Molecular Cloning And Characterization Of Five Key Genes Involved In Lignan Biosynthesis Pathway From Isatis Indigotica Fort.

Isatis indigotica Fort. has been used in traditional Chinese medicine since ancient times. Its leaves and roots, known as Daqingye and Banlangen, respectively, are frequently used as anti-leukemia, antipyretic, anti-inflammatory and anti-influenza agents. By investigation on its chemical constituents and antivirus activity study, we found that the lignans inâ… . indigotica, including Lariciresinol and Larch Lignan glycosides, showed significant antivirus activity. However, their content inâ… . indigotica is very low. Until now, there is few research report on the lignan biosynthesis inâ… . indigotica, and many important enzymes involved in the pathway have not been isolated.In the present study, five genes involved in the lignan biosynthesis pathway inâ… . indigotica have been firstly cloned, using the method of rapid amplification of cDNA ends (RACE). Characterization, structure prediction and bioinformatics analysis of these five novel genes, as well as expression of Iiccr in E.coli were performed and reported as follows:1. Iic4h gene:Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) is the first P450 upstream in the phenylpropanoid pathway, which catalyses the hydroxylation of cinnamic acid to coumaric acid. The full-length cDNA of Iic4h was 1368 bp (GenBank accession no. GQ872418) long with an open reading frame (ORF) of 1026 bp encoding a polypeptide of 341 amino acid residues. The comparison of Ii4h genomic DNA sequences and Iic4h cDNA sequence revealed that the Iic4h genomic DNA contained two introns. Southern-blot analysis indicated that Iic4h is a multiple copy gene. Iic4h expression could be detected in all tissues at different expression levels, with the strongest expression in roots. Further expression analysis revealed that the signaling components of defense/stress pathways, such as ultraviolet-B radiation (UV-B), methyl jasmonate (MeJA), abscisic acid (ABA)and Gibberellins (GA3) could up-regulate the Iic4h transcript levels compared with the control.2. Iiccr gene:Cinnamoyl coenzyme A reductase (CCR, EC 1.2.1.44), one of the key enzymes in the biosynthesis of lignins monomers, catalyzes the NADPH-dependent reduction of cinnamoyl-CoA esters to their corresponding cinnamaldehydes. The full-length cDNA of Iiccr was 1674 bp (GenBank accession no. GU014562) long with an ORF of 1233 bp encoding a polypeptide of 411 amino acid residues. The analyses of genomic DNA sequences revealed that Iiccr cDNA sequence and the genomic sequence were identical, which indicated that the Iiccr gene did not contain introns. Southern-blot analysis indicated that Iiccr is a multiple copy gene.IiCCR expression could be detected in all tissues at different expression levels, with the strongest expression in roots. Further expression analysis revealed that UV-B, MeJA and GA3 could up-regulate the Iiccr transcript levels compared with the control, while ABA down-regulate the Iiccr transcript levels. The expression construct of Iiccr with pET-32a(+) was transformed into E.coli BL21(DE3), and the recombinant IiCCR protein was detected by SDS-PAGE analysis.3.Iiugt gene:UDP-glucosyltransferase (UGT, EC 2.4.1.91) catalyzes the addition of the glycosyl group from a UTP-sugar to a small hydrophobic molecule. The full-length cDNA of liugt was 1576 bp (GenBank accession no. GU434222) long with an ORF of 1431 bp encoding a polypeptide of 476 amino acid residues. The analyses of genomic DNA sequences revealed that Iiugt cDNA sequence and the genomic sequence were identical, which indicated that the liugt gene did not contain introns.4.1i4cl gene:4-coumarate:CoA ligase (4CL, EC 6.2.1.12) is the last enzyme of the general phenylpropanoid pathway, which catalyzes the conversion of 4-coumarate and other 4-hydroxycinnamates to their corresponding CoA thiol esters. The full-length cDNA of Ii4cl was 1967 bp (GenBank accession no. GU937875) long with an ORF of 1632 bp encoding a polypeptide of 543 amino acid residues. The comparison of Ii4cl genomic DNA sequences and Ii4cl cDNA sequence revealed that the Ii4cl genomic DNA contained five introns.5.Iicad gene:Cinnamyl alcohol dehydrogenase (CAD, EC1.1.195) catalyzes the synthesis of cinnamyl alcohols from corresponding cinnamaldehydes. The full-length cDNA of Iicad was 1402 bp (GenBank accession no. GU937874) long with an ORF of 1083 bp encoding a polypeptide of 360 amino acid residues. The comparison of Iicad genomic DNA sequences and Iicad cDNA sequence revealed that the Iicad genomic DNA contained three introns.Five key genes involved in the lignan biosynthesis pathway were isolated from medicinal plant 1. indigotica, and their stuctural and bioinformatic analyses were carried out, which will help us to further illuminate this pathway. The research also provides a possibility to elevate the content of lignans inâ… . indigotica by plant metabolic engineering.