| Lotus root (Nelumbo nucifera Gaertn), a member of Nymphaeaceae, is a perennial aquatic herbaceous plant. Lotus root originated in India and China, is wildly cultivated in china as a kinds of vegetable for a long history. With richness in starch, protein, vitamins etc, lotus products are generally accepted by consumers. Starch is the main component of Lotus root, and its composition, content and structure affect lotus product quality. Therefore, the study of key genes regulating starch synthesis shows very significant to explore the molecular mechanism of starch biosynthesis, new variety breeding, and promotion for the development of lotus industry in fulture.Many enzymes are involved in starch biosynthesis, among which, pyrophosphorylase (AGPase) is believed as one of the key enzymes in catalyzing the first step in starch synthesis in plant. Plant AGPase is a four heterologous dimmer including two large subunits and two small subunits. The large subunit, encoded by multiple genes, is believed as regulation center, and the small subunit is the catalytic center encoded by a single gene. In this study, the full-length cDNA sequences, genomic DNA of lotus AGPase was isolated, and the temporal and spatial expression of these genes was also analyzed. In addition, the activity of AGPase during rhizome swelling was studied. We found the following results:1. The immature leaves of "Mei Ren hong" was chosen as material to get the total RNA. A small subunit gene encoding AGPase was isolated using RACE technique. The full-length of this gene cDNA is 1945 bp, and the open reading frame is 1572 bp encoding 524 amino acids. Its relative molecular weight is 57.40 kDa.Its theoretical PI is 6.43 and the instability coefficient is 36.98, and considered as bestable protein. This gene showed 99%similarity with goldenseal (AHZ08828.1) in nucleotide acid, and also had 91%,90%and 90%homolog with rice, sweet potato and barley. Gene phylogenetic tree analysis in nucleotide or amino acid showed that lotus AGPase has a close phylogenetic relation with goldenseal, suggesting that AGPase between’Mei renhong’and goldenseal has a similar function in starch synthesis.2. Genomic DNA was extracted from immature leaves of cultivar ’Mei ren hong’.The gDNA sequence is 4169 bp including 9 exons and 8 introns. The first intron and the second exons is the largest of 1175 bp and 298 bp, and the second intron and seventh exon is the smallest for only 92 bp and 97 bp respectively.3.The temporal and spatial expression of AGPase gene during rhizome development was analyzed using semi quantitative RT-PCR method. We found that this gene was expressed in leaf, petiole and rhizome, however, the expression is highest in leaves, then followed by petiole, the underground stems was the lowest. In addition, the mRNA level of AGPase gene was observed to be highest in initial swelling stage, then followed by middle and late swelling stages, the stolon stage is least. The activity of AGPase was identified during rhizome swelling. We found that the activity of this enzyme was slowly enhanced from stolon stage to initial swelling stage, and the activity in initial stage has 1.86 times than that of stolon stage. Otherwise, the activity of AGPase was quickly increased from initial swelling stage to middle swelling stage, and the activity in middle swelling stage had 5 times than that of middle swelling stage. The activity of AGPase was decreased from middle swelling stages to late swelling stage. We found that the gene expression and the change of enzyme activity during rhizome swelling showed high consistency. |
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