【作者】 王忠；

【导师】 杨清；

【作者基本信息】 南京农业大学 ， 细胞生物学， 2009， 博士

【摘要】 茄子黄萎病(Verticillium Wilt)俗称半边疯、黑心病,是由大丽轮枝菌(Verticillium dahliae Kleb)侵染引起的茄子重要病害之一,常常造成大幅度的减产。在已有相关经济作物抗病的理论研究和生产实践中,选育和种植抗病品种是控制病害最为经济有效的措施。但是目前茄子抗黄萎病的育种尚无突破性进展,究其原因,除了栽培品种中缺乏抗病材料外,还与茄子黄萎病的基础性研究不够深入有关。开展茄子抗黄萎病机制及其抗黄萎病相关基因的克隆研究,不仅能够为茄子抗黄萎病育种提供理论依据,而且可能为抗病育种提供抗病基因,具有十分重要的研究价值。本研究以野生茄子托鲁巴姆(Solanum torvum)和苏崎茄为主要材料,通过比较两种茄子在大丽轮枝菌(Verticillium dahliae Kleb)侵染过程中体内的生理生化指标变化,分析托鲁巴姆对黄萎病的防卫响应机制；同时运用cDNA-AFLP技术构建分析托鲁巴姆感染黄萎病前后的差异基因表达谱；并进一步结合RT-PCR和RACE技术,分离抗黄萎病相关的三个全长基因,对其进行诱导表达分析。研究结果如下：1.托鲁巴姆对黄萎病菌胁迫的响应机制。采用黄萎病菌侵染方法对托鲁巴姆的抗性进行了分析,发现：托鲁巴姆植株表现出很强的自我防御和自我修复能力。对托鲁巴姆体内一些相关的生理指标分析结果表明,(1)在侵染前,托鲁巴姆体内存在的活性氧清除系统,如：SOD、POD、CAT等酶的活性均高于苏崎茄；而侵染后,托鲁巴姆体内各种酶的活性快速增加,其幅度高于苏崎茄；MDA的变化却恰恰相反。这个结果提示,托鲁巴姆具有较高活性氧清除基础能力,而黄萎病菌胁迫进一步增强了托鲁巴姆体内活性氧清除系统,加快了某些防御物质(如木质素和抗菌物质绿原酸等)的形成,同时减缓或降低MDA等有害物质在植株体内的积累,最终把病菌侵染造成的危害降到最低。(2)侵染后各生理指标的响应时间表现出差异。托鲁巴姆中POD、PAL的活性和可溶性蛋白含量在侵染后的12 h内就迅速作出响应(POD增加、PAL和可溶性蛋白减少)；而SOD、PPO、CAT的活性和MDA的含量则在处理后初始阶段(至少12 h)进行了一些调整,随后才进入持续性的增加或减少阶段。由此可见,托鲁巴姆对黄萎病病菌侵染的响应具有时序性,其体内POD酶、PAL酶和可溶性蛋白首先参与植物的防卫反应来应对黄萎病的胁迫,其它酶随后参与响应,它们的共同作用形成对黄萎病菌的有效防卫。2.黄萎病菌胁迫下托鲁巴姆植株根部的基因差异表达谱分析。(1)利用cDNA-AFLP技术分析黄萎病菌侵染前后S. torvum根部的基因差异表达,获得118条TDFs (transcript-derived fragments)。根据基因的表达特征,可将其分为4种类型：诱导表达、抑制表达、上调表达、以及瞬时表达。(2)在118条TDFs中,共有50条TDFs与已知功能基因同源。根据功能推测分为7类：①与代谢相关的；②与细胞自救和防御相关的；③与细胞周期和DNA加工相关的；④与细胞组分的生物学合成相关的；⑤与蛋白质合成相关的；⑥与结合功能蛋白有关的；⑦与转运相关的。这些结果表明,托鲁巴姆对黄萎病的防卫反应是一个多方面、多层次的复杂的生物学过程。3. Stcyp450、Strpll6和StDAHP基因的克隆。采用同源序列克隆和RACE的方法从托鲁巴姆中克隆了Stcyp450、Strpll6和StDAHP基因：(1) Stcyp450 cDNA序列含有一个1536bp的开放阅读框,编码一个511个氨基酸残基构成的蛋白质分子,分子量为58.08 kD,等电点为9.17。采用DNAMAN对Stcyp450和其他同源蛋白的氨基酸序列比对,结果表明：Stcyp450氨基酸序列与拟南芥、大豆等四个CYP蛋白的氨基酸序列有较高的一致性；表达分析显示：Stcyp450受黄萎病病菌的诱导上调表达,侵染后期恢复。(2) Strpll6 cDNA序列含有一个498 bp的开放阅读框,编码一个由165个氨基酸残基组成的蛋白,其分子量为42.16kDa,等电点为5.2。DNAMAN软件对Strpl16编码的蛋白和其他四个RPL16进行氨基酸序列比对,结果表明：Strpl16和来自野生马铃薯、番茄、莳萝、辣椒的同源蛋白在氨基酸水平有较高的一致性,在蛋白的第91氨基酸位点出有一个保守的核糖体蛋白L16的标志性特征域Ⅱ；进化分析表明：Strpl16与野生马铃薯、番茄、和辣椒三种茄科植物的对应同源蛋白的亲缘关系更近。半定量RT-PCR分析结果显示：Strpl16属于组成型表达,表达量随病菌侵染时间延长有所增加。(3) StDAHP cDNA含有一个1683 bp的开放阅读框,编码一个由560个氨基酸残基组成的蛋白,该蛋白分子量为62.53 kD,等电点为9.1。DNAMAN软件对StDAHP和其他同源蛋白的氨基酸序列比对,结果表明：该蛋白与来自马铃薯、番茄、烟草和水稻四种植物的同源蛋白在氨基酸水平有较高的一致性；且在靠近N-末端和C-末端的氨基酸都比较保守；采用半定量RT-PCR方法对StDAHP mRNA水平分析结果显示：StDAHP属于诱导表达,病菌侵染后期持续表达。更多还原

【Abstract】 Eggplant Verticillium wilt is a disastrous plant vascular disease causing severe yield and quality losses in eggplant. Practice shows that resistant variety is the most effective way for V. wilt control. However, little progress has been made in eggplant breeding for resistance to V. wilt. The main reasons are the lack of full understanding about the mechanism of Verticillium resistance and the rare resistant materials. Therefore, study on the mechanisms of V. wilt resistance and cloning of resistance-related genes in Solanum torvum could provide theoretical and practical basis for eggplant breeding in resistance to V. wilt.In this thesis, firstly, the physiological mechanisms in response to V. wilt were analyzed by comparing the physiological parameters of Solanum torvum and Suqi eggplant infected with V. dahliae, then the differentially expressed genes were identified using cDNA-AFLP in S. torvum infected by V. dahliae, finally, three pathogenesis-related genes were isolated by homology cloning and RACE, and their expression were analyzed by RT-PCR. The main results are as follows:1. The mechanisms of S. torvum in response to V. dahliae infection. We analyzed the resistance of S. torvum to V. dahliae, and found that the S. torvum had strong self-defense and self-repair capacity. A number of related physiological parameters in vivo analysis showed that:(1) The active oxygen scavenging system (such as:SOD, POD, CAT enzymes activity) in S. torvum was higher than in Suqi during the treatment. MDA and soluble protein content in S. torvum decreased after treatment, but increased in the Suqi. The results suggested that infection might activate active oxygen scavenging system in vivo, then producing certain defensive substances (such as lignin and chlorogenic acid et al.) quickly, while reducing the accumulation of MDA in the body, and then caused to a minimum harm. (2) The response time of the physiological parameters in S. torvum in response to V. dahliae was different:Activity of POD, PAL and content of soluble protein in S. torvum change rapidly within 12h, and then, change in activity of SOD, PPO, CAT and MDA content. Eventually, S. torvum defense against V. wilt effectively by combinations of the enzymes.2. Anaylise of differential geneexpression profiling in roots of S. torvum after V. dahliae inoculation. Optimization of cDNA-AFLP technology system applications in S. torvum. (1) Analysis of differential gene expression profiling in S. torvum after V. dahliae inoculation, a total of 118 differential transcript-derived fragments (TDFs) were isolated, which were divided into four kinds based on expression pattern: induced expression, inhibited expression, inducible up-regulated expression, and transient expression. (2) Functional analysis on showed that 50 of these TDFs belonged to function-known genes which mainly involved in seven biolgical processes:Metabolism, Cell rescue, defense and virulence, Cell cycle and DNA processing, Biogenesis of cellular components, Protein synthesis, Proteins with binding functions, and transport et al. The results showed that response of S. torvum resistance to V. wilt was a multifaceted, multi-level complex physiological process.3. Cloning and Expression of Stcyp450、Strpll6 and StDAHP from S. torvum. (1) A gene named Stcyp450 (Solanum torvum cytochrome P450) was isolated from S. torvum, the full length of the cDNA contained an open reading frame of 1536 bp coding a protein of 511 amino acids, corresponding to a 58.08 kD polypeptide with an isoelectric point of 9.17. Alignment of the amino acid sequence of Stcyp450 and that of other homologues showed that Stcyp450 had a high identity with CY77A1、CY77A3 and CY77A4 from Arabidopsis and Glycine at amino acid level. Analysis of Stcyp450 mRNA level by semi-quantity RT-PCR showed that it was induced up-regulated by V. dahliae infection, and restorated at the late infection. (2) A gene named Strp116(Solanum torvum ribosomal protein L16) was isolated from S. torvum, the full length of the cDNA contains an open reading frame of 498 bp coding a protein of 165 amino acids, corresponding to a 42.16 kD polypeptide with an isoelectric point of 5.2. Alignment of the amino acid sequence of Strpl16 and that of other homologues showed that Strpl16 had a high identity with RPL16 from five kinds of plants at amino acid level. In addition, the conserved 12-amino acid Ribosomal protein L16 signature 2 (RMGsGKGspeyW) was observed in N-terminal end of this protein. The deduced amino acid sequence and clustering analysis revealed that Strp116 was colse to RPL16 in S. tuberosum, S. lycopersicum, and C. annuum. Analysis of Strpl16 mRNA level by semi-quantity RT-PCR showed that it belongs to constitutive expression, and its expression increased during infection. (3) A gene named StDAHP (Solanum torvum 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase) was isolated from S. torvum, the full length of the cDNA contains an open reading frame of 1683 bp coding a protein of 560 amino acids, corresponding to a 62.53 kD polypeptide with an isoelectric point of 9.1. Alignment of the amino acid sequence of StDAHP and that of other homologues showed that StDAHP had a high identity with DAHP from four kinds of plants at amino acid level (S. tuberosum, S. lycopersicum, N. tabacum, and O. sativa). In addition, the amino acids are more conservative near the N-terminal and C-terminal in StDAHP. Analysis of StDAHP mRNA level by semi-quantity RT-PCR showed that its expression was induced by V. dahliae infection.更多还原