Molecular cloning and characterization of granule-bound starch synthase I (GBSSI) alleles from potato and sequence analysis for detection of cis-regulatory motifs
Granule-bound starch synthase (GBSS), an isoform of starch synthase, is responsible for amylose synthesis in all starch containing tissues of potato (Solanum tuberosum L.), and in other plant species. In potato, the importance of GBSSI gene function is realized through a number of published reports on both basic and applied research. Here genomic DNA was isolated from six high-yielding Indian potato cultivars, and the cultivar Desiree. Polymerase chain reactions (PCR) were carried out at varying annealing temperatures using GBSSI gene-specific primers and genomic DNA from the individual potato cultivars. Analysis of PCR amplification products provided an overview of GBSSI allelic composition of these potato cultivars which was hitherto unknown. Two partial GBSSI alleles were isolated from one of the Indian potato cultivars. The distinguishing sequence features between them were shown. Multiple sequence alignment of GBSSI alleles from different potato cultivars provided a clear view for comparison between their different regions. Base composition analysis revealed a notably higher GC rich region immediately upstream of the transcription start site of the GBSSI alleles, unlike other potato genes. Preliminary sequence analysis detected a number of different cis-regulatory sequence motifs in the GBSSI alleles which were not documented in earlier reports. The GBSSI sequences from different potato cultivars reported to date were sorted out into four groups as shown in the phylogenetic tree. A promoter was isolated from one GBSSI allele of this study, and found to be strong and tuber-specific in the potato cultivar under field conditions as examined by histochemical GUS assay.
KeywordsIndian potato cultivars GBSSI alleles Promoters Multiple sequence alignment cis-Regulatory motifs Histochemical GUS assay
We gracefully thank the Department of Biotechnology (DBT), Govt. of India for providing research funding to N. Das and fellowships to A. Bansal and D. Taneja. Also thanks to Council of Scientific & Industrial Research (CSIR), Govt. of India for providing fellowship to V. Kumari.
- Dai WL, Deng W, Cui WY, Zhao SY, Wang XM (1996) Molecular cloning and sequence of potato granule-bound starch synthase gene. Acta Bot Sinica 38:777–784Google Scholar
- Kuipers GJ, Vreem JTM, Meyer H, Jacobsen E, Feenstra WJ, Visser RGF (1992) Field evaluation of antisense RNA mediated inhibition of GBSS gene expression in potato. Euphytica 59:83–91Google Scholar
- Nielsen TH, Wischmann B, Enevoldsen K, Moller BL (1994) Starch phosphorylation in potato tubers proceeds concurrently with de novo biosynthesis of starch. Plant Physiol 105:111–117Google Scholar
- Rohde W, Becker D, Kull B, Salamini F (1990) Structural and functional analysis of two waxy gene promoters from potato. J Gen Breed 44:311–315Google Scholar
- Sadasivam S, Manickam A (1996) Biochemical methods. New Age International, New DelhiGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
- Wang T, Bogracheva TY, Hedley CL (1998) Starch: as simple as A, B, C? J Exp Bot 49:481–502Google Scholar