IDENTIFICATION OF GROUP-II GLYCININ GENES IN SOYBEAN AND THE MOLECULAR CHARACTERIZATION OF A GROUP-II GLYCININ NULL ALLELE (DNA, SOUTHERNS, MUTATIONS, CLONES)
Abstract
Two cDNA clones that encode the group-II glycinin subunits, A(,5)A(,4)B(,3) and A(,3)B(,4), were isolated from a cDNA library constructed from polyA('+) RNA of mid-maturation stage soybean (Glycine max L., Merr) embryos. These group-II clones hybridize weakly to group-I clones at low stringency, but fail to hybridize to them at moderate or high stringency. The cDNA clones were used as probes in Southern hybridizations to leaf DNA. This showed that the genes encoding A(,5)A(,4)B(,3) (Gy(,4)) and A(,3)B(,4) (Gy(,5)) were contained on 13 kb and 9 kb EcoRI fragments, respectively. Genomic reconstruction analysis suggested there was one copy of the group-II genes per haploid genome. A genomic clone containing the complete Gy(,4) gene was isolated from a genomic library of soybean leaf DNA (cultivar 'Dare'). RNA protection experiments and DNA sequence analysis revealed that this group-II gene had the same basic intron/exon structure as the group-I genes. Northern hybridizations showed that the sizes of the different glycinin mRNAs were correlated with the sizes of the subunits they encoded. RNA for both groups of glycinin subunits were shown to appear at the same developmental stages of embryogenesis. To identify the genetic lesion responsible for the absence of the A(,5)A(,4)B(,3) subunit in the cultivar 'Raiden', a genomic library was constructed from Raiden leaf DNA and used to isolate Gy(,4). The complete sequence of this gene was determined and compared to the complete sequence of the same gene from a normal cultivar. Only three nucleotide differences were observed. While two of the differences would be expected to have no effect on the expression of the Raiden gene, one caused the elimination of the normal translation start codon. Northern hybridizations indicated that Raiden Gy(,4) RNA was either absent or present in very low amounts in polyA('+) RNA isolated from polysomes. RNA dot blot hybridizations, nuclear run-off transcription experiments and S1 nuclease protection experiments showed that Gy(,4) RNA was present in Raiden embryos. This indicated that the Gy(,4) gene in Raiden is transcribed but the Gy(,4) RNA is not translated into glycinin protein.
Degree
Ph.D.
Subject Area
Molecular biology
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