【作者】 陈啸天； 赵磊； 李伟伟； 许玉娇； 王云生；

【Author】 CHEN Xiao-tian;ZHAO Lei;LI Wei-wei;XU Yu-jiao;WANG Yun-sheng;School of Biology Science,Anhui Agricultural University;Key Laboratory of Tea Biochemistry and Biotechnology,Ministry of Education,Anhui Agricultural University;

【机构】 安徽农业大学生命科学学院； 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；

【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。类类黄酮3,5-羟化酶(flavonoid 3’5’-hydroxylase,F3’5’H)是催化类黄酮B环5’羟基化反应的唯一酶系,与B环三羟基儿茶素合成积累密切相关。本文采用RACE技术克隆了推测的CsF3’5’H的全长序列,通过氨基酸序列分析,推测CsF3’H和CsF3’5’H的N段30个氨基酸左右为信号肽序列。构建了pYES-dest52-CsF3’5’H、pYES-dest52-VvF3’5’H、pYES-dest52-PhF3’5’H-1、pYES-dest52-PhF3’5’H-2重组质粒,成功实现了不同来源的F3’5’H基因在酿酒酵母中的表达,并测定了部分酶对不同底物的活性,其中含pYES-dest52-VvF3’5’H、pYES-dest52-PhF3’5’H-1、pYES-dest52-PhF3’5’H-2重组质粒的菌株均对柚皮素和圣草酚具有明显催化活性。通过优化了HPLC条件,建立了茶树F3’H和F3’5’H粗酶酶活的检测体系,证实茶树粗酶中确实存在F3’5’H酶的活性。通过实时荧光定量PCR技术技术研究了类黄酮B环羟基化相关基因在茶叶不同发育阶段的表达量。研究发现,F3’5’H在根中几乎检测不到表达量,说明其表达组织器官特异性。该研究初步揭示了F3’5’H的最适底物水平和CsF3’5’H基因的表达差异。更多还原

【Abstract】 Catechins are the most prominent characteristic and functional compounds of tea.And the B ring 3’ 4’5’-hydroxy catechins are the most prominent flavonoid compounds in tea leaf,constituting about 65% of the total amount of catechins.Flavonoid 3’,5’hydroxylase(F3’5’H) is a key enzyme of flavonoids synthetic way in tea plant[Camellia sinensis(L.) O.Kuntze].RACE was used to obtain the full length cDNA sequence of F3’5’H in tea plant.30 amino acids in the both N-terminal of the predicted CsF3’ H and CsF3’5’H proteins are signal peptide sequences.Recombinant plasmids pYES-dest52-CsF3’5’H,pYES-dest52-VvF3’5’H,pYES-dest52-PhF3’5’H-1 and pYES-dest52-PhF3’5’H-2 were successfully constructed,which achieving F3’5’H from different species expressed in S.cerevisiae.Enzyme activity on different substrates had been detected.YES-dest52-VvF3’5’H,pYES-dest52-PhF3’5’H-1 and pYES-dest52-PhF3’5’H-2 had apparent catalytic activity to naringeninand eriodictyol.Enzyme activity detection system of flavonoid 3’-hydroxylase and flavonoid 3’,5’-hydroxylase were established by optimizing the HPLC conditions.It is showed that the tea crude enzyme might have the activity of flavonoid 3’,5’-hydroxylas.Real-time PCR was performed to identify the different experssion level of relevant B-ring-hydroxylation flavonoid genes in different matural tea leaves.The results showed that CsF3’5’H had rare expression level in roots and belonged to induced expression.更多还原