药学学报, 2018, 53(10): 1743-1752
引用本文:
董先娟, 冯莹莹, 刘晓, 齐博文, 闫雅如, 丁宁, 吴云, 高博闻, 王晓晖. 白木香S-腺苷甲硫氨酸合成酶基因的克隆与表达分析[J]. 药学学报, 2018, 53(10): 1743-1752.
DONG Xian-juan, FENG Ying-ying, LIU Xiao, QI Bo-wen, YAN Ya-ru, DING Ning, WU Yun, GAO Bo-wen, WANG Xiao-hui. Cloning and expression analysis of S-adenosylmethionine synthetase gene from Aquilaria sinensis[J]. Acta Pharmaceutica Sinica, 2018, 53(10): 1743-1752.

白木香S-腺苷甲硫氨酸合成酶基因的克隆与表达分析
董先娟1, 冯莹莹1, 刘晓1, 齐博文1, 闫雅如1, 丁宁1, 吴云1, 高博闻2, 王晓晖1
1. 北京中医药大学中药学院中药现代研究中心, 北京 100029;
2. 包头医学院药学院, 内蒙古 包头 014060
摘要:
S-腺苷甲硫氨酸合成酶(S-adenosylmethionine synthetase,SAMS)是植物代谢过程中的一个关键酶,在植物响应生物与非生物胁迫中发挥着重要作用。本研究根据白木香转录组高通量测序结果结合RACE、RT-PCR技术从白木香愈伤组织中首次克隆得到1个SAMS基因,命名为AsSAMS1,并对其进行生物学信息分析、原核表达与纯化、组织特异性分析及非生物胁迫和激素诱导表达分析。白木香AsSAMS1基因的开放阅读框(ORF)长1 183 bp,编码393个氨基酸,其蛋白分子质量是43.13 kDa。生物学信息分析表明AsSAMS1蛋白含有3个SAMS的特征序列,系统进化树显示AsSAMS1蛋白与野大豆(Glycine soja)SAMS蛋白具有较高的同源性。构建原核表达载体pET28a-AsSAMS1并在大肠杆菌BL21(DE3)菌株中成功表达AsSAMS1重组蛋白,利用Ni2+亲和色谱纯化得到可溶性AsSAMS1重组蛋白。实时荧光定量PCR检测结果表明AsSAMS1基因在茎中表达最高,叶和茎尖次之,在根中表达最低。盐、干旱、低温以及重金属胁迫均能够提高AsSAMS1的表达量和其产物S-腺苷甲硫氨酸的含量;茉莉酸甲酯、水杨酸、脱落酸、赤霉素也能够诱导白木香愈伤组织中AsSAMS1基因表达。本研究可以为进一步研究S-腺苷甲硫氨酸合成酶在白木香结香机制和植物防御反应中的作用奠定基础。
关键词:    白木香      S-腺苷甲硫氨酸合成酶      生物学信息分析      原核表达      表达分析     
Cloning and expression analysis of S-adenosylmethionine synthetase gene from Aquilaria sinensis
DONG Xian-juan1, FENG Ying-ying1, LIU Xiao1, QI Bo-wen1, YAN Ya-ru1, DING Ning1, WU Yun1, GAO Bo-wen2, WANG Xiao-hui1
1. Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China;
2. School of Pharmacy, Baotou Medical College, Baotou 014060, China
Abstract:
S-adenosylmethionine synthetase, a key enzyme in plant metabolism, plays an essential role in the plant defence system. In present study, a full length cDNA sequence of AsSAMS1 gene was cloned by RACE and reverse transcription PCR from Aquilaria sinensis calli. Meanwhile, the bioinformatics, prokaryotic expression, tissue-specific expression analysis, and expression analysis under different abiotic stresses and hormone treatments were performed. The open reading frame (ORF) of AsSAMS1 gene was 1 183 bp, encoding a protein of 393 amino acids with a calculated molecular mass (MW) of 43.13 kDa. Bioinformatic analysis indicated that AsSAMS1 contained 3 SAMS characteristic sequences. The phylogenetic analysis indicated that AsSAMS1 protein had the highest level of homology with SAMS protein from Glycine soja. The recombinant AsSAMS1 protein was successfully expressed in Escherichia coli BL21 (DE3) cells using the prokaryotic expression vector pET28a-AsSAMS1 and the recombinant AsSAMS1 was purified by Ni2+ affinity chromatography. Expression analysis results in different tissues indicated that AsSAMS1 was primarily observed in stems, and then stem tips and leaves, following by roots. The transcript level of AsSAMS1 and the content of S-adenosylmethionine (SAM) were induced by various abiotic stresses including salt, drought, cold, and heavy metal stress. Furthermore, AsSAMS1 expression level was enhanced upon methyl jasmonate (MeJA), salicylic acid (SA), gibberellin (GA3), and abscisic acid (ABA) treatment. These results provided valuable insights for further study on the role of SAMS in the mechanism of agarwood formation and plant resistance.
Key words:    Aquilaria sinensis    S-adenosylmethionine synthetase    bioinformatics analysis    prokaryotic expression    expression analysis   
收稿日期: 2018-06-27
DOI: 10.16438/j.0513-4870.2018-0591
基金项目: 国家自然科学基金青年基金资助项目(81503182);内蒙古自然科学基金资助项目(2017BS0803);包头医学院科学研究项目(BYJJ-YF201608).
通讯作者: 高博闻,Tel:86-10-64286180,E-mail:wangxhui2014@163.com;王晓晖,Tel:86-472-7167795,E-mail:gaobw001@163.com
Email: wangxhui2014@163.com;gaobw001@163.com
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