【作者】 张春秋；

【导师】 谢丙炎；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2010， 博士

【摘要】 WRKY转录因子广泛参与植物生长发育及对外界生物和非生物的胁迫应答,在植物抗病防卫反应中起着重要的调控作用。近来的研究表明,WRKY转录因子参与了植物与线虫互作应答反应,CaRKNIF1是Me3基因介导辣椒与根结线虫不亲和互作中表达的WRKY转录因子,深入研究CaRKNIF1基因的功能对于揭示Me3基因介导辣椒抗根结线虫的分子机理具有重要意义。本研究以含Me3基因的辣椒HDA149为材料分离得到了CaRKNIF1基因,并通过TRV载体诱导的基因沉默和农杆菌介导的异源超表达,对该基因的生物学功能进行了深入研究。主要结果如下:1.根据GenBank中已经公布的CaRKNIF1基因(DQ180384)序列信息,分离得到CaRKNIF1基因的全长。CaRKNIF1基因gDNA全长为2387 bp,由3个外显子和2个内含子组成;包含1,473 bp开放阅读框,编码490个氨基酸的蛋白,含有2个保守的WRKY结构域和2个C2H2型锌指结构,属于第Ⅰ组WRKY转录因子。CaRKNIF1基因能够表达出带有His-tag 58 kDa的融合蛋白;洋葱表皮瞬时表达证实CaRKNIF1基因的编码产物是作为核蛋白起转录调控作用的。2.CaRKNIF1基因的表达具有组织特异性,并且可被多种生物胁迫诱导。RT-qPCR分析发现,CaRKNIF1基因在辣椒HDA149中的表达具有组织特异性,根和叶中表达量较高,花和果中次之,茎中表达量最低;该基因可被根结线虫、青枯病菌、疫霉病菌和烟草花叶病毒等生物胁迫诱导,但表达模式存在差异,疫霉病菌和青枯病菌诱导表达峰出现较早(分别为3 h和6 h),其次是根结线虫(12 h),烟草花叶病毒诱导表达峰出现较晚(48 h)。该基因可能参与辣椒对病原线虫、细菌、真菌和病毒等的胁迫应答反应。3.构建了CaRKNIF1基因基于TRV的沉默载体,获得了CaRKNIF1基因沉默的辣椒HDA149植株。在CaRKNIF1基因的保守区和特异区设计了3个靶标片段,分别构建了该基因的TRV载体对CaRKNIF1基因进行表达沉默,结果发现TRV载体能够成功侵染辣椒植株并有效引起CaRKNIF1基因沉默,但该基因沉默并没有引起辣椒HDA149对南方根结线虫的抗性发生变化,推测该基因可能与WRKY转录因子家族的其他成员存在功能冗余或者VIGS不彻底而该基因极低转录本即能维持正常的功能。4.构建了CaRKNIF1基因的植物表达载体,通过农杆菌介导的植物转化方法获得了转基因番茄植株,抗性鉴定结果表明转基因植株对南方根结线虫的抗性和耐盐能力增强。利用pBI121和pCHF3两套载体系统分别构建了CaRKNIF1基因的植物表达载体,通过农杆菌介导的转化方法将CaRKNIF1基因导入番茄Moneymaker、Micro-tom和丽春的基因组中。PCR和Southern杂交结果表明,CaRKNIF1基因已成功整合到番茄基因组中;RT-qPCR分析发现CaRKNIF1基因在转基因植株中高量表达。抗性鉴定结果显示:超表达CaRKNIF1基因的番茄植株对南方根结线虫的抗性提高,且耐盐能力增强,表明该基因作为正调控因子参与植物对生物和非生物胁迫的应答。CaRKNIF1基因的超表达能够引起下游PR(PR1/PR2/PR5/NP24)基因的高量表达,推测转基因番茄植株的抗性提高可能与PR基因的组成型表达有关。5.构建了辣椒HDA149的BAC文库,PCR筛选得到了含有CaRKNIF1和CaMe基因的阳性BAC克隆。辣椒HDA149的BAC文库包含200,000个BAC克隆,平均插入片段大小为95 kb,覆盖辣椒基因组约7倍,预计从文库中筛选到单拷贝基因的机率为99.9%;利用特异引物PCR的方法,从文库中筛选得到了含CaRKNIF1和CaMe基因的阳性BAC克隆,为研究目标基因的结构与互作调控关系奠定了基础。综合上述研究结果,WRKY转录因子CaRKNIF1可能作为正调控因子参与了辣椒对生物和非生物胁迫应答反应,利用CaRKNIF1基因来改良辣椒的综合抗病性将是一种颇具潜力的分子育种途径;研究中构建的高质量的辣椒HDA149 BAC文库为辣椒重要基因的克隆和功能分析提供了技术平台,同时也为深入开展辣椒分子标记开发和物理图谱构建工作奠定了基础。更多还原

【Abstract】 WRKY transcription factors involved in many plants processes including plant development, responses to biotic/abiotic stresses and play important roles in plant defense responses. Recently evidences indicated that WRKY transcription factors also involved in the plant responses to nematode. CaRKNIF1 was the specific expression WRKY transcription factor in the Me3-mediated incompatible interaction between root-knot nematode and pepper. So study the function of CaRKNIF1 gene may be important to reveal the molecular mechanism of Me3-mediated root-knot nematode resistance. In this paper, we isolated the CaRKNIF1 gene from pepper HDA149 which contains root-knot nematode resistant gene Me3 and studied its function by using TRV-based gene silencing and Agrobacterium- mediated heterologous overexpression. The main results were as follows:1. CaRKNIF1 gene was isolated based on the published sequence(DQ180384). The full length of CaRKNIF1 gDNA is 2387 bp with three exons and two introns. CaRKNIF1 contained an open reading frame of 1,473 bp encoding 490 amino acids. Its amino acids sequence contained two conserved WRKY domains and two C2H2 zinc-finger motifs, and belongs to the group I WRKY gene family. The recombinant CaRKNIF1 can expression a 58 kDa fusion protein with His-tag, and the CaRKNIF1 protein was localized in the nucleus of epidermal cells which confirmed by Agrobacterium-mediated transient expression.2. The RT-qPCR results showed that the expression of CaRKNIF1 gene was tissue-specific, with relative higher in roots and young leaves, next in flowers and fruits, and lower in stems. The CaRKNIF1 gene can be induced by M.incognita, R.solanacearum, P.capsici and TMV, but the expression patterns were different. P.capsici and R.solanacearum induced the CaRKNIF1 gene expression earlier (3 h and 6 h, respectively), followed by M.incognita (12 h), TMV induced expression peak appeared later (48 h). The results suggested that CaRKNIF1 gene involved in plant defense responses to different pathgens including nematodes, bacteria, fungi and viruses.3. The CaRKNIF1 gene silenced HDA149 plants were obtained by TRV-based gene silencing. The TRV vectors were constructed by using three target segments from conseved and specific regions of CaRKNIF1 gene respectively. The RT-PCR results showed that TRV vectors were successfully infected pepper plants and caused CaRKNIF1 gene silence effectively, but the resistance of pepper HDA149 to M.incognita did not change. These results indicated that functional redundancy may be existed between CaRKNIF1 gene and other WRKY transcription factors or VIGS were not thorough and the pepper HDA149 was able to maintain its resistance to M.incognita with low transcription of CaRKNIF1 gene.4. Plant expression vector of CaRKNIF1 gene were successfully constructed using pBI121 and pCHF3, and Kanamycin resistant tomato plants of Moneymaker, Micro-tom and Li Chun were obtained by Agrobacterium-mediated transformation. PCR and Southern blot analysis proved that CaRKNIF1 gene were successfully integrated into the genomes of the tomato plants. RT-qPCR results showed that the target gene was expressed in tomato plants. The transgenic tomato plants that overexpressing CaRKNIF1 gene displayed enhanced resistance to M.incognita and tolerance against salt. The results indicating that CaRKNIF1 gene may act as a positive regulator of plant responses to biotic and abiotic stresses. Overexpression of CaRKNIF1 gene activated the expression of the under-stream PR gene under normal growth conditions, suggesting that the increased resistance of transgenic tomato plants may be related to constitutive expression of PR (PR1/PR2/PR5/NP24) gene.5. The BAC library of pepper HDA149 was constructed which consisted of 200,000 clones with an average insert size of 95 kb, and the positive clones of CaRKNIF1 and CaMe gene were obtained by screening the BAC library. Based on the pepper haploid genome size of 2,702 Mb, the BAC library was estimated to contain approximately 7 genome equivalents and represent at least 99.9 % of the pepper genome. The results indicated that the library was highly reliable and laid good foundation for cloning important gene of pepper and studying their regulation.In conclusion, WRKY transcription factor CaRKNIF1 gene may act as a positive regulator of plant responses to biotic and abiotic stresses. It is a potential way to improve plant resistance to different stresses by expression of CaRNKIF1 gene. The high-quality pepper HDA149 BAC library provides a technology platform for cloning and function analysis of important gene, and laid good foundation for developing new molecular markers and physical mapping of pepper.更多还原