Qualitative and quantitative analysis of storage proteins in single and double mutants
--Azevedo, RA

The Ask1 mutant (dominant mutation), which leads to an overproduction of threonine due to an altered aspartate kinase that is less sensitive to lysine inhibition, was transferred to near isogenic conversions to the inbred line Cat100-1 in normal and o2 versions. Endosperms of the single mutants Ask1 Ask1, o2 o2, double mutant Ask1 Ask1 o2 o2, and the wild type were used for protein extraction. Storage proteins were extracted in albumins + globulins, zeins, and glutelins fractions.

The effect of the o2 mutation in reducing the synthesis of the zein fraction from 57.6% to 27% was observed, whereas albumins + globulins increased from 9.5% to 22.6% and glutelins from 32.9% to 50.7%. With the introduction of the Ask1 mutation this effect was intensified since the double mutant Ask1 Ask1 o2 o2 showed a further reduction in the zein fraction from 27% to 20.9% and increases in albumins + globulins from 22.6% to 25.3% and glutelins from 50.7% to 53.8%.

The storage protein fractions were also applied to PAGE-SDS and the pattern of bands analysed. Zein in the o2 mutant presented 3 bands and although the introduction of the Ask1 mutation had caused an alteration in the concentration of zein compared to the o2 mutation, this alteration did not alter the distribution of the bands. The same result was also observed for the other protein fractions indicating that the effect of the Ask1 mutation on the o2 mutant is not related to a specific polypeptide. These results were confirmed by testing protein fractions by conventional isoelectric focusing in amphoteric buffers. 

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