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茶树类黄酮3’,5’-羟化酶在大肠杆菌的表达和优化
陈啸天,高丽萍,赵磊,刘亚军,王云生,夏涛
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(安徽农业大学生命科学学院,合肥 230036;安徽农业大学教育部茶叶生物化学与生物技术重点实验室,合肥 230036)
摘要:
儿茶素是茶叶主要特征和功能成分之一,其中B环三羟基儿茶素约占儿茶素总量的65%以上。类黄酮3’,5’-羟化酶是类黄酮合成中B环5’羟基化的唯一酶系,与B环三羟基儿茶素合成积累密切相关。采用RACE技术,克隆了茶树类黄酮3’,5’-羟化酶基因的1 944 bp的全长序列,开放阅读框区域为55~1587 bp,编码510个氨基酸,推测其分子量为57.1 kDa,等电点为9.06。该基因重组至pET-32a(+)表达载体上,在大肠杆菌Rosetta中进行原核表达;优化了原核表达中温度诱导条件;利用钴离子吸附柱纯化了目的蛋白。该研究为开展F3’5’H酶的反应动力学、酶的器官组织定位等研究奠定了基础。
关键词:  茶树  类黄酮3’,5’-羟化酶  RACE  原核表达
DOI:
基金项目:国家自然科学基金(30972401, 31170647, 31170282和31000314)共同资助。。
Expression and optimization of flavonoid 3’, 5’hydroxylase of tea plant [Camellia sinensis (L.) O. Kuntze] in Escherichia coli
CHEN Xiao-tian,GAO Li-ping,ZHAO Lei,LIU Ya-jun,WANG Yun-sheng,XIA Tao
(School of Biology Science, Anhui Agricultural University, Hefei 230036;Key Lab of Tea Biochemistry and Biotechnology, Ministry of Education, Anhui Agricultural University, Hefei 230036)
Abstract:
Catechins are the most prominent characteristic and functional compounds of tea, of which, the type of B ring 3', 4’ 5'-hydroxy catechins account for 65% of the total content. Flavonoid 3’,5’ hydroxylase (F3’5’H) is a key enzyme for flavonoid synthesis in tea plant(Camellia sinensis (L.) O. Kuntze). The cDNA sequence of F3’5’H gene was cloned by rapid amplification of cDNA ends(RACE), and its full length was 1 944 bp with ORF (open reading frame) beginning from 55 nt to 1 587 nt , encoding 510 amino acids. Bioinformatics analysis results showed that the deduced protein molecular weight and theoretical isoelectric point were 57.1 kDa and 9.06, separately. The gene was cloned into the expression vector pET32a (+) for expression in prokaryotic cells. The SDS-PAGE results showed that the flavonoid 3’, 5’ hydroxylase protein was expressed in Escherichia coli rosetta. The temperature for protein expression was optimized, and the induced protein was purified by Cobalt ion purification column. This research will lay a foundation for the enzymatic kinetics and histochemical localization of flavonoid 3’, 5’ hydroxylase.
Key words:  Camellia sinensis  flavonoid 3’, 5’hydroxylase  RACE  prokaryotic expression

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