The Prokaryotic Expression Of Pest-resistant Genes In Nicotiana Tabacum And The Interaction With ChIFN-? Protein

Chicken interferon gamma(ChIFN-?)is the cytokines,which has the effect of Immunoregulation,antiviral activity,antibacterial action and antiparasitic immunity,and ChIFN-? produced by activated Th cells and NK cells and belongs to type?interferon.The ChIFN-? was successfully expressed in Nicotiana tabacum in the early stage of the experiment in Our laboratory,and it was found that the resistance was significantly enhanced in transgenic plants,putting forward a new insight into the function of ChIFN-? on the insect resistance.Based on this discovery,the wild type tobacco was used as control in the early stage of laboratory,and the differentially expressed genes in transgenic tobacco were screened by the whole genome expression microarray technology.The candidate genes,CAF1,HD and cop8/CSN4,related to resistance were used to screen in tobacco by GO and KEGG Pathway.The target gene HD,CAF1 and Cop8/CSN4 were constructed into prokaryotic expression vector pET28 a,and were transformed into BL21(DE3).The Cop8 protein was detected by SDS-PAGE and Western blotting.HD and CAF1 were optimized and reconstructed into prokaryotic expression vector pET30 a and transformed into the host bacteria Rosetta(DE3)and BL21(DE3)respectively.The target proteins were induced to express with 0.25 mM IPTG at 15? for 16 h,and recombinant fusion proteins of HD,CAF1 and Cop8 were obtained by ultrasonic disruption,inclusion body deformation,renaturation,NI-IDA nickel column,dialysis.The Cop8 protein concentration was 0.476mg/mL and its purity was up to 95%,and the HD protein concentration was 0.295mg/mL and its purity was up to 90%,and CAF1 protein concentration was 0.213mg/mL and its purity was up to 90%.Isothermal titration method was used to study the interaction between ChIFN-?protein and HD protein,CAF1 protein and Cop8 protein.The results showed thatChIFN-? protein and HD protein,CAF1 protein and Cop8 protein interaction had the reaction.The affinity between CAF1 protein and ChIFN-? protein is 0.781 ?M.The affinity between HD protein and ChIFN-? protein is 0.168 ?M.The affinity between Cop8 protein and ChIFN-? protein is 0.269 ?M.The resistance mechanism in transgenic tobacco ChIFN-?-mediated may regulate the expression of downstream specific genes HD,CAF1 and Cop8/CSN4 induced the plant defense response depending on JA ?SA or ABA,and the transgenic plants finally had the function of resistant insect.