Isolation、Expression And Function Analysis Of The WRKY Transcription Factor Gene CaWRKY6and CaWRKY30in Pepper

Pepper(Capsicum annuum L.) is one of the most economically important vegetables in China, which is susceptible to the adverse phytopathogens during production, and then leads to yields losses and quality decline. In recent years, the root-knot disease (RKN), one of the plant parasitic nematodes has become a major and seriously disease in pepper production. Due to lack of cultivars with the high-level resistance to RKN, Many methods were used to control this disease, however series of problems on pesticides residues, ecosystem environment pollution and soil deteriorated were brought from highly toxic and high residue chemicals in the field. Thus emergency problems must face up in the vegetable production.The mechanism on compatible and incompatible interaction between pepper planta and RKN were performed on molecular biology in this study. Moreover relative key resistant genes were isolated and functioned from host plant pepper, the aims to provide novel strategy for pepper resistance-breeding program in the future.In this paper, to understand the key processes governing resistance mechanisms in pepper HDA149(Capsicum annuum L.), which contains Me3nematode-resistance gene that confers the pepper resistance to the nematodes, upon infection with RKN(Meloidogyne incognita), two trancriptomes, from the root tips infested with virulent and avirulent M. incognita, were sequenced by llumina/Solexa high-throughput deep-sequencing technology. On this basis, two WRKY transcription factors were isolated from the pepper root tissue during the incompatible interaction between pepper HDA149and M. incognita by RACE method. Then, the characterization、expression and function were analysed. The main results are as following: 1. The phenotypic differences were identified after the pepper HDA149were innoculated with virulent and avirulent M. incognita, respectively. Based on this, after trancriptomes were sequenced by llumina/Solexa high-throughput deep-sequencing technology, two transcriptome cDNA databases were constructed, named HR transcriptome and SR transcriptome, respectively. A total of214909ESTs (190213in HR and177407in SR) were clustered by a computational pipeline.24696and37502unigenes were unique expressing in compatible and incompatible, respectively, among of them382in SR and187in HR were selected to future analysis. Among of the commonly transcripts,3,615and1,982unigenes were differentially up-regulation expression in SR and HR transcriptome, respectively. Several genes from the HR sample, i,e., fatty acid hydroperoxide lyase, peroxidase, pathogen-related proteins, and from SR samples,i,e., auxin-induced protein, ethylene-response transcription factor, phenylalanine ammonium-lyase, were selected and their differential expression in the SR and HR samples collected from different time points after nematode inoculation was confirmed by quantity RT-PCR process.2. Two WRKY transcription factor, named CaWRKY30and CaWRKY6, were isolated from the pepper HDA149root tissue. The nucleotide sequence of CaWRKY30cDNA is1,533bp long and contains an open reading frame (ORF) encoding a polypeptide of364amino acids. The molecular mass of the predicted protein is41.2kDa, and the isoelectric point was calculated to be6.57. The nucleotide sequence of CaWRKY6cDNA is1,944bp long, and contains an ORF encoding a polypeptide of553amino acids. The molecular mass of the predicted protein is60.2kDa and the isoelectric point was calculated to be6.96.3. The CaWRKY30and CaWRKY6genomic sequences were obtained. The genome DNA length of CaWRKY30was1,743bp, consisted of2introns and3exons, and the genome DNA length of CaWRKY6was2,530bp, contained5introns and6exons. Southern blot demonstrated that CaWRKY30and CaWRKY6only have single copy in pepper genome.4. The CaWRKY30and CaWRKY6expression patterns were analysied after the pepper plants treatment with varous pathogens and signaling molecules, i,e,. salicylic acid and methyl jasmonate. The CaWRKY30transcripts were up-regulate after the pepper plants innoculated with signaling molecules SA, different pathogens, such as Ralstonia solanacerum, Tobacco mosaic virus (TMV), Phytophthora capsici and avirulent M. incognita, but the transcripts were suppressed by the MeJA. Similarly, the transcripts of CaWRKY6were up-regulate by the SA, R.solanacerum, TMV, P.capsici, avirulent M. incognita, and suppressed by the MeJA and virulent M. incognita. The phylogenetic relationship of CaWRKY proteins were analysis after the consensus maximum parsimony (MP) tree were constructed.5. we demonstrated that the transiently expressed both the CaWRKY30::GFP and CaWRKY6::GFP fusion proteins were localized exclusively to the nuclei of onion epidermal cells. With the control vector alone, the GFP signal was distributed in both the nucleus and cytoplasm. The nuclear localization of CaWRKY30and CaWRKY6protein supports its role as a transcriptional regulator.6. TRV vectors which contain specific regions of CaWRKY30and CaWRKY6gene were constructed, and then innoculated to the pepper HDA149, respectively. RT-PCR showed that the virus were successfully infected pepper plants and induced WRKY gene silence. Pepper HDA149which CaWRKY30gene was silenced didn’t shown any changes against o the avirulent M.incogntia infection compared to the control, these results may be interpretated by the functional redundancy of WRKY genes or silence is not thorough and lower transcripts of CaWRKY30gene can maintain its roles. In contrast, silenceing of CaWRKY6in pepper HDA149increases the number of root knot and egg masses, suggested that CaWRKY6have an important role in RKN resistance.7. Plant expresssion vector of pCAMBIA2300-CaWRKY30and pCAMBIA2300-CaWRKY6were constructed, and kanamycin resistant tomato (Moneymaker) plant of CaWRKY30, tobacco plants of CaWRKY6were obtained by Agrobacterium mediated transformation approach. CaWRKY30overexpressing transgenic plants showed increased the growth of aivrulent M. incognita, and more root-knot and egg masses compared to the control were observed after infected with M. incognita in CaWRKY30overexpressing transgenic tomato plants, CaWRKY30protien is a negative regulator of RKN defense.In a word, comprehensive of the nematode-infested characterization by transcriptome sequencing would provides a rich cDNA resource for further analyzing the function of key regulatory genes involved in nemaotdes resistance in pepper. CaWRKY30and CaWRKY6have an important role in pepper plants responses to pathogens infection, it is a potential way to improve the plant resistance by the use of specific WRKY transcription factor, i,e., CaWRKY30and CaWRKY6.