水稻冷胁迫响应蛋白 OsCML16 的外源表达、纯化及质谱验证.

【Objective】 The present paper aimed to overexpress and purify rice cold stress response protein OsCML16 in Escherichia coli, and prepare OsCML16 protein antibody to provide antigen, and provide reference for further study of biological function of rice OsCML16 protein by using the antibody. 【Method】 At first, the transmembrane region of the target protein OsCML16 was analyzed online using TMHMM 2.0 software, and then the fragment of target gene was amplified by high fidelity PCR, the prokaryotic expression vector pET30a-OsCML16 was constructed by gene recombination method, and the recombinant plasmid was transformed into expression E.coli line BL21 (DE3). OsCML16 protein expression was induced by IPTG, and purified by affinity chromatography, then the authenticity of the protein sequence was verified by mass spectrometry. 【Result】 By analyzing the transmembrane region of OsCML16 protein, it was found that the protein did not contain transmembrane region, and it was suitable for heterologous expression of the full-length protein. The prokaryotic expression vector PET30A-OSCML16 was successfully constructed by linking OsCML16 gene to pET30a vector. The result showed that the target gene had no mutation site and could be used for expression of the target protein. Exogenous expression OsCML16 protein was analyzed by SDS-PAGE electrophoresis, and results showed that OsCML16 protein could be induced expression at 28 ℃.The fusion protein His6-OsCML16-His6 could be purified by affinity chromatography, and the 30 kD fusion protein was obtained successfully. The purified protein was validated by mass spectrometry, and a total of three peptides were identified as OsCML16 protein, indicating that the exogenously expressed protein was the OsCML16 protein. 【Conclusion】 The rice cold stress response gene OsCML16 was expressed in E.coli by prokaryotic expression method. The fusion expression protein His6-OsCML16-His6 was purified by affinity chromatography according to the histide-carrying label of the fusion protein, and the sequence of the protein was verified by protein spectroscopy. Rice OsCML16 protein can be used for antibody preparation and protein function study in the future. [ABSTRACT FROM AUTHOR]