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Isolation and characterization of a gene encoding cinnamoyl-CoA reductase from Isatis indigotica Fort.

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Abstract

A novel cinnamoyl-CoA reductase gene, designated as Iiccr (GenBank Accession No. GQ872418) was cloned from Isatis indigotica Fort. The full-length cDNA of Iiccr was 1368 bp with an ORF of 1026 bp that putatively encoded a polypeptide of 341 amino acids, with a predicted molecular mass of 37.50 kDa. The deduced amino acid sequence of IiCCR shared high homology with other known CCRs. No intron was detected in the genomic sequence of Iiccr. Southern-blot analysis revealed that Iiccr was a high-copy gene and real-time quantitative PCR analysis indicated that Iiccr was constitutively expressed in roots, stems and leaves of I. indigotica, with the highest expression level in roots. The results from treatment experiments using different signaling components for plant defense responses including methyl jasmonate (MeJA), gibberellins (GA3), abscisic acid (ABA) and ultraviolet-B revealed that expression of IiCCR had a prominent diversity. The full-length of ORF was sub-cloned into prokaryotic expression vector pET32a(+), which was then transferred into E. coli BL21(DE3). The recombinant protein had high expression level in E. coli BL21(DE3) with IPTG induction. A 2.6 kb long promoter sequence was isolated and its putative regulatory elements and potential specific transcription factor binding sites were analyzed. This study will enable us to further understand the role of IiCCR in the synthesis of phenylpropanoid compounds in I. indigotica Fort. at the molecular level.

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Abbreviations

ORF:

Open reading framed

RACE:

Rapid amplification of cDNA ends

PCR:

Polymerase chain reaction

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

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Acknowledgments

This research was financially supported by National Natural Science Foundation of China (30900786); Modernization of traditional Chinese medicine foundation (08DZ1971502) and Domestic science and technology cooperation projects (10495801400, 10395820200), Shanghai Science and Technology Committee.

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Authors and Affiliations

  1. Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai, 200003, China

    Yongsheng Hu, Peng Di, Junfeng Chen, Ying Xiao & Wansheng Chen

  2. Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai, 200433, China

    Lei Zhang

  3. Department of Pharmacy, The 118th Hospital of PLA, Wenzhou, 325000, China

    Yongsheng Hu

  4. Modern Research Center for Traditional Chinese Medicine, Second Military Medical University, Shanghai, 200433, China

    Peng Di, Junfeng Chen, Ying Xiao & Wansheng Chen

  5. Department of Plant Sciences, University of California, Davis, CA, 95616, USA

    Lei Zhang

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  1. Yongsheng Hu
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Fig. S1

The full-length cDNA sequence and deduced amino acid sequence of Iiccr. The start codon (ATG) is in italics and the stop codon (TGA) is in bold (JPG 398 kb)

Fig. S2

Alignment of the deduced amino acid sequences of IiCCR and other known plant CCRs. The completely identical residues and the conserved residues among the aligned sequences were black and gray shaded, respectively. The common signature CCR catalytic site and the putative NADP binding domain are boxed. IiCCR (I. indigotica, GQ872418); AtCCR (A. thaliana, AAL37194.1); RsCCR (R. sativus, BAC58030.1); TaCCR (Triticum aestivum, ABE01883.1); PtCCR (Pinus taeda, AAL47684.1); OsCCR (Oryza sativa, CAD21520.1) (JPG 367 kb)

Fig. S3

The secondary structure of the deduced IiCCR protein. Alpha helix, extended strand and random coil were represented by the longest, the second longest and the shortest vertical bars respectively (JPG 185 kb)

Fig. S4

The three-dimensional structure of the deduced of IiCCR protein established by homology-based modeling. The a-helix and b-sheet were indicated in red and blue, respectively. Turns and loops were indicated in silver (JPG 149 kb)

Fig. S5

Phylogenetic relationships of CCR proteins from different species. Sequences were identified by the names of species. Eucalyptus (Eucalyptus globulus, AAT74889.1); Corymbia (Corymbia citriodora subsp. variegata, ABQ95557.1); Codonopsis (Codonopsis lanceolata, BAE48787.1); Scutellaria (Scutellaria baicalensis, ACB45437.1); Solanum (Solanum tuberosum, AAN71761.1); Lycopersicon (Lycopersicon esculentum, AAY41880.1); Arabidopsis (A. thaliana, AAL37194.1); Raphanus (R. sativus, BAC58030.1); Isatis (I. indigotica, GQ872418); Triticum (T. aestivum, ABE01883.1); Zea (Zea mays, AAO42630.1); Oryza (O. sativa, CAD21520.1); Mesorhizobium (Mesorhizobium loti MAFF303099, NP_103432.1); Streptomyces (Streptomyces avermitilis MA-4680, NP_821682.1); Aspergillus (Aspergillus flavus NRRL3357, XP_002372662.1); Talaromyces (Talaromyces stipitatus ATCC 10500, XP_002486183.1) (JPG 88 kb)

Fig. S6

Iiccr promoter sequence. A 2.6 kb genomic DNA fragment flanking the 5′-end of the gene contains several putative regulatory elements including a TATA box (sequences are boxed), CAAT box (sequences are underlined) and Gbox (sequences are yellow background). Potential specific transcription factor binding sites were identified by PLACE. MYBST1 sites (sequences are red), MYBPZM sites (sequences are green). The deduced transcription factor binding sites by the computer-based TFSEARCH program are blue background (JPG 192 kb)

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Hu, Y., Di, P., Chen, J. et al. Isolation and characterization of a gene encoding cinnamoyl-CoA reductase from Isatis indigotica Fort.. Mol Biol Rep 38, 2075–2083 (2011). https://doi.org/10.1007/s11033-010-0333-6

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