甘蓝型油菜BnaCIPK15基因的表达分析与功能鉴定

Expression Analysis and Functional Identification of BnaCIPK15 Gene in Brassica napus L.

CBL-CIPK是高等植物中广泛存在的一类解析Ca^(2+)信号的蛋白。该研究在前期工作基础上,对甘蓝型油菜(Brassica napus L.)的BnaCIPK15基因进行了亚细胞定位、双分子荧光互补(BiFC)、酵母双杂交和qRT-PCR检测等一系列分析,以探究BnaCIPK15蛋白在ABA激素响应中的作用。结果显示:(1)亚细胞定位发现,BnaCIPK15蛋白定位于细胞质和细胞核中; BiFC分析发现,BnaCIPK15蛋白与BnaCBL1/3/4/9蛋白之间的互作较强,与BnaCBL10仅有微弱互作。(2)qRT-PCR检测发现,BnaCIPK15基因受ABA和冷胁迫的诱导极显著上调表达,而对百草枯(Paraquat)、活性氧(H_2O_2)和热胁迫的诱导较弱,表明BnaCIPK15基因很可能参与ABA和冷胁迫的调控过程。(3)酵母滴定实验结果显示,BnaCIPK15蛋白与脱落酸(ABA)信号通路中的BnaHAB1蛋白(属于蛋白磷酸酶PP2C家族)存在明显的互作,而与BnaABFs/AREB3/ABI5转录因子无明显互作;BiFC验证显示,BnaCIPK15与BnaHAB1蛋白之间存在互作信号,而BnaCIPK15与BnaHAB2组合没有观察到信号,证明BnaCIPK15与BnaHAB1磷酸酶具有特异互作特征,推测BnaCIPK15可能参与调控ABA信号转导。研究认为,甘蓝型油菜中可能存在基于BnaCIPK15-BnaHAB1的互作模块,并参与ABA的信号转导和网络调控。

CBL-CIPKs are universal proteins in higher plants that decode Ca2+ signals. Base on the previous work, a series of investigations of BnaCIPK15 gene in Brassica napus L., including subcellular localization, bimolecular fluorescence complementation(BiFC), yeast two-hybrid(Y2 H) and qRT-PCR, were carried out to explore its role in ABA hormone response.(1) Subcellular localization result showed that BnaCIPK15 protein was localized in the cytoplasm and nucleus. BiFC assay demonstrated that BnaCIPK15 interacted strongly with BnaCBL1/3/4/9 proteins, and interacted weakly with BnaCBL10 protein as well.(2) Using qRT-PCR, the expression pattern of BnaCIPK15 gene was examined under various phytohormone and abiotic stress treatments, and the results revealed that abscisic acid(ABA) and cold stress significantly induced its expression. Moreover, a mild up-regulation of BnaCIPK15 gene was also observed by paraquat, reactive oxygen species(H2O2) burst and heat stress treatments, suggesting that BnaCIPK15 gene may be involved in the regulation of ABA and cold stress.(3) Y2 H assay discovered that BnaCIPK15 protein interacted with BnaHAB1, which belongs to the protein phosphatase type 2 C family negatively regulating ABA signaling pathway, and did not show any evident interaction with BnaABFs/AREB3/ABI5 transcription factors. At the same time, BiFC assay also showed that there were interaction signals between BnaCIPK15 and BnaHAB1 proteins, rather than BnaHAB2 protein, which proved that they had specific interaction, suggesting that BnaCIPK15 protein might be involved in regulating ABA signal transduction. In conclusion, this study identified that BnaCIPK15-BnaHAB1 module may be involved in ABA signal transduction and regulatory network in Brassica napus.