Identification of the cysteine proteinase inhibitor, cystatin, in mungbean (Vigna radiata L. wilczek)

Abstract

We have identified and analyzed the cysteine proteinase inhibitor (CPI), cystatin, in mungbean (Vigna radiata). Cystatin cDNA encoding an 89 amino acid coding region was isolated using RT-PCR and sequenced (GenBank Accession Number AF454396). The cystatin sequences from C. chinensis-resistant and susceptible lines showed a high degree of similarity, 90-95%. The cystatin nucleotide sequence from Jangan mungbean, a resistant breeding line, shows 95%, 86%, and 74% homology compared to that of cowpea, oryza, and soybean respectively. The deduced coding region contains a QXVXG putative reactive domain in the central region and a PW motif near the C-terminus, both of which are conserved among members of the cystatin superfamily. No signal sequence motif was found in the N-terminal region, indicating that mungbean cystatin may be an intracellular protein similar to cowpea cystatin and oryzacystatin. Using 3′-RACE PCR analysis, a polyadenylation signal was detected in the 3′ UTR. Quantitative RT-PCR and Northern blot analysis from mature seeds of resistant and susceptible mungbean lines showed similar cystatin mRNA expression levels. These results strongly suggested that cystatin is expressed in mungbean seeds during maturation. Southern blot analysis of the cystatin gene using BamHI, EcoRI, PstI, HindIII and XhoI performed on both resistant, and susceptible mungbean lines showed no significant difference. We concluded that mungbean cystatin functions in plant regulation rather than for defense as in the case of the cowpea cystatin.