异叶天南星苯丙氨酸解氨酶基因的克隆与蛋白结构分析

施圆圆; 张声祥; 赵德蕊; 王晨凯; 马克龙; 吴家文

doi:10.11913/PSJ.2095-0837.2019.20221

异叶天南星苯丙氨酸解氨酶基因的克隆与蛋白结构分析

施圆圆^1,2,
张声祥^1,2,
赵德蕊^1,2,
王晨凯^1,2,
马克龙³,
吴家文^2,4,5, ,

1. 安徽中医药大学研究生院, 合肥 230012;
2. 安徽中医药大学科研实验中心、新安医学教育部重点实验室, 合肥 230038;
3. 安徽中医药大学中西医结合临床学院, 合肥 230012;
4. 安徽省中医药科学院, 合肥 230012;
5. 安徽道地中药材品质提升协同创新中心, 合肥 230012

基金项目:

名贵中药资源可持续利用能力建设项目（2060302）；安徽高校自然科学研究重大项目（KJ2018ZD028）；安徽省自然科学基金项目（1608085MH177）；安徽省留学人员科技活动启动项目（20151x024）；国家级大学生创新创业训练项目（201810369050）；国家自然科学基金（81373598）。

详细信息

作者简介:
施圆圆(1993-),女,硕士研究生,研究方向为中草药基因研究(E-mail:871735748@qq.com)。

通讯作者:
吴家文,E-mail:wujiawen@ahtcm.edu.cn

中图分类号: Q943.2
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出版历程
- 收稿日期: 2018-10-24
- 修回日期: 2018-11-13
- 网络出版日期: 2022-10-31
- 发布日期: 2019-04-27

Gene cloning and structure characterization of phenylalanine ammonia-lyase from Arisaema heterophyllum

1. Graduate School of Anhui University of Chinese Medicine, Hefei 230012, China;
2. Key Laboratory of Xin'an Medicine, Ministry of Education, Anhui University of Chinese Medicine, Hefei 230038, China;
3. Clinical College of Anhui Integrated Traditional Chinese and Western Medicine, Anhui University of Chinese Medicine, Hefei 230032, China;
4. Anhui Academy of Chinese Medicine, Hefei 230012, China;
5. Synergetic Innovation Center of Anhui Authentic Chinese Medicine Quality Improvement, Hefei 230012, China

Funds:

This work was supported by grants from the Sustainable Utilization of Famous Traditional Chinese Medicine Resources (2060302), Natural Science Research Grant of Higher Education of Anhui Province (KJ2018ZD028), Natural Science Foundation of Anhui Province of China (1608085MH177), Anhui Province Scientific Research Foundation for the Returned Overseas Chinese Scholars (20151x024), National Students' Platform for Innovation and Entrepreneurship Training Program (201810369050), and National Natural Science Foundation of China (81373598).

摘要

摘要: 以异叶天南星（Arisaema heterophyllum Blume）为材料，采用逆转录PCR（RT-PCR）法扩增其苯丙氨酸解氨酶（Phenylalanine ammonia-lyase，PAL）基因AhPAL，获得该基因的开放读码框（ORF），并通过系统的生物信息学软件分析AhPAL的结构和理化性质。结果显示，AhPAL的ORF全长为2184 bp，编码727个氨基酸；AhPAL与郁金香（Tulipa fosteriana W.Irving）PAL的亲缘关系最近，序列相似性达88%。空间结构模型分析结果显示，AhPAL为同型四聚体，每个单体由3个结构域组成，其中MIO结构域含有PAL酶家族的保守序列和Ala-Ser-Gly三肽活性中心，是AhPAL酶活性的决定性结构域。利用荧光定量PCR方法检测3个AhPAL Unigene在根、块茎和叶中的表达情况，发现它们在根中表达量均最高，而在叶和块茎中表达较低。
- 异叶天南星 /
- 苯丙氨酸解氨酶 /
- 基因克隆 /
- 生物信息学分析
Abstract: Phenylalanine ammonia-lyase (PAL) is the first key enzyme in the synthesis of flavonoids and the rate-limiting enzyme of the phenylpropanoid pathway, which is associated with secondary metabolites, growth and development, as well as defense of plants. In this study, the mRNA of Arisaema heterophyllum Blume was used as a template and the full-length open reading frame (ORF) of the phenylalanine ammonia-lyase gene (AhPAL) was amplified by real-time polymerase chain reaction (RT-PCR). The structure and physicochemical properties of AhPAL were then analyzed by bioinformatics. Results showed that the ORF of AhPAL was 2,184 bp in length, encoding a protein with 727 amino acids. Furthermore, AhPAL had the closest relationship with PAL of Tulipa fosteriana W. Irving, with a sequence identity of 88%. Structure analysis indicated that AhPAL was a homotypic tetramer, and each monomer consisted of three domains. Among these domains, the MIO domain was highly important for the activity of AhPAL, containing a conserved sequence of the PAL family and Ala-Ser-Gly triad residues. The expression levels of three AhPAL unigenes in the roots, tubers, and leaves were detected by quantitative RT-PCR (qRT-PCR), with higher expression found for all three in the roots compared with that in the leaves and tubers.
- Arisaema heterophyllum /
- Phenylalanine ammonia-lyase /
- Gene cloning /
- Bioinformatics analysis

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