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

CONSTITUTION OF MAIZE TRANSGENIC LINES AND IDENTIFICATION OF MU-TAGGED MUTANTS WITH ALTERED EXPRESSION OF RPD3-TYPE HISTONE DEACETYLASE

LOCATELLI S., LANZANOVA C., ROSSI V.

Istituto Sperimentale per la Cerealicoltura – sezione di Bergamo - Via Stezzano 24, 24126 Bergamo

isc@spm.it

Histone deacetylase, gene expression, Zea mays, Mutator element

Transcriptional control is the primary mechanism for regulating eukaryotic gene expression during development and in response to environmental stimuli. Evidence indicates that eukaryotic transcription is an interlaced network of transcription factor and chromatin-modifying complexes. One important mechanism for the dynamic alteration of chromatin structure is the acetylation and deacetylation of histones, which is effected by two enzymes activities, histone acetyltransferase (HAT) and histone deacetylase (HDAC). Recent findings, indicating that several transcriptional co-activators and co-repressors possess HAT and HDAC activities, revealed a direct mechanistic link between chromatin modification and transcriptional regulation. Multiprotein complexes containing HAT and HDAC activities have been identified in a wide variety of fungi and animal cells. We have identified and characterised a maize Rpd3-like histone deacetylase (ZmRpd3I). Comparison with its mammalian homologues indicates conserved and unique features. Although a role for ZmRpd3I in controlling cell cycle progression has been proposed, the biological significance of these enzymes in plants is still poorly understood.

To analyse the biological role of Rpd3-type HDACs in plants gain- and loss-of-function maize mutants were constituted. Furthermore, since ZmRpd3I is a member of a gene family, ZmRpd3I gene-specific mutants for insertion of the Mu transposon were identified.

Maize lines were stably transformed with plasmids carrying the PAT (phosphinotricin acetyltransferase) gene and expressing sense or antisense orientated ZmRpd3I cDNA under the control of the ubiquitin constitutive promoter. A total of 56 ZmRpd3I antisense and 46 sense positive calli were identified both for their resistance to the BASTA herbicide and by means of PCR screening and corresponding plants were regenerated (T0). Progeny recovered from backcrossed transgenic lines (T1) were analysed to determine the segregation, expression, and stability of introduced genes. RT-PCR and Northern blot techniques were employed to analyse modification in the level of transcript for different ZmRpd3 members. Currently, we are investigating the abundance of ZmRpd3I protein and changes in the amount of histone acetylation in the transgenic lines. Meanwhile we are observing if there are some phenotypic changes, in particular in the male and female flowers development.

In order to identify a ZmRpd3I single gene mutant we screened a maize Mu-tagged collection. One hundred pools of genomic DNA, each representing 50 “Mutator plants” was screened in duplicate by PCR using ZmRpd3I specific, and one mutator specific oligonucleotide primers. A secondary screen was carried out on the positive pools using genomic DNA extracted from single plant and the same primer combination. To confirm the insertion event, the amplification products was hybridised with a ZmRpd3I probe and subsequently cloned and sequenced to identified the exact site of insertion of the mutator transposon. One mutant containing a Mu insertion in the 5’ region of ZmRpd3I gene was identified. Progeny seeds from the positive plant were germinated and will be backcrossed to a Mu-minus line (B73) several times to reduce Mu activity prior to selfing and characterisation.

In addition, a “prototype” MuArray has been screened to identify other ZmRpd3I mutants. A nylon membrane was spotted with amplified Mu flanking sequences from over 700 plants. Individual plants with an insertion within ZmRpd3I gene will be identified by hybridising this membrane with radiolabelled cDNA.

The maize transgenic lines and Mu tagged mutants mentioned above will constitute important tools for the identification of ZmRpd3I target genes as well as to gain deep inside the role of ZmRpd3I in cell cycle and DNA replication.