Isolation And Function Analysis Of Me3 Mediated Root-knot Nematode Resistant WRKY Gene CaRKNIF1 Of Pepper

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.