【作者】 宋莉；

【导师】 赵德刚；

【作者基本信息】 贵州大学 ， 农药学， 2009， 博士

【摘要】 植物病毒病是严重危害农业生产的重要病害之一,抗病品种的培育是防治植物病毒病的有效措施,为了揭示动物细胞因子对植物病毒的抗性作用及其在植物体内的保护机制,本论文报道了编码高效和广谱抗病毒干扰素的ChIFN-α基因遗传转化植物及提高烟草抗病性的研究。试验构建含有ChIFN-α基因的植物双元表达载体,采用农杆菌介导法将ChIFN-α基因导入植物细胞中,以组织化学鉴定和分子生物学鉴定技术筛选出转基因植株,研究转基因植株表达干扰素蛋白的生物学活性,并对转基因烟草对花叶病毒（TMV）的抗性及其生化、分子、细胞学机制和ChIFN-α基因表达对烟草生长及抗逆性的调控作用进行了较为全面深入的研究,取得结果如下:（1）植物表达载体构建及验证。构建了由35S驱动ChIFN-α基因的植物双元表达载体pSFChIFNR和pSFRLH,构建了以Napin驱动的ChIFN-α基因的pSWNRN植物双元表达载体。表达载体经生菜真空渗透瞬时表达系统检验目的基因的表达,通过GUS组织化学染色、ChIFN-α基因的PCR和RT-PCR检测,以及表达蛋白的ELISA和细胞病变抑制实验（CPE）,证明35S和Napin启动子驱动的ChIFN-α基因均能在植物中获得表达,具有体外抗病毒的生物学活性,重组蛋白约占生菜总可溶蛋白的0.0004%,体外抗滤泡性口炎病毒（VSV）活性为8.2×10² AU·mg^-1。（2）植物遗传转化体系的建立。首次建立以油菜种子为材料的超声波辅助农杆菌介导的遗传转化方法。适宜的种子处理和转化条件为:超声波振幅/超声时间/侵染时间=5μm/0.5h/20h,获得转基因植株的周期（?）2月,该方法不需要复杂和消毒严格的组织培养操作,具有快速、简单、成本低廉和易于大规模转化等特点。建立百脉根真叶叶片、主茎和花茎作为外植体的遗传转化体系,优化MS+3.0mg·L^-1 6BA+0.1mg·L^-1 NAA为百脉根愈伤诱导培养基,MS+0.3mg·L^-1 KT为芽分化培养基,1/2MS+0.4mg·L^-1 IBA为生根培养基,45.0mg·L^-1Kan作为抗性筛选浓度。该体系克服子叶和胚轴外植体细小、柔软而不易操作等不足,获得再生植株的周期为2.5-4.0个月,比报道的子叶和胚轴外植体转化体系短0.5-5.0个月。（3）ChIFN-α基因的植物转化与表达。通过农杆菌介导法将含ChIFN-α基因的植物表达载体pSFChIFNR、pSFRLH和pSWNRN分别导入烟草、油菜和百脉根植物中,经报告基因GUS组织化学染色及ChIFN-α基因的PCR、Southern blot和RT-PCR检测,ChIFN-α基因插入受体植物染色体基因组中,进行了正确转录表达。筛选出转基因烟草11株,转基因百脉根15株,转基因油菜26株。经Western blot、ELISA和CPE检测,重组ChIFN-α蛋白在转基因植物体内得到正确翻译表达,其在烟草和百脉根植物中分别约占总可溶蛋白的0.0003-0.0033%和0.00002-0.0001%:植物表达的ChIFN-α分子量为29.51kD:CPE实验证明转基因植株粗提蛋白具有抗病毒的生物学活性,能够保护鸡胚成纤维细胞（CEF）免受VSV的攻击,其体外抗病毒活性为5.01×10³ IU·g^-1。（4）转基因烟草对TMV的抗性。对T₀代转基因及同批非转基因烟草对照苗进行烟草花叶病毒TMV接种试验,转基因烟草全部出现花叶病症的时间比非转基因对照推迟3-6d:转基因烟草在病毒侵染10d时的发病率为0,对照为60%,此时转基因植株的平均病情指数为50,非转基因对照为77.78;26d试验期内转基因烟草对TMV的相对防效平均为34.18%。说明转基因烟草植株在病毒侵染后表现出发病率低、发病时间推迟和病症减轻,首次证明转ChIFN-α基因烟草对TMV初期侵染具有一定抗性。（5）TMV接种对转基因烟草的防御酶活性影响。侵染前转基因烟草叶片中的过氧化物酶（POD）和超氧化物歧化酶（SOD）活性均高于对照植株,二者在转基因植株中的活性分别为10.6和77.67g^-1·min^-1,在对照中为和46.63g^-1·min^-1;TMV接种后POD活性在转基因烟草中先降低后升高,而对照中呈持续下降趋势,且低于转基因组;转基因组的SOD活性在侵染10d时平均为40.13g^-1·min^-1,高于对照的25.24g^-1·min^-1。多酚氧化酶（PPO）活性在TMV接种0d时的转基因烟草叶片中为11.97g^-1·min^-1,对照中为31.1g^-1·min^-1;在侵染10d和28d的转基因叶片中分别为22.35和23.95g^-1·min^-1,对照中为14和50g^-1·min^-1;TMV接种前转基因烟草叶片中多酚氧化酶（PPO）的活性平均低于对照,但TMV接种0-10d内其酶活性在转基因植株中呈上升趋势,对照则逐渐下降,转基因植株表现出在病毒侵染初期没有产生应激和PPO合成增加现象。TMV接种后转基因植物中苯丙氨酸解氨酶（PAL）活性相对增幅低于对照,降幅快于对照。研究说明,ChIFN-α基因的导入促进了烟草体内POD、SOD和PPO三种防御酶活性的增高,但是和PAL活性的相关性不明显。（6）TMV接种对转基因烟草中抗病相关基因表达的影响。对接种TMV前后的烟草植株中的病程相关蛋白基因PR-la、N基因和促分裂原活化蛋白激酶基因MAPK的表达差异进行了荧光实时定量检测。TMV接种后烟草植株体内PR-la基因的转录活性呈先上升后下降趋势;侵染6d时,转基因植株中PR-la基因的转录活性较对照植株分别增强276.00倍和33.75倍;15d时转基因植株中PR-la的表达量分别比对照提高1.95倍和82.95倍。TMV接种后烟草植株体内抗病基因N基因和信号转导基因MAPK基因的表达均呈持续上升趋势:侵染6d时,转基因植株中N基因表达量分别为对照的1.20倍和0.01倍,15d时转基因植株中N基因的表达量分别比对照提高3.19倍和20.10倍;侵染6d时,转基因植株中MAPK基因表达量分别较对照提高5.51倍和2.63倍,15d时转基因植株中MAPK的表达量分别较对照组提高3.96倍和7.94倍。研究说明ChIFN-α基因参与了植物防御反应的调节过程,ChIFN-α基因的导入通过上调防卫基因（PR-la基因）、植物抗病基因（N基因）和信号转导基因（MAPK基因）的表达,从多个途径综合实现转基因烟草对TMV的抗性。对TMV侵染后烟草植株中ChIFN-α基因的表达检测得到,TMV接种后15d,转基因植株中ChIFN-α基因的相对表达量分别是接种前的17.89和2.09倍,表明ChIFN-α基因的表达受病毒侵染的诱导,植物体中ChIFN-α基因的作用途径与动物机体相似,病毒感染激活细胞中干扰素基因的表达,进一步促进机体抗病基因表达,从而建立机体的抗病毒状态。（7）TMV接种对转基因烟草叶绿体的影响。接种10d内转基因与对照植株中的叶绿素含量均呈现下降趋势,但对照组降幅大,降速快。侵染10d时对照叶片中叶绿素a含量下降40.94%,转基因组下降12.21%;对照中叶绿素b含量下降43.33%,转基因组中则增加了3.42%;叶绿素总量的下降在对照和转基因植株中分别为41.64%和9.77%。病毒侵染10d后对照植株中叶绿素含量开始上升并逐渐接近转基因组,但28d时其平均含量仍低于转基因组。转基因植株在受到病毒侵染后,其体内叶绿素含量变化幅度不大;对照植物体内叶绿素含量的迅速降低,说明病毒侵染后其叶绿体可能被破坏。以8-10叶期的烟草组培苗接种TMV,对病毒侵染15d和25d的病叶进行细胞超微结构观察,样品细胞内的叶绿体、线粒体和细胞壁等细胞器或组织都出现不同程度的解体,但与转基因烟草细胞的损伤相比较,对照发病重,发展快。对照细胞中出现病毒粒子从细胞质向叶绿体的转移,表明转基因烟草叶片中,叶绿体受到病毒攻击时间晚于对照,转基因植株可能通过推迟病毒对叶绿体的破坏和损伤而延迟花叶病症的出现。植物叶片中叶绿素的含量变化和细胞学的研究说明,ChIFN-α对受体植物叶绿体具有一定的保护作用,在病毒侵染初期能抑制病毒的复制。（8）ChIFN-α基因表达对植物的调控作用。通过对转基因烟草植株农艺性状（株高、发芽和生根）及非生物胁迫影响（水涝和高温环境）的研究,结果表明转入ChIFN-α基因对植物生长没有产生有害影响,受体烟草品系种性未发生改变。ChIFN-α基因的导入能够促进植物生长和早期根系发育以及具有抗逆调控作用。转ChIFN-α基因烟草的早期根系形成快,发育好,促进植物营养吸收和生长健壮,提高了烟草的抗病性。转基因烟草对水涝和高温的耐受性优于对照植株,ChIFN-α基因的表达可以改善水涝条件下的种子萌发和促进转基因植株合成热激蛋白,增加对植物的抗逆保护作用,提高植物抗病性。（9）ChIFN-α蛋白的生物学活性分析。采用生物信息学手段,在APD和NCBI两个数据库进行抗性预测和同源性比较,发现ChIFN-α具有两亲性,倾向于形成跨膜结构、α螺旋以及β折叠结构,和植物防御素等抗性蛋白结构相似,具有作为植物抗性药物筛选的潜力。ChIFN-α氨基酸序列与APD数据库中的9个防御素及抗菌植物蛋白等具有较高相似性（＞25%）,表明在植物体内表达ChIFN-α会对植物产生一定的保护作用。ChIFN-α与植物DNA损伤修复、核糖核酸酶、病程相关蛋白和抗病毒蛋白等均有一定的相似性,也说明其具有抗植物病毒病的药物运用潜力。ChIFN-α蛋白序列与生物体中参与生命活动和信号转导的一些蛋白质具有30%以上的同源性,提示ChIFN-α的作用方式可能通过促进植物体的代谢过程或参与诱导植物系统防御的信号转导而间接发生作用。生物信息学分析证实了本研究关于转ChIFN-α基因烟草对TMV的生理生化、分子水平、细胞学水平及植物调控的抗性机制等方面的研究结果。以上研究表明ChIFN-α是一类在植物体内具有抗病毒信息的候选药物,ChIFN-α基因导入植物细胞后,对叶绿素的稳定及POD、SOD和PPO三种植物防御酶活性具有调节作用,在TMV侵染时可以上调PR-la、N和MAPK三种抗病或调节基因的表达,通过促进植物早期生长,提高其非生物胁迫耐性,增强其抗病力,提高了转基因烟草植株对TMV的抗性。本研究为转基因生物农药的研制提供了一定依据,为培育自身具有抗逆作用,同时又可以提高动物免疫力的农作物或牧草新品种,以及转基因植物生物反应器的研究奠定了一定基础。更多还原

【Abstract】 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.更多还原