Establishment Of Efficient Nicotiana Benthamiana Transformation System And Stable Expression Of 126kDa Replicase In Plants

Oil-Seed Mosaic Virus?ORMV?is a classical Tobaccovirus,which infects the Brassica family,such as Brassica napus and ararbidopsis thaliana.Its genome encodes four proteins,the 126kDa replicase,the 183kDa replicase,movement protein,coat protein,respectively.The 126kDa protein size-specifically binds to 21nt small RNAs.In this study,the ORMV-126kDa replicase in Arabidopsis thaliana and Nicotiana benthamiana?N.ben?were overpressed,which laid a foundation for further study on the enrichment of the protein on the small RNA in plants and the effect of the protein on the function and metabolism of small RNA.In this experiment,the leaves of wild-type N.ben were used as explants.Effects of different hormone ratios and concentrations on tobacco regeneration in vitro,different concentration of AgNO₃ and gellan gum on sprouting,and different NAA also were studied.The in vitro regeneration system of N.ben was preliminarily optimized.The screening concentrations of different kanamycin?Kan?and herbicides?Basta?,the bacteriostatic concentration of cephalosporin,the infestation time,the pre-culture and co-culture time,and Acetosyringone?AS?were also studied.Finally,a highly efficient N.ben genetic transformation system was established.On this basis,an inducible expression system of126kD replicase protein in N.ben was established.At the same time,the Arabidopsis lines in the laboratory were maintained,propagated,screened and phenotypically observed.Among them,two different phenotypes related to development were discovered.The main findings are as follows:1.The T1 transgenic Arabidopsis thaliana was screened and propagated in the early stage.Five SUC2-126kDa double-resistant Arabidopsis plants and Four 35S-126kDa double-resistant Arabidopsis plants were obtained.Two homogenous lines with phenotypes were obtained.2.Using wild type N.benthamiana as experimental material,we established an efficient regeneration.The 40 d seedling sterile tobacco leaves were selected as explants,and the best combination of hormones for inducing callus formation in tobacco leaves was6-BA 4 mg/L,NAA 0.2 mg/L,3 mg/L AgNO₃ and 9 g/L Gum is more conducive to promoting sprouting,and 0.001 mg/L NAA promotes rooting.3.The optimized the genetic transformation system based on the establishment of a tobacco regeneration system.Different with most of the studies,we found that the transient conversion efficiency was the highest when co-cultured for three days at 18? for tow days after pre-incubation at 25?.4.By now,ten PCR-positive tobacco kanamycin resistant strains?including activation elements?have been obtained;four PCR-positive tobacco Basta resistant strains?including response elements?have been obtained.