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

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 (GA(3)), 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.

Characterization of Developmental- and Stress-Mediated Expression of Cinnamoyl-CoA Reductase in Kenaf (Hibiscus cannabinusL.)

“…Particularly in 48 h ZmCCR2 showed 10fold upregulation compared to control. MeJA treatment showed high induction of IiCCR gene from Isatis indigotica , especially at 4 and 8 h after treatment [13]. ABA treated plant showed downregulation of IiCCR transcript, which is similar to our cold treated sample.…”

“…Among them CCR2 and CCR6 showed highest expression in leaf tissues. Some CCR genes from Isatis, Ginkgo, and Norway spruce highly expressed not only in lignified tissues like stems, but also in other tissues [13, 30, 31]. Those results suggest that different CCR homologs might be involved in different cellular function.…”

“…Among them Gossypium hirsutum (ACQ59094) is the highest with 90% identity. The alignment result also showed two highly conserved motifs: NAD(P)-binding domain at N-terminal (VTGAGGFIASWMVKLLLEKGY) and probable catalytic domain (NWYCYGK) [12, 13]. Consensus sequence for NAD(P) binding domain is VTGA(G/A)G(F/Y)(I/L)ASW(I/L/M)VK(L/I)LL(E/D)(K/R)GY.…”

“…However, substrate specificity varies between different CCR enzymes from different species, even in between isoenzymes from the same species [11]. CCR orthologs were identified from various plants [12, 13]. Plants with downregulated CCR and CCR -like genes caused various phenotypic and developmental abnormalities: dwarfism, reduced number of seeds, small stem diameter, shorter floral stems, dark green leaves, growth delay, and collapsed xylem vessels [11–14].…”

Developmental- and stress-mediated expression analysis of cinnamoyl-CoA reductase 1 (CCR1) fromHibiscus cannabinus

“…The presence of slightly different sequences and structural features may lead to differential function, which might be due to the different mode of action and biochemical properties. The motif (TVCVTGAGGF IASWMVKLLLEKGYIVKGTV) has a sheet-helix-sheet structure (Figure 1a), common to the βαβ-dinucleotide binding fold of NAD (H) and NADP(H)-dependent reductases and dehydrogenases (Larsen 2004;Hu et al 2011). HcCCR1 and HcCCR2 have identical NADPHbinding motifs and catalytic domains, but showed differential expression patterns.…”