甘蓝型油菜CIPK22基因的特性分析与互作蛋白CBL的鉴定

Characterization of CIPK22 gene and identification of interacting CBL proteins of CIPK22 in rapeseed(Brassica napus)

甘蓝型油菜(Brassica napus)作为重要的油料作物之一,其产量与品质常受到环境因素的影响。钙离子是生物体内重要的应答内外刺激的第二信使。作为植物特有的钙信号解码蛋白,类钙调磷酸蛋白B(calcineurin B-like protein,CBL)以及与之互作的CIPK(CBL-interacting protein kinase)家族部分成员的功能与调控机制已经在模式植物拟南芥中被广泛报道,但是在甘蓝型油菜中却报道寥寥。本研究从甘蓝型油菜中克隆到一个CIPK家族的基因序列,分析表明它与拟南芥中CIPK22基因序列高度相似,故命名为BnaCIPK22。亚细胞定位结果显示BnaCIPK22定位在细胞质与细胞核。实时荧光定量(qRT-PCR)技术检测BnaCIPK22对多种非生物逆境和激素处理后的响应,发现BnaCIPK22的表达受脱落酸(abscisic acid,ABA)、水杨酸(salicylic acid,SA)、冷害、渗透胁迫和盐害处理诱导。同时,利用酵母双杂交体系,筛选到可能与BnaCIPK22互作的BnaCBL蛋白,并通过双分子荧光互补(BiFC)技术进行了验证。本研究结论为深入了解CIPK22的功能与调控机制提供参考。

Rapeseed (Brassica napus) is one of the important oil-producing crops,and its yielding and quality are often affected by environmental factors.Calcium ions can respond to internal and external stimuli and act as an important second messenger in eukaryotes.The function and regulatory mechanism of plant-specific calcineurin B-like proteins (CBLs) and interacting protein CIPKs (CBL-interacting protein kinases),which are two associated families decoding calcium signals,have been widely reported in model plants,but few reports have been published with rapeseed.In this research,we cloned the cDNA sequence of a CIPK from rapeseed,and it was found to show the highest sequence similarity to CIPK22 in Arabidopsis,so it was named BnaCIPK22.Subcellular localization study showed that BnaCIPK22 was localized in the cytoplasm and nucleus of leaf epidermal cells.After a variety of abiotic stress and phytohormone treatments at three time points,quantitative RTPCR (qRT-PCR) was used to examine the transcriptional changes of BnaCIPK22.The results showed that BnaCIPK22 transcript abundance was increased by abscisic acid (ABA),salicylic acid (SA),cold,PEG8000 and NaCl treatments.Using the yeast two-hybrid (Y2H) system,this study screened the interaction pattern between BnaCIPK22 and multiple CBLs of rapeseed,and found that there was significant interaction between BnaCIPK22 and BnaCBL1/2/9.The authenticity of the interaction was verified by bimolecular fluorescence complementation (BiFC).The conclusion of this study lays a foundation for understanding the function and regulatory mechanism of BnaCIPK22 in rapeseed.