青花菜钙依赖蛋白激酶基因BoCDPK1的克隆与表达

Cloning and expression analysis of a calcium-dependent protein kinase gene BoCDPK1 from Brassica oleracea var. italica

钙依赖蛋白激酶(calcium-dependent protein kinase,CDPK)为Ca^2+传感蛋白,在植物生长发育和逆境响应中起着重要作用。在克隆青花菜BoCDPK1基因的基础上,开展序列分析、系统发育分析和表达分析,为后续的基因功能鉴定和抗逆育种奠定基础。该研究以青花菜为材料,利用PCR法克隆1个CDPK基因,利用生物信息学对序列进行分析,并采用qRT-PCR研究该基因在霜霉菌和核盘菌侵染下的表达模式。测序结果表明,BoCDPK1的基因组DNA全长为2 414 bp,具6个内含子,编码区全长为1 647 bp,编码548个氨基酸;BoCDPK1有1个STKc和4个EF手性结构域。多序列比对结果表明,BoCDPK1与芸薹属植物同源序列的相似性最高,仅个别氨基酸残基存在差异,它们在系统发育树上聚于一组。qRT-PCR结果表明,BoCDPK1的表达受霜霉菌和核盘菌的诱导,表达量均呈现先上升后下降的规律。在霜霉菌的诱导下,BoCDPK1的表达量在72 h达最大值,为对照的3.4倍;而在核盘菌侵染下,BoCDPK1的表达量在36 h达最大值。该研究明确了青花菜BoCDPK1基因的序列特点、系统发育关系和表达特征。

Calcium-dependent protein kinases(CDPKs) are Ca2+ sensor proteins, and they play an important role in plant growth, development and stress responses. On the basis of BoCDPK1 gene isolation, sequence analysis, phylogenetic analysis and expression analysis were performed to provide a foundation for both gene function identification and stress-resistance breeding in the future. PCR method was applied to isolate a CDPK gene from broccoli, and sequence analysis was performed by using bioinformatics. Expression patterns of this CDPK gene during Hyaloperonospora parasitica and Sclerotinia sclerotiorum infection were obtained by qRT-PCR. Sequencing results indicated that the full genomic DNA of BoCDPK1 was 2 414 bp in size with six introns. The complete coding sequence was 1 647 bp in length, encoding 548 amino acids. BoCDPK1 contained one STKc and four EF-hand domains. Multiple sequence alignment results revealed that BoCDPK1 shared the highest similarity with homologous sequences from Brassica plants and had a few amino acid differences, and they grouped to the same clade. qRT-PCR results showed that the expression of BoCDPK1 was induced by both H. parasitica and S. sclerotiorum, and the expression levels increased firstly and then decreased with the increasing of inoculation time. The highest expression level of BoCDPK1 was observed at 72 h after H. parasitica inoculation, with 3.4 fold higher than the control. When inoculated by S. sclerotiorum, the highest level was observed at 36 h after inoculation. Sequence characteristics, phylogenetic relationships and expression patterns of BoCDPK1 gene isolated from broccoli were confirmed.