Screening Of Cadmium Transport And Nornicotine Metabolism-related Genes Mutation In Tobacco Using HRM Method

Tobacco is a typical cadmium-rich crop.The cadmium will enter into the human body form the smoke,which is harmful to people.Nornicotine is the precursor of N’-nitrosonornicotine（NNN）which is one most abundant carcinogenic tobacco-specific nitrosamines in tobacco.Thus,development of tobacco with low cadmium and low-nornicotine is important for the safety of tobacco breeding and production.This study established a method for detecting mutations using high resolution melting（HRM）curve analysis in tobacco.Besides,this study identify some mutations of cadmium transporter gene HMA4,MRP4,and nornicotine metabolism-related genes CYP82E4,CYP82E5v2,CYP82E10 target gene using established HRM technology in tobacco cultivar Guiyan No.1 M₂ population treated by EMS.Furthermore,the phenotype of some homozygous mutants was identified.The main results are as follows:1.The gene pool of wild-type samples:mutant ratio of 2:1 to 8:1 can be distinguished well in mutation detection of tobacco;HRM not only can detect SNP,but also for small fragment insertion/deletion;Optimized HRM can detect 1000 bp fragment in tabacco.2.Mutations of cadmium transporter-related gene HMA4,MRP4 and nornicotine metabolism-related genes CYP82E4,CYP82E5v2 and CYP82E10 were screened from tobacco cultivar Guiyan No.1 M₂ population treated by EMS using established HRM method.Furthermore,combined with DNA sequencing,69 materials were confirmed,which include 27 cadmium transporter-related gene mutations and 42 mutations.The mutation frequency of Cd transporter-related gene is about 1/458 kb while its nornicotine metabolism-related gene is 1/328 kb.Besides,the sensitivity of HMA4,MRP4 and nornicotine metabolism-related gene are 58%,40%and 49.6%,respectively.3.The 13 homozygous Cd transporter-related gene mutants were grown in hydroponic condition with cadmium treatment.The Cd concentration was determined by ICP-MS method.The results showed that,the Cd concentration of 8 from selected 13 materials are significant lower than control.And the decline is between 16.19%⁴2.08%.The results lay the foundation for development of new tobacco varieties with low cadmium concentration.