山葡萄VaCIPK18原核表达与多克隆抗体制备

Prokaryotic Expression and Polyclonal Antibody Preparation of Calcineurin B-like Proteins (CBLs) Interacting Protein Kinases Protein Kinase VaCIPK18 From Vitis amurensis

类钙调磷酸酶B亚基蛋白(CBLs)互作蛋白(CIPKs)作为类丝氨酸/苏氨酸蛋白激酶,在植物响应非生物胁迫信号转导中起着重要作用。基于前期山葡萄响应低温胁迫转录组测序结果,发现低温胁迫引发的早期伤害及感知阶段的激酶基因中涉及CBL-CIPK信号通路的VaCIPK18表达显著上调。为进一步研究山葡萄(Vitis amurensis)VaCIPK18激酶参与低温胁迫的功能,采用同源克隆获得了VaCIPK18基因,其开放阅读框为1 320 bp,编码439个氨基酸。基于对VaCIPK18蛋白生物信息学分析,获取胞外结构域中抗原表位丰富的肽段,并将其C端调控结构域(230~439 aa)构建到原核表达载体pET28a-SUMO。将重组表达载体转化至大肠杆菌(E.coli Rosetta)中,经0.8 mmol·L-1 IPTG、37℃诱导4 h表达出大小为42 kDa的包涵体蛋白。将重组蛋白作为抗原免疫日本大耳白兔,获得anti-VaCIPK18多克隆抗体,经检测具有高效价及特异性。Western Blot结果表明,该抗体可以与葡萄内源CIPK18特异性结合,且在50 kDa位置出现与预期一致的条带。同时,CIPK18在低温胁迫后葡萄叶片中蛋白表达水平与室温下相一致,但两种状态下均存在可能的磷酸化与泛素化修饰现象。本研究结果为进一步探究VaCIPK18的蛋白定位、表达及其功能奠定了基础。

Calcineurin B-like proteins(CBLs) interacting protein kinases(CIPKs) act as a class of serine/threonine protein kinases, which play critical roles in response to abiotic stress signaling in plants. Based on our previous RNA-seq data that Vitis amurensis responded to cold stress, seven CIPK members were observed to be involved in the early injury and perception phase of the CBL-CIPK signaling pathway, among which VaCIPK18 was significantly up-regulated. To further investigate the function of the kinase in cold stress, VaCIPK18 was cloned by homologous cloning. The ORF of VaCIPK18 was 1320 bp in length, encoding 439 amino acids. Based on the bioinformatics analysis of VaCIPK18 protein, the peptides rich in epitopes in the extracellular domain were obtained, and the C-terminal regulatory domain(230-439 aa) was constructed into the prokaryotic expression vector pET28 a-SUMO. The recombinant expression vector was further transformed into E. coli Rosetta strain, and the inclusion body protein of 42 kDa was expressed under the condition of 0.8 mmol·L-1 IPTG and induction at 37℃ for 4 h. The recombinant protein was used as an antigen to immunize white rabbits, and an anti-VaCIPK18 polyclonal antibody was obtained with high titer-specificity. Western blotting indicated that the antibody specifically binds to grape endogenous CIPK18, and the band was coincided with the 50 kDa position as expected. The results showed that the protein expression level of CIPK18 in grape leaves responding to cold stress was consistent with that at normal temperature condition, but there were possible phosphorylation and ubiquitination under both conditions. The current study provides a basis for further investigations of protein localization, expression, and function of VaCIPK18.