Proceedings of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

CLONING AND SEQUENCING OF STRUCTURAL GENES INVOLVED IN ANTHOCYANINS BIOSYNTHESIS IN BLOOD ORANGES

LO PIERO A. R.*, PUGLISI I.*, CONSOLI A.*, LA ROSA M.**, REFORGIATO RECUPERO G.**, PETRONE G.*

* Dipartimento di Scienze Agronomiche, Agrochimiche e delle Produzioni Animali, Università di Catania

alopiero@mbox.fagr.unict.it

** Istituto Sperimentale per L’Agrumicultura, corso Savoia 190, 95024 Acireale, Catania

breedcitr@mail.gte.it

anthocyanins, cDna cloning, flavonoids, sweet orange flesh, Citrus sinensis

The development of red colour in orange flesh is due to the presence of water-soluble pigments belonging to anthocyanins, and represents a pivotal feature in determining Italian product acceptability in the worldwide markets. In addition blood oranges are characterized by a greater aroma and, in some cases (Tarocco), show an high content of C vitamin. Moreover, along with these characteristics, anthocyanins containing fruits exhibited antioxidant and anti-inflammatory properties as well as high activity against free radicals. Due to their biological and agricultural importance, anthocyanins biosynthesis-related genes have been isolated from maize, petunia, snapdragon, Japanese morning glory, Perilla frutescens, poplar, pea and have been extensively investigated at molecular level. The cDNA clones implicated in anthocyanins biosynthesis have been isolated from fruits trees such as Vitis, Malus, Juglans and Casuarina glauca. CHS, CHI and F3H, enzyme of the early steps of flavonoid biosynthesis, have been analyzed during satsum mandarin fruit development. Furthermore, the same genes have been cloned in Valencia orange [Citrus sinensis (L.) Osbeck] seeds. We report here the first results on characterization of cDNAs encoding structural genes required for anthocyanins biosynthesis in the Moro flesh which is the blood orange with the highest content of anthocyanins. A cDNA 8TriplEx expression library has been constructed using total blood oranges flesh RNA as starting material. The cDNA first strand was synthesized using Superscript II RNAase H^-reverse transcriptase, while for the second strand Taq polymerase was used. The titer of the resulting amplified library was 0.82 x 10⁹ pfu with percentage of recombination efficiency ≥ 95%. Partial cDNA clones coding for CHSI, CHSII, CHI, F3H, DFR, ANS and UFGT were isolated from the library by PCR amplification using heterologous primers. In order to achieve full lenght clones, restriction digests of PCR products have been labelled with ³²P and used as probes to screen the cDNA 8TriplEx library. Sequencing of the positive clones and expression of the single genes are at present in progress.