Effect And Mechanism Of ChIFNs Gene On Tobacco Development And TMV Resistance

The breeding and application of disease-resistant varieties is an effective way to control tobacco virus diseases.In the previous research of the research group,the chicken interferon coding genes（Ch IFNs）,which have a wide range of biological activities such as inducing gene expression,antiviral and immune regulation,were used to create Ch IFN-α,Ch IFN-γand their hybrids tobacco（Ch IFN-α-γ）,Three types of genetically modified tobacco（TP）.This thesis uses ELISA,RNA-seq,q RT-PCR and other technologies to study the effects of Ch IFNs gene expression on the growth and development and TMV resistance of the above-mentioned transgenic tobacco,and its plant regulation mechanism,so as to provide a basis for the application of transgenic Ch IFNs.The main research contents and results of the thesis are as follows:1.The genetic stability of Ch IFNs transgenic tobacco.Using GUS histochemical staining,genome PCR amplification and ELISA experiments,the T₂ generation Ch IFN-α,Ch IFN-γand F₂ generation Ch IFN-α-γTP tobacco were identified.The results show that the three TP tobaccos have good genetic stability,the GUS reporter gene is positive,and the recombinant protein（r Ch IFN）expressed by the target gene translation in the three TP tobaccos are r Ch IFN-αis 27.05 ng·L^-1,r Ch IFN-γis 29.52 ng·L^-1,r Ch IFN-α-γis 52.64 ng·L^-1.2.Growth and development performance of transgenic Ch IFNs tobacco.The growth and development of TP5_{Ch IFN-}α,TP4_{Ch IFN-}γ,TP2 _{Ch IFN-}α_-γTP tobacco and wild-type parent（WT）on the transplanting soil for 25 days were observed and compared.It was found that the plant heights of the three TP tobaccos were increased by 5.18%,23.29%,and 44.71%（P<0.05）,and the leaf length increased by 2.74%,2.56%,and2.91%.The leaf width of the TP2_{Ch IFN-}α_-γtobacco was increased by 1.14%than that of the WT.TP tobacco is more robust and has better growth advantages.The expression of Ch IFNs gene promotes the growth and development of recipient tobacco.3.TMV resistance analysis of transgenic tobacco.The TMV virus solution stored at-80℃was diluted with PBS solution,and the virus was inoculated on tobacco leaves at the 7～8 leaf stage by mechanical friction method for proliferation.The virulence of TMV virus was identified and analyzed by ELISA,and it was found that at 24 h of inoculation The OD₄₅₀ value of TMV in the leaves is 0.390,and the virus inoculated has strong vigor.Inoculation experiments of TP and WT tobacco separated leaves of TMV found that the virus content in TP5_{Ch IFN-}α,TP4_{Ch IFN-}γ,TP2_{Ch IFN-}α_-γat 24 h was lower than that of WT type,with TP5_{Ch IFN-}αand TP2_{Ch IFN-}α_-γis the most significant,lower than WT blades 14.87%and 14.36%respectively（P<0.05）.Transgenic tobacco with Ch IFNs has good resistance to TMV.4.Photosynthetic properties of transgenic tobacco with Ch IFNs.The chlorophyll content in TP5_{Ch IFN-}α,TP4_{Ch IFN-}γ,and TP2 _{Ch IFN-}α_-γtobacco leaves transplanted for 25days were 0.33%,4.03%and 4.58%higher than that of WT,respectively;the net photosynthetic rate of the above three kinds of TP tobacco and WT tobacco,that are4.62 umol/（m²·s）,4.91 umol/（m²·s）,4.90 umol/（m²·s）and 5.08 umol/（m²·s）,respectively.The stomatal conductances are 0.13 umol/（m²·s）,0.10 umol/（m²·s）,0.10umol/（m²·s）.umol/（m²·s）and 0.09 umol/（m²·s）,the transpiration rate is 5.83umol/（m²·s）,5.68 umol/（m²·s）,5.59 umol/（m²·s）and 5.64 umol/（m²·s）.Ch IFN gene expression has no significant effect on the chlorophyll and photosynthetic parameters of the recipient tobacco.The chlorophyll measurement of detached leaves infected by TMV at the 7～8 leaf stage showed that the total chlorophyll content of TP2_{Ch IFN-}α_-γincreased by 7.115%at 12 h of TMV infection;TP5_{Ch IFN-}α,TP4_{Ch IFN-}γ,TP2_{Ch IFN-}α_-γat24 h Chlorophyll a in TP2_{Ch IFN-}α_-γand WT decreased by 5.361%,3.511%,5.952%and13.897%,respectively.The total chlorophyll of TP5_{Ch IFN-}αincreased by 4.546%,while the total chlorophyll of WT decreased by 16.580%;at 36 h,The chlorophyll a content of TP5Ch IFNα_-γ,TP4_{Ch IFN-}γ,TP2_{Ch IFN-}α_-γand WT all decreased,with WT being the most significant at 21.608%（P<0.01）.The chlorophyll of TP leaves can be effectively maintained after TMV infection,which has a good basis for light contraction.5.The effect of TMV infection on the germination and growth of tobacco seeds.The TP5_{Ch IFN-}α,TP4_{Ch IFN-}γ,TP2_{Ch IFN-}α_-γTP and WT tobacco seeds were immersed in TMV virus solution for germination experiments.The germination rates of the three TP and WT seeds treated with TMV were 44.67%,58.00%,60.00%and 8.00%,respectively,and the germination vigor was 19.33%,28.67%,32.00%and 0.67%,respectively.TMV treatment had little effect on the germination of TP tobacco seeds;compared with WT,three TP tobacco seeds showed whiteness 3 days earlier,and cotyledons formed 5 days earlier.Three TP tobacco seeds showed certain resistance to TMV during germination and growth.6.The growth and development and molecular regulation mechanism of T MV resistance in transgenic tobacco with Ch IFN-αgene.The c DNA library of Ch IFN-αgene and WT tobacco leaves infected with TMV on 0d,6d,9d was constructed and transcriptome sequencing was performed.Data analysis found t hat there were differences in the number of differentially expressed genes invol ved in growth or disease resistance regulation in TP and WT tobacco in the m iddle and late stages of virus infection;GO and KEGG analysis found that 12and 19 were enriched in TP and WT tobacco,respectively Pathway:Nitab4.5＿0000152g0350.1,Nitab4.5＿0000397g0020.1,Nitab4.5＿0000527g0160.1,Nitab4.5＿0001190g0040.1,Nitab4.5＿0001925g0090.1 and Nitab4.5＿0012714g0020.1 were excavated from tobacco co-expressed differential genes during the three stages of TMV infection and other 6 lines key genes for growth and disease resistanc e regulation.