黑果枸杞原花青素合成相关无色花色素还原酶基因和花青素还原酶基因的克隆及表达分析

doi:10.5246/jcps.2014.06.050

中国药学(英文版) ›› 2014, Vol. 23 ›› Issue (6): 369-377.DOI: 10.5246/jcps.2014.06.050

黑果枸杞原花青素合成相关无色花色素还原酶基因和花青素还原酶基因的克隆及表达分析

沈笑飞¹, 曾少华², 吴敏², 刘春朝³, 王瑛^1,2*

1. 中国科学院武汉植物园, 湖北武汉 430074
2. 中国科学院华南植物园, 广东广州 510650
3. 中国科学院过程工程研究所, 北京 100190

收稿日期:2014-05-05 修回日期:2014-05-20 出版日期:2014-06-28 发布日期:2014-05-30
通讯作者: Tel./Fax: 86-20-37082052
基金资助:
National Natural Science Foundation of China (Grant No. 31100223), Scientific Research Equipment Project of Chinese Academy of Sciences (Grant No. YZ201227)

Characterization of proanthocyanin-related leucoanthocyanidin reductase and anthocyanidin reductase genes in Lycium ruthenicum Murr.

Xiaofei Shen¹, Shaohua Zeng², Min Wu², Chunzhao Liu³, Ying Wang^1,2*

1. Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China
2. Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China
3. National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China

Received:2014-05-05 Revised:2014-05-20 Online:2014-06-28 Published:2014-05-30
Contact: Tel./Fax: 86-20-37082052
Supported by:
National Natural Science Foundation of China (Grant No. 31100223), Scientific Research Equipment Project of Chinese Academy of Sciences (Grant No. YZ201227).

摘要/Abstract

摘要： 原花青素是黑果枸杞中一类重要的植化产物, 但其生物合成机制仍不清楚。本研究利用黑果枸杞EST数据库, 克隆了无色花色素还原酶基因(LrLAR)和花青素还原酶基因(LrANR)。PCR结果表明, LrLAR和LrANR分别由333个氨基酸残基、338个氨基酸残基组成的蛋白编码。进化分析表明, 相应蛋白LrLAR、LrANR分别聚类于LAR和ANR簇。实时定量PCR结果表明, 从果实未成熟时期到变色期, LrLAR、LrANR的表达量均急剧上升, 但在随后的发育时期中逐渐下降; 与在果实中相比, 在嫩叶、成熟叶、茎、根中, 两基因的表达量极低。植化分析发现, 果实中总原花青素含量高于嫩叶、成熟叶、茎、根中的含量, 并且在果实成熟过程中呈现先增加后基本趋于稳定的趋势。本研究将为揭示黑果枸杞原花青素生物合成机制及促进其工程改良奠定基础。

关键词: 花青素还原酶, 无色花色素还原酶, 黑果枸杞, 原花青素

Abstract:

Proanthocyanidins (PAs) are a set of important phytochemical compounds in Lycium ruthenicum Murr. However, PA biosynthesis in L. ruthenicum remains unclear. In this study, leucoanthocyanidin reductase (LAR) and anthocyanidin reductases (ANR) involved in PA biosynthesis wereretrieved from L. ruthenicum EST database. Polymerase chain reaction (PCR) results confirmed that LrLAR and LrANR encoded a protein composed of 333 and 338 amino acids, respectively. Phylogenetic analysis showed that LrLAR and LrANR were clustered with known LAR and ANR proteins of other plants, respectively. Quantitative reverse-transcription PCR (qRT-PCR) results indicated that both genes expressed in a sharp increase manner from unripe stage to color break stage and were down-regulated increasingly hereafter, and that both expression levels are extremely low in young leaves, mature leaves, stems, and roots compared with fruits. Phytochemical assay revealed that the content of total PA in fruit was higher than that in young leaves, mature leaves, stems, and roots, and that the total PA level was increased sharply and then relatively stable hereafter during fruit ripening. Our results lay a foundation for uncovering the PA biosynthesis and further engineering manipulation in L. ruthenicum.

Key words: ANR, LAR, Lycium ruthenicum, Proanthocyanidin

中图分类号:

R284

Supporting: National Natural Science Foundation of China (Grant No. 31100223), Scientific Research Equipment Project of Chinese Academy of Sciences (Grant No. YZ201227)

沈笑飞, 曾少华, 吴敏, 刘春朝, 王瑛. 黑果枸杞原花青素合成相关无色花色素还原酶基因和花青素还原酶基因的克隆及表达分析[J]. 中国药学(英文版), 2014, 23(6): 369-377.

Xiaofei Shen, Shaohua Zeng, Min Wu, Chunzhao Liu, Ying Wang. Characterization of proanthocyanin-related leucoanthocyanidin reductase and anthocyanidin reductase genes in Lycium ruthenicum Murr. [J]. Journal of Chinese Pharmaceutical Sciences, 2014, 23(6): 369-377.

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黑果枸杞原花青素合成相关无色花色素还原酶基因和花青素还原酶基因的克隆及表达分析

Characterization of proanthocyanin-related leucoanthocyanidin reductase and anthocyanidin reductase genes in Lycium ruthenicum Murr.

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