Cloning And Functional Analysis Of NPR1 And NPR1-like Genes In Plants

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.