Cloning Of Large Conductance Ca²⁺ Activated K⁺ Channel α Subunit Gene And Construction Of Stable Cell Lines Of Ditylenchus Destructor

The large conductance calcium activated potassium channel(BK)is located on the cell membrane of organism,which has high permeability to K~+,can be activated by calcium ion and membrane voltage,and plays critical roles in regulating cell excitability.At present,there are few studies on the large conductance calcium-activated potassium channel of plant parasitic nematodes,and its structure and function have not yet been determined.The purpose of this study was to cloning BK channelαsubunit gene of Ditylenchus destructor.The experimental results show that the full length of Dd-slo1 c DNA is 3456 bp encoding 1124 amino acids,and the 5’UTR is 45 bp,the 3’UTR is 36 bp,Gen Bank accession number is MW015082.Through bioinformatics analysis of amino acid sequence of BK channel subunit,it is known that the BK channelαsubunit of D.destructor has high homology with invertebrates,and the functional domains in organisms are basically the same,which is highly conserved in the evolutionary process.By analyzing its protein structure,it can be seen that theαsubunit of BK channel of D.destructor has seven transmembrane domains(S0-S6),among which S1-S6 is the transmembrane domain of all voltage-gated potassium channels,S0 is the unique transmembrane domain of BK channels,and it also contains hydrophobic domains of S7-S10,which form a large carboxyl end,is also the unique structure of BK channel.The full-length coding sequence of slo1 gene of BK channel of D.destructor was optimized according to the optimization of mammalian codon,and then connected to p IRESneo3 plasmid to construct p IRESneo3-Dd-slo1 eukaryotic expression vector.The eukaryotic expression vector was successfully constructed by NotⅠand NheⅠdouble digestion.The p IRESneo3-Dd-slo1 vector and GFP were co-transfected into HEK 293 cells by liposome transfection method,and HEK 293 cell lines stably expressing p IRESneo3-Dd-slo1 were screened through G418 resistance.The results showed that after 14 days of G418resistance screening,cells expressing green fluorescent protein were selected for monoclonal cell culture,and HEK 293 single cell line expressing fluorescent protein was observed under fluorescence inverted microscope.According to the expression of fluorescent protein,it was preliminarily confirmed that BK channelαsubunit gene of D.destructor existed in HEK 293single cell line.