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

ANALYSIS OF DFR EXPRESSION BY REAL TIME PCR IN TRANSGENIC L. CORNICULATUS PLANTS IMPAIRED IN LEAF TANNIN SYNTHESIS

PAOLOCCI F., TURCHETTI V., DAMIANI F.

Istituto di Ricerche sul Miglioramento Genetico delle Piante Foraggere CNR, Via Madonna Alta 130, 06128 Perugia

Francesco.Damiani@irmgpf.pg.cnr.it

real time PCR, quantitative expression, DFR, Condensed tannins, Lotus

We have previously showed that the bHLH maize Sn gene can both transactivate and suppress the condensed tannin (CT) synthesis in leaves of Lotus corniculatus plants. Molecular analyses also showed that the transactivated phenotype correlated with high steady state transcript levels of the single copy introduced transgene. Conversely, mature Sn transcript was never detected into CT negative transgenic lines harbouring multicopies of the transgene, corroborating the hypothesis of a gene silencing phenomenon. Since DFR is the last enzyme shared between the anthocyanin and CT metabolic pathways and dfr is a target gene of Sn in maize, we have cloned both the full-length cDNA(s) and a dfr genomic fragment of L. corniculatus. Extensive sequence analysis revealed the presence of cDNAs clones differing for few nucleotides and aminoacids. Northern analysis on CT enhanced and suppressed plants revealed an up and down regulation, respectively, of leaf dfr expression with respect to the control untransformed or GUS- transformed mother plants.

In order to achieve a better estimation of the transcript level of genes putatively involved on CT biosynthesis in our mutants, we have started a relative quantification analysis of dfr by a real time PCR apparatus and a two-step RT-PCR protocol. Therefore, cDNAs from control and CT mutated plants were amplified both with a dfr –specific primer pair, designed on a strictly conserved region among the different cDNAs cloned, and a primer pair specific to the L. corniculatus α-elongation factor, as reference gene. Preliminary results showed a marked (> 200%) increase of dfr expression into CT + mutants, as well as its dramatic reduction (ranging from 50 to 90%) in CT- plants with respect to the controls. Although the rate of dfr expression may change according to the environmental conditions as well as to the physiological status of plants, we have observed a tight and positive correlation between dfr expression and the amount of tannin-containing leaf cells as revealed by DMACA staining. Therefore the level of dfr transcript is likely to be a marker of tannin biosynthesis in Lotus leaves. Interestingly, the dissociation curves relative to the dfr amplicons obtained through real-time PCR assays on control and CT- plants displayed two peaks, while only one was clearly detected in CT enhanced plants, suggesting the presence of polymorphic cDNA amplicons. It is therefore conceivable that Sn exerts its transactivation activity either on a single or only towards specific dfr gene(s) masking or preventing the expression of the other one(s). The polymorphic amplicons will be sequenced as first step to identify these hypothetically different dfr sequences.

Acknowledgements: work carried out in the framework of EU project FAIR CT 98-4068