| 瓜叶菊F3＇5＇H基因cDNA的克隆、序列分析及其原核表达 |
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| 引用本文： | 孟丽,戴思兰.瓜叶菊F3＇5＇H基因cDNA的克隆、序列分析及其原核表达[J].分子植物育种,2005,3(6):780-786. |
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| 作者姓名： | 孟丽 戴思兰 |
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| 作者单位： | 北京林业大学园林学院,北京100083 |
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| 基金项目： | 本课题由国家林业局948项目（2001-24）;教育部振兴行动计划项目资助. |
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| 摘 要： | 蓝色花的形成主要由于是花瓣中3＇,5＇-羟羟基花青素（飞燕草色素及其衍生物）的相对含量较高,类黄酮-3＇,5＇-羟基化酶（F3＇5H）是合成这类色素的关键酶.不同物种F3＇5＇H同源基因的分离与特征解析能够为蓝色花的分子育种工作提供更多的理论依据.瓜叶菊花色多变,舌状花有白色、红色和蓝紫色等.根据已知F3＇5＇H保守的氨基酸序列设计了3条简并引物,通过RT-PCR从蓝色的瓜叶菊舌状花中扩增出了保守序列;利用RACE方法得到了两端序列.设计全长引物RT-PCR分离出了瓜叶菊F3＇5＇H同源基因（PCFH）.cDNA全长1 660bp,编码505个氨基酸.序列分析表明PCFH编码的氨基酸序列中包含有已确定的保守基序,包括血红素结合区、Ⅰ螺旋区和膜插入点的信号序列等.与其它物种的F3＇5＇H基因的序列相似性也较高.与翠菊、拟南芥和矮牵牛的序列相似系数分别为76%、59%和50%.序列相似性表明PCFH基因与茄子、矮牵牛和草原龙胆等同属于CYP75亚家族.利用pET28a载体PCFH在大肠杆菌BL21中实现了表达,IPTG诱导的差异蛋白大约为57kD,PCFH编码的多肽分子量相近,这将为进一步研究PCFH的酶学特性奠定了基础.
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| 关 键 词： | 瓜叶菊 F3＇5＇H 序列分析 原核表达 蓝色花 |
Cloning, Sequencing and Prokaryotic Expression of F3'5'H cDNA from Pericallis cruentia (L.) Herit. |
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| Meng Li ,Dai Silan.Cloning, Sequencing and Prokaryotic Expression of F3'5'H cDNA from Pericallis cruentia (L.) Herit.[J].Molecular Plant Breeding,2005,3(6):780-786. |
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| Authors: | Meng Li Dai Silan |
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| Abstract: | Blue flowers generally contain 3',5'-hydroxylated anthocyanins(delphinidin derivatives) as pigments, which were formed only in the presence of flavonoid-3',5'-Hydroxylase (F3'5'H). The isolation and characteristic analysis of novel F3'5'H from different species should provide more probability to blue flower molecular breeding. According to the conserved amino acid sequence three degenrate primers were designed. F3'5'H gene was cloned from Pericallis cruentia (L'Herit. (PCFH) with RT-PCR and RACE. The cDNA sequence was 1 660bp and included a whole ORF, which encode 505aa. Sequence alignment results showed that PCFH had three known motif such as I-helix, P450 haem-binding and "halt-transfer"signal domain. PCFH and DGFH shared 45%~80% of amino acid sequence identity with other CYP75s. PCFH was expressed with pET28a in Escherichia cilo BL21. The inducible proteins with His-Tag by IPTG could be detected with SDS-PAGE and their molecular weight was about 57kD, approximate to that of the deduced protein, which had prepared for its enzymatic character study. |
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| Keywords: | Peicallis cruentia F3'5'H Sequence analysis Prokaryotic expression Blue flowers |
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