Molecular Evolution And Differential Expression Regulation Of Chalcone Synthase Genes From Phalaenopsis Orchid

Phalaenopsis, native to tropical Asia, are among the most rewarding orchids to grow. Flavonoids are the major flower pigments in plants and play important roles in many other biological functions, including serving for UV protection, plant-microbe interaction and male fertility. Chalcone synthase (CHS, EC2.3.1.74) is the key enzyme in flavonoid biosynthesis pathway, which catalyses the condensation of three acetate residues from malonyl-CoA with 4-coumaroyl-CoA to form naringenin chalcone.In this study, three novel genes encoding Phalaenopsis chalcone synthase were isolated by a series of PCR amplifications. They were designated as phchs3, phchs4 and phchs5, respectively. Firstly, the cDNA fragment of phchs3 and phchs5 as well as DNA fragment of phchs4 were isolated by RT-PCR or routine PCR. To obtain full-length gene, inverse PCR were performed using primers that designed according to the sequences of known regions of individual genes.Phchs3 (GeneBank accession no. AY954515) contained an open reading frame of 1173 bp disrupted by one 103bp intron, which was located at the conserved site, i.e., between the first and the second nucleotide of the conserved cystein codon. CHS protein predicted from phchs3 showed 61-65% identities with CHS of other plant species and retained most of the conserved residues. 684bp 5' un-translated region were obtained. And some putative cis-elements characteristic of CHS gene, including G-box, W-box, P-box and TACPyAT motif, were found at the promoter region of phchs3.Phchs4 (GeneBank accession no.AY825502) also contained an intact open reading frame of 1173 bp with one 109 bp intron at the conserved site. The deduced polypeptide (PHCHS4) from phchs4 comprised 390 amino acids, which showed 61～65% identities with CHS from other plants and retained most of the conserved residues. Some putative cis-regulatory elements responsible for flexible regulation of plant CHS genes were present at the 5' and 3' flanking region of phchs4.Phchs5 (GeneBank accession no. DQ089652) contained an open reading frame of 1185bp with one 159 bp intron at the conserved site. Phchs5 showed higher identity with CHS genes from other orchid plants and other plant families than with Phalaenopsis CHSs on both nucleotide and amino acid levels.Southern blot analysis predicted at least four chs-like genes indicating thepresence of a small multigene family in Phalaenopsis.The inferred phylogeny of the chs genes of Phalaenopsis with other two orchid plants, Bromoheadia finlaysoniana and Dendrobium hybrid, suggested that orchid CHS genes have diverged into two subfamilies before divergence of these three genera. Phchs5 belonged to the branch, which is slowly evolved; Phchs3 and phchs4 belonged to the other branch, which is evolved in accelerated rate. Amino acid sequence comparison between Orchid CHSs indicated that several amino acids, which are indispensable for CHS enzyme activity, are absent in sequences of fast evolved.Relatively quantitative RT-PCR analysis identified expression patterns of these three CHS genes in different floral tissues at different developmental stages. Phchs5 was the most abundantly expressed chs gene in floral organs and it was specially transcribed in petal and lip at the stages when anthocyanin accumulated (stage 1 to 4). The peak level of phchs5 mRNA was in petal tissue at stage3 to 4 when the anthocyanin was accumulated at the highest rate. Phchs5 is evidently the sole chs gene responsible for the accumulation of pigment in petals. Phchs3 and phchs4 were expressed at much lower levels than phchs5. Phchs3 was expressed in pigmented tepal tissue (including lip, petal and sepal) at early and middle stages (stage 2 to 4) and in colorless reproductive tissue at late stage (stage 5). Like phchs5, phchs4 was another CHS gene, which is specially expressed in petal and lip. It was weakly expressed in petals at earlier stages (stage 1 to 3) and in lips at middle stage (stage 4). These results present new data on differentiation of gene expression among duplicate copies of CHS genes in Phalaenopsis.Expression analysis of phchs5 in different Phalaenopsis cultivars who show different flower color type further suggested that phchs5 was specially expressed in petal and lip tissue and the expression level of it was apparently consistent with anthocyanin accumulation. The expression level of phchs5 in petal of Mau(who has mauve petals) was much higher than those in petal of both Yel(who has yellow petals) and Wht(who has white petals).Expression of exon2 sequence of phchs3 in tobacco can lead to darker flower limbs than controls. These data confirm the identity and function of phchs3 and further suggest that over-expression of Phalaenopsis CHS gene could be used to manipulate flower color of other plants.