【作者】 赖燕；

【导师】 何水林；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 辣椒（Capsicum annuum L.）是一种具有重要经济价值的茄科作物，而在生产过程中包括病害在内的各种生物和非生物逆境的发生严重地影响辣椒的产量和质量。培育，推广及应用抗逆品种是当前和今后解决辣椒品质问题的最为根本有效的技术对策，辣椒抗病分子机制的阐明则有利于促进现代生物技术和常规育种相结合，进而有效的促进辣椒抗逆遗传改良。大量的研究表明，包括病原菌在内的逆境胁迫可激活植物防御反应，这种诱导抗性在植物抗逆过程中起重要作用，这一过程受逆境胁迫下信号传递途径的控制，其中转录因子通过整合外界信号和协调基因表达的过程中起重要作用参与了该过程。开展辣椒应答逆境的重要转录因子的结构和功能鉴定有利于揭示辣椒抗逆分子机制，促进辣椒抗逆遗传改良。因此，本研究为了分离获得若干可能在辣椒抗病或抗逆防御反应中起重要调节作用转录因子，首先构建了外源SA处理的辣椒均一化cDNA文库，从中分别分离获得2个NAC和1个ERF转录因子阳性克隆，对所分离阳性克隆进行了与特定顺式作用原件互作的瞬间表达、亚细胞定位以及在不同逆境下转录表达的荧光定量PCR分析，并对ERF在烟草中超表达对青枯菌接种抗性和高温抗性的影响进行了分析，主要研究结果如下：1.采用DSN均一化技术与SMART建库技术相结合的方法，构建了在SA处理下辣椒全长均一化cDNA文库，该文库含有1.8×10~6个独立克隆，插入片段平均长度为1. 5 kb，重组率为99%，经测序获得unigenes数目为817，冗余度为5.5%，文库的均一化效果良好，质量符合要求。2.采用文库稀释池法从全长均一化cDNA文库中分离获得了两个辣椒NAC阳性克隆CaNAC6和CaNAC2。两个NAC蛋白都包含有NAC类转录因子特有的NAC域和C末端的可变域。蛋白结构域和分子进化分析表明CaNAC6属于第Ⅲ（stress-related NAC genes, SNAC）亚族，CaNAC2属于第Ⅰ-5（NAC2）亚族。酵母实验表明，CaNAC6和CaNAC2的C末端均具有转录激活活性。两个蛋白均可结合NAC类转录因子的识别序列NACRS序列。基因枪介导的洋葱表皮转化实验表明，35S::CaNAC6-GFP融合蛋白定位于细胞核中，35S::CaNAC2-GFP融合蛋白在细胞核和细胞膜中均有表达。荧光定量结果表明，NAA处理下CaNAC6上调表达，CaNAC2在短时间内表现为下调。CaNAC6可由SA，MeJA，ETH，ABA，机械损伤，青枯菌及干旱诱导表达。CaNAC2由SA，MeJA，青枯菌，干旱诱导表达，ABA作用下无明显变化，在乙烯利作用下表现为一定程度的下调。两个基因的表达水平在4℃冷处理时均在处理后48 h上调，在NaCl和42℃高温处理一定时间内都表现出下调。4.从均一化cDNA文库中分离了一个辣椒ERF阳性克隆CaERF5。洋葱表皮转化实验表明35S::CaERF5-GFP融合蛋白定位于细胞核中。瞬时表达结果表明CaERF5结合GCC-box而不结合CRT/DRE元件。分子进化和序列分析表明CaERF5属于ERF转录因子bⅨ亚族家族成员。5.表达模式分析表明，CaERF5在青枯菌侵染后24-96 h间上调表达，并受SA，MeJA，ETH，机械损伤、低温、高温作用诱导，但不受ABA诱导，在干旱处理下下调表达。相对于K326，除NtNPR1外，包括NtPR2，Ntosmotin，NtACS，NtPR1b，NtPR3，NtPRQ，NtMLP2及NtGST1在内的防御反应相关基因在过量表达CaERF5转基因株系中表达量显著上调。CaERF5过量表达的转基因烟草在接种青枯菌后一系列防御反应相关基因Ntosmotin，NtNPR1，NtPR3，NtPR1b，NtPRQ，NtMLP2，NtCAT1，NtGST1及NtACC均在48 h和96 h表达量无明显组间差异，而在野生型K326中，接种青枯菌96 h后这一系列基因依然呈诱导表达状态。此外，青枯菌接种48h和96h后过量表达CaERF5植株中两个热激蛋白基因（NtHSP和NtHSP18）表达水平较K326均有一定程度的提高。42℃高温处理下CaERF5转基因株系在高温胁迫下萌芽率明显高于K326，并且幼苗表现出较强的高温耐受能力。综上所述，本研究构建了一个外源SA处理的辣椒均一化cDNA文库，从中分离获得2个NAC（CaNAC6和CaNAC2）和1个ERF（CaERF5）转录因子阳性克隆，初步分析表明，CaNAC6和CaNAC2可能参与了辣椒响应不同生物和非生物逆境的过程。定位于细胞核可与GCC盒结合的CaERF5的转录表达同样受各种生物和非生物逆境不同程度诱导，过量表达CaERF5显著提高转基因烟草一系列防御反应相关基因的表达，并介导了转基因烟草对高温和青枯病的抗性。更多还原

【Abstract】 Pepper (Capsicum annuum L.) is a Solanaceae crop with important economic value. Adverse factors including biotic and abiotic stresses like frequent diseases during pepper production have seriously negative effects on yield and quality. The development, promotion and application of disease-resistant cultivars are efficient technological strategy to overcome the problem of yield and quality. Illumination on molecular mechanism of disease resistance in pepper is helpful to boost combination of modern biotechnology and conventional breeding, and promote genetic improvement of stress resistance in pepper. Previous studies indicate that defense reaction can be triggered by biotic and abiotic stresses like pathogenicbacteria. This induced resistance plays an essential role in the process of stress resistance in plants. This process is controlled by signal pathways under stresses. Transcription factors, such as WRKY,NAC,ERF and so on play vital roles in the signal integration and coordination of transcriptional regulation of various genes expression. So structure and functional analysis of transcription factors in response to stresses is a feasible approach to elucidate the molecular mechanism of stress resistance in plants. In this paper, to isolate several transcription factors possibly playing important role in disease-resistant or stress-resistant response, a normalized cDNA library enriched in full-length sequences was constructed using DSN (duplex-specific nuclease)-normalization method combined with SMART (switching mechanism at 5′end of RNA transcript) technique. Full-length cDNA of two NAC transcription factors (TFs) and one ERF TF were isolated from the normalized cDNA library. The structure, expression pattern, subcullar localization and their binding to coresponding cis-elements of the three candidate transcription factors were characterized, and the effect of ERF overexpression in T1 transgenic tobacco lines on resistance to Ralstonia solanacearum inoculation and resistance to high temperature stress were also analyzed. The main results were as followings:1. A full-length normalized cDNA library of pepper seedling under Salicylic acid treatment was constructed using DSN-normalization method combined with SMART technique. The normalized cDNA library contained 1.8×10~6 independent clones and the average insertion size of cDNA was 1.5 kb with 99% recombination rate. After sequencing, 817 unigenes were obtained. The cDNA library was well normalized with 5.5% redundancy.2. Two full-length cDNA clones encoded NAC TFs were isolated from pepper normalized cDNA library based on pooling strategy combined with PCR-based screening method. Both NAC proteins contained highly conserved NAC domain accompanied by diverse C-terminal domains. Protein structure and sequence alignment analysis suggested that CaNAC6 belonged to groupⅢ(stress-related NAC genes, SNAC) and CaNAC2 belonged to the fifth subgroup of the groupⅠ(NAC2).β-gal assay suggested that the transactivation region of CaNAC6 and CaNAC2 were located in the C-terminals. Both NAC proteins could bind to the NACRS sequence to activate the GUS gene expression. Transient expression analysis in onion epidermal cells suggested 35S::CaNAC6-GFP fusion protein was localized in the nucleus, whereas 35S::CaNAC2-GFP was localized in both cytoplasm and nucleus.According to Real time PCR analysis, the expression of CaNAC6 was up-regulated, and down-regulated for CaNAC2 in a short time under 1- Naphthaleneacetic acid (NAA)treatment. CaNAC6 could be induced by SA, methyl-jasmonic acid (MeJA), Ethephon (ETH), Abscisic acid ( ABA), mechanical wounding,drought and Ralstonia solanacearum inoculation. CaNAC6 could be induced by SA, MeJA, drought and Ralstonia solanacearum inoculation. However,CaNAC6 showed down-regulated expression under ETH treatment and no significant difference was observed under ABA treatment. In addition,the expression of two NAC genes showed up-regulated after 48 h under 4℃condition and down-regulated under high salinity and heat stresses.3. A full-length cDNA clone encoded ERF TF was isolated from pepper normalized cDNA library. Transient expression analysis in onion epidermal cells indicated that 35S::CaERF5-GFP fusion protein was localized in the nucleus, and CaERF5 could bind to the GCC-box but no DNA binding activity was observed with CRT/DRE motif. Protein structure and sequence alignment analysis suggested that CaERF5 belonged to theⅨb subfamily of the ERF TF superfamily.4. According to Real time PCR analysis, CaERF5 was up-regulated from 24 h to 96h after R.asolanacearum infection and also could be induced by SA, MeJA, ETH, mechanical wounding, extreme temperatures, but no transcript accumulation was observed under ABA treatment. Furthermore, CaERF5 transcript was repressed by dehydration.The T1 generation of ERF overexpression transgenic tobacco was obtained, and the expression patterns of several representative stress-responsive genes were monitored in 8-weeks-old transgenic plants. In contrast to K326 plant, the NtPR2, Ntosmotin, NtACS, NtGST1, NtMLP2 and also JA/ET pathway related genes including NtPR1b, NtPR3 and NtPRQ transcripts were found to be up-regulated in the overexpression CaERF5 transgenic line. No transcript alteration of NPR1 was observed between K326 and ox-CaERF5-5 transgenic line. The Ntosmotin, NtNPR1, NtPR3, NtPR1b, NtPRQ, NtMLP2, NtCAT1, NtGST1 and NtACC transcripts were found to have no significant difference between 48 h and 96 h postinoculation with R.asolanacearum in ox-CaERF5-5 transgenic line. In K326, these genes show higher expression level at 96h after R.asolanacearum infected than that at 48h.NtHSP and NtHSP18 were up-regulated in ox-CaERF5-5 transgenic line after 48 h and 96 h postinoculation with R.asolanacearum.Furthermore, the germination and seedling growth of K326 plants was significantly inhibited by 42℃heating treatment than ox-CaERF5 transgenic lines.Taken together, a full-length normalized cDNA library of pepper seedling under Salicylic acid treatment was constructed. Full-length cDNA of two NAC (CaNAC6 and CaNAC2) TFs and one ERF (CaERF5) TF were isolated from the normalized cDNA library. A preliminary analysis showed that CaNAC6 and CaNAC2 play roles in pepper response to both abotic and biotic stresses. CaERF5 located in the nucleus and binding to the GCC-box was also induced by different abotic and biotic stresses. The expression of CaERF5 was also induced by different abotic and biotic stresses. A series of defense related genes transcripts were found to be up-regulated in the overexpression CaERF5 transgenic line.Transgenic tobacco plants over-expressing CaERF5 showed improved tolerance against high temperature and R.asolanacearum.更多还原