【作者】 柴友荣；

【导师】 王三根； 唐克轩；

【作者基本信息】 西南农业大学 ， 作物遗传育种， 2003， 博士

【摘要】 由大丽轮枝菌(Verticillium dahliae)等引起的黄萎病是一种世界范围的顽固性维管束病害，严重为害棉花、番茄、茄子、马铃薯、苜蓿等重要农作物的生长。作为一种土传病害，黄萎病的寄主广，危害重，化学防治成本高、效果差，并且存在食品安全性和生态安全性问题。选育抗黄萎病作物品种是黄萎病防治的根本途径，但却面临育种周期长和抗黄萎病资源严重匮乏的限制，利用基因工程技术分离植物抗黄萎病有关的基因并通过遗传转化创造作物抗黄萎病新材料，具有基因型资源广泛、周期相对较短和不受生殖障碍限制等优点，是抗黄萎病遗传改良的一个重要方向。 根据基因对基因学说(Gene-for-gene Theory)，植物抗病基因R的产物即受体蛋白R与来自病原菌无毒基因Avr的直接或间接产物即Avr配体之间的原初识别，是介导植物抗病反应的根本分子机制。许多抗病和感病植物材料之间的本质差异并不在于个别下游防卫反应基因的有无，而在于负责原初识别的抗病基因的有无或启动抗病信号传导的机制上存在差异，所以克隆和利用受体蛋白类的抗病基因是植物抗病基因工程的核心内容。迄今已有6大类植物抗病基因克隆成功，它们绝大多数均符合基因对基因学说并编码一种介导抗病信号传导的胞内或跨膜受体蛋白，这些抗病基因在转化感病植物以后均成功地取得了抗病性的提高。 除R-Avr识别以外，由PG-PGIP识别而介导的抗病信号传导同样能激活寄主植物的系统性抗病反应。真菌等病原菌在入侵寄主植物时的一个首要致病机理就是分泌多聚半乳糖醛酸酶(polygalacturonase，PG)等水解酶来降解植物细胞壁，而植物在长期的协同进化过程中也针对性地进化出了一种多聚半乳糖醛酸酶抑制蛋白(polygalacturonase-inhibiting protein, PGIP)，它能特异性、可逆性、可饱和性地与PG发生结合，抑制PG的活性，阻止和延缓病原菌对寄主细胞壁的瓦解，同时延长了具有激发子活性的、适当聚合度的寡聚半乳糖醛酸类寡糖素的产生与滞留时间。这种寡糖素进入植物细胞膜并与膜上的寡糖素受体产生识别作用，启动抗病信号传导，激活植保素合成有关的 柴友荣—一植物抗大丽轮枝菌受体类蛋白基因及甘露糖结合型凝集素基因的克隆与表达酶、蛋白酶抑制剂、病程相关蛋白等一系列防卫相关蛋白的基因转录表达。PGIP同抗病基因产物R蛋白一样也是主要由富亮氨酸重复（leucine－rich。epeat，LRR）构成的细胞表面受体类糖蛋白，均属于LRR超家族成员，主要功能是负贡与外源分子的识别作用，介导防卫相关的信号传导，位了LRR重复单元内p链／B转角（pstrand／pt。rn）上保守性亮氨酸两侧的氨基酸残基是决定识别特异性的核心，其上发生的点突变足以改变识别特异性。 棉花等植物抗黄萎病基因】程长期以来进展不大，但番茄抗黄萎病研究则已取得突破。采用图位克隆法（map－based cloning）己成功分离出番茄（Lycopemcon eSCUentorMill．）抗大丽轮枝菌 1号小种的地基因家族的两个抗病基因Vel和yeZ。它们并不局限于小种特异性，而是可以介导跨越轮技菌属物种间的抗黄萎病性，比如它们转化马铃薯后可以提高马铃薯抗黑白轮枝菌的能力。茄科内番茄的一些近缘物种如茄属的野生种托鲁巴姆等存在对黄萎病、枯萎病、立枯病、线虫等病害的接近免疫的a性，由于黄萎病菌的小种分化性复杂，寄主广泛，在这些近经物种中克隆Ve基因的同源基因是寻找新型抗黄萎病基因型尤其是抗大丽轮枝菌2号小种的抗病基因的一个重要途径，同时也有助于揭示ye基因的系统发生学和抗病作用机制。本研究选择与番茄属亲缘关系最近的茄属植物类番茄茄（Solanor ］NmpjcoideSDun．）为对象，克隆其与番茄 yeM对应的两个同源基因 Slyel和 AfVeZ。以此为跳板，使挪18po更容易地从茄科内其它物种或更远缘的物种中克隆具有不同识别特异性的Ve同源基因。 海岛棉（auioh—— L．）与陆地棉（c ——iM L．）同为棉属的四倍体栽培种，亲缘关系很近，但陆地棉普遍不抗黄萎病，而海岛则普遍高抗黄萎病并在与陆地棉杂交时表现为显性单基因的遗传特点，说明海岛棉中存在陆地棉所不具有的由质量性状基因控制的抗黄萎病性。它们在受大丽轮枝菌侵染时的抗病反应速度上存在巨大差别，海岛棉的防卫反应早而快，陆地棉迟而慢，表明由抗病基因产物R蛋白或KIP介导的抗病反应速度是决定它们抗病差异性的本质因素。水杨酸诱导后从陆地棉茎中分离的PGIP蛋白对大丽轮枝菌和黑曲霉的m酶活性具有抑制能力，番茄果实中纯化的PGIP蛋白也对黑白轮枝菌的PG酶活性具有抑制能力，表明oIP在植物界广泛参与了对黄萎病菌的抗性作用。植物Pgp基因家族仍处于活跃的进化过程中，通过活性位点氨基酸的正向选择性进化、基因加倍、启动于变异等，使植物界产生了识别特异性各异、活性水平不同、调控方式多样的Pgp基因家族成员。探讨海岛棉与陆地棉之间在基因的活性氨基酸位点和启动子功能上存在的差异性将十分有助于阐明基因与棉花抗黄萎病性的相关性。因此本研究克隆了海岛棉基因（的赠中 的全长。DNA序列、基因组序列、启动于序列和终止于?更多还原

【Abstract】 Caused by Verticillium pathogens such as V. dahliae etc., verticillium wilt is a worldwide disastrous plant vascular disease infecting many important crops such as cotton, eggplant, tomato, potato and clover etc. with heavy yield loss. As a soil-born disease, it has a very broad host range. Chemical control of this disease is of high cost and less efficient accompanied with food safety and environment safety problems. Selecting verticillium resistant cultivars is the basic strategy to control this disease, but it is confined by long term of selection and deficiency of resistance genotypes. Gene engineering is an adequate strategy to create new breeding germplasms by isolating and transforming useful verticillium resistance-related genes with short term and without confinement from reproductive barriers.According to Gene-for-gene Theory, the core mechanism of plant resistance to pathogen invasion lies in the primary recognition between the receptor-like protein of the resistance gene (R) product of the host and me ligand (or elicitor) of the avirulence gene (Avr) product of the pathogen. Many resistant and susceptible plant genotypes are not typically differed in downstream defensive genes but in whether or not possessing an adequate resistance gene and in the mechanism of defense-related signal transduction. To date 6 types of plant resistance genes have been cloned, most of which conform to Gene-for-gene Theory and encode cytoplasmic or transmembrane receptor-like proteins which can mediate signal transduction related to disease resistance. Expression of these R genes in susceptible plants resulted in acceptable enhancement of disease resistance.Besides R-Avr recognition, more and more evidences indicate that the PG-PG1P recognition on the plant cell wall can also mediate systemic defense reactions. A primary pathogenetic strategy of fungal pathogens is to secrete hydrolytic enzymes, the most primary of which is polygalacturonase (PG), to destruct the plant cell wall. On the other hand, the plant kingdom also evolves a molecular arm which is called polygalacturonase-inhibiting protein (PGIP) to restrict the activity of PG PGIP can specifically, reversibly andsaturably bind to PG and prevent or delay the degradation of the cell wall ingredient polygalacturonic acid, which increases the concentration and prolongs the half life of the oligogalacruronide (OGA). OGA with a certain DP (degree of polymerization) is a kind of elicitor-active oligosaccharin, which enters the cell and is recognized by the membrane-associated oligosaccharin receptor and thus certain defensive signaling pathways are activated to induce the transcriptional expression of phytoaiexin synthesis enzymes, proteinase inhibitors (Pis), pathogenesis-related proteins (PRs) and other defensive-related proteins. PGIP as well as the plant R proteins are all members of the LRR (leucine-rich repeat) type receptor-like glycoprotein superfamily, which recognize foreign molecules and mediate defensive reactions. The variable amino acid residues flanking the conserved leucine residues within the strand/ turn motif of the LRR unit is characterized as the determinant factor of recognition specificity. Single residue mutations of them are enough to change the specificity.Verticillium resistance engineering on most plant species such as cotton has poorly yielded, but a striking advancement has been achieved on tomato. By using map-based cloning method, the family members Ve\ and Vel of verticillium wilt resistance gene locus have been isolated from tomato (Lycopersicon esculentwn Mill.). One extinct feature of them is mat they are not typically race-specific, e.g., transformation of them into potato can enhance resistance to V. albo-atrum. But these 2 genes are not resistant to V. dahlias race 2, so it is still uigent to seek new genes to suppress this highly toxic race. Some relative species of tomato from family Solanaceae such as the wild eggplant species Solomon torvum shows almost immunity to Verticillium wilt and other diseases. Because更多还原