ChIFN–γ Regulated Function To Key Genes Associated With The Development Of Tobaccos Glandular Trichomes

As a kind of cytokine,Chicken interferon-gamma(ChIFN-γ)has high antiviral activity,antibacterial activity and anti-parasite activity.The previous research found Transgenic ChIFN-γ increased obviously in density of glandular trichome and the number of the glandular secretion,and it speculatled that characters improved resistance to herbivores attack of tobacoo.Bioinformatics analysis and microarray gene expression,speculated that three key gene on the signal pathway(CyP71,LBM1 and IspH)developed significant correlations with tobacco glandular hairs.Based on IspH function research,with the remaining two genes to study and purify the target protein by prokaryotic expression,the interaction of ChIFN-γ with 3 proteins was studied by isothermal calorimetry(ITC),and the results were as follows: in glandular secretion.Plant overexpression vector pSH737-CyP71 and pSH737-LBM1 was constructed,and transformed into Arabidopsis by Agrobacterium-mediated method.Seeds was collected and screened after genetic transformation of Arabidopsis,then many resistant plants for 50 and 65,The result of genome PCR rapid identification showed that positive.GUS staining and microscopic observation showed that the epidermal hair cells were not blue and the number of trichome in transgenic CyP71 Arabidopsis thaliana had more than doubled.increase fifty percent.all epidermal hair cells were blue and the number of trichome in transgenic LBM1 Arabidopsis thaliana had increase one hundred and twenty percent.indicating that the expression of LBM1 and the development of trichome were positively associated.ChIFN-γ and IspH was constructed into the prokaryotic expression vector of BL21(DE3)/pET28a-ChIFN-γ and BL21(DE3)/pET28a-IspH.CyP71 and LBM1 codonwas disposed by password optimized software.Removal the signal peptide of CyP71 and the 5’ twenty-four nucleotides in the sequence about LBM1,and construct into the prokaryotic expression vector of BL21(DE3)/pET30a-CyP71 and BL21(DE3)/pET30a-LBM1,The conditions for induced expression of four types recombinant bacterias were respectively:pET28a-ChIFN-γ/BL21(DE3):0.25 mmol/L IPTG 3L LB 37 ℃ 14 h,pET28a-IspH/BL21(DE3)0.5 mmol/L IPTG 3L LB 28 ℃ 14 h,pET30a-CyP71/BL21(DE3)and pET30a-LBM1/BL21(DE3)0.25 mmol/L IPTG 3L LB15 ℃ 14 hrecombinant bacteria.After expressed,four types of recombinant bacterias were treated by ultrasonication 、 inclusion body protein renaturation 、 NI-IDA nickel column dialysis and were detected using SDS-PAGE、Western blot、Bradford methods to obtain four types of corresponding protein.The concentration and purity of four types corresponding protein were respectively: Soluble protein ChIFN-γ0.616 mg/mL、95%,Soluble protein IspH 0.410 mg/ml、90%,inclusion bodies refolded protein LBM1 0.390mg/ml、90%,inclusion bodies refolded protein CyP71 0.248 mg/ml、90%.The interaction of ChIFN-γ and IspH,CyP71 and LBM1 was verified by ITC.ChIFN-γ used as titration protein,IspH,CyP71 and LBM1 had been titrated,the result was that the interaction between ChIFN-γ protein interacts with IspH protein via hydrophobic interaction,the appetency was 0.074 μmol/L;While ChIFN-γ and CyP71 were hydrogen bond and VDW,the appetency was 0.141 μmol/L;while ChIFN-γand LBM1 had no interaction.The above analysis results,ChIFN-γ may be involved in the interaction of IspH and its downstream CyP71 proteins in the MEP pathway,Or ChIFN-γ causes changes in conformation or activity of the interacting proteins,lead to an increase in trichome numbers of tobacco trichome or Arabidopsis thaliana,the detailed mechanism of action needs further study.