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MOLECULAR STUDIES OF THE HIGH LYSINE GENES OPAQUE 2 AND OPAQUE 6 IN MAIZE

Objective

CEREAL GRAINS HAVE A LOW PROTEIN CONTENT AND THESE PROTEINS ARE OF POORLY NUTRITIONAL VALUE FOR HUMANS AND MONOGASTRIC ANIMALS. THE LATTER IS DUE MAINLY TO THE HIGH CONTENT OF PROLAMINS WHICH ARE POOR IN ESSENTIAL AMINOACIDS. THE DISCOVERY OF HIGH LYSINE MUTANTS, PARTICULARLY THE HIGH LYSINE MUTANT OPAQUE-2 IN MAIZE, INDICATED NEW POSSIBILITIES FOR THE IMPROVEMENT OF PROTEIN QUALITY. HOWEVER, THE MOLECULAR AND PHYSIOLOGICAL BASIS OF THE HIGH LYSINE MUTATIONS IS LARGELY UNKNOWN.

THE INTEREST OF THE PROJECT THEREFORE LIES IN THE ELUCIDATION OF MOLECULAR MECHANISMS CONTROLLING THE LEVEL OF PROLAMIN IN NORMAL AND MUTANT MAIZE LINES. BY UNDERSTANDING THE WAY IN WHICH TWO KEY GENES (02 & 06) PERFORM THEIR REGULATORY FUNCTIONS, NEW POSSIBILITIES TO DEVELOP HIGH-LYSINE VARIETIES MAY ARISE.
The molecular mechanisms regulating the accumulationof the major maize storage proteins, zeins, have been investigated with a view to improving the nutritional value of maize kernels. Strategies have been proposed for the molecular cloning of 2 high lysine mutants (opaque-2 and opaque-6). These genes may possess regulatory functions used in different combinations: the O2 locus affects O6 which is reported to control positively the synthesis of 20 and 22 kDa zein proteins. However, by itself, O2 only acts positively on the 22 kDa class. The study of these genes may prove decisive in understanding the role of the O2 and O6 loci in regulating the deposition of zein.

The complementary deoxyribonucleic acid (cDNA) and genomic sequences coding for the b-32 protein, an albumin expressed in maize endosperm cells under the control of O2 and O6 loci were cloned and the complete amino acid sequence of the protein derived. The structure of the b-32 protein, a putative regulatory factor of zein expression, has a central acid region separated by 2 domains covered by secondary structure motifs. Genetic analysis suggested that the O2 locus in maize acts as a positive transacting, transcriptional activator of the zein gene expression. This regulatory locus was successfully cloned through transposon tagging. The structure of this locus was determined by sequence analysis of genomic and cDNA clones. The O2 protein contains a domain similar to the leucine zipper motif identified in deoxyribonucleic acid (DNA) binding proteins of animal protoocogenes and in transcriptional activators. In conclusion, these studies led to an understanding of the mechanisms controlling zein gene expression, which may prove useful for practical application of genetic engineering of these seed proteins.
THE OBJECTIVE OF THE PROJECT IS THE ELUCIDATION OF THE MOLECULAR MECHANISMS CONTROLLING THE ACCUMULATION OF ZEIN, THE MAJOR STORAGE PROTEIN OF THE MAIZE ENDOSPERM (60% OF TOTAL GRAIN PROTEINS AT MATURITY). TWO GENES INTERACT EACH OTHER IN CONTROLLING THE ZEIN LEVEL : 06 BY PRODUCING A CYTOPLASMIC PROTEIN RELATED TO ZEIN ACCUMULATION, 02 BY CONTROLLING 06 THROUGH AN UNKNOWN EFFECTOR. THE COOPERATING LABORATORIES WILL AIM AT THE ISOLATION OF THE TWO GENES :

- 06 GENES WILL BE SEARCHED BY PROBING A DNA BANK WITH CDNA OBTAINED FROM THE PRODUCT SYNTHESIZED BY THE GENE;

- 02GENES SHOULD BE ISOLATED FROM 02 MUTANTS, RESULTING FROM THE INSERTION OF WELL-KNOWN TRANSPOSABLE ELEMENTS INTO THE REGION OF THE GENE ITSELF.

WHEREAS THE LABORATORY IN BERGAMO, COORDINATOR OF THE PROJECT, WILL BE MORE DIRECTLY INVOLVED IN FORMAL GENETICS OF MAIZE (CROSSES, FIELD EXPERIMENTS) INCLUDING FIRST STEPS OF GENE CLONING, THE LABORATORY IN COLOGNE WILL PROVIDE EXPERTISE IN THE CONSTRUCTION OF A GENOMIC LIBRARY AND IN SEQUENCING THE CLONES, GIVING ACCESS TO THOSE REGULATORY SEQUENCES WHICH CONTROL GENE EXPRESSION.

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Coordinator

ISTITUTO SPERIMENTALE PER LA CEREALICOLTURA
EU contribution
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Address
Via Cassia 176
00191 ROMA
Italy

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