Cloning And Expression Analysis Of WRKY Transcription Factor Gene CaRKNIF1 In Pepper

WRKY proteins play an important role in plant. They are involved in developmental and metabolic processes, as well as in biotic and abiotic stress responses. To study the function of WRKY transcription factor in hot pepper, a WRKY gene CaRKNIF1 was cloned. Its amino acids sequence, copy number in genome, intron and expression patterns were analyzed. Main results are summarized as follows:1. Using homology-based cloning approach,four new hot pepper WRKY genes were isolated from pepper HDA149 after challenging inoculation with J2 root-knot nematodes. Using RACE technique, the new WRKY gene CaRKNIF1 was cloned. The nucleotide sequence of CaRKNIF1 cDNA is 2,139 bp long, and contains an open reading frame (ORF) of 1,473 bp encoding a putative polypeptide of 490 amino acids. The molecular mass of the predicted protein is 53.6 kDa and the isoelectric point was calculated to be 8.09. The GenBank number is DQ180348.2. To determine the intron of CaRKNIF1 gene, we performed a PCR amplification of the hot pepper genomic DNA using a pair of the full-length CaRKNIF1 cDNA primer. The result showed that CaRKNIF1 gene has no intron. Genomic DNA samples were digested with EcoRI, EcoRâ…¤and BamHI. Southern blot was performed with a partial cDNA fragment (not containing the conserved WRKY domains) labeled with DIG-High prime as a probe. The result indicated that CaRKNIF1 likely presents as multi-copy gene in genome .3. The bioinformatic analysis showed that the amino acid sequence of CaRKNIF1 protein contains two conserved WRKY domains belonging to the groupâ… of WRKY protein. It also showed that the protein localizes in the nucleus. The secondary and tertiary structure were predicted. The putative functions were predicted, e.g. it may have transcription, transcription regulation, signal transducer and stress response activity.4. Total RNA samples were isolated from the leaf tissues after different treatment. Northern blot analysis revealed that CaRKNIF1 transcription factor is induced by salicylic acid, root-knot nematode, wound treatment, and expresses differently at various time point after treatment. The transcription factor is induced upon inoculation with ethephon and NaCl. Jasmonic acid doesn't have influence. The results indicated that CaRKNIF1 transcription factor should be involved in salicylic acid and ethylene transduction pathways.