【作者】 马进；

【导师】 温孚江； 朱常香；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 水稻条纹叶枯病(Rice stripe disease)是水稻上普遍发生、危害严重的一种病毒病,其病原为水稻条纹病毒(Rice stripe virus, RSV)。近年来,随着种植方式的改变,耕作栽培技术的变化,导致水稻条纹叶枯病在黄淮稻区迅速回升,1999年后暴发成灾,目前己成为该稻区水稻生产上最主要的病害。水稻感染RSV后,造成水稻心叶褪绿,捻转,并弧圈装下垂,严重的心叶枯死,导致水稻产量大幅降低,甚至绝产。RSV主要由介体灰飞虱持久性经卵传播,电镜下观察病毒粒子呈丝状粒体,以折叠、分枝和超螺旋等形式存在。RNA介导的病毒抗性(RNA-Mediated Virus Resistance, RMVR)是近年来发展起来的一种新的抗病毒基因工程策略,其通过对入侵的病毒RNA进行降解,使入侵的病毒不能在植物中积累,从而赋予转基因植物病毒抗性。本研究探求了RSV致病性增强的分子机理,并培育了高抗且稳定遗传到T2代的抗水稻条纹病毒的转基因水稻,对于提高水稻的产量,保护水稻生产的可持续发展,具有重大意义。研究结果和主要结论如下：(1)水稻条纹病毒山东济宁分离物的分子变异分析传统的生物学测定发现RSV不同分离物之间存在着化学组成和致病性上的差异。近些年的研究表明,这种差异同样表现在核苷酸的水平。通过对部分分离物的RNA3、RNA4或部分编码基因的序列分析发现,RSV的基因序列和5′端及3′端非编码区序列相当保守,变异主要发生在基因间隔区(IR),但目前尚缺乏对不同类型病毒种群的遗传变异的系统分析。从山东济宁感病的水稻上,分离到强致病性水稻条纹病毒,命名为RSV-SD-JN2。为了从分子水平探讨RSV-SD-JN2变异及其致病性增强的原因,采用RT-PCR技术,克隆RSV-SD-JN2的RNA3.RNA4区段cDNA。序列测定结果显示,RSV-SD-JN2的RNA3和RNA4核苷酸序列长度分别为2487bp、2157bp；RNA3中,NS3基因长为636bp,IR为725bp,CP基因为969bp；RNA4中,SP基因长为537bp,IR为654bp,NSvc4基因为861bp。将RSV-SD-JN2与不同时期、不同区域和不同致病性的辽宁盘锦PJ、北京双桥SQ、江苏洪泽HZ、云南昆明KM、山东济宁SD-JN1,江苏江阴JY、浙江ZHEJIANG分离物的RNA3、RNA4全序列或部分序列进行比较分析,发现RNA3、RNA4序列5′和3′末端非编码区具有高度保守,仅存在个别碱基的差异；基因编码区保守性较高,核苷酸序列同源性均在93%以上,氨基酸序列同源性高达97%以上,且大部分碱基变异为无意义变异；IR易于变异,IR4的核苷酸序列同源性仅在91.6%-94.3%之间,而IR3的同源性为85.8%-96.7%。IR的变异导致RNA二级结构一发夹结构的稳定性提高是病毒致病性增强的重要原因。同时发现,SD-JN2分离物的RNA3内部各个序列可能存在不同的亲缘关系；NS3基因和CP基因与HZ分离物有很高的同源性,分别为98.9%和99.1%；而IR仅为86.3%,推测是由不同亚群的分离物的基因组相应片段经过交换重配引起的。(2)抗RSV两种分离物转基因水稻新种质的培育目前,种植抗条纹叶枯病品种是防治RSV最根本、有效的方法。而粳稻是黄淮稻区主要的种植品种,由于粳稻中缺少抗条纹叶枯病基因,病害的流行和发生已经给水稻生产造成重大损失。常规的培育水稻抗病品种的方法周期长、效率低。RNA介导病毒抗性是目前培育抗病毒作物最为有效而且快捷的方式,其具有抗性表型近乎免疫、抗性持久、生物安全性高等特点,是一种具有实际应用价值的植物抗病毒基因工程策略。但是RNA介导的病毒抗性还具有抗病性谱窄的缺点。为了克服这种缺点,可以通过构建包含同一病毒不同基因的嵌合基因片段的表达载体,导入植物,既可以提高抗性水平,又能增强对同一病毒不同分离物抗性范围及抗性遗传的稳定性。本研究基于RNAi的原理,同时为了克服因病毒突变造成的抗性逃逸,培育了高抗、广谱并且稳定遗传的转基因水稻。首先改造了pCAMBIA1300表达载体,加入了玉米泛素蛋白基因Ubi启动子,胭脂碱合成酶基因3′端Nos终止子,将选择标记基因Hygromycin B替换为生物安全性更高的Bar基因,构建了新的表达载体pUNB。利用RSV-SD-JN2的CP、SP基因部分片度构建了包含CP/SP嵌合基因及单CP、单SP基因的RNAi载体p1300CP/SP p1300CP、p1300SP；冻融法导入农杆菌EHA105,利用农杆菌介导的转化方法,转化水稻豫粳6号愈伤组织,分别获得了TO代转基因植株23、24和17棵。自交结实获得T1代株系,每个载体的T1代转基因株系各选取100棵,用来自山东济宁和江苏淮安RSV分离物接种,病毒抗性的检测结果显示：转化p1300CP/SP的转基因株系中有CS1、CS3、CS6、CS7、CS10、CS13、CS15、CS16、CS19株系,转化p1300CP的转基因株系中有CP1、CP3、CP4、CP7、CP9、CP12株系转化,p1300SP的转基因株系中中有SP2、SP5、SP6、SP7、SP13株系对两种病毒分离物的感病率都在6%以下,远远低于转化空载体转基因株系野生型株系的感病率,表现为抗性株系。三个载体其余株系的感病率都在12%以上,表现为中抗和感病株系。表明p1300CP/SP、p1300CP、p1300SP的T1代转基因株系中抗病株系的比率分别为39.13%、25.00%、29.41%。含CP/SP嵌合基因的转基因株系的抗病性和广谱性要好于转化单CP基因和单SP基因的转基因株系,说明同时干扰RSV多个基因的表达,可以更加有效的抵御RSV病毒的入侵,并且对不同地区分离物具有广谱抗性。T1代抗性株系的Southern杂交显示,外源基因以不同拷贝整合于水稻的基因组中,并进行了有效表达,且转基因植株的抗病性与转基因的拷贝数之间无明显的相关性；T1代抗性株系的总RNA和siRNA的Northern杂交显示,抗病植株中RNA的积累量明显低于同类型的感病植株,转基因植株中有siRNA存在,表明病毒抗性是由RNAi引发的。选取转化p1300CP/SP、p1300CP、p1300SP载体的T1代单拷贝抗性植株CS1、CS6、CP1、CP3、CP9、SP5、SP7进行T2代遗传分析,结果表明,转基因及其介导的抗性可以稳定的遗传至T2代,并且获得CSl和CP9两个不在发生分离的纯合体株系。更多还原

【Abstract】 Rice stripe disease, with the pathogen Rice stripe virus (RSV), is one of the most widespread and severe virus diseases. In recent years, a quick spread of this disease had caused great losses in Huanghuai paddy fields due to changes in farming cultivation practices and technologies. Since 1999, the disease has broken out and become the major disease of this area. Infection of rice plants by RSV leads to the appearance of chlorotic stripes or mottling and necrotic streaks on leaves, with subsequent premature wilting which results in considerable decrease in grain yield. RSV is a representative of the genus Tenuivirus that is transmitted by a small brown planthopper, Laodelphax striatellus Fallen (Hemiptera, Delphacidae), in circulative-propagative and transovarial manners. RSV has filamentous particles which appear folded, branched and supercoiled in an electron microscope. A new powerful type of resistance, based upon the presence of RNA, known as RNA-mediated virus resistance (RMVR), was characterized by a high level of resistance that was not easily overcome by a high dosage of inoculums. In this study, we explore the molecular mechanism of RSV severer pathogenicity, and plant strong resistant and stably inherited T2 transgenic rice. The significant result can help increase the rice harvest and keep the sustainable development of rice product. The main results and conclusions presented in this thesis are as follows:(1) The analysis of molecular variability of RSV isolate (RSV-SD-JN2) in Jining ShandongTraditonal biology assay found that there is chemical composition and pathogenicity difference among different isolates of RSV. The difference expressed on the nucleotide level in recent years’study. According to the sequence analysis of RNA3, RNA4, or part encoding gene of some isolates, RSV gene sequence,5’or 3’untranslated regions are very conserved, the main variability occur in the intergenic region(IR). However, there is no systematic analyse of genetic variation among different virus population.A highly pathogenic Rice stripe virus was isolated from susceptible rice in Jining, Shandong Province, China, and was designated as RSV-SD-JN2. In order to explore the reason of RSV molecular variation and severer pathogenicity on the molecular level, we cloned the RNA3 and RNA4 cDNA fragments of RSV-SD-JN2 by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequenced them. The results showed that the length of RNA3 and RNA4 of SD-JN2 were 2487bp and 2157bp respectively; the NS3 gene, IR and CP gene in RNA3 were 636bp,725bp and 969bp, respectively; the SP gene, and IR and NSvc4 in RNA4 was 537bp,654bp,861bp, respectively.Compared with the complete or part sequences of the RSV isolates from Panjin (PJ) in Liaoning province, Shuangqiao (SQ) in Beijing, Hongze (HZ) in Jiangsu, Kunming (KM) in yunan provice, Jining (SD-JN1) in Shandong, Jiangyin (JY) in Jiangsu province, ZHEJING in Zhejiang province that had been reported previously with different period, different area, different pathogenicity, we found that the most highly conserved regions located in 5’and 3’ untranslated regions, which had only few base difference; the highly consensus of encoding regions also revealed more than 93% identity at the nucleotide sequence level, and more than 97% identity at the amino acid level, and most bases mutation was nonsense mutation; however, the most variable regions located in the intergenic region(IR). The variability of IR leaded to enhancement of RNA secondary structures-hairpin structures stability, which was the important reason of severer pathogenicity. Moreover, there is possible different genetic relationship among internal RNA3 different sequences of SD-JN2 isolate; the high homology of NS3 gene and CP gene compared with HZ isolate, is 98.9% and 99.1% respectively, but IR is 86.3%. It suggests that exchanged recombinant of different subgroup isolate cause the genome relevant fragment cause the result.(2) Production of transgenic rice new germplasm against two isolations of RSVCultivating a resistant breed is one of the most economical and efficient methods in preventing rice virus disease. However, a conventional method of breeding resistant varieties is a long-term and low-efficiency process. With the development of genetic engineering, transgenic technology has become a fast and efficient method of developing virus resistant varieties. RMVR, which has the advantage of high resistance (almost immunity), resistance durability, and high biosafety, has become a more tangible strategy to develop virus resistant plants. Although RMVR has a narrow resistance spectrum, transgenic plants can be produced by cloning effective nucleic acid fragments of different genes of the same virus, constructing plant expression vectors, and inserting into plants to extend resistance against different isolates of the same virus, apart from enhancing and stabilizing its genetic resistance. In this study, we generated stable, RSV-resistant, transgenic lines. The success in producing new germplasms of highly resistant transgenic rice plant will lay the theory foundation for breeding RSV-resistant rice to prevent resistance loss due to virus mutation.Expression vector pCAMBIA1300UNB was constructed with inserting Ubiquitin promoter and Nos-3’terminator, Changing hygromycin B resistance gene with Bialaphos resistance gene (Bar) based on vector pCAMBIA1300. Following RNA interference (RNAi) theory, we constructed three RNAi binary vectors based on coat protein(CP), special-disease protein(SP) and chimeric CP/SP gene sequence. Transgenic lines of rice CV. Yujing6 were generated through Agrobacterium-mediated transformation. We got T0 transgenic plants 23, 24 and 17, respectively.The self-fertilized seeds from T0 generation transgenic plants of transformed p1300CP/SP, p1300CP, and p1300SP vectors were planted to obtain T1 generation lines. In every transgenic line,100 positive plants were selected and inoculated with two RSV isolates SD-JN2 and JS-HA using viruliferous vector insects. Wild types (cv. Yujing 6) were used as control and then monitored daily for the appearance of symptoms. After four weeks of inoculation, lines CS1, CS3, CS6, CS7, CS10, CS13, CS15, CS16, and CS19 of p1300CP/SP transgenic plants; lines CP1, CP3, CP4, CP7, CP9, and CP12 of p1300CP transgenic plants; and lines SP2, SP5, SP6, SP7, and SP13 of p1300SP transgenic plants exhibited resistant phenotype, which had a susceptible score below 6%, far lower than the susceptible ratio of wild types. The susceptible ratios of other lines of the three vectors were all above 12%, exhibiting moderately resistant lines or susceptible lines. In these resistance assays chimeric CP/SP RNAi lines showed stranger resistance against two isolates than CP or SP single RNAi lines. The ratios of resistant transgenic lines were 39.13%,25.00%, and 29.41% in p1300CP/SP, p1300CP, and p1300SP, respectively. The result verifies that the ratio of T1 transgenic resistant lines containing CP/SP chimeric gene is higher than that contained the single CP or SP gene; furthermore, resistant lines containing CP/SP chimeric gene have the same high resistance to two RSV isolates.Stable integration and expression of RNAi transgenes were confirmed by Southern and Northern blot analysis of independent transgenic lines. In the resistant transgenic lines, relative to the susceptible transgenic plant, lower levels of transgene transcripts and specific short interference RNAs were observed, which showed that virus resistance was increased by RNAi.we selected T2 self-fertilized progeny of resistant transgenic plants which were Southern blotted proved have one copy from lines CS1, CS6, CP1, CP3, CP9, SP5, and SP7 to study the segregation pattern and stable inheritance of RNA-mediated multiple virus resistance. In the T2 generation, some resistant transgenic plants were stably homogenous. The resistance of transgenic plants would not separate at all, he results showed that transgene and RNA-mediated virus resistance can be stably inherited until T2.更多还原