【作者】 张颖；

【导师】 郭兴启；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 NPR1（nonexpressor of PR gene）基因可调控植物广谱抗性的发生,在植物系统抗性中起着关键作用。植物NPR1基因已成为植物抗病信号传导途径,以及植物抗病基因工程领域的研究热点。从不同植物中克隆NPR1基因及NPR1-like基因,并进行基因表达特点和抗病功能的研究,对于丰富NPR1理论基础和进一步在植物抗病基因工程中的应用都具有重要的意义。本文分别以棉花品种鲁棉22和心叶烟为材料,从棉花植株中首次克隆出1个NPR1同源基因（GhNPR1）,从心叶烟中首次克隆出2个NPR1同源基因（NgNPR1、NgNPR3）,在此基础上,分别对它们的表达特点及其抗病的生物学功能进行了分析。具体结果如下:1.根据同源序列设计简并引物,通过RT-PCR和RACE-PCR的方法从棉花（Gossypium hirsutum）中克隆到一个NPR1基因,命名为GhNPR1,GenBank注册号为EF988657。通过对其分子结构和氨基酸结构的对比分析,证明该基因为NPR1的同源基因。Southern杂交说明在棉花基因组中GhNPR1为单拷贝。Northern杂交发现GhNPR1在棉花中的表达不仅具有组织特异性而且还具有时间特异性。此外,植物抗病反应相关信号分子SA、MeJA、ET可以诱导GhNPR1的表达,同样给棉花接种棉花枯萎病菌和角斑病菌后,发现GhNPR1的表达量也逐步增加,进一步证表明GhNPR1可能参与了棉花抗病反应的基因表达调控。2.从心叶烟（Nicotiana glutinosa）中同源克隆了NPR1基因,命名为NgNPR1,GenBank注册号为EU139477。利用同源序列对比及分析,证明该基因为NPR1的同源基因。并且通过NgNPR1-GFP亚细胞定位的研究,可以证明NgNPR1蛋白的结构特点与AtNPR1蛋白最为相近。RT-PCR检测NgNPR1表达特性时,发现NgNPR1基因可以被信号分子SA、MeJA、H₂O₂和INA诱导表达,而且细菌性病害青枯病和真菌病害烟草立枯病菌、黑茎病菌和赤星病菌接种心叶烟后,也可以不同程度的增加NgNPR1的表达量。这表明NgNPR1基因可能是SA依赖的信号转导途径的成员,参与心叶烟抗病反应的基因表达调控。3.根据已知的NPR1及NPR1-like基因的同源序列,设计简并引物,通过RT-PCR和RACE-PCR的方法从心叶烟（Nicotiana glutinosa）中克隆得到了一个NgNPR1-like基因,命名为NgNPR3,GenBank注册号为EF077161。同源对比分析发现,NgNPR3与拟南芥中AtNPR3同源性最高,且氨基酸结构中具有几个高度保守的典型序列结构。除此之外,NgNPR3基因组外显子-内含子结构位置的特征,也与已知的AtNPR3基因一致。因此推测NgNPR3基因是AtNPR3的同源基因。利用绿色荧光蛋白在洋葱表皮瞬时表达的实验,证明了NgNPR3蛋白可以通过SA、INA诱导而引起细胞内氧化还原电位势的变化,从细胞质运动到细胞核之中。Southern杂交说明在心叶烟基因组中NgNPR3为单拷贝。RT-PCR检测发现分别利用信号分子SA、MeJA、H₂O₂和INA诱导,以及接种青枯病、立枯病菌、黑茎病菌和赤星病菌后,NgNPR3在心叶烟中的表达量都会出现明显的增加,这意味着NgNPR3具有与NgNPR1基因相似的信号传导和调控体系。4.将NgNPR3构建正义植物表达载体pBI121-NgNPR3,采用农杆菌介导的方法,转化烟草NC89,同时转空载体作为对照。经卡那霉素筛选获得若干再生植株。经过PCR鉴定,并选取部分转基因植株进行了Northern和Western杂交分析,结果证明NgNPR3在转基因烟草中已成功得到表达。并且转基因植株中存在三种不同的表达量,分别为高表达（H）、中表达（M）、低表达（L）。5.选取具有代表性的三个T0代转基因株系（H、M、L）进行自交种子扩繁,得到T1代转基因植株。在分子鉴定基础上,对T1代转基因植株进行了生物学功能分析。抗真菌实验中,转基因植株在接种了烟草白粉病菌后,其抗病能力与NgNPR3基因的表达量成正比关系。即NgNPR3表达量高的植株（H、M）具有较高的抗性,低表达（L）植株与对照植株抗性很低。通过RT-PCR鉴定侵染后植株中PR基因的表达含量,证明高表达植株可以加快提高PR基因的表达,从而提高抗病能力。实验结果说明SAR诱导后NgNPR3基因也是诱导下游PR基因表达的重要调控因子。6.构建原核表达载体pET-NgNPR3,并在大肠杆菌BL21（DE3）中表达融合蛋白,将特异诱导带切下,溶于PBS中获得抗原,免疫小鼠,制备抗体,从而对转基因植株进行Western杂交。更多还原

【Abstract】 The NPR1 （nonexpressor of PR gene） gene is a key regulator of acquired resistance responses. And it is shown to be a workable target for genetic engineering of nonspecific resistance in plants. It is very valuable to isolate and characterize the NPR1 and its paralogous gene from different species. In this thesis, a series of studies have been conducted on the isolation, sequence and expression analysis, function identification of NPR1 from cotton and tobacco, and NPR3 from tobacco, respectively. The main results are as follows:1. A novel gene, termed Gossypium hirsutum NPR1 （GhNPR1）, was isolated from cotton. Amino acid sequence alignment revealed that GhNPR1 had the closest association with the NPR1 group. Southern blot analysis indicated that there was a single GhNPR1 gene in the cotton genome. Northern blot analysis revealed that GhNPR1 mRNA was expressed at different levels during different development stages in roots, stems and leaves. Furthermore, the results showed that GhNPR1 could be markedly induced by SA, MeJA and ET, which play important roles in signaling defense responses, as well as pathogen attacks. These results suggest that the increased expression of GhNPR1 induced by pathogens and defense signal molecules may be critical in the activation of the plant defense responses.2. A novel gene, termed Nicotiana glutinosa NPR1 （NgNPR1）, was isolated from tobacco. Amino acid sequence alignment revealed that NgNPR1 had the closest association with the NPR1 group. As indicated in our subcellular location of NgNPR1, the structure and function of NgNPR1 showed the highest identity to AtNPR1. Northern blot analysis revealed that NgNPR1 was upregulated by the exogenous signaling molecules, such as SA, MeJA, H₂O₂ and INA, as well as pathogen attacks, such as fungal pathogens Rhizoctonia solani, Phytophthora parasitica, and Alternaria alternata, and bacterial pathogen Pseudomonas solanacearum. These results suggest that the increased expression of NgNPR1 induced by pathogens and defense signal molecules may be critical in the activation of the plant defense responses.3. Based on the conserved region of plant NPR1-like genes,we isolated a novel gene from tobacco, termed Nicotiana glutinosa NPR3 （NgNPR3）. Sequence comparison revealed that NgNPR3 had high sequence similarity with AtNPR3. It means that NgNPR3 belongs to the plant NPR1-like superfamily. In addition, the intron-exon structure is also conserved among NPR1 and NPR1-like genes. Location prediction analysis showed that NgNPR3 expression in onion epidermal subcellar sited in response to activators of SAR. Southern blot analysis indicated that NgNPR3 gene has only one copy in the cotton genome. And Northern blot analysis revealed that NgNPR3 was induced by the exogenous signaling molecules, such as SA, MeJA, H₂O₂ and INA, as well as pathogen attacks, such as fungal pathogens Rhizoctonia solani, Phytophthora parasitica, and Alternaria alternata, and bacterial pathogen Pseudomonas solanacearum. All the results indicate that NgNPR3 is a signaling molecule-responsive gene, which can be involved in the defense responses.4. Construct a sense expression vector pBI-NgNPR3, and transformed it in to the tobacco plant （NC89）. We also transformed the empty vector into tobacco at the same time as control. We carried out Northern blot and Western blot on some transgenic plants, and found that NgNPR3 has been expressed successfully in the tobacco plants. Based on the results, we can found that the expression level of the NgNPR3 gene in the transgenic lines varied to some extent: high, middle and low.5. On the basis of the molecular identification, we analyzed the biological function of progeny T1 generation plants. The transgenic lines H and M exhibited higher resistance than the L, Vec and nontransgenic lines against Erysiphe cichoracearum DC. The induction of PR genes after pathogen infections is faster and stronger in NgNPR3-overexpressing plants. In conclusion, the results of our study demonstrated that expression of NgNPR3 in tobacco was effective in activating certain defense signal transduction pathways, leading to enhanced resistance to a wide range of important fungal and bacterial pathogens.6. Constructed an E.coli expression vector pET-NgNPR3 and induced it to express in E.coli strain BL21 （DE3）. The strong induced fusion protein bands were collected into PBS solution and immuned little mouse to obtain antiserum.更多还原