Abstract：WRKY transcription factors constitute a class of proteins involved in growth, development, stress and defense responses in plants. In this study, the cDNA sequence of WRKY69 was cloned through reverse transcription PCR (RT-PCR) from oilseed rape (Brassica napus) (GenBank No. KC246583). Sequence analysis indicated the encoded protein had 279 amino acids with a single WRKY domain and a C2H2-type zinc finger motif, which belonged to GroupⅡ. Yeast transcriptional activity analysis showed that BnaWRKY69 might have transcriptional activation activity, and its activity was mainly determined by the carboxyl terminus. GFP-based subcellular localization showed that BnaWRKY69 was located not only in the nucleus but also at the cell membrane. A prediction of BnaWRKY69 protein sequence suggested it might undergo 2 types of post-translational modifications, myristoylation and farnesylation, which possibly determined its special membrane localization. Under hydroxylamine (NH2OH) treatment, the BnaWRKY69 signals at the plasma membrane decreased gradually and even disappeared. The transcriptional level of BnaWRKY69 under various abiotic stresses, hormone and Sclerotinia sclerotiorum treatments was analyzed by quantitative real-time PCR (qRT-PCR). It was found that BnaWRKY69 was specifically induced by jasmonic acid (JA) and S. sclerotiorum. Further detection by qRT-PCR showed that transient overexpression of BnaWRKY69 gene in tobacco (Nicotiana benthamiana) leaves caused significant up-regulation of Pathogenesis-related 1a (PR1a), PR2 and Non-expressor of PR1 (NPR1) and down-regulation of Plant defense 1.2 (PDF1.2). In conclusion, this study preliminarily analyzed the basic features of BnaWRKY69 in different hormones and abiotic stress treatments and molecular mechanism responding to JA and S. sclerotiorum, which could provide a reference to breed oilseed rape varieties with improved disease resistance.
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