα1G,MgCα1D,MgCα1G and MgCα1A all had four transmembrane domains and sixαtransmembrane helices in each transmembrane domain,which belonged to the basic structure of calcium channelα1 subunit protein.Based on the amino acid sequence comparison ofβsubunit andα₂δsubunit of M.graminicola,it was found that its important functional domain had high homology with beautiful hidden rod nematode.The functional domains of the two auxiliary subunits were determined by using C.elegans as a template.The relative expression of voltage-gated calcium channelα1 andβsubunits in M.graminicola was preliminarily determined by fluorescence quantitative PCR.The results showed that the expression of each gene was the highest in the J4,and decreased in the J2,J3and male after infection.The transgenic plants of voltage-gated calcium channelα1 subunit of upland rice cyst nematode have been obtained by RNAi technique.