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Characterization and expression of a novelglobulin1 null allele of maize

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Title: Characterization and expression of a novelglobulin1 null allele of maize
Author(s): Bhattramakki, Dinakar
Doctoral Committee Chair(s): Kriz, Alan L.; Sachs, Martin M.
Department / Program: Agronomy
Discipline: Agronomy
Degree Granting Institution: University of Illinois at Urbana-Champaign
Degree: Ph.D.
Genre: Dissertation
Subject(s): Agriculture, Agronomy Biology, Molecular Biology, Genetics
Abstract: The maize globulin1 (glb1) gene encodes an abundant embryo storage protein. This gene is highly polymorphic and several protein size alleles as well as a null allele have been described and the molecular basis for the differences has been explained. This study extends the analysis of glb1 variants to further explore the nature of polymorphism at this locus.Analysis of the endosperm proteins by SDS-PAGE indicated a general trend in the level of different polypeptides in different protein selection strains. As percent total protein increased as a function of selection, the level of zein also increased. Embryo protein profiles of Illinois High Protein (IHP) and Illinois Low Protein (ILP) by SDS-PAGE and immunoblot analysis indicated that these populations lack detectable glb1 protein. However PCR amplification of DNA from IHP plants and subsequent Southern hybridization with radiolabeled oligo-nucleotide probe specific for glb1 intron2 confirmed that glb1 gene exists in the IHP population. The glb1 gene expression patterns were analyzed using embryos from different developmental stages of maize kernels of both IHP and ILP strains. There was more than 10-fold decrease in the glb1 steady state transcript level at 26 DAP compared to the standard functional glb1 alleles from W64A and Va26. The reduction in steady state transcripts in ILP embryos was more than 30-fold to that of the standards. However, transcript levels of glb2 and kd16 genes were not affected in these populations. Analysis of expression of various storage protein genes in the endosperm also revealed that, the IHP and ILP populations lack detectable glb1 steady-state transcripts. There was no significant difference in the 19 kD transcript accumulation pattern in the IHP and ILP endosperm. Possible reasons for this have been suggested. The expression pattern of transcriptional regulator opaque2 has also been studied. Probably this is one of the first studies that explains the expression of major embryo and endosperm storage protein genes in the IHP and ILP strains.The null allele Glb1-N1Hb derived from the IHP strain of maize was studied at molecular level by nucleotide sequence analysis. Genomic DNA extracted from the IHP maize leaves showed that Glb1-N1Hb allele is larger in size compared to the alleles characterized earlier. The difference was mainly due to nucleotide insertions. The sequence analysis also revealed a single-base insertion in the third exon at position 950 bp leading to a change in the reading frame and resulting in premature termination codon. The predicted size of the polypeptide that could be translated from the Hb transcript is around 46 kD. However the IHP embryos do not accumulate GLB1 protein. It is probable that the insertion results in mRNA destabilization and leads to drastic reduction in globulin1 protein prevalence. The possible mechanism of defective translation machinery affecting the steady state transcript accumulation is discussed in detail.
Issue Date: 1994
Type: Text
Language: English
URI: http://hdl.handle.net/2142/23415
Rights Information: Copyright 1994 Bhattramakki, Dinakar
Date Available in IDEALS: 2011-05-07
Identifier in Online Catalog: AAI9512301
OCLC Identifier: (UMI)AAI9512301
 

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