Cloning and expression analysis of chalcone synthase gene from Koelreuteria paniculata

  Objective  Chalcone synthase (CHS) is one of the rate-limiting enzymes of phenylpropanoid pathway which plays superior roles in the production of secondary metabolites. In this study, by cloning and bioinformatics analysis of CHS gene and analyzing the relationship between CHS gene expression and flavonoid synthesis of Koelreuteria paniculata, we hope to provide reference for further study of flavonoid biosynthesis pathway related genes, evolution of CHS gene family and coloration mechanism of Koelreuteria paniculata ‘Jinye’.

  Method  CHS genes were isolated and characterized by RT-PCR from Koelreuteria paniculata. And the expression patterns of CHS gene in different tissues of Koelreuteria paniculata and in the leaves of Koelreuteria paniculata and Koelreuteria paniculata ‘Jinye’ in May, July and September were analyzed by qRT-PCR; the differential flavonoid metabolism between Koelreuteria paniculata and Koelreuteria paniculata ‘Jinye’ was screeidues ether.

  Result  Two full-length DNA of CHS genes were cloned named KpCHS1 and KpCHS2 . The KpCHS1 gene sequence was found to be 2 492 bp and comprised an open reading frame of 1 173 bp, encoding for 390 amino acid residues, the KpCHS2 gene sequence was found to be 1 321 bp and comprised an open reading frame of 1 182 bp, encoding for 393 amino acid residues ether. Alignment of the predicted amino acid sequence of KpCHS2 had been shown to have high sequence similarity with KpCHS1, with four CHS specific conserved motifs and one chalcone synthase active site. Furthermore, KpCHS1 and KpCHS2 were generally expressed in roots, stems, leaves and seeds of Koelreuteria paniculata. Among them, the expression of KpCHS2 was the highest in seeds, while that of KpCHS1 was higher in leaves. In roots and stems, the expression levels of the two genes were similar and lower. The expression pattern analysis showed that in Koelreuteria paniculata and Koelreuteria paniculata ‘ Jinye’, the expression of KpCHS1 decreased with the increase of months, while the expression of KpCHS2 did not show obvious regularity. In the July plant samples, the expression of KpCHS1 gene in Koelreuteria paniculata ‘Jinye’ was higher than that in Koelreuteria paniculata. Besides, we analyzed the metabonomics of Koelreuteria paniculata and Koelreuteria paniculata ‘Jinye’ leaves in July, and screened out the different flavonoids. It was found that kaempferol-7-o-glucoside, 7-hydroxycoumarin, quercetin-3β-D-glucoside, and kaempferol, naringin, which were important intermediate products in flavonoid biosynthesis, were significantly increased in Koelreuteria paniculata ‘Jinye’ leaves.

  Conclusion  KpCHS1 and KpCHS2 belong to the chalcone synthase family of Koelreuteria paniculata and are highly homologous, but they are distributed in far branches of the phylogenetic tree. It is speculated that the two proteins may have great differences in the catalytic function of amino acid activity. KpCHS1 and KpCHS2 are expressed in roots, stems, leaves and seeds, and higher in leaves and seeds of Koelreuteria paniculata. Our results indicate that the expression of KpCHS1 gene is highly related to the synthesis of flavonoids in Koelreuteria paniculata.