Xu Ben-Bo, Li Jia-Na, Zhang Xue-Kun, Wang Rui, Xie Ling-Li, Chai You-Rong
Chongqing Rapeseed Technology Research Center, Chongqing Key Laboratory of Crop Quality Improvement, Beibei, Chongqing, PR China.
J Plant Physiol. 2007 Mar;164(3):350-63. doi: 10.1016/j.jplph.2006.03.001. Epub 2006 Apr 17.
A flavonoid 3'-hydroxylase (F3'H) gene, denoted BnF3'H-1, was cloned from oilseed rape (Brassica napus). The gene of 3038 base pairs (bp) contains 3 introns. The complementary DNA (cDNA) consists of 1820bp and has an open reading frame of 1536bp encoding a polypeptide of 511 amino acids with a molecular weight of 56.62kDa and an isoelectric point of 7.08. BnF3'H-1 shows high homology to known F3'H genes, especially F3'H from Arabidopsis thaliana. Untranslated regions (UTRs) may play important roles in regulating the expression of BnF3'H-1. Besides containing a Kozak sequence, the first 77-bp region is C-rich but G-poor, and the 26-bp 5'-UTR contains 3 sites of ACCACT-like sequences. Alternative polyadenylation in the 3'-UTR is adopted by this gene to generate heterogeneous transcripts. Conserved domain search and motif characterization identified BnF3'H-1 as a cytochrome P450. All F3'H-featured motifs, VVVAAS, GGEK and VDVKG, are unchanged in BnF3'H-1. The N-terminal signal peptide/anchor and 3 transmembrane helices were predicted in BnF3'H-1, and its subcellular localization is most probably at the endoplasmic reticulum. Since 16 phosphorylation sites could be predicted, phosphorylation may be a necessary post-translational modification of BnF3'H-1. The secondary structure is dominated by alpha-helices and random coils. Most helices are located in the middle region, while extended strands mainly intersperse in terminal regions. DNA gel blot analysis indicated that 2 different F3'H genes might exist in B. napus. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and RNA gel blot analysis showed that flowers have the highest F3'H expression, followed by pericarp and seed, and lower levels in some other organs. This species-featured expression pattern is in obedience to multiple functional roles that F3'H gene(s) play(s) in various organs of B. napus. The BnF3'H-1 coding region was expressed in Escherichia coli, and enzyme activity of the His-tagged protein was demonstrated by monitoring the conversion of the substrate naringenin using high-performance liquid chromatography (HPLC), suggesting that BnF3'H-1 is catalytically functional. RT-PCR analysis suggests that transcription level of the F3'H gene(s) is not the reason for the different seed colorations found in near-isogenic lines (black-seeded L1 and yellow-seeded L2) of B. napus.
从油菜（甘蓝型油菜）中克隆出一个类黄酮3'-羟化酶（F3'H）基因，命名为BnF3'H-1。该基因全长3038个碱基对（bp），包含3个内含子。互补DNA（cDNA）由1820bp组成，具有1536bp的开放阅读框，编码一个511个氨基酸的多肽，分子量为56.62kDa，等电点为7.08。BnF3'H-1与已知的F3'H基因具有高度同源性，尤其是与拟南芥的F3'H基因。非翻译区（UTR）可能在调节BnF3'H-1的表达中发挥重要作用。除了含有一个科扎克序列外，前77bp区域富含C但G含量低，26bp的5'-UTR包含3个ACCACT样序列位点。该基因采用3'-UTR中的可变聚腺苷酸化来产生异质转录本。保守结构域搜索和基序特征鉴定表明BnF3'H-1是一种细胞色素P450。所有F3'H特征基序，VVVAAS、GGEK和VDVKG，在BnF3'H-1中均未改变。在BnF3'H-1中预测到N端信号肽/锚和3个跨膜螺旋，其亚细胞定位很可能在内质网。由于可以预测到16个磷酸化位点，磷酸化可能是BnF3'H-1必要的翻译后修饰。二级结构以α-螺旋和无规卷曲为主。大多数螺旋位于中间区域，而延伸链主要散布在末端区域。DNA凝胶印迹分析表明，甘蓝型油菜中可能存在2个不同的F3'H基因。半定量逆转录-聚合酶链反应（RT-PCR）和RNA凝胶印迹分析表明，花中F3'H的表达最高，其次是果皮和种子，在其他一些器官中的表达水平较低。这种物种特有的表达模式符合F3'H基因在甘蓝型油菜各器官中发挥的多种功能作用。BnF3'H-1编码区在大肠杆菌中表达，通过高效液相色谱（HPLC）监测底物柚皮素的转化，证明了His标签蛋白的酶活性，表明BnF3'H-1具有催化功能。RT-PCR分析表明，F3'H基因的转录水平不是甘蓝型油菜近等基因系（黑籽L1和黄籽L2）种子颜色不同的原因。
