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

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Oral Communication Abstract

GENES DIFFERENTIALLY EXPRESSED IN ISOGENIC MUTANTS OF LOTUS CORNICULATUS

TOSTI N., TURCHETTI V., RAGANO-CARACCIOLO M., DAMIANI F.

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

Francesco.Damiani@irmgpf.pg.cnr.it

cDNA subtraction, Condensed tannins, Lotus, cDNA-AFLP

Transformation of one genotype of L.corniculatus with the maize gene Sn under the constitutive promoters CAMV35S produced individuals that resulted highly divergent for the level of Condensed Tannins (CT) in leaves. Since the isolation of the genes coding for CT is of strong interest for improving forage quality in legumes, these mutants were further characterised for transgene copy number, type of insertion, transgene and endogene expression.

Four plants up and down regulated were selected, together with controls, in order to identify and eventually clone differentially expressed genes.

Two methods of selection were adopted: “Subtraction PCR select” and “cDNA-AFLP”. Both utilised PCR amplification and therefore need low amount of material. This is extremely important because allow to careful select the starting material avoiding old and infected leaves that can produce unreliable results.

“Subtraction PCR select” is a method proposed by Clontech consisting on the subtraction of cDNA of the subject (tester), previously linked to adaptors, through hybridisation with cDNA of a control (driver) followed by the amplification of the residual cDNA. From the subtraction between CT up-regulated plants (tester) and CT down-regulated plants (driver) several sequences were produced. They were sequenced and through RT PCR with sequence specific primers confirmed for their differential expression. A very low percentage was confirmed, indicating that the method produced large amounts of escapes. In a second round of subtraction, a preliminary screen of amplified putatively subtracted sequences was performed using northern reverse hybridisation of amplified sequences with labelled cDNA of parental plants, as preliminary screening it was also added the hybridisation with cDNA from alfalfa, a CT negative species, which improvement is the long term target of the project.

With this analysis it resulted that the other than qualitative subtraction, meaning “yes or not” expression, the most of polymorphic sequences resulted quantitatively differentially expressed. Obviously quantitative differential expression is more difficult to be assessed, and PCR techniques and tissue sampling can affect plant expression.

In parallel with “Subtraction PCR select”, cDNA-AFLP was also performed. With this method we were able to compare in the same experiments all the 4 transgenic genotypes and the control, thus reducing the number of escapes. However, in some cases, also non-orthodox pattern of expression, yielded interesting sequences. At the moment through this methods 144 out of the 256 possible 2x2 protruding nucleotides combination were performed and about 80 polymorphic fragments were sequenced.

In summary several expressed sequences were isolated and cloned, some of these showed homology with genes known to be related with the flavonoid pathway, some other showed homology with genes that is difficult to associate with CT expression, and the most interesting ones resulted homologous to unknown proteins or showed no homology on the gene bank.

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