Cloning and transcription analysis of flavonoid synthesis gene TrCHI from white clover

The aim of this study was to identify the role of Trifolium repens chalcone flavonone isomerase (TrCHI) in white clover flower color formation. Transcription analysis and CHI enzyme activity determination were performed for TrCHI obtained from a white clover wild type and safflower mutant by homologous cloning. The open reading frame of TrCHI in white clover was 660 bp, encoding 219 amino acids, and 669 bp, encoding 222 amino acids, in the safflower mutant. Among the seven amino acid mutations, glutamic acid at position 13 was mutated to aspartic acid, glutamic acid at position 75 was mutated to glutamine, serine at position 154 was mutated to threonine, lysine at position 218 was mutated to arginine, and glycine, methionine, and lysine were added at the end. We found that the evolution of CHI was species-conserved; TrCHI was type II. In the petals, roots, and petioles, the transcription level of the safflower mutant TrCHI was significantly higher than that of the wild type. However, in the stems, leaves, and pedicels, the transcription level of the safflower mutant TrCHI was lower than that of the wild type. CHI enzyme activity in the petals of the mutant was significantly higher than that in the wild-type. We speculate that the safflower phenotype of the white clover mutant is related to the TrCHI gene mutation and the regulation of flavonoid synthesis in clover. This study provides valuable information for further studies on the potential function of TrCHI in white clover.