cDNA cloning and SSCP analysis on Ss5MaT1 gene in Salvia splendens

In the present study, nested polymerase chain reaction-single-strand conformation polymorphism (nested PCR-SSCP) was used for detection of point mutations in flower color genes in Salvia splendens. Totally 916 bp of Ss5MaT1 cDNA segments were obtained in eight varieties of Salvia splendens flowers by RT-PCR. Four sets of specific primers were synthesized according to the obtained cDNA cloning for SSCP analysis. Results from assays of nested PCR-SSCP showed the presence of three kinds of polymorphic DNA bands using primers SS3. Six and four DNA bands were displayed in samples of Rose and Salmon flowers, respectively, compared to only two bands in other six varieties of Salvia splendens flowers. DNA sequencing showed particularly high homology in nucleotide sequences of partial Ss5MaT1 segment in eight varieties of Salvia splendens flowers. However, the point mutant appeared at size of 744 bp in Rose flower with a single base changed in nucleotide sequence from thymine (T) to cytosine (C) and at 678 bp in Salmon changed flower with from cytosine (C) to thymine (T). Consequently, these changes resulted in the amino acid mutations from isoleucine (Ile) to threonine (Thr) in Rose flower and from valine (Val) to alanine (Ala) in Salmon flower. In conclusion, the consistency of the result analyzed by DNA sequencing with that by nested PCR-SSCP suggests that PCR-SSCP could provide an accurate and rapid tool for detecting gene point mutations in ornamental flowers. Key word: Cloning and sequencing, flower color gene, Salvia splendens, nested PCR, point mutation detection, SSCP analysis, Ss5MaT1. Abbreviations: AMP, Ampicillin; DEPC, Diethylpyrocarbonate; EDTA, Ethylene diaminetraacetic acid; MPSV, mutations, polymorphisms and sequence variants; PCR-SSCP, polymerase chain reactionsingle-strand conformation polymorphism; Ss5MaT1, malonyl-CoA:anthocyanin 5-O-glucoside-6O-malonyltransferase; VPAT, versatile plant acyltransferase. Introduction Flower color is a complex trait which is mainly determined by three kinds of plant pigments: flavonoids, carotenoids and alkaloids 1. Flavonoids, a class of widely distributed pigments in plants, fulfil many functions including production of yellow, red or blue pigmentation in flowers. They are mostly found in the vacuoles of plant cells and represent a very important group of plant natural products such as anthocyanidin and xanthein. Anthocyanidins are responsible for different flower colors from nacarat to carmine red or from blue to purple. Carotenoids are a class of natural fatsoluble pigments principally accumulated in plant plastids. They are split into two classes: xanthophylls and carotenes. Carotenoids take the responsibility for many hues of the red, yellow, blue and purple in plant organs such as leaves, fruits and flowers. Another group of plant pigments, alkaloids, is of limited distribution in the plant kingdom, and only restricted to Chenopodium album. They have not yet been found in plants containing anthocyanin pigments 2. Alkaloids contain many compounds, such as betalain, berberine and narceine. Flavonoids, carotenoids and alkaloids are three main classes of plant secondary metabolites of which the molecular weights are usually less than 30 Da. The biosyntheses of the three plant secondary metabolites are very complex since their metabolisms comprise several overlapping pathways. Many enzymes, structural genes and regulator genes are involved in the biosynthetic pathways. It is often not understood why a certain compound is produced. In recent years, researches focus on the genetic engineering of flower color in ornamental plants, including gene structures, gene functions and interactions in anthocyanidin biosynthesis. Most pigmentation from orange to blue in flowers attributes to anthocyanins, a class of plant flavonoids that exist as glycosylated, acylated and/or methylated forms 3, 4. In Salvia splendens, major anthocyanins are savlinin, monodemalonylsalvianin, pelargonidin 3,5-diglucoside and delphinidin 3,5-diglucoside (Fig. 1). The shisonin biosynthetic pathway has been well described in Salvia splendens flowers in the study by Suzuki 5 (Fig. 2). Two distinct malonyltransferase (Ss5MaT1 and Ss5MaT2) activities were identified in the crude extracts of Salvia splendens flowers. Ss5MaT1 catalyzed the monomalonylation of bisdemalonylsalvianin (first malonylation) 3, whereas Ss5MaT2 catalyzed the further malonylation of the 4’’’-hydroxyl group of