羽衣甘蓝中一个突变型肉桂酸- 4 - 羟化酶基因的克隆及分析

 以羽衣甘蓝为材料, 克隆了全长2 431 bp的肉桂酸- 4 - 羟化酶基因BoC4H。它有两个内含子, mRNA为1 715 bp, 编码一个481个氨基酸的推导蛋白, 与其它植物C4H有广泛同源性。它含有细胞色素P450保守域和特征基序, 如血红素结合域、E - R - R三联体、含T的结合槽基序、酶正确定向必需的铰链基序。BoC4H具有C4H /CYP73A5的大部分特征性底物结合位点基序( SRSs) 和酶活性位点残基,但与拟南芥等植物C4H相比其编码区3′发生了C₂₂₄₂单碱基缺失和相应移码, 导致其蛋白C - 末端缺失/变异了36个残基, SRS6基序和其上的活性位点残基也相应缺失, 因此BoC4H可能无功能或活性低。BoC4H为膜蛋白, 可能定位于内质网, 其二级结构主要为α - 螺旋和随机卷曲。Swiss-Model不能预测出其三级结构。甘蓝中存在一个至少由5个成员构成的C4H基因家族。

Cloning and Sequence Analysis of a Mutation-type Cinnamate-4-hydroxylase Gene from Brassica oleracea L. var. acephala DC.

A 2 431-bp full-length cinnamate-4-hydroxylase gene, BoC4H, was cloned from Brassica oleracea L. var. acephala DC. It contains 2 introns. ItsmRNA is 1 715 bp, encoding a deduced 481-amino-acidpolypeptide with wide homologies to C4Hs from other plants. It possesses cytochrome P450 conserved domainsand motifs such as the haem-iron binding motif, the E - R - R triad, the T-containing binding pocket motifand the hinge motif necessary for optimal orientation of the enzyme. It also has most of the canonical C4H /CYP73A5-featured substrate-recognition sites ( SRSs) and active site residues. But owing to a single2base deletion at C₂₂₄₂ and subsequent frame shiftwithin the 3′coding region as compared with C4H genes from Arabidopsis thaliana and other p lants, BoC4H shows a 36-aa deletion /variation at its C-terminus, and the SRS6 motif together with active site residues therein are absent. Thus BoC4H may be of no function or low activity.BoC4H is a membrane p rotein and is probably associated with endop lasmic reticulum. Its secondary structureis dominated by alpha helices and random coils. Swiss-Model could not p redict its tertiary structure. B. oleracea contains a C4H gene family with at least 5 members.