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2021, v.46(19) 4950-4958

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茅苍术硫解酶AIKAT基因的克隆及原核表达分析
Cloning and prokaryotic expression of 3-ketoacyl-CoA thiolase gene AIKAT from Atractylodes lancea

徐睿;单婷玉;吴君贤;刘梦丽;余函纹;查良平;彭华胜;
XU Rui;SHAN Ting-yu;WU Jun-xian;LIU Meng-li;YU Han-wen;ZHA Liang-ping;PENG Hua-sheng;School of Pharmacy, Anhui University of Chinese Medicine;Institute of Conservation and Development of Traditional Chinese Medicine Resources, Anhui Academy of Chinese Medicine;National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences;Research Unit of Dao-di Herbs (2019RU57), Chinese Academy of Medical Sciences;

摘要(Abstract):

对茅苍术脂肪酸β-氧化途径中关键酶3-酮脂酰-CoA硫解酶(KAT)基因进行克隆并开展生物信息学分析、原核表达和基因表达分析,为研究茅苍术脂肪酸β-氧化机制奠定基础。根据茅苍术转录组数据中KAT基因序列信息,设计特异性引物,采用RT-PCR方法克隆获得茅苍术3-酮脂酰-CoA硫解酶基因全长序列,命名为AIKAT(GenBank登录号为MW665111)。结果分析表明,AIKAT基因的开放阅读框(ORF)为1 323 bp,编码440个氨基酸,推测其相对分子质量为46 344.36,蛋白理论等电点为8.92。通过在线工具预测AIKAT为稳定的碱性蛋白,跨膜结构域分析表明AIKAT无跨膜结构,二级结构显示其主要由α-螺旋结构组成,利用同源建模进行三级结构预测;同源氨基酸序列分析表明茅苍术与黄花蒿、洋蓟、地黄、拟南芥KAT编码的氨基酸序列具有较高的同源性;系统进化树分析显示AIKAT与洋蓟CcKAT2聚为一支,证实了菊科3-酮脂酰-CoA硫解酶基因的同源性;构建原核表达载体pET-32a-AIKAT并转化至大肠杆菌BL21 (DE3)表达感受态进行诱导表达,在64 kDa处成功表达出目的蛋白;利用实时荧光定量PCR技术检测了茅苍术3个组织中的AIKAT基因表达量,结果显示AIKAT在根状茎中表达量最高,其次是叶、茎。该研究首次克隆得到AIKAT基因,使其重组蛋白在大肠杆菌中成功表达,并验证了其在不同组织中存在差异表达,为进一步研究茅苍术脂肪酸β-氧化途径奠定了基础。
In this study, the gene encoding the key enzyme 3-ketoacyl-CoA thiolase(KAT) in the fatty acid β-oxidation pathway of Atractylodes lancea was cloned. Meanwhile, bioinformatics analysis, prokaryotic expression and gene expression analysis were carried out, which laid a foundation for the study of fatty acid β-oxidation mechanism of A. lancea. The full-length sequence of the gene was cloned by RT-PCR with the specific primers designed according to the sequence information of KAT gene in the transcriptomic data of A. lancea and designated as AIKAT(GenBank accession number MW665111). The results showed that the open reading frame(ORF) of AIKAT was 1 323 bp, encoding 440 amino acid. The deduced protein had a theoretical molecular weight of 46 344.36 and an isoelectric point of 8.92. AIKAT was predicted to be a stable alkaline protein without transmembrane segment. The secondary structure of AIKAT was predicted to be mainly composed of α-helix. The tertiary structure of AIKAT protein was predicted by homology modeling method. Homologous alignment revealed that AIKAT shared high sequence identity with the KAT proteins(AaKAT2, CcKAT2, RgKAT and AtKAT, respectively) of Artemisia annua, Cynara cardunculus var. scolymus, Rehmannia glutinosa and Arabidopsis thaliana. The phylogenetic analysis showed that AIKAT clustered with CcKAT2, confirming the homology of 3-ketoacyl-CoA thiolase genes in Compositae. The prokaryotic expression vector pET-32 a-AIKAT was constructed and transformed into Escherichia coli BL21(DE3) for protein expression. The target protein was successfully expressed as a soluble protein of about 64 kDa. A real-time quantitative PCR analysis was performed to profile the AIKAT expression in different tissues of A. lancea. The results demonstrated that the expression level of AIKAT was the highest in rhizome, followed by that in leaves and stems. In this study, the full-length cDNA of AIKAT was cloned and expressed in E. coli BL21(DE3), and qRT-PCR showed the differential expression of this gene in different tissues, which laid a foundation for further research on the molecular mechanism of fatty acid β-oxidation in A. lancea.

关键词(KeyWords): 茅苍术;3-酮脂酰-CoA硫解酶;基因克隆;原核表达;表达分析
Atractylodes lancea;3-ketoacyl-CoA thiolase;gene cloning;prokaryotic expression;expression analysis

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81703633,82071412);; 中国医学科学院医学与健康科技创新工程项目(2019-I2M-5-065);; 安徽省自然科学基金青年基金项目(1808085QH290);; 中央本级重大增减支项目(2060302)

作者(Author): 徐睿;单婷玉;吴君贤;刘梦丽;余函纹;查良平;彭华胜;
XU Rui;SHAN Ting-yu;WU Jun-xian;LIU Meng-li;YU Han-wen;ZHA Liang-ping;PENG Hua-sheng;School of Pharmacy, Anhui University of Chinese Medicine;Institute of Conservation and Development of Traditional Chinese Medicine Resources, Anhui Academy of Chinese Medicine;National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences;Research Unit of Dao-di Herbs (2019RU57), Chinese Academy of Medical Sciences;

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