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Title: PROTEOMIC AND GENOMIC CHARACTERIZATION OF KUNITZ TRYPSIN INHIBITORS IN WILD AND CULTIVATED SOYBEAN GENOTYPES

Author
item Natarajan, Savithiry - Savi
item XU, CHENPING - UNIVERSITY OF MARYLAND
item Bae, Hanhong
item Bailey, Bryan

Submitted to: Journal of Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/5/2006
Publication Date: 5/5/2006
Citation: Natarajan, S.S., Xu, C., Bae, H., Bailey, B.A. 2006. Proteomic and genomic characterization of kunitz trypsin inhibitors in wild and cultivated soybean genotypes. Journal of Plant Physiology. 164(6):756-763.

Interpretive Summary: Soybean is a rich and inexpensive source of proteins for humans and animals. But it also contains many compounds which are considered allergenic or anti-nutritional. One such anti-nutritional protein in soybean is the Kunitz trypsin inhibitor (KTI), which inhibits the gastrointestinal enzymes of animals. We investigated the natural variation of KTI in soybean and also looked for variation in the structure of the KTI genes (DNA) in the soybean. We observed that wild soybeans have more KTI proteins than cultivated soybeans. Moreover, our studies revealed differences in DNA patterns for the KTI genes in wild and cultivated soybeans. The results establish the range of KTI concentration that naturally occurs in soybean and can be used by scientists and industry as the basis of comparison to assess KTI in transgenic soybean that may be developed in the future. These data can be used to assure that unwanted levels of KTI are not present in soybean cultivars that are released to the grower.

Technical Abstract: In this study, we investigated protein and genetic profiles of Kunitz trypsin inhibitors (KTIs) in seeds of sixteen different soybean genotypes that included four groups consisting of wild soybean (Glycine soja), the cultivated soybean (G. max) ancestors of modern N. American soybean cultivars, modern N. American soybean, and Asian cultivated soybean landraces that were the immediate results of domestication from the wild soybean. Proteins were well separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and stained protein cut from a 2D-PAGE indicated that KTI exists as multiple isoforms (spots) in soybean. Protein spots of KTI were identified and characterized using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Although overall distribution patterns of the KTI protein spots appeared similar, the number and intensity of the protein spots between wild and cultivated genotypes varied. Three KTI peptides were identified in three of the wild genotypes, PI 393551, PI 407027 and PI 407282 in which KTI3 peptide showed highest intensity. The remaining wild genotype, PI 366120 showed four protein spots. In contrast, the ancestors, modern and Asian landrace genotypes showed only two protein spots corresponding to KTI. Based on DNA blot analysis, there is one copy of the KTI3 gene in all sixteen genotypes. Polymorphism was detected in one of the wild genotypes (PI 366120) both in proteomic and genomic analyses. Our data suggest that the major variation of protein profiles were between wild and cultivated soybean genotypes rather than among genotypes in the same group. Genetic variation of KTI1 and KTI2 and KTI3 related genes were detected within and between groups.