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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (2): 18-29    DOI: 10.13523/j.cb.20180204
研究报告     
中间锦鸡儿CCR2和CCR3基因的克隆和功能鉴定 *
刘坤,崔爽,杨飞芸,韩晓东,王瑞刚,张子义()
内蒙古农业大学生命科学学院 呼和浩特 010018
Clone and Functional Identification of Cinnamoyl CoA Reductase Genes from Caragana intermedia
Kun LIU,Shuang CUI,Fei-yun YANG,Xiao-dong HAN,Rui-gang WANG,Zi-yi ZHANG()
College of Life Sciences, Inner Mongolia Agricultural University, Hohhot 010018, China
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摘要：

肉桂酰辅酶A还原酶(cinnamoyl-CoA reductase, CCR)是催化木质素合成特异途径的第一个限速酶,对木质素的合成起关键作用。从中间锦鸡儿中克隆了两个CCR基因,CiCCR2和CiCCR3,其中CiCCR2基因开放阅读框为897bp,编码299个氨基酸,CiCCR3基因开放阅读框为966bp,编码322个氨基酸。过表达CiCCR2和CiCCR3转基因拟南芥株系幼苗期和成熟期木质素含量均高于野生型,组织化学染色也表明转基因株系木质素积累较野生型拟南芥多,且转基因株系鲜重和干重显著高于野生型。
关键词： 肉桂酰辅酶A还原酶;  克隆;  木质素;  中间锦鸡儿    
Abstract:

Cinnamoyl-CoA reductaseis the first rate limiting emzyme which catalyzes the biosynthesis of lignin and plays an important role in lignin biosynthesis. Two cinnamoyl-CoA reductase encoding genes, CiCCR2 and CiCCR3, were cloned from Caragana intermedia through PCR. The open reading frame(ORF) of CiCCR2 is 897bp which encoding 299 amino acids, and ORF of CiCCR3 is 966bp, which encoding 322 amino acids. Quantification assay and Histochemical staining showed that the lignin content increased by overexpressing of CiCCR2 and CiCCR3 in Arabidopsis compared with the wild type during both seedling and mature stages. At the same time, the fresh and dry weight of overexpressing plants were higher than that of the wild type.
Key words: Cinnamoyl-CoA reductase    Clone    Lignin    Caragana intermedia
收稿日期: 2017-09-19 出版日期: 2018-03-21
ZTFLH:  Q819  
基金资助: * 国家自然科学基金(31360169);内蒙古自治区科技创新团队资助项目(3201503004)
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刘坤
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杨飞芸
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王瑞刚
张子义

引用本文:

刘坤,崔爽,杨飞芸,韩晓东,王瑞刚,张子义. 中间锦鸡儿CCR2和CCR3基因的克隆和功能鉴定 *[J]. 中国生物工程杂志, 2018, 38(2): 18-29.

Kun LIU,Shuang CUI,Fei-yun YANG,Xiao-dong HAN,Rui-gang WANG,Zi-yi ZHANG. Clone and Functional Identification of Cinnamoyl CoA Reductase Genes from Caragana intermedia. China Biotechnology, 2018, 38(2): 18-29.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180204    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I2/18

引物名称 Primer name 引物序列Primer sequence (5'→3')
F-CiCCR2-oe GTCGAC(Sal I)ATGGCACCTTCTTTCGACAT
R-CiCCR2-oe GAGCTC(Sac I)GAACAGCTAAGGTTATTGCCC
F-CiCCR3-oe GTCGAC(Sal I)ATGGCAACAAGTGGGGAAGG
R-CiCCR3-oe ACTAGT(Spe I)AACATTGGCAAGTCCAGTCTGTG
F-CiCCR2-q CTGTCGCAAATTCAAGTTATGGC
R-CiCCR2-q CAGCAGCTCCTCTCAAGTAAGGGT
F-CiCCR3-q AAACACAGGCCACTGGAGGAA
R-CiCCR3-q TGACATTTATTTTGAATCGAAGAGACC
qCiEF1αF TGGGTGGGACATTCTCTGATT
qCiEF1αR GCACGGTTCACTTCTTCTTAGC
qAtEF1αF AGAAGGGTGCCAAATGATGAG
qAtEF1αR GGAGGGAGAGAGAAAGTCACAGA
表1  本实验所用引物
图1  CiCCR2和CiCCR3基因cDNA扩增结果
图2  CiCCR2和CiCCR3与其它物种CCR蛋白多重序列比对
图3  CiCCR2和CiCCR3与其他物种CCR的系统进化分析
图4  qRT-PCR检测中间锦鸡儿CiCCR2和CiCCR3不同组织部位表达水平
图5  CiCCR2和CiCCR3过表达载体双酶切鉴定
  
图6  实时荧光定量PCR检测过表达株系CiCCR2和CiCCR3的表达水平
图7  CiCCR2 和CiCCR3转基因株系和野生型拟南芥重量
图8  木质素含量标准曲线
图9  CiCCR2 和CiCCR3转基因株系和野生型拟南芥木质素含量
图10  CiCCR2转基因株系和野生型拟南芥组织化学染色
图11  CiCCR3转基因株系和野生型拟南芥组织化学染色
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