【作者】 唐前君；

【导师】 肖启明； 张德咏； 谢丙炎； 刘勇；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2010， 博士

【摘要】 双生病毒在世界范围内广泛发生,严重危害多种重要经济作物。近年来,随着B型烟粉虱的入侵和扩散,番茄黄化曲叶病毒(tomato yellow leaf curl virus, TYLCV)在我国番茄产区不断蔓延传播且逐年加重,在部分地区大面积爆发,危害严重,难以防治,对农业生产构成严重的威胁。近年发展起来的RNA沉默(RNA silencing)技术可能是解决此问题的有效途径之一本研究以中国番茄黄化曲叶病毒Y10外壳蛋白2个不同氨基酸同源性150bp左右短片段和3个不同长度的外壳蛋白基因片段构建dsRNA植物表达载体,转化烟草和番茄,以求获得抗性强、抗性范围广的抗病转基因植株,并试图找出能诱导RNA介导的病毒抗性的最佳片段；同时,以B型烟粉虱传毒相关蛋白GroEL基因片段构建dsRNA植物表达载体,并转化烟草和番茄,探索利用RNAi切断病毒传播途径的可能性。本论文的研究为进一步构建双价基因,从病毒侵染和介体传毒两条途径切断双生病毒的侵染和流行,建立双生病毒分子调控技术体系打下基础。研究的结果和主要结论如下：1、利用中国番茄黄化曲叶病毒(tomato yellow leaf curl China virus, TYLCCNV)的CP基因片段构建dsRNA转化本氏烟和番茄及其抗病性研究1.1不同氨基酸同源性CP基因片段dsRNA介导的抗病性研究对大部分TYLCV的CP基因序列与TYLCCNV的CP的基因序列进行氨基酸同源性比对,分别选取同源性为94.94%的159bp的CP片段和81.32%的158bp的CP片段,成功构建含CP片段的dsRNA植物表达载体pBIN19-2HCP和pBIN19-2LCP,以本氏烟和番茄Moneymaker为转基因材料,通过农杆菌介导法分别将基因转入烟草和番茄中,得到含2HCP的转基因烟草56株、转基因番茄59株；得到含2LCP的转基因烟草43株、转基因番茄61株。通过对TO代扩繁株攻毒实验后表型观察和PCR检测发现,来自同一母株的TO代扩繁株抗性完全一致,转低同源CP片段dsRNA的转基因本氏烟TO代植株抗病型的比率为47.1%,而转高同源CP片段dsRNA的转基因本氏烟TO代植株抗病型的比率为72.7%。1.2不同长度CP基因片段dsRNA介导的抗病性研究分别选取长度约为150bp、300bp和450bp的CP片段,成功构建CP片段dsRNA植物表达载体pBIN19-2CP159(即pBIN19-2HCP)、pBIN19-2CP302和pBIN19-2CP457,以本氏烟和番茄Moneymaker为转基因材料,通过农杆菌介导法将基因转入烟草和番茄中,分别得到含2CP159、2CP302和2CP457转基因烟草56株、57株和38株,转基因番茄植株59株、77株和54株。通过对T0代扩繁株攻毒实验后表型观察和PCR检测发现,来自同一母株的T0代扩繁株抗性完全一致；但转3个不同长度CP片段dsRNA的转基因本氏烟T0代植株抗病性存在差异,分别为72.7%、80.7%和89.6%。我们从转pBIN19-2CP159 T0代转基因烟草抗病植株中随机选取4株繁殖,进行T1代抗病性分析,结果发现T1代植株抗病株比率分别为12.5%、22.5%、10.0%和17.5%。2、利用B型烟粉虱GroEL基因片段构建dsRNA转化本氏烟和番茄干扰烟粉虱传毒的研究随机选取长度约为300和500bp的GroEL基因片段,成功构建GroEL基因片段dsRNA植物表达载体pBIN19-2G502和pBIN19-26306。以本氏烟和番茄Moneymaker为转基因材料,通过农杆菌介导的转基因策略将基因转入并整合到烟草和番茄中,分别得到转基因烟草73株和46株,转基因番茄91株和66株；农杆菌介导的植株接种和烟粉虱传毒的接种方法对T0代扩繁株攻毒实验,表型观察和PCR检测结果表明,来自同一母株的T0代扩繁株抗性完全一致,转2个不同长度GroEL基因片段dsRNA的转基因本氏烟T0代中没有发现抗病型植株,两种不同接种方法所得的耐病型比率有一定差别,前一种接种方法得到的耐病型比率分别为56.6%和57.0%,而后一种接种方法得到的耐病型比率则分别为69.9%和65.0%。将带毒并在TO代转基因烟草扩繁株取食2天后的烟粉虱取食非转基因植株,通过PCR检查发现,转含26502和26306的转基因植株对烟粉虱的传毒能力干扰率分别为22.0%和16.0%。3在本氏烟中瞬时表达TYLCCNV CP基因dsRNA的抗病性初步分析为了初步确定TYLCCNV CP基因dsRNA能否诱导病毒抗性,减少基因操作的盲目性,我们构建植物表达载体pBIN19-GFP和pBIN19-CP-GFP,瞬时表达本氏烟,均可见GFP荧光。预先渗入pBIN19- 2CP457植物表达载体,然后再瞬时表达pBIN19-GFP和pBIN19-CP-GFP,前者GFP正常表达,而后者GFP荧光不可见；或渗入实验后16天用TYLCCNV Y10农杆菌侵染性克隆进行再接种,处理植株均表现为免疫。更多还原

【Abstract】 Geminiviruses occurred worldwide, and cause severe diseases in several economically important crops. In recent years, with bio-invasion of B biotye Whitefly Tomato Yellow Leaf Curl Virus (TYLCV) has outbreaked in a large area of the tomato-producing areas, and serious threats had been posed to agricultural production. Whitefly is the transmission vector of TYLCV, and the GroEL chaperone protein, which produced by the symbiotic bacteria of vector, can protect the circulation of the virus in the insect. Once the disease occurs, it is difficult to control. Recently the development of RNA silencing technique in the plant may be an effective way to solve this problem.In this study, a comparison has been made between TYLCV Y10 coat protein and other isolates. Different sequences of two 150bp segments and about 3 different length coat protein gene segments of TYLCV are selected to construct dsRNA plant expression vector respectively, then they are transformed into tobacco and tomato, on the purpose of obtaining resistance plants with strong and wide resistance to TYLCV, furthermore, to find the optimal length of inducing RNA-mediated virus resistance. Furthermore, to explore the use of RNAi to cut off the transmission of the virus, dsRNA plant expression vector is also constructed using virus transfer-mediated or GroEL protein gene segments in Whitefly. These studies provided a basis for further construction of dual-gene, infection and epidemic are cut off in two ways, which are virus infection and virus-transferring mediator, thus laying the foundation work on establishing the technical system of molecular mechanism for geminivirus. Study results and main conclusions are as follows:1 RNA Mediated Transgenic Resistance to TYLCCNV by transforming N. benthamiana and tomato with the CP gene segments dsRNA1.1 Studies on different homologous in amino acid sequence of CP gene dsRNA-mediated virus resistanceThe homology of 94.94% of the 159bp segment and 81.32% of the 158bp segment of the CP were selectec for construnction of plant expression vector based on the comparision of mino acid homology of most CP gene sequence of TYLCV and TYLCCNV, CP segment is successfully constructed dsRNA expression vector pBIN19-2HCP plant and pBIN19-2LCP, And introduced into N. benthamiana and tomato via Agrobacterium tumefaciens-mediated transformation. After tissue culture we got 56 and 43 transgenic N. benthamiana,61and 56 transgenic tomato. Through PCR testing and observation of TO progeny phenotype after TYLCCNV inoculation, it revealed that the TO progeny from the same mother plants have exactly the same resistant.The resistance ratio of transgenic N. benthamiana of TO progeny to TYLCCNV is 47.1%and 72.7%, respectively.1.2 Studies on CP gene segment of different length dsRNA-mediated virus resistanceAbout 150bp,300bp and 450bp length of the CP segment are selected, and The CP segment is successfully constructed dsRNA expression vector pBIN19-2CP159(pBIN19-2HCP), pBIN19-2LCP302 and pBIN19-2CP457. These vectors were further introduced into N. benthamiana and tomato via Agrobacterium tumefaciens-mediated transformation.56,57 and 38 transgenic N. benthamiana,59, 77 and 54 tomato plants were achieved. Through PCR testing and observation of TO progeny phenotype after TYLCCNV inoculation, it revealed that the TO progeny from the same mother plants have exactly the same resistant. The resistance ratio of transgenic N. benthamiana of TO progeny to TYLCCNV is 72.7,80.7%and 89.6%, respectively.Tl progeny from resistance plants are used to analyze the resistance, and it revealed that the resistant strain ratio of T1 progeny is 12.5%,22.5%,10%and 17.5%, respectively.2 RNAi interfere Whitefly transmission capacity by transforming N. benthamiana and tomato with the GroEL gene segments dsRNAAbout 300bp and 500bp of the GroEL gene segment are selected, GroEL gene segment is successfully constructed dsRNA expression vector pBIN19-2G502 and pBIN19-2G306, And introduced into N. benthamiana and tomato.via Agrobacterium tumefaciens-mediated transformation. After tissue culture we got 46 and 73 transgenic N. benthamiana,66 and 91 transgenic tomato. Through PCR testing and observation of TO progeny phenotype after TYLCCNV inoculation, it revealed that the TO progeny from the same mother plants have exactly the same resistant. No resistant type and higher rates of disease tolerance have been found in transgenic N. benthamiana of TO progeny, which has been transferred two different length of GroEL gene segment dsRNA, and contemporary, the ratio of the virus-tolerance plants is different in two inoculation method. Feed the whitefly two days with the infected in TO generation of transgenic tobacco first and then non-transgenic plants, and PCR examination showed that transgenic plants with transferred GroEL gene have a certain interference to whitefly’s transmission capacity.3. The preliminary analysis of the resistance in the transient expression of dsRNA TYLCCNV CP gene in N. benthamianaIn order to initially ensure whether TYLCCNV CP gene dsRNA can induce viral resistance, and prevent blindness of trans-gene. We were constructed successfully plant expression vector pBIN19-GFP and pBIN19-CP-GFP. These genes transiently expressed after agro-infiltration in N. benthamiana, GFP fluorescence was observed in N. benthamiana leaves. We first infiltrated A.tumefaciens with pBIN19-2CP457in N. benthamiana leaves, followed by infiltration of pBIN19-CP-GFP, the result was a shutdown of GFP expression; or followed, after 16 days, by inoculation of TYLCCNV, the result was immunity to TYLCCNV.更多还原