Cloning and characterization of the genes encoding the small and large subunit of the ADP-glucose pyrophosphorylase in lotus (Nelumbo nucifera Gaertn)

In higher plants, ADP-glucose pyrophosphorylase (EC 2.7.7.27, AGPase), a heterotetrameric enzyme comprised of two small and two large subunits, carries out the first committed step of starch synthesis. To characterize the AGPase genes in lotus, we cloned a small subunit and a large subunit of AGPase using the rapid amplification of cDNA ends (RACE) technique, designated NnAGPS1 and NnAGPL1 genes, respectively. The full-length cDNA of NnAGPS1 was 2,168 bp which codes for a protein of 524 amino acids and of NnAGPL1 was 2,304 bp encoding a protein of 528 amino acids. Complete genomic structures of NnAGPS1 (comprising 9 exons and 8 introns) and NnAGPL1 (comprising 14 exons and 13 introns) were distributed over 3.8 and 4.8 kb, separately. Phylogenetic tree divided the AGPase subunits into two classes: small subunit (SSU) and large subunit (LSU). Sequence alignment of these two genes with other plant species displayed a vast dissimilarity in the N-terminal regions. Results of quantitative real-time RT-PCR (qRT-PCR) showed that both genes were expressed in all tissues, including leaf, leafstalk, rhizome and root. The highest expression of NnAGPS1 was observed in leaves and NnAGPL1 seemed to be preferentially expressed in rhizomes. Besides, transcripts of NnAGPS1 decreased significantly while NnAGPL1 kept a steady level during the early to middle rhizome-swelling stages. Our study will provide important molecular information for the genetic modification and regulation of starch content in lotus.