CLONING & EXPRESSION OF TWO SOYBEAN GENES ENCODING SIMILAR ASPARTOKINASE- HOMESERINE DEHYDROGENASE ISOFORMS

Authors: GEBHARDT, JOAN - MISC.; WEISEMANN, JANE - MISC.; Matthews, Benjamin - Ben

Submitted to: Plant Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/1/1998
Publication Date: N/A
Citation: N/A

Interpretive Summary: The amino acids, lysine, threonine, and methionine are essential amino acids required in the diets of non-ruminant animals. These amino acids are low in seeds of grains and soybean. Supplements of these amino acids are added to soybean and corn meal fed to pigs and chickens. We are cloning genes encoding enzymes that synthesize these amino acids with the intent of genetically engineering plants to make more of these amino acids in order that feed grains will provide a more nutritious diet. We cloned two similar genes encoding the bifunctional protein, aspartokinase-homoserine dehydrogenase. This important bifunctional enzyme is involved in the synthesis of these amino acids. The genes are large and complex. The genes appear to be expressed more in leaves and cotyledons grown in the light as compared to the dark. This information is important to scientists involved in genetically engineering plants to synthesize higher quantities of essential amino acids, because this gene is likely to be a target for modification.

Technical Abstract: A cDNA encoding one aspartokinase-homoserine dehydrogenase (AK-HSDH) and a genomic clone encoding a second AK-HSDH were isolated from soybean libraries. Comparison of the two reading frames from soybean indicated that they were two separate, but highly similar, sequences encoding bifunctional AK-HSDH isoforms. There were only ten amino acid differences between the two encoded proteins out of 916 amino acids. Four of these differences were located in the 85 amino acid transit peptide. Seventeen introns interrupted the coding region. Of these, fifteen were in the coding region of the mature polypeptide. Although the size of the introns were much larger in soybean than those found in the gene encoding AK-HSDH in Arabidopsis, the locations of the fifteen introns interrupting the coding region were identical. The soybean amino acid sequence of AK-HSDH had 78, 75, and 80% identity with those from maize 1,maize 2 and Arabidopsis, respectively. Northern blot analysis revealed increased transcript levels of AK-HSDH in light-grown than in dark-grown leaves. The transcript was also detected in cotyledons of soybean. In addition, the data suggest the existence of another possible AK transcript. This information is important to scientists involved in genetically engineering plants for synthesizing higher quantities of essential amino acids, since this gene is likely to be a target for modification.