ORMV-P125 Over-expression In Nicotiana Benthamiana And Establishment Of EMS Mutant Library For Restored Flowering In Arabidopsis Thaliana

Oil-Seed Mosaic Virus(ORMV)is a kind of rod-shaped monomer genome virus,which belongs to single-stranded ribonucleic acid and Tobamovirus,which can infect rape and other cruciferous crops.Its structural gene contains at least four open reading frames and can encode four proteins.The expressed 125 KDa replicase protein(P125)is not only a pathogenic protein of plant mosaic disease caused by rape mosaic virus,but also an inhibitor of gene silencing.P125 protein can bind to 21 nt RNA in vitro.Zhao Xianlei and others observed that the expression of P125 protein caused the late flowering phenotype of Arabidopsis thaliana.In this study,P125 protein was expressed in Nicotiana benthamiana by Agrobacterium tumefaciens-mediated transformation,so as to explore the effect of P125 protein on the normal growth and development of Nicotiana benthamiana,so as to make an comparison with the experimental results in Arabidopsis thaliana.And it is helpful to carry out protein interaction,plant disease interaction and other experiments.At the same time,the late flowering line Line31-1-1 was mutagenized by EMS(Ethyl methanesulfonate),and the flowering recovery mutants were screened in M1 and M2 populations respectively.In addition,the feasibility of miniaturized planting of Nicotiana benthamiana was explored,and a complete miniaturized planting method of Nicotiana benthamiana was established to improve the spatial multiple cropping index of Nicotiana benthamiana and provide favorable conditions for large-scale planting of Nicotiana benthamiana under laboratory conditions.The main results are as follows:1.A total of 19 Monoclonal antibody T1 lines were screened for Kan resistance,and a total of 15 lines accorded with 3:1 segregation ratio were obtained,and 7 Kan resistant pure lines were obtained in T2 generation.2.After secondary infection with Agrobacterium GV3101(p MDC160-P125),167 strains of T0 double-antibody Nicotiana benthamiana were obtained.69 T1 lines were screened for Basta resistance by plate screening,of which 56 accorded with3:1 segregation.139 T2 lines were screened by Kan plate and Basta plate,and the genotype data were obtained.Among them,26 lines were double-resistant homozygous lines,and 18 monoantibody homozygous lines containing only ORMV-P125 gene were isolated.3.A preliminary phenotypic observation of double-resistant Nicotiana benthamiana was carried out,and a notched leaf phenotype and a branching phenotype were found.4.Late flowering Arabidopsis thaliana was mutagenized by EMS,and a candidate for resuming flowering was found in M1 population.The Kan and Basta resistance of its progeny were identified by MEF-1,It was determined that the MEF-1 mutant was a double-resistant homozygous line,and the flowering time of its offspring was the same as that of wild-type Arabidopsis thaliana.5.In this paper,the feasibility of miniaturized planting of Nicotiana benthamiana was explored,a complete miniaturized planting method of Nicotiana benthamiana was established,and the multiple cropping index was increased by 8 times.All in all,in this study,the ORMV-P125 gene was expressed in Nicotiana benthamiana by Agrobacterium tumefaciens transformation,and the transgenic double-resistant homozygous lines were obtained and preliminary phenotypic observation;in addition,late flowering Arabidopsis thaliana was mutagenized by EMS,and a candidate mutant MEF-1 for resuming flowering was found and preliminarily identified.