【作者】 张余洋；

【导师】 叶志彪；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2006， 博士

【摘要】 作物遗传改良依赖于向植物基因组导入外源DNA片段。虽然基因工程赋予植物很多通过常规育种途径无法实现的优良性状，人们对转基因作物及其植物产品心存疑虑。植物转基因研究中抗性标记基因可以提高转化体的筛选效率。其中70％转基因植物中采用卡那霉素抗性标记基因。然而，对抗性标记基因潜在的生物安全性疑虑阻碍了植物转基因研究的发展，同时现有可利用的标记基因种类有限性也阻碍了转基因作物的长期利用。解决抗性标记基因安全性问题的策略有标记回避和标记剔除两类，前者是在转化阶段不使用抗性标记基因或采用安全标记基因筛选转基因植株；后者则采用抗性标记基因筛选获得转化体后剔除标记基因。在剔除策略中，可诱导的自主剔除途径显现出较好的利用前景。棉铃虫是一种寄主极广的农业大害虫，在蔬菜生产区主要危害番茄。苏云金芽孢杆菌（Bacillus thuringiensis）毒蛋白（insecticidal crystal protein，ICP）基因是目前应用最为广泛的抗虫基因。本研究内容之一旨在以番茄为受体，通过构建化学诱导剔除载体系统用于从抗虫转Bt基因植物中剔除标记基因。对作物遗传改良的另外一个疑虑是关于外源基因。对于这种顾虑使得研究人员考虑是否能够向植物基因组导入植物自身基因，而不含其他物种基因，即顺化基因植物（cisgenic plant），以区别于转基因植物（Transgenic plant）。植物病毒病给世界各地的农作物生产造成严重损失。随着植物翻译起始因子特别是eIF4E的研究深入，植物抗病毒研究将面临新的课题。病毒侵染寄主植物并在植物体内进行自我复制和增殖需要借助寄主自身蛋白质合成机制。通过植物基因工程途径，干扰病毒利用植物mRNA翻译蛋白质的过程，可抑制病毒在寄主植物内的复制和增殖，不仅可获得抗病毒植物材料，而且可加深对植物-病毒互作分子机理的认识。鉴于此，本研究第二部分内容通过克隆真核翻译起始因子4E相关基因，利用基因失活和正义表达顺式调控植物自身eIF4E表达，提高植物对马铃薯Y病毒属病毒的抗性，探讨eIF4E介导的抗病毒性的作用机理。本实验主要开展以下研究：1．利用位点特异重组系统Cre／lox和化学诱导系统XVE相结合构建标记基因剔除载体p35C，在该系统中，反式激活因子XVE、重组酶基因cre、标记基因nptⅡ构建于识别序列loxP正向重复之间，AVE由位于左侧loxP上游的花椰菜花叶病毒35S启动子驱动，cre由β-雌二醇特异诱导的启动子驱动，在右侧loxP下游构建无启动子的cryIAc基因。β-雌二醇诱导处理后，β-雌二醇与反式激活因子XVE结合，启动cre基因表达，Cre重组酶识别loxP正向重复位点，发生重组反应，导致两个loxP之间的cre、nptⅡ以及XVE同时剔除，将剩下一个loxP位点，同时将cryIAc基因置于35S启动子驱动下。在载体p35G中，目的基因为gfp，用于通过绿色荧光蛋白检测标记基因的剔除。2．通过农杆菌介导的方法进行番茄遗传转化。p35G载体转化番茄中，19个抗性芽，经过于β-雌二醇诱导后，有2个再生芽可以观测到绿色荧光蛋白的表达，表明该诱导自主剔除系统适用于番茄转化。通过p35C载体将cryIAc导入番茄品种“中蔬五号”获得抗性芽，将抗性芽转移至含有2μMβ-雌二醇诱导培养基中生根。选取携带单拷贝转基因番茄植株进行鉴定，对4个转基因株系的T1进行分析，重组频率一般在12％～39％之间，完全重组的频率8％～30％，将标记基因nptⅡ和cre基因剔除。重组区域序列分析与预期的重组反应一致。其中株系C4所发生的重组皆为不完全重组。另外，利用λ噬菌体的两个重组位点attP构建正向重复序列，置于标记基因两侧，结果表明在转基因当代和T1代均未检测到重组植株，可能是由于重组加强序列影响其重组效率。3．部分转cryIAc基因植株的Northern blot结果表明，多数转基因植株cryIAc的正常转录。4．在转基因番茄叶片中表达量为3500 ng g^-1 FW～6300 ng g^-1 FW，而在果实中CryIAc含量较叶片低，介于2400 ng g^-1 FW～4400 ng g^-1 FW之间。对转基因植株的抗虫性初步鉴定表明，对棉铃虫具有较强的抗性。幼虫校正死亡率在70％～90％之间，转基因株系的抗虫指数在80％～90％之间。5．分别从克隆了番茄品种“中蔬五号”和抗病辣椒的真核翻译起始因子4E编码基因。中蔬五号番茄所获得的eIF4E与报道的序列有3个碱基差异，而辣椒eIF4E与报道序列一致。6．构建了辣椒eIF4E正义（超量）表达载体（pCA4S）及番茄eIF4E RNAi抑制表达载体（pLE4D）。7．以番茄品种“中蔬五号”为受体，通过农杆菌介导的方法用pCA4S和pLE4D两种载体进行遗传顺化，获得卡那霉素抗性顺化植株13株。PCR检测和Southernblot结果表明，目标基因已整合到番茄基因组中。pCA4S载体同时用于辣椒品种苏椒五号的遗传顺化，4株再生辣椒PCR检测呈阳性，初步确认基因已整合到番茄基因组。由于在此实验中所用目的基因eIF4E来自番茄和辣椒自身，我们暂且称所获得的基因工程植株为基因顺化植株（cisgenic plants），尽管它们还携带非植物自身基因。8．通过半定量RT-PCR对基因顺化番茄进行eIF4E表达分析，表明pCA4S顺化番茄中eIF4E得到超量表达，而pLEAD顺化番茄中eIF4E表达受不同程度抑制。9．利用摩擦接种对番茄顺化植株进行PVY和CMV攻毒感染。通过对接种后的病毒病病情调查和病毒RNA的半定量RT-PCR分析，结果表明，pLE4D和pCA4S顺化植株相对于对照均获得不同程度的病毒抗性。其中pCA4S顺化植株的抑制病毒效果较差，而pLE4D顺化植株的效果较好。就不同病毒而言，无论是正义表达eIF4E或者eIF4E RNA干涉，对PVY抗性的调控效果比CMV调控效果好。10．对本实验中获得的基因顺化辣椒材料以及本实验室所创建转基因顺化辣椒进行PVY和CMV攻毒感染。结果表明pLE4D和pCA4S转化植株相对于对照均获得不同程度的病毒抗性。更多还原

【Abstract】 Crop genetic engineering relies on the introduction of foreign DNA into plant genomes. Although genetically engineered traits provide valuable alternatives to those available through conventional breeding, there is public concern about the consumption of foods derived from transgenic plants. The selectable marker genes are required to ensure the efficient plant genetic transformation, 70 percent of which are genes conferring kanamycin resistance. The presence of antibiotic or herbicide resistant selective marker genes in transgenic plants causes the concerns of the potential hazards on ecological environment and human health. At the same time, the limited number of selectable marker gene prevents the retransformation for genetic engineering. Basically, there are two strategies available for generating marker-free transgenic plants, that is, marker gene avoidance or marker gene excision. The first approach is carry out plant transformation using safer markers or without markers at all. The second approach is marker gene elimination after selections. Among the marker gene elimination strategies, inducible auto-excision presents the most promising choice. Cotton bollworm （Helicoverpa armigera Hubner） is severe insect pest, mainly destroying tomatoes. Insecticidal crystal protein from Bacillus thuringiensis is the most widely-used endotoxin for insect-control. One of the main purposes of this thesis was to construct a chemical-inducible marker-free system for tomato and eliminate the selectable marker gene from Bt transgenic tomato.Another major concern raised by transgenic crops is related to exogenous genes of interest. This concern educes the question of whether crops can be improved by inserting only native DNA into their genomes and to generated cisgenic plants. The virus disease cause severe losses in crop production worldwide. The growing knowledge on eukaryotic initiation factor 4E has provided the novel strategy for genetic engineering for virus resistance. The protein translation mechanism in host is required for virus infection and multiplication. Interfering the process of virus utilizing of the protein translation via genetic engineering not only improves the virus resistance of host plants, but also helps to deepen insights into molecular interaction of plant-virus. So another part of this thesis was to clone the eukaryotic initiation factor 4E from tomato and pepper, and regulate the elF4E expression in tomato and pepper via sense and RNAi strategy. This would not only improve virus resistance in plants but also help to elucidate the function of elF4E in plant virus interaction.Main experiments were carried out as follows:1 A chemical-inducible auto-excision marker free vector system, p35C, was constructed with Cre/loxP site-specific recombination system and an inducible expression system XVE. In vector p35C, three elements including the transactivator XVE, cre and the coding sequence of the neomycin transferaseⅡwere located between the two direct repeats of loxP. XVE was driven by cauliflower mosaic virus 35S promoter upstream of loxP. Cre was driven byβ-estradiol-inducible promoter. Downstream of the next loxP was the crylAc. Upon induction byβ-estradiol, the XVE was activated, and drive the cre expression and DNA recombination between two loxP sites, leading to excision ere, nptll and XVE. Only one intact loxP site would be left and cryIAc would be put directly downstream the CaMV 35S promoter. In the vector p35G, the gfp serves as the trait gene for detect maker gene excision by visualizing green fluorescence.2 Agrobacterium-mediated transformation was carried out on tomato （ZS5） transformation with p35G. Among 19 kanamycin resistant shoots, 2 shoots exhibited green fluorescence, indicating the suitability of this vector for tomato transformation. CrylAc was introduced into ZS5 tomato via p35C, and kanamycin resistant shoots were obtained. The shoots were transferred to inductive rooting media containing 2μMβ-estradiol. Trangenic tomato with single transgene locus was selected for further analysis. T1 progeny analysis was carried out, showing that Cre/loxP-mediated DNA recombination occurrence was 12%～39%, and complete recombination frequency was 8%～30%. Sequence analysis of the recombination region showed the precise and complete recombination at the loxP sites. But in some transgenic lines, e. g. C4 had incomplete recombination. In addition, the attP site fromλbacteriophage was constructed on both sides of nptⅡand introduced into tomato genome for investigation of the recombination occurrence. However, the results showed that the recombination did not happen either in primary transformants or T1 progeny probably because of absence of transformation boost sequence.3 Northern blot analysis was carried out to detect crylAc expression level in transgenic tomato with DNA recombination. And results showed that the cryIAc was significantly transcripted in all transgenic lines tested.4 The CryIAc content in leaves and fruit were assayed by ELISA, and the content was 3500 ng g^-1 FW～6300 ng g^-1 FW in leaves and 2400 ng g^-1 FW～4400 ng g^-1 FW in fruits. In vitro insect assay showed that the transgenic lines were conferred improved resistance to cotton bollworm （Helicoverpa armigera Hubner） larva with resistance index between 80%-90%, and insect adjusted mortality was 70%-90%.5 Eukaryotic initiation factor 4E （eIF4E） from tomato ZS5 and pepper Yolo Y was cloned. Tomato elF4E had three nucleotide differences from the reported gene, and pepper eIF4E sequence was the same as the reported gene.6 Sense construct for pepper eIF4E （pCA4S） and RNAi construct for tomato eIF4E （pLE4D） was respectively constructed.7 Agrobacterium-mediated transformation was carried out on tomato （ZS5） with pCA4S and pLE4D respectively. Thirteen kanamycin resistant plants were obtained. PCR and Southern blot analysis showed the integration of the exogenous gene in tomato genome, pCA4S was also used for pepper transformation, and PCR analysis provided the preliminary confirmation of exogenous gene integration in 4 regenerated plants. Because the trait gene, eIF4E, used in this study was from tomato and pepper’s own genome, we called the genetically modified plants obtaind as cisgenic plants, although they still contain non-plant-native genes.8 Semi-quantitative RT-PCR was carried out to analysis eIF4E expression in cisgenic plants. Overexpression was observed in cisgenic plants with pCA4S, while gene silence was detected in cisgenic plants with pLE4D.9 Cisgenic tomato plants were challenged with PVY and CMV through rub-inoculation. Through the symptom investigation and semi-quantitative RT-PCR of virus RNA, it was showed that two kinds of cisgenic plants were conferred improved virus resistance. And the cisgenic plants with pLE4D showed more resistance than that with pCA4S. As to different virus, cisgenic plants showed more resistance to PVY than to CMV.10 Cisgenic pepper plants with pCA4S were challenged with PVY and CMV through rub-inoculation. It was shown that cisgenic plants were conferred improved resistance to virus.更多还原