柠条锦鸡儿肉桂酰辅酶A还原酶基因克隆和分析

Cloning and Analysis of the Gene Encoding Cinnamoyl-CoA Reductase from Caragana korshinkii Kom

肉桂酰辅酶A还原酶(cinnamoyl-CoA reductase,CCR)是木质素特异合成途径中的关键酶。根据已报道的植物CCR基因序列设计简并引物,利用RACE技术,首次从柠条锦鸡儿(Caragana korshinkii Kom.)中克隆得到CCR基因全长cDNA序列,命名为CkCCR,GenBank登录号为HQ829859。该序列长1 270bp,具备长度为1 014bp的完整开放阅读框(ORF)。推导该基因编码的蛋白质有434个氨基酸,预测等电点6.69,分子量36.76kDa。序列分析发现,柠条锦鸡儿CCR基因推导的氨基酸序列与其它植物来源的CCR序列高度相似,并且具有植物CCR共有的氨基酸序列"KNWYCYGKA"以及NADPH的结合序列。系统进化分析显示,柠条锦鸡儿CCR与拟南芥CCR2处于同一分支,与同属豆科的银合欢CCR亲缘关系最近。利用荧光定量PCR技术对柠条锦鸡儿一个月龄幼苗基因转录水平进行检测,结果显示CkCCR基因在根、茎、叶中广泛表达,并且干旱处理初期表达量有所下降,处理后期又恢复到未处理时的表达水平。

Cinnamoyl-CoA reductase （ CCR, EC 1.2.1.44） plays an important role in the phenylpropanoid pathway. A gene coding for CCR was isolated from Caragana korshinkii Kom. by rapid amplification of eDNA ends technique, designated as CkCCR （GenBank accession no. HQ829859）. Full-length eDNA is 1 270 bp in length with a 1 014bp open reading frame. The protein deduced from eDNA is 434 amino acids with a calculated molecular weight of 48kDa and an isoelectric point of 6.42. Sequence alignments revealed that CkCCR had close similarity with CCRs from other species, it contains NADPH binding sequence and a conserved region ＂KNWYCYGKA＂. Phylogenetic analysis indicated that the CkCCR stayed at the same evolutionary branch with the AtCCR2 and shared the closest homology with the CCR from Leucaena glauca. The results of real- time- quantitative analysis showed that the transcripts of CkCCR expressed constitutively in root, stem and leaves. In addition, the expression levels of CkCCR decreased after drought treatment at the beginning, then returned to the expression level of the control at late time points under drought condition.