| The control of viral disease in plants has become an importance-area of research in plant breeding by genetic engineering technology.ChIFN-αis a pleiotropic cytokine that possess powerful and wide-range of antiviral properties through multiple pathways.In this thesis, ChIFN-αgene was transformed into tobacco,Brassica napus L.and Lotus japonicus plants for obtaining transgenic lines with antiviral activity.Expression of ChIFN-αgene in transgenic tobacco plants conferred immunity to tobacco mosaic virus(TMV) infection.The possible resistance mechanism of biochemistry,molecule,cell morphology and plant regulation against TMV were studied.The results were list following.(1) The construction and verification of plant expression vector with ChIFN-αgene.pSFChIFNR,pSFRLH and pSWNRN of plant expression vectors carrying ChIFN-αgene were constructed.Then they were transformed into lettuce using an agro-infiltrated transient expression system.The results of GUS histochemical staining,reverse transcriptase polymerase chain reaction(RT-PCR),enzyme-linked immunosorbent assay(ELISA) and cytopathic effect (CPE) inhibition assay showed that the agro-infiltrated lettuce expressed ChIFN-αwith the maximum antiviral activity of about 8.2×102 AU·mg-1 of total soluble protein.The expression level was 0.0004%of total soluble protein in lettuce plants.(2) Establishment of transformation systems in Brassica napus L.and Lotus japonicus plants with ChIFN-αgene.A simplified genetic transformation method was set use seed as the explant in Brassica napus L.firstly.It shows that the transgenic Brassica napus L.could be easily obtained in a short of time without tissue culture procedure.A nother genetic transformation system of Lotus japonicus to obtain transgenic plants only 2.5-4.0 months was optimized using leaves,main stem and flower stalk explants.(3) Plant transformation and expression of ChIFN-αgene.Expression vectors of pSFChIFNR,pSFRLH and pSWNRN carrying ChIFN-αgene were transformed to tobacco,Brassica napus and Lotus japonicus plants by Agrobacterium-mediated transformation.The results of GUS histochemical staining,genome polymerase chain reaction (PCR),southern blot,RT-PCR,western blot,ELISA and CPE inhibition assay showed that 11 transgenic tobaaco lines,15 transgenic Lotus japonicus lines,and 26 transgenic Brassica napus L. lines had been selected respectively.ChIFN-αin transgenic plants had the markedly mRNA and protein expression and ChIFN-αwas integrated into plants genome.The results of ELISA showed that the expression level of recombinant protein of ChIFN-αwas 0.0003-0.0033%and 0.00002-0.0001%of total soluble protein in transgenic tobacco and Lotus japonicus plants respectively.The biological activity of ChIFN-αprotein from transgenic tobacco plants was 5.01×103 IU·g-1 of tobacco tissue in vitrol.(4) TMV resistance research in transgenic tobacco plants.The disease resistance of transgenic plant was further studied for plant protection.Transgenic tobacco had a certain resistance to tobacco mosaic virus(TMV) infection.After infection with TMV,the transgenic plants enhanced the disease resistance,delayed disease symptoms and reduced leaf lesions to different extent.(5) Effect of TMV infection on activities of defense enzymes in transgenic plants.Expression of ChIFN-αgene improved the activities of defense enzymes of peroxidase (POD),superoxide dismutase(SOD),and polyphenol oxidase(PPO) in transgenic tobacco plants. The activity of the POD and SOD in transgenic tobacco leaves was better than the CK before infection with TMV.When inoculated by the TMV,the POD activity in transgenic tobacco leaves increased at first and then decreased and it was found to be continuously decreased at the same time as the CK plants.The 40.13g-1·min-1 of SOD activity in transgenic tobacco leaves was higher than the 25.24g-1·min-1 of CK after 10 days infection with TMV.The PPO level was lower than that of CK,however,the POD activity in transgenic tobacco leaves increased continuously and it continuous decrease in CK after infection by TMV.The results also showed that PAL activity had no significant correlation with the expression of ChIFN-αgene.(6) Effects of TMV infection on related resistance genes in transgenic tobacco plants.PR-la,N and MAPK genes of y3 and y12 transgenic lines at different periods of TMV infection were all up-regulated and they were higher than in non-transgenic controls.When inoculated by the TMV,the PR-la expression in tobacco plants increased at first and then decreased and the enhanced amplitudes in transgenic plants were higher than CK plants.The PR-la expression levels of y3 and y12 lines were 276.00,33.75 times and 1.95,82.95 times of control lines in 6 and 15 days of TMV infection respectively.The transcriptional quantity of N and MAPK genes in tobacco increased continuously after infection by TMV.However,comparison of transgenic lines vs control of non-transgenic lines showed that up-regulated amplitudes of the former was better than the latter.The N expression levels of y3 and y12 lines were 1.20,0.01 times and 3.19,20.10 times of control lines in 6 and 15 days of TMV infection respectively.The MAPK expression levels of y3 and y12 lines were 5.51,2.63 times and 3.96,7.94 times of control lines in 6 and 15 days of TMV infection respectively.Therefore,it was considered that the ChIFN-αgene had a close relationship with the resistance or regulative genes in tobacco plants. The ChIFN-αgene might regulate the related gene expression after TMV infection.The continuous elevation of transcriptional level of ChIFN-αgene was also observed in transgenic tobacco infected with TMV.The ChIFN-αexpression levels in y3 and y12 transgenic lines were 17.89 and 2.09 times of control line in 15 days of TMV infection respectively.This result suggested that there had same mechanisms between the cell protection mechanism of ChIFN-αin plant and animal bodies.(7) Effects of TMV infection on chloroplast changes in the transgenic tobacco plants.Transgenic tobacco had a certain resistance to tobacco mosaic virus(TMV) in the infection initial stage.After infection with TMV,the chlorophyll content was found to be continuously decreased in the transgenic or non-transgenic lines.Nevertheless,the change of chlorophyll content in transgenic plant was obviously superior to the CK.The decrease of chlorophyll a of 10d TMV infection was 12.21%and 40.94%in transgenic and CK tobacco plants respectively. Meanwhile,the changes of chlorophyll b were 43.33%(reduction) and 3.42%(increase),and the decline of total chlorophyll were 41.64%and 9.77%in CK and transgenic tobacco plants respectively.The chlorophyll content of CK was found to be continuously increased and close to the transgenic plants after 10d of TMV infection,however,the CK average content was still lower than the chlorophyll of transgenic plants.The result showed that chloroplast of CK was damaged rapidly when inoculated by the TMV.The electron microscopic observation results also showed that ChIFN-αmight protect chloroplast of transgenic tobacco leaves.The cell ultrastructure of the transgenic plant from 15 and 25days TMV infection showed the disease course was severe and rapid in control plants of 8-10 leaves stage.In addition,virus transfer was observed from cytoplasm to chloroplast in cells of control leaf but not in transgenic leaves. (8) The regulatory effects of ChIFN-αgene expression in transgenic tobacco plants.Agronomic characters determination of plant height and root growth showed,introduced ChIFN-αgene had any harmful effect on tobacco cells,in contrary to promote the plant growth and root system development.ChIFN-αalso improves the ability to resist waterlogging for ameliorating the germination status under stress with water and resist high temperature by up-regulating the synthesis of heat shock proteins in transgenic tobacco plants.(9) The bioinformatics analysis of ChIFN-αactivity.ChIFN-αcan use as one potential antibacterial or antiviral drug in plant.The results of bioinformatics analysis showed that ChIFN-αwas one kind of transmembrane protein withα-helical andβfolding structure,and was similar to some of defensins or other antibacterial proteins in plants.In addition,the amino acid sequence of ChIFN-αwas also similar to some of regulatory or resistance proteins such as DNA damage repair,ribonuclease,pathogenesis-related proteins,antiviral protein and so on.ChIFN-αmay participate in the metabolic process and signal transduction of system defense for that there was certain similarity among ChIFN-αand other related proteins in plant body.Findings of above resistance mechanism of biochemistry,molecule, cell morphology and plant regulation against TMV in transgenic tobacco were confirmed by the bioinformatics analysis.The above results would prove helpful to produce antiviral resistant plant.It also provides some information about potential animal pharmaceutical applications in transgenic plants,which may lead a new way to prevent infectious diseases or improve the immune function of birds by direct oral administration in poultry fields. |
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