甘蓝SI相关基因BoCDPK14的克隆与分析

Cloning and analysis of BoCDPK14 in self-incompatibility Brasscia olerace

甘蓝自交不亲和性(self-incompatibility,SI)是柱头对相同单倍型的花粉产生的排斥或抑制反应。钙依赖蛋白激酶(calcium-dependent protein kinase,CDPK)是植物面对逆境信号时参与抗逆反应的重要元件。本文通过甘蓝自花授粉0~60 min的柱头转录组数据分析,成功地筛选到一个受自花授粉诱导上调表达的基因BoCDPK14,该基因与拟南芥中参与植物逆境信号传导的钙依赖蛋白激酶基因高度同源。BoCDPK14基因开放阅读框1599 bp,编码一种具有533个氨基酸残基的亲水性蛋白,可在大肠杆菌胞质中被诱导表达,其相对分子质量为60.4 kD,表明BoCDPK14为活性胞质蛋白。该基因起始密码子上游2000 bp的核苷酸序列中含有胁迫反应、激素反应、代谢调节等应答元件。BoCDPK14在甘蓝柱头、花粉、花蕾、花瓣和叶片中表达,且柱头中的表达量低于花粉。荧光定量PCR结果证实,BoCDPK14在0~60 min的表达变化趋势与转录组分析结果一致。通过酵母双杂交发现,BoCDPK14蛋白激酶结构域与谷氨酸受体通道蛋白BoGLR2.8d存在相互作用,表明BoCDPK14可能是参与SI反应过程的新蛋白。本研究结果表明BoCDPK14可能作为Ca2+信号元件参与甘蓝柱头响应花粉刺激的分子过程,这为甘蓝自交不亲和的进一步研究和利用提供了新内容。

Brasscia oleracea self-incompatibility(SI)is the reaction of rejection or inhibition of stigma to pollens from the same haplotype.Calcium-dependent protein kinase(CDPK)is an important component of plants involved in resistance to stress signals.In this study,we identified an up-regulated gene named BoCDPK14 based on the stigma transcriptome data in 0–60 min self-pollination.The gene was highly homologous to the calcium dependent protein kinase gene involved in stress signaling in Arabidopsis lyrata.BoCDPK14 gene contained an open reading frame(ORF)with the length of 1599 bp,encoding a protein with the length of 533 amino acid residues.It was a hydrophilic protein expressed in cytoplasm,and could be induced to express a 60.4 kD protein in the cytoplasm of E.coli Transetta(DE3).It was suggested that BoCDPK14 is an active cytoplasmic protein.The 2000 bp upstream of BoCDPK14 translation initial codon contained elements for responses to stress,hormone,and metabolic regulation.BoCDPK14 was expressed in stigma,pollen,flower bud,petal,and leaf,with lower expression level in stigma than in pollen.The results of qRT-PCR revealed that BoCDPK14 mRNA expression level after self-and cross-pollinations for 0 min to 60 min was consistent with that of RNA-seq data.The interaction between BoCDPK14 protein kinase domain and glutamate receptor channel protein BoGLR2.8d was identified by yeast two-hybrid,suggesting that BoCDPK14 might be a novel protein involved in SI reaction.These results suggest that BoCDPK14 may act as a Ca2+signal element to participate the process of response to pollen stimulation in the stigma of Brasscia oleracea,which provides a new insight into the further research and utilization of self-incompatibility of Brasscia oleracea.