Potato AGPase Gene Clone

ADP-glucose catalyzes an important regulatory step in the biosynthesis of starch in plant. The plant enzyme is stimulated and suppressed by 3-PGA and PPi, respectively. The rate of starch biosynthesis is controlled by 3-PGA/PPi. Starch is a major component of the potato tuber, and thus directly has an impact on its application and process. Cloning AGPase gene, constructing expression vector and trnasforming potato will increase the expression yield of the enzyme and then increase it's catalyzing activities in molecular level. Therefore the rate of potato starch biosynthesis will be greatly increased.AGPase is also an induced enzyme. We performed a series of experiments of using various concentration of sucrose to induce it's expression. Under the conditions of elevated sucrose (6%, w/v), the activities and expression yields of AGPase are both maximum.Detached leaves were dealt with sucrose solution (6%, w/v), then abstracted total RNA. According to known AGPase gene sequence, we cloned a 1.58 kb distinctive fragment by RT-PCR using artificial synthetic special primer and combined it with pGEM-T Easy Vector and tRNAsformed into E.Coil DH5 a . Digesting by enzyme and sequence analysis indicated this fragment is composed by 1589 bp open read fragment (ORF), which codes a 530 AA fragment. This fits in with reports of AGPase gene well. The recombined plasmid is named as pGEMA. Cutting off the aimed fragment from pGEMA and replacing GUS gene of plasmid pBI121 in directive direction, we constructed the plant expression vector pBIAGP of AGPase gene and integrated it into agrobacterium LBA4404.