药学学报, 2021, 56(5): 1478-1485
魏小艳, 李勇, 谭宇萍, 郭娟, 王雅南, 黄璐琦. 薏苡delta-12脂肪酸去饱和酶基因FAD2的克隆和表达分析[J]. 药学学报, 2021, 56(5): 1478-1485.
WEI Xiao-yan, LI Yong, TAN Yu-ping, GUO Juan, WANG Ya-nan, HUANG Lu-qi. Cloning and expression analysis of delta-12 fatty acid desaturase gene in Coix lacryma-jobi L.[J]. Acta Pharmaceutica Sinica, 2021, 56(5): 1478-1485.

魏小艳1,2, 李勇2, 谭宇萍2, 郭娟2, 王雅南2*, 黄璐琦1,2*
1. 江西中医药大学院士工作站, 江西 南昌 330004;
2. 中国中医科学院中药资源中心, 道地药材国家重点实验室培育基地, 北京 100700
本文克隆了薏苡delta-12脂肪酸去饱和酶基因FAD2(fatty acid desaturase),并对其分子结构特征和功能进行了研究。结果表明,薏苡FAD2基因的cDNA全长序列为936 bp,编码311个氨基酸残基。生物信息学预测结果显示,该基因编码蛋白为碱性亲水不稳定蛋白,分子质量34.87 kDa,含有3个跨膜螺旋结构域,不含信号肽剪切位点,最可能定位在质体膜。与其他植物中的同类蛋白相比,其仅具有一个组氨酸保守位点His Box Ⅲ(HXXHH),由此推测其活性可能会降低。系统发育树分析显示,薏苡FAD2与单子叶植物的亲缘关系较近,尤其是玉米和粳稻,而与双子叶植物的亲缘关系较远,由此推测其与玉米和粳稻中的同类基因可能具有相似功能。此外,基因表达分析结果显示,在高油脂含量薏苡中可以检测到该基因的表达变化,但在低油脂含量薏苡中未检测到该基因的表达。为明确薏苡FAD2的功能,在圆红冬孢酵母中对该基因进行了异源表达,结果显示该基因编码蛋白并未催化C18∶1不饱和脂肪酸形成C18∶2不饱和脂肪酸,由此推测FAD2基因组氨酸保守位点的缺失可能造成其蛋白活性降低甚至失活。本研究为深入了解脂肪酸去饱和酶的分子结构特征提供了一定参考。同时,为阐明薏苡脂肪酸类物质的生物合成途径奠定基础。
关键词:    薏苡      delta-12脂肪酸去饱和酶      克隆      生物信息学分析      表达特性     
Cloning and expression analysis of delta-12 fatty acid desaturase gene in Coix lacryma-jobi L.
WEI Xiao-yan1,2, LI Yong2, TAN Yu-ping2, GUO Juan2, WANG Ya-nan2*, HUANG Lu-qi1,2*
1. Academician Workstation, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China;
2. State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
In this study, the fatty acid desaturase gene FAD2 was cloned from Coix lacryma-jobi L. and its molecular structure and function were studied. The results showed that the full-length cDNA sequence of FAD2 gene was 936 bp encoding 311 amino acid residues. Bioinformatics prediction results showed that the protein encoded by the FAD2 gene was an alkaline hydrophilic unstable protein with a molecular weight of 34.87 kDa. It contained three transmembrane helix domain, and did not contain the signal peptide splicing site, and was most likely to be located in plasmid membrane. Compared with other similar genes in plants, it has only a histidine conserved site, His Box Ⅲ histidine site (HXXHH), suggesting its activity may be reduced. Phylogenetic tree analysis showed that FAD2 was closely related to monocotyledonous plants, especially Maize and Oryza sativa japonica Group, but farther from dicotyledonous plants. Therefore, it was inferred that FAD2 might have similar functions with similar genes in Maize and Oryza sativa japonica Group. In addition, the expression of FAD2 gene could be detected in Coix lacryma-jobi L. with high oil content, but not in low oil content of Coix lacryma-jobi L. In order to clarify the function of FAD2, the gene was heterologously expressed in sporomyces cerevisiae. The results showed that the protein encoded by FAD2 gene did not catalyze the formation of C18:1 unsaturated fatty acid into C18:2 unsaturated fatty acid. Therefore, it was speculated that the deletion of histidinine conserved site of FAD2 gene might lead to the decrease of protein activity or even inactivation. This study provides reference value for further understanding the molecular structure characteristics of fatty acid desaturase. At the same time, it laid a foundation for elucidating the biosynthetic pathway of Coix lacryma-jobi L.
Key words:    Coix lacryma-jobi L.    delta-12 fatty acid desaturase    cloning    bioinformatic analysis    expression characterization   
收稿日期: 2020-12-08
DOI: 10.16438/j.0513-4870.2020-1880
基金项目: 国家自然科学基金青年基金项目(81703657);中央本级重大增减支项目(ZZXT201702,ZZ13-YQ-085,20160302,2060302).
通讯作者: 王雅南,Tel:86-10-64087469,E-mail:wyn19870127@126.com;黄璐琦,Tel:86-10-84044340,E-mail:huangluqi01@126.com
Email: wyn19870127@126.com;huangluqi01@126.com
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李勇  在本刊中的所有文章
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郭娟  在本刊中的所有文章
王雅南  在本刊中的所有文章
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