《植物生理学报》 2018, 54(1): 173-181

苹果N-乙酰基-5-羟色胺-O-甲基转移酶基因的克隆及原核表达

吴成成, 李保华, 练森, 梁文星, 王彩霞^*

青岛农业大学植物医学学院, 山东省植物病虫害综合防控重点实验室, 山东青岛266109

通信作者：王彩霞；E-mail: cxwang@qau.edu.cn

摘　要：

以‘富士’和‘嘎啦’苹果(Malus pumila)叶片总RNA为模板, 利用反转录PCR (RT-PCR)技术, 克隆N-乙酰基-5-羟色胺-O-甲基转移酶(ASMT)基因, 通过DNAMAN、MEGA 5.1、ProtParam等生物信息学软件对基因cDNA序列、系统进化关系、理化性质等进行分析, 将克隆的苹果ASMT基因与表达载体pET32a连接, 构建原核表达载体并转入大肠杆菌(Escherichia coli)表达菌株Rosetta (DE3)中, 优化原核表达条件, 用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)检测融合蛋白的可溶性, 用蛋白免疫印迹法(western blot)和酶活性测定验证目的蛋白的表达。结果表明, 获得了苹果ASMT的全长cDNA序列, 命名为MpASMT1 (GenBank登记号MF135479)。该基因开放阅读框(ORF)为1 077 bp, 编码358个氨基酸。MpASMT1编码蛋白的理论分子量约为39.7 kDa, 等电点为5.42, 是非分泌型、疏水的稳定蛋白; 其263位组氨酸(His)为催化位点, 225位谷氨酸为底物S-腺苷甲硫氨酸结合位点。多序列比对及系统发生树分析表明, MpASMT1编码的氨基酸序列与珠美海棠(M. zumi) MzASMT1 (KJ123721)同源性最高, 相似性达99.2%, 其次为葡萄(Vitis vinifera) ASMT (LOC100248671), 氨基酸序列相似性为66.4%。原核表达结果显示, 经0.25~2.0 mmol·L^-1异丙基-β-d-硫代半乳糖苷(IPTG)诱导的重组质粒可特异性表达分子量约58 kDa的融合蛋白, 且低温条件(15和25°C)能够提高该蛋白的可溶性表达; western blot分析表明融合表达蛋白能与His单克隆抗体特异性结合, 纯化蛋白的酶活性为7.8 pmol·mg^-1 (蛋白), 进一步证实MpASMT1编码蛋白成功表达。

关键词：苹果; MpASMT1; 克隆; 生物信息学; 原核表达

收稿：2017-07-10   修定：2017-11-22

资助：现代农业产业技术体系建设专项资金(CARS-28)、国家自然科学基金(31272001)和山东省“泰山学者”建设工程专项。

Cloning and prokaryotic expression of N-acetylserotonin O-methyltransferase gene in Malus pumila

WU Cheng-Cheng, LI Bao-Hua, LIAN Sen, LIANG Wen-Xing, WANG Cai-Xia^*

College of Plant Health and Medicine, Key Lab of Integrated Crop Pest Management of Shandong, Qingdao Agricultural University, Qingdao, Shandong 266109, China

Corresponding author: WANG Cai-Xia; E-mail: cxwang@qau.edu.cn

Abstract:

N-acetylserotonin O-methyltransferase gene (MpASMT1) was amplified by reverse transcription PCR from leaves of Malus pumila Mill. ‘Fuji’ and ‘Gala’. Full-length cDNA sequence, phylogenetic relationship and physicochemical properties of this gene were analyzed by a variety of bioinformatics softwares such as DNAman , MEGA 5.1 and ProtParam. Then, a recombinant plasmid pET-MpASMT1 was constructed using pET32a as a fused expression vector. Optimal conditions for recombinant protein expression were determined in Escherichia coli Rosetta (DE3) including inducing time, isopropyl β-d-thiogalactopyranoside (IPTG) concentration and inducing temperature. Recombinant protein expression and solubility of MpASMT1 were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blot and enzyme activity assay were used to confirm the successful expression of MpASMT1 in E. coli. The results show that fulllength cDNA sequence of MpASMT1 from M. pumila was obtained and its GenBank accession number is MF135479. Its open reading frame (ORF) of 1 077 bp encodes 358 amino acid residues with a predicted molecular weight of 39.7 kDa and an isoelectric point value of 5.42. MpASMT1 is a non-secretory and hydrophobic stable protein. The 263 aa histidine is a catalytic site and 225 aa glutamate is a substrate binding site of S-adenylyl methionine. Multiple sequence alignment and phylogenetic analysis showed that the encoded protein had the closest genetic relationship with M. zumi MzASMT1 (GenBank: KJ123721) and Vitis vinifera ASMT (GenBank: LOC100248671), and the similarities of amino acid sequences were 99.2% and 66.4%, respectively. Prokaryotic expression results showed that efficient expression of recombinant MpASMT1 protein about 58 kDa was realized when induced by 0.25–2.0 mmol·L^-1 IPTG in E. coli. However, lower temperature (15 and 25°C) could increase the solubility of recombinant protein. Western blot analysis showed that the recombinant protein could be specifically recognized by His monoclonal antibody, and the enzyme activity of purified MpASMT1 protein was 7.8 pmol·mg^-1 (protein), which further confirms that MpASMT1 protein was successfully expressed.

Key words: apple; MpASMT1; cloning; bioinformatics; prokaryotic expression