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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2014, Vol. 34 Issue (1): 50-56    DOI: 10.13523/j.cb.20140108
技术与方法     
柠条锦鸡儿肉桂酰辅酶A还原酶基因克隆和分析
李高1,2, 杨杞1, 张烨1,3, 王颖1, 张涛1, 李国婧1, 王瑞刚1
1. 内蒙古农业大学生命科学学院 呼和浩特 010018;
2. 中国林业科学研究院林业研究所 北京 100091;
3. 鄂尔多斯市食品药品检验研究中心 鄂尔多斯 017000
Cloning and Analysis of the Gene Encoding Cinnamoyl-CoA Reductase from Caragana korshinkii Kom
LI Gao1,2, YANG Qi1, ZHANG Ye1,3, WANG Yin1, ZHANG Tao1, WANG Rui-gang1, LI Guo-jing1
1. College of Life Sciences, Inner Mongolia Agricultural University, Hohhot 010018, China;
2. Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China;
3. The Ordos Food and Drug Inspection and Research Center, Ordos 017000, China
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摘要: 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。
关键词: 柠条锦鸡儿CCR RACE序列分析    
Abstract: 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 cDNA ends technique, designated as CkCCR (GenBank accession no. HQ829859). Full-length cDNA is 1 270 bp in length with a 1 014bp open reading frame. The protein deduced from cDNA 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.
Key words: Caragana korshinskii Kom.    CCR RACE    Sequence analysis
收稿日期: 2013-01-22 出版日期: 2014-01-25
ZTFLH:  Q78  
基金资助: 教育部新世纪优秀人才支持计划(ECNT-11-1020);教育部博士学科点基金博导类联合资助项目(20111515110001);国家自然科学基金(31060105);内蒙古自然科学基金重点(2010Zd13)资助项目
通讯作者: 王瑞刚,E-mail:wungruigang@imau.edu.cn     E-mail: wungruigang@imau.edu.cn
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引用本文:

李高, 杨杞, 张烨, 王颖, 张涛, 李国婧, 王瑞刚. 柠条锦鸡儿肉桂酰辅酶A还原酶基因克隆和分析[J]. 中国生物工程杂志, 2014, 34(1): 50-56.

LI Gao, YANG Qi, ZHANG Ye, WANG Yin, ZHANG Tao, WANG Rui-gang, LI Guo-jing. Cloning and Analysis of the Gene Encoding Cinnamoyl-CoA Reductase from Caragana korshinkii Kom. China Biotechnology, 2014, 34(1): 50-56.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140108        https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I1/50

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