Abstract
In order to clone the cDNA sequence of water chestnut AGPase large subunit gene ( EdAGPL1 ) and analyze its structural characteristics and its expression in different tissues and corm development of water chestnut. Using water chestnut 'Guilin water chestnut ' as research material. The EdAGPL1 gene was cloned by RT-PCR and analyzed by bioinformatics. The expression of EdAGPL1 in different tissues and corm development process was analyzed by real-time fluorescence quantitative PCR technology. The results showed that the length of the cloned EdAGPL1 gene was 1 744 bp, its open reading frame was 1 599 bp, it encoded 532 amino acids, the protein molecular mass was 58.84 kD, the isoelectric point was 7.54, and it had NTP_transferase and PbH1 domains. The secondary structure was composed of helix (13.72%), for the strand (25.19%) and the loop (61.09%). The tertiary structure consists of 17 α-helices, 34 β-sheets, and 51 β-turn (curl) composition. Homology and phylogenetic analysis showed that the homology of amino acid sequence of EdAGPL1 and other plants AGPL protein was 57.54%~61.64%, and has far evolutionary kinship with other plants. Fluorescence quantitative PCR analysis showed that the expression of EdAGPL1 in different tissues was corm> leaf stem > stolon > root. The expression of EdAGPL1 was the highest in the initial stage of corm development, and then it decreased and remainedstable. The study of this gene will help to clarify the regulation mechanism of starch synthesis in water chestnut and provide a theoretical basis for the breeding of high-starch varieties.
Highlights
IntroductionThe activity of ADP-glucose pyrophosphorylase (AGPase) affects the synthesis rate of starch
At least 4 enzymes are involved in plant starch biosynthesis, including ADP-glucose pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzyme (BE) and debranching enzyme (DBE) (Abe et al, 2014)
1.1 The gene squence encoding the large subunit gene AGPase was cloned The cDNA obtained by reverse transcription of total RNA of water chestnut corm was used as the template for PCR amplification and the target gene fragment with length of 1 744 bp was obtained (Figure 1)
Summary
The activity of AGPase affects the synthesis rate of starch. It was significantly positively correlated with starch content (Min et al, 2010). Due to the importance of AGPase in starch biosynthesis, many studies have focused on improving the catalytic activity of AGPase to improve starch content and yield (Tuncel and Okita, 2013). There are few studies on starch biosynthesis in water chestnut, and only a few annotated information of genes involved in starch biosynthesis was obtained by high-throughput sequencing of leaves, corms and other. The expression of EdAGPL1 gene in different corm development stages and tissues was detected by real-time quantitative PCR, which provided theoretical basis for studying the starch synthesis mechanism of water chestnut
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