水稻冷胁迫响应蛋白OsCML16的外源表达、纯化及质谱验证  

Exogenous expression,purification and mass spectrometry validation of rice cold stress responsive protein OsCML16

作　　者：卿冬进 邓国富[2] 戴高兴[2] 潘英华[1] 陆春菊 李经成[1] 周维永[1] 陈韦韦[1] 梁海福[1] 陈荣林[2] QING Dong-jin;DENG Guo-fu;DAI Gao-xing;PAN Ying-hua;LU Chun-ju;LI Jing-cheng;ZHOU Wei-yong;CHEN Wei-wei;LIANG Hai-fu;CHEN Rong-lin(Rice Research Institute,Guangxi Academy of Agricultural Sciences/Guangxi Key Laboratory of Rice Genetics and Breeding,Nanning 530007,China;Guangxi Academy of Agricultural Sciences,Nanning 530007,China)

机构地区：[1]广西农业科学院水稻研究所/广西水稻遗传育种重点实验室,南宁530007 [2]广西农业科学院,南宁530007

出　　处：《西南农业学报》2023年第6期1150-1156,共7页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31960059);中央引导地方科技发展专项资金项目(桂科ZY20198015、桂科ZY19183020);广西水稻优质化育种研究人才小高地项目(水稻人才小高地QN-27);广西农业科学院科技发展基金项目(2021JM23)。

摘　　要：【目的】在大肠杆菌中过表达、纯化水稻冷胁迫响应蛋白OsCML16,同时制备OsCML16蛋白抗体提供抗原,为采用该抗体研究水稻OsCML16蛋白的生物学功能提供参考依据。【方法】采用TMHMM 2.0在线分析软件对目的蛋白OsCML16进行跨膜区分析,利用高保真PCR扩增目的基因片段,以基因重组方法构建原核表达载体pET30a-OsCML16,并将重组质粒转入大肠杆菌表达菌株BL21(DE3)中;通过IPTG诱导OsCML16蛋白表达,然后采用亲和层析方法纯化获得目的蛋白,最后运用质谱验证法分析验证蛋白序列的真实性。【结果】通过对OsCML16蛋白跨膜区分析,发现该蛋白不包含跨膜结构,适合外源表达全长蛋白。将OsCML16基因链接至pET30a载体能成功构建原核表达载体pET30a-OsCML16,目的基因序列无突变位点,可用于表达目标蛋白。外源表达的OsCML16蛋白经过SDS-PAGE电泳分析,结果表明,OsCML16蛋白在28℃下可诱导表达,采用亲和层析方法纯化融合蛋白His6-OsCML16-His6,能成功获得分子量约30 kD的目的蛋白。对纯化的蛋白进行质谱验证分析,结果鉴定到3条多肽属于OsCML16蛋白,表明外源表达的蛋白即为OsCML16蛋白。【结论】采用原核表达方法将水稻冷胁迫响应基因OsCML16在大肠杆菌中表达,运用亲和层析技术根据融合蛋白携带组氨酸标签纯化获得融合表达蛋白His6-OsCML16-His6,并以蛋白质谱技术验证该蛋白的序列完全正确,后续可将水稻OsCML16蛋白用于抗体制备及蛋白功能研究。[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 afinity chromatography,then the authenticity ofthe 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 trans-membrane region,and it was suitable for heterologous expression of the full-length protein.The prokaryotic expression vector PET30A-OSC-ML16 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 C.The fusion protein Hise-OsCML16-His6 could be purified by affinity chromatography,and the 30 kD fusion protein was obtained successfully.The purified protein was validated by masspectrometry and a total of three peptides were identified as OsCML16 protein,indicating that the exogenously expressed protein was the OsCML16 pro-tein.[Conclusion]The rice cold stress response gene OsCML16 was expressed in E.coli by prokaryotic expression method.The fusion expres-sion protein His-OsCML16-His6 was purified by affinity chromatography according to the histide-carrying label of the fusion protein,and the sequence of the protein was veri

关 键 词：水稻 冷胁迫 OsCML16蛋白 外源表达 纯化 

分 类 号：S511[农业科学—作物学]