Cloning and Expression of 4CLs in Impatiens uliginosa

  Objective  Genes of the enzyme in the phenylpropane metabolic pathway associated with anthocyanin synthesis of Impatiens uliginosa were studied for breeding purposes.

  Method  RT-PCR was employed to isolate and clone Iu4CL1,Iu4CL2, Iu4CL3, and Iu4CL4 for bioinformatic analysis. Expressions of the genes in the flowers of different colors (i.e., white, pink, red, and deep red) at the budding (S1), initial flowering (S2), blooming (S3), and withering (S4) stages of I. uliginosa were determined using qRT-PCR.

  Result  Iu4CL1, Iu4CL2, Iu4CL3, and Iu4CL4 had the cDNA lengths of 1620 bp, 1653 bp, 1698 bp, and 1638 bp encoding 539, 550, 565, and 545 amino acids, respectively. Iu4CL1 contained 2 introns and Iu4CL2, 4, but Iu4CL3 and Iu4CL4 had none. All 4 genes were signal peptide-free hydrophobic proteins, but only Iu4CL3 was unsterilized. Iu4CL2 had 3 transmembrane structures, but the other 3 had none. All of them belonged to the AMP-binding enzyme superfamily and the adenylate-forming domain class I superfamily with the highest homology with I. uliginosa. Iu4CL1 and Iu4CL2 were in one large branch, while Iu4CL3 and Iu4CL4 in another, suggesting a paraphyletic homology. The expressions of Iu4CL1 and Iu4CL3 peaked in S3 on the white flowers, Iu4CL2 on red flowers, and Iu4CL4 on dark red I. uliginosa.

  Conclusion   It was evident that Iu4CLs played a crucial role in the anthocyanin biosynthesis of I. uliginosa.