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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2018, Vol. 38 Issue (2): 18-29    DOI: 10.13523/j.cb.20180204
Orginal Article     
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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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     
Received: 19 September 2017      Published: 21 March 2018
ZTFLH:  Q819  
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Kun LIU
Shuang CUI
Fei-yun YANG
Xiao-dong HAN
Rui-gang WANG
Zi-yi ZHANG
Cite this article:

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.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180204     OR     https://manu60.magtech.com.cn/biotech/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
Table 1 Primers used in this study
Fig.1 Amplification results of cDNA of CiCCR2 and CiCCR3 (a)CiCCR2 cDNA (b)CiCCR3 Cdna M: Fermentas GeneRuler TM 1kb DNA Ladder
Fig.2 Multiple alignment of CiCCR2 and CiCCR3 along with other species CCR proteinsThe box indicates the conserved domain; the underline indicates the catalytic site of CCR
Fig.3 The phylogenetic analysis of CiCCR2 and CiCCR3 with other species CCRsThe phylogenetic tree is constructed by the neighbor-joining method. Bootstrap values based on 1 000 replications
Fig.4 The expression level of CiCCR2 and CiCCR3 in Caragana intermedia assayed by real-time quantitative PCR(a)CiCCR2 (b)CiCCR3 Reference gene was CiEF1α. The result was calculated by 2-ΔCT
Fig.5 Identification by double digestion of CiCCR2 and CiCCR3 over-expression binary vector(a) Identification by double digestion of CiCCR2 M:TaKaRa DL5000 Marker; 1: Vector control; 2: Digest with Sal I and Sac I (b)Identification by double digestion of CiCCR3 M:TaKaRa DL5000 Marker; 3: Vector control; 4: Digest with Sal I and Spe I
 
Fig.6 The expression level of CiCCR2 and CiCCR3 in transgenic lines assayed by real-time quantitative PCR(a)CiCCR2 (b)CiCCR3 Reference gene was AtEF1α. The result was calculated by 2-ΔCT
Fig.7 Weight of the CiCCR2 and CiCCR3 transgenic lines and wild-type Arabidopsis thaliana(a)and(c)for fresh weight;(b)and(d)for dry weight
Fig.8 Standard curve of lignin content
Fig.9 Content of lignin in CiCCR2 and CiCCR3 transgenic lines and wild-type A.thaliana(a)and(c)for young seedlings; (b)and(d)for mature seedlings
Fig.10 Histochemical staining of the CiCCR2 transgenic lines and wild-type Arabidopsis thalianaXY: Xylem; PH: Phloem
Fig.11 Histochemical staining of the CiCCR3 transgenic lines and wild-type Arabidopsis thaliana XY: Xylem; PH: Phloem
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