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根据转录组测序获得的钙依赖蛋白激酶基因的EST序列设计特异引物,利用RT-PCR和RACE技术从油菜中克隆获得Bn CDPK1基因全长序列,NCBI登录号为KF740477,其c DNA和g DNA全长分别为2115 bp和2857 bp,含有11个内含子和12个外显子。生物信息学分析表明其开放阅读框为1581 bp,编码527个氨基酸,具有CDPKs家族典型的特征,含有1个激酶区域和4个钙离子手型结构域,与拟南芥At CDPK28同源性最高,属于第Ⅳ亚家族。Bn CDPK1在油菜的根、茎、叶、花、种子中均有表达,但表达丰度存在差异,叶片中表达量最高,其次为茎、根和种子,花中的表达量最低;在高抗和高感油菜品种中,菌核菌胁迫均能够诱导Bn CDPK1上调表达,但是高抗品种比高感品种反应更迅速,表达量更高。接种前、接种后12 h、接种后24 h,高抗品种的表达量相比高感品种分别提高1.4倍、4倍和3倍,推测其可能参与油菜的生长发育以及油菜对菌核菌侵染的应答和防御反应。
Specific primers were designed based on the EST sequence of the calcium-dependent protein kinase gene obtained by transcriptome sequencing. The full-length Bn CDPK1 gene was cloned from Brassica napus by RT-PCR and RACE technology. The NCBI accession number was KF740477, 2115 bp and 2857 bp in length, containing 11 introns and 12 exons. Bioinformatics analysis showed that the open reading frame was 1581 bp encoding a protein of 527 amino acids with the typical characteristics of the CDPKs family and contained one kinase region and four calcium finger motifs and had the highest homology with Arabidopsis At CDPK28 , Belongs to the fourth subfamily. Bn CDPK1 was expressed in roots, stems, leaves, flowers and seeds of rapeseed, but its expression abundance differed. The expression of Bn CDPK1 was highest in leaves, followed by stems, roots and seeds, And high susceptible rape cultivars, the sclerotinia could up-regulate the expression of Bn CDPK1, but the high-resistant cultivars responded more rapidly and expressed higher than the susceptible cultivars. Before inoculation, 12 h after inoculation and 24 h after inoculation, the expression level of high resistant varieties increased 1.4 times, 4 times and 3 times higher than the high susceptible ones, respectively, suggesting that they may participate in the growth and development of rapeseed Dyed response and defensive response.