Cloning and Partial Nucleotide Sequence Analysis of PPO and CYP Genes in Celosia argentea Callus

Abstract

Betalains are phenolic compounds found in various plant species, possessing significant biological activities such as antioxidant, anti-inflammatory, antimicrobial, and anticancer properties. These compounds are typically present in plants of the Amaranthaceae family, including Celosia argentea var. plumosa, an ornamental species with the ability to produce betalains in both the betaxanthin group (yellow to orange) and the betacyanin group (red to purple). The biosynthetic pathway of betalains in plants involves several key genes, especially polyphenol oxidase (PPO) and cytochrome P450 76AD1 (CYP76AD1; CYP), which function in the early stages of the pathway. This study aimed to clone and analyze partial nucleotide sequences of the PPO and CYP genes in C. argentea var. plumosa. Degenerate primers were designed based on nucleotide sequences from related species within or closely related to the Amaranthaceae family. For PPO, the primers were designed using sequences from Amaranthus tricolor and Beta vulgaris, while for CYP, sequences from B. vulgaris were used. Total RNA was extracted from red callus of C. argentea, and RT-PCR was performed to amplify cDNA of the target genes. PCR products were confirmed by agarose gel electrophoresis, ligated into the pGEM®-T Easy vector, and transformed into Escherichia coli strain JM109 for cloning and preliminary sequencing analysis. The cloned PPO cDNA fragment was 471 nucleotides in length and showed 74.82% similarity to the PPO gene of A. tricolor. The CYP cDNA fragment was 270 nucleotides long and shared 98.90% similarity with Celosia cristata, as well as 87.41%, 86.67%, and 84.44% similarity to Amaranthus cruentus, A. tricolor, and Chenopodium quinoa, respectively. These findings provide foundational information for the design of gene-specific primers to study gene expression related to betalain biosynthesis in plants.