CLONING AND SEQUENCING OF POTATO trnI AND trnA PLASTID DNA FRAGMENTS FOR DEVELOPING HOMOLOGOUS PLASTID TRANSFORMATION VECTOR

Abstract

For developing plastid transformation vector, based on homologous targeting regions of potato (Solanum tuberosum L.), the trnI and trnA intergenic regions of the plastome from the commercial potato cultivar Kufri Bahar was PCR amplified, cloned and sequenced. Isolation and cloning of potato trnI and trnA cpDNA fragments into pDrive cloning vector yielded plasmid pDtrnI and pDtrnA with cpDNA fragments of approximately 800 bp and 1000bp, respectively. Sequence alignment using Clustal W revealed 98% sequence homology with the corresponding fragment of Nicotiana tabacum L. (GenBank Accession no. Z00044). The most remarkable difference was 9 bp insertion from 647-655 in potato plastome that was missing in tobacco. Similarly, sequence alignment of potato trnA region showed 100% identity to the respective fragment of Nicotiana tabacum L., except an insertion of 102 nucleotides from 700-801. The observed differences in the sequence homology between potato and tobacco plastome might be responsible for lower transformation efficiency in potato using tobacco-based vector. The presence of unique restriction enzyme sites within the intergenic region of both fragments will make possible the construction of potato specific plastid transformation vector. The cloned trnI/trnA region of the potato plastome will contribute towards designing plastid transformation vector with better transformation efficiency.