Characterization of sporamin gene in transgenic Brassica oleracea var. alboglabra and Arabidopsis thaliana

• Main Author: 陳懷如
• Other Authors: Kai-Wun Yeh
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/72387967612787198712

碩士 === 國立臺灣大學 === 植物學研究所 === 90 === Sporamin is an abundant storage protein in tuberous roots of sweet potato. It accounts for about 80% of total soluble proteins in sweet potato tubers, and no expression in leaves and a little expression in stems at the growing stage. In 1997’s, Yeh et al., constructed a recombinant plasmid with sporamin cDNA and expressed it’s in Escherichia coli cells as fusion proteins. This protein was identified by strong inhibitory activity to trypsin in vitro and was proved to be pest-resistant in transgenic tobacco plants. The transgenes driven by CaMV 35S promoter usually have gene silencing. To compare the gene expression driven by CcaMV 35S promoter and sporamin promoter, we selected prospoA promoter to drive it’s own cDNA sporamin and genomic sporamin, then established three different constructs: 35S::sporamin, prospoA::sporamin and prospoA::sporamin 3’-flanking region. They were introduced into Brassica oleracea var. alboglabra and Arabidopsis thaliana plants by Agrobacterium tumefaciens-mediated transformation. Genomic sporamin has longer 3’-flank region than cDNA sporamin, and contains one MAR T-box, three E-box, one SEF4 box. All transformants were screened for kanamycin resistance and confirmed by PCR. Arabidopsis thaliana transformants showed 2~5 % transformation frequency, and Brassica oleracea var. alboglabra expressed 5 % transformation ration. We used western analysis, trypsin inhibitory gel activity analysis and biochemical analysis to detect sporamin protein expression level in the transformants. Among the three-constructs, prospoA::sporamin 3’-flanking region shows better trypsin inhibitory activity than other two constructs. We also tested insecticidal activity of these transformants by the feeding test of Helicoverpa armigera Hubner. It was demonstrated the genomic transgenic plants, with a genomic construct expressing better trypsin inhibitory activity, had better pest-resistant than wild type plants.