Molecular characterization of the Glb2 gene

Molecular characterization of the Glb2 gene --Nancy H. Wallace and Alan L. Kriz Normal maize embryos contain high levels of saline-soluble, water-insoluble globulin storage proteins. The most abundant globulin component is the Mr 63,000 product of the Glb1 gene, which has recently been characterized at the molecular level (above report). The second most abundant globulin is designated GLB2, which is encoded by the Glb2 gene (Kriz, Biochem. Genet. 27:239, 1989). We have isolated and characterized at the nucleotide sequence level cDNA clones corresponding to Glb2. Clones were isolated from an embryo-specific cDNA library by using antiserum raised against whole embryo globulin. Immunoreactive plaques were picked and purified by a secondary screen. A tertiary screening was performed using as probe the radiolabelled insert from a Glb1-specific cDNA clone. Those plaques reacting with whole globulin antiserum but not with the Glb1 probe were considered to be potential Glb2 cDNA clones. The clone with the largest insert (1600 bp) was radioactively labelled and used as a probe in a Southern blot analysis of five other potential Glb2 clones. All five showed hybridization with the radiolabelled probe. The largest clone was subjected to nucleotide sequence analysis. To obtain the 5' untranslated region for analysis, a PstI fragment of ~180 bp from the 5' end of the 1600 bp clone obtained above was used to screen the cDNA library for longer clones. Such a clone was isolated and subjected to nucleotide sequence analysis. This 1638 bp clone, designated pcGlb2, contains a 1350 bp open reading frame corresponding to a polypeptide of 450 amino acids. The translated region contains a putative signal peptide of 23 amino acids, followed by 15 amino acids which correspond to the amino terminus of GLB2 as determined by direct protein sequencing of the mature polypeptide. The predicted molecular weight of the mature polypeptide (47,380 daltons) is in good agreement with the value of 45,000 daltons empirically determined from SDS gel analysis. We are currently characterizing genomic clones corresponding to the Glb2 gene.

Tissue specificity of Glb2 expression has been examined by Northern blot analysis of total RNA from various plant tissues. Glb2 transcripts are found only in the developing embryo and not in endosperm, seedling or unfertilized ear. Western blot analysis comparing the globulin protein profiles of embryo and endosperm of the maize inbred line Va26 shows the GLB2 protein to be present only in the embryos. This is in contrast to the situation observed for Glb1 transcripts and proteins, which are present at low levels in endosperm tissues. It has also been determined by Northern blot analysis that Glb2 transcript is present between 21 DAP to 36 DAP and absent in mature dry embryos. This expression pattern also differs from that of Glb1 in that Glb1 transcripts, first detectable at 15 DAP, persist in mature embryos. Lastly, embryos homozygous for a Glb2 null allele lack the Glb2 transcript as shown by Northern blot analysis. Isolation of genomic clones corresponding to the null allele will eventually be performed.

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