Molecular cloning and characterization of the cinnamate 4-hydroxylase gene from Eupatorium adenophorum
Molecular cloning and characterization of the cinnamate 4-hydroxylase gene from Eupatorium adenophorum
复制标题
紫茎泽兰肉桂酸4-羟化酶基因的分子克隆和表征
DOI：
10.1111/wbm.12044
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发表时间：
2014
影响因子：
1.4
通讯作者：
Cheng Hongmei
中科院分区：
文献类型：
--
作者：
Chen Lijuan;Guo Huiming;Lin Yi;Wu Yulun;Cheng Hongmei
肉桂酸-4-羟化酶 (CA4H) 是一种细胞色素 P450 依赖性单加氧酶，在苯丙素代谢和植物防御中发挥着至关重要的作用。此前，作者表明 CA4H 的表达是响应紫茎泽兰中的化感物质而被诱导的。在此，通过使用 cDNA 末端的快速扩增克隆了 EaCA4H 的全长 cDNA。推测EaCA4H的1518bp开放阅读框编码505个氨基酸残基的蛋白质。与其他 CA4H 蛋白一样，预测的 EaCA4H 多肽包含细胞色素 P450 的保守结构域。 Southern印迹分析表明大肠杆菌基因组中至少存在5个EaCA4H拷贝。 紫茎泽兰。亚细胞定位揭示了洋葱表皮细胞中核定位的 EaCA4H-绿色荧光蛋白融合蛋白。通过保守的EaCA4H片段对烟草内源CA4Hin进行异源沉默，导致关键酶基因的表达减少以及苯丙素途径中下游类黄酮的产生。有趣的是，在过表达 EaCA4H 的转基因烟草植物中观察到了类似的效果。总而言之，结果表明植物中CA4H的表达受到干扰导致关键酶基因的表达水平相对较低以及参与苯丙素代谢的黄酮类化合物的积累。
Cinnamate‐4‐hydroxylase (CA4H), a cytochrome P450‐dependent monooxygenase, plays crucial roles in phenylpropanoid metabolism and plant defense. Previously, the authors showed that the expression ofCA4Hwas induced in response to an allelopathic substance inEupatorium adenophorum. Here, the full‐length cDNA ofEaCA4Hwas cloned by using rapid amplification of cDNA ends. The 1518 bp open reading frame ofEaCA4Hwas deduced to encode a protein of 505 amino acid residues. Like other CA4H proteins, the predicted EaCA4H polypeptides contained conserved domains of cytochrome P450. A Southern blot analysis indicated that at least five copies ofEaCA4Hexists in the genome ofE. adenophorum. Subcellular localization revealed nuclear‐localized EaCA4H–green fluorescent protein fusion protein in onion epidermal cells. Heterologous silencing of endogenousCA4Hin tobacco by a conservedEaCA4Hfragment resulted in reduced expressions of key enzymatic genes and the production of downstream flavonoids in the phenylpropanoid pathway. Intriguingly, similar effects were observed in transgenic tobacco plants overexpressingEaCA4H. Altogether, the results indicate that the disturbed expression ofCA4Hin plants leads to relatively low expression levels of key enzymatic genes and the accumulation of the flavonoids that are involved in phenylpropanoid metabolism.