Isolation And Expression Of Floral Color Genes In Cattleya And Their Anthocyanins Composition

Cattleya Alliance, is a epiphyte of perennial herb, which is rare, exotic, and exprensive in orchids, growing wild in central and South America. Although Cattleya have been cultivated over 200 years, there is little report about color chemical theory and molecular aspect of flavonoid biosynthesis. In this study, two key genes of full length cDNA of anthocyanin biosynthesis in Cattleya hybrid ‘Pink Lady’ was fistly isolated, and we analysed their sequence information. Later on, we studied different stages expression patterns in ‘Pink Lady’ and different petals’ tissues in the fully opened stages of ‘Pink Lady’and ‘Suzie’, aiming to provied theoretical principle for patterns of molecular biology and gene engineering in Cattleya. Meanwhile, the different floral organ in blooming stage of ‘Pink Lady’ were used to investigate the flavonoid profile by HPLC-DAD and HPLC-ESI-MSn, aiming to provide a theoretical basis for Cattleya’s color chemistry. The main conclusions were as follow:1. The full-length cDNA sequences of chalcone synthase(CHS) and dihydroflavonol 4-reductase(DFR) genes were cloned from Cattleya hybrida ‘Pink lady’by using RT-PCR and RACE, named ChCHS1 and ChDFR1. Their GenBank accession was No. KP171693 and No. KP171694. The bioinformatics analysis indicated that ChCHS1 is 1508 bp in full length and encoding a polypeptide of 394 amino acids while ChDFR1 is 1250 bp in full length and encoding a 350 predicted amino acids residues. The sequence and homology of GenBank revealed that ChCHS1 and ChDFR1 had their conserved active sites and the family signture. Sequence and phylogenetic analysis indicated a high degree homeotic between Cattleya and Cymbidium or Dendrobium, and in different family ChCHS1 and ChDFR1 shared more than 70% homology with Liliaceae.2. The expressions of ChCHS1 and ChDFR1 were detected in five different stages of purple flowers’ petal by using relateive real-time PCR. The five stages was when the bud size was 1.5 cm, 2.5 cm, 4 cm, 5cm and open, respectively. Relative real-time PCR analysis showed that the highest expression of ChCHS1 was when flower was in bloom. When the flower is nearly open, ChDFR1 had a highest abundant transcript. In bud stage, ChCHS1 and ChDFR1 had the low level expression.3. The expressions of these two genes were detected in different floral organ(fully opened stage) of ‘Pink Lady’ and ‘Suzie’. Relative real-time PCR analysis showed that these two genes in two kind of cultuvated species presented similar trend. The purple foral organ in ‘Pink Lady’(petal, sepal and lip) had a obvious higher expression than the white foral organ in ‘Suzie’, expecially in petal. In ‘Pink Lady’, these two genes declined in petal, sepal, throat and lip while the expression of ChCHS1 and ChDFR1 in throat of ‘Suzie’ was the highest than other floral organ.4. Eighteen flavonoids were identified by HPLC-DAD and HPLC-ESI-MSn in ‘Pink lady’of petal. They were Isorhamnetin 3-O-sambubiose(Is3Sam), Kaempferol 3-succinylglucoside(Km3SuG), Kaempferol 3-O-galloylsambubiose(Km3GloSam), Kaempferol 3-O-rutinoside-7- O-rhamnoside(Km3Ru7R), Isorhamnetin 3-O-succinylrutinoside(Is3SuRu), Kaempferol 3-O-glucoside(Km3G), Quercetin 3-O-rutinoside(Qu3Ru), Kaempferol 3-O- rutinoside(Km3Ru), Isorhamnetin 3-O- rutinoside(Is3Ru), Cyanidin 3-O- disaccharide(Cy3Dis), Cyanidin 3-succinylglucoside(Cy3SuG), Cyanidin galloylsambubiose(CyGlo Sam), Cyanidin 3-O-rutinoside-7- O-glucoside(Cy3Ru7G), Malvidin Peonidin 3-O- rutinoside-7-O- glucoside(Pn3Ru7G), Cyanidin 3-O-glucoside(Cy3G), Peonidin 3-O-rutinoside(Pn3-Ru), Cyanidin 3-O-rutinoside(Cy3Ru) and Petunidin 3-O-rutinoside(Pt3Ru).