Cloning of the ACC synthase gene from Curcuma alismatifolia Gagnep. And its use in transformation studies

Abstract

The goal of this work was to suppress the expression of the ACC synthase gene in the Siam tulip, Curcuma alismatifolia Gagnep. A cDNA fragment encoding ACC synthase from C. alismatifolia Gagnep. was isolated and expression studies were done. To isolate this gene a pair of primers was designed from the highly conserved motif of ACC synthases in various plant species. The PCR product of 600 bp. in C. alismatifolia Gagnep. was subcloned into the pGEM-T easy vector resulting in pCa-ACSI. After sequencing, the sequence and its deduced amino acid sequence were highly homologous to the ACC synthases from other plants. To determine expression patterns of pCa-ACSI, a Northern blot analysis showed that the expression of the pCa-ACSI gene was in the bracts of curcuma, the highest expression was observed 2 days after cutting the flowers. Ca-ACSI was subcloned in pBI121 resulting in pBI121-Ca-ACSI and transformed into leaf tissues of Torenia foumieri and retarded shoots of C. alismatifolia Gagnep via Agrobacterium tumefaciens strain AGLO. Putative transformants, with the gene in an antisense orientation, were investigated by PCR analysis, GUS assays and Southern blotting. The transgenic plantlets were transferred to pots containing soil for cultivation in growth chamber. At present, the transgenic plants grow happily in the greenhouse and their phenotypic is under investigation.