Construction Of A Normalized Full-length CDNA Library And Expressional Or Functional Analysis Of Several Important Genes In Pepper

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.