Characterization of two Acanthoscelides obtectus alpha-amylases and their inactivation by wheat inhibitors.

Wheat alpha-amylase inhibitors represent an important tool in engineering crop plants against bean bruchids. Because Acanthoscelides obtectus is a devastating storage bean insect-pest, we attempted to purify and characterize its gut alpha-amylases, to study their interaction with active proteinaceous inhibitors. Two digestives alpha-amylases (AoA1 and AoA2) were purified from gut larvae, showing molecular masses of 30 and 45 kDa for each one, respectively. The stoichiometry interaction between these alpha-amylases with two wheat inhibitors (0.19 and 0.53) showed a binding complex of 1:1 enzyme:inhibitor. In vivo activities of these inhibitors against A. obtectus were also evaluated using a rich ammonium sulfate inhibitor fraction (F(20)(-)(40)) and purified inhibitors after reversed phase high-performance liquid chromatography columns. Incorporation of three different inhibitor concentrations (0.25, 0.5, and 1.0% w/w) into artificial seeds showed that addition of the purified 0.19 inhibitor at the highest concentration (1.0%) reduced the larval weight by 80%. Similar data were observed when 0.53 inhibitor was incorporated at 0.5%. When the concentration of purified 0.53 was enhanced to 1.0%, no larvae or adult emergence were observed. Our data suggest that these alpha-amylase inhibitors present great potential for use in Phaseolus genetic improvement programs.

Molecular cloning and differential expression of corm-specif cDNAs in Colocasia esculenta

A CDNA library was constructed using mRNA isolated from mature corm tissue of taro (Colocasia esculenta). By differential screening, four cDNA clones, pCE1, pCE2, pCE3 and pCE4, complementary to moderately abundant corm mRNAs, were selected. These were used as probes to study the expression of the corresponding genes in different taro tissue. Northern analysis of transcripts indicated that their expression is highly enhanced in the corm and that they encode mRNAs with 0.70 kb, 0.80 kb, 0.75 kb and 1.20 kb, respectively. Dot blot hybridizations revealed that clones pCE1 to 4 bear inserts homologous to mRNAs that accumulate to 1.5 percent, 1.0 percent, 0.40 percent and 0.20 percent respectively, of the total poly (A)+ mRNA present in mature corms. The four genes are differentially regulated in taro tissue. Their transcripts were detected at lower levels in the steady state mRNA of petiole, lamina and roots, except in the case of pCE3 whose mRNA could not be detected neither in petiole nor in lamina.