The analysis and reduction of starch in sugarcane by silencing ADP-glucose pyrophosphorylase and over-expressing β-amylase

[摘要] Sugarcane is cultivated because of the high levels of sucrose it stores in itsinternodes. Starch metabolism has been a neglected aspect of sugarcane researchdespite the problems caused by it during sugarcane processing. Currently there is noinformation available on the starch content in different South African commercialsugarcane varieties. This project had two main aims of which the first was todetermine the starch content in the internodal tissues of six commercial sugarcanevarieties. The activities of ADP-Glucose Pyrophosphorylase (AGPase) and β-amylase were also determined. The second aim of the project was to manipulatestarch metabolism in sugarcane using transgenesis. To achieve this, transformationvectors for the down-regulation of AGPase activity and over-expression of β-amylaseactivity were designed. These vectors were then used to transform sugarcane calliand the results were analysed in suspension cultures. Starch levels in sugarcaneinternodal tissue increased more than 4 times from young to mature internodes.There were also large differences between varieties. When mature tissues ofdifferent varieties were compared, their starch concentration varied between 0.18and 0.51 mg g-1 FW, with the majority of the varieties having a starch concentrationbetween 0.26 and 0.32 mg g-1 FW. NCo376's starch concentration was much lowerthan the rest at 0.18 mg g-1 FW and N19's was much higher at 0.51 mg. g-1 FW.There was also a very strong correlation between starch and sucrose concentration(R2 = 0.53, p ≤ 0.01) which could be due to the fact that these metabolites aresynthesized from the same hexose-phosphate pool. No correlation was evidentbetween starch concentration and AGPase activity. This was true for correlationsbased on either tissue maturity or variety. β-amylase activity expressed on a proteinbasis was almost 5 times higher in the young internodes compared to mature internodes, suggesting that carbon might be cycled through starch in theseinternodes. AGPase activity in the transgenic suspension cultures was reduced bybetween 0.14 and 0.54 of the activity of the wild type control. This reduction led to areduction in starch concentration of between 0.38 and 0.47 times that of the wild typecontrol. There was a significant correlation between the reduction in AGPase activityand the reduction in starch (R2 = 0.58, p ≤ 0.05). β-amylase activity in the transgenicsuspension cultures was increased to 1.5-2 times that of the wild type control. Thisled to a reduction in starch concentration of between 0.1 and 0.4 times that of thewild type control. Once again the increase in β-amylase activity could be correlated tothe reduction in starch concentration of the transgenic suspension cultures (R2 =0.68, p ≤ 0.01). In both experiments there was no significant effect on sucroseconcentration.

[发布机构] Stellenbosch University