Characterization of transgenic sugarcane lines with perturbed pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity

Spracklen, Ashley Lindsay
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Stellenbosch : University of Stellenbosch
Pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP) is an important glycolytic enzyme and catalyses the reversible conversion of fructose-6-phosphate (Fr-6-P) and pyrophosphate (PPi) to fructose 1,6-bisphosphate (Fr-1,6-P2) and inorganic phosphate (Pi). Sugarcane PFP has been inversely correlated with sucrose content across segregating F1 varieties. The down-regulation of PFP in cultivar NCo310 in a previous study led to an increase in sucrose accumulation and fibre content in immature tissue. Several potential transgenic sugarcane lines from genotypes 88H0019 and N27, transformed with the untranslatable sense sugarcane PFP-β gene, were characterized in this study. Initial screening for transgenesis was determined by slot blot and Southern blot analysis to confirm the presence of the co-transformed selectable marker npt II transgene. Northern blot analysis confirmed expression of the 1.2 kb PFP-β transcript in 7 of 9 lines analyzed. Sugar analysis using standard South African Sugarcane Research Institute (SASRI) mill room practices and HPLC was performed on 12 month old pot grown stalks divided into immature and mature tissue sections. The analysis of wild type 88H0019 showed an average sucrose content of 17.84 and 30.76 g sucrose/stalk in immature and mature tissue, respectively. However, no significant difference between the putative transgenic plant values and wild type controls was seen. PFP specific activity was determined in these tissues using enzymatic assay analysis and although levels obtained in immature tissue were between 5-18 nmol/min/mg protein, they were less than values previously reported in sugarcane. The results indicated that no down-regulation of PFP in immature tissue occurred when comparing transgenic and wild type plants. A more discrete internodal tissue sampling method was used to overcome the difficulty of detecting small changes in PFP enzyme activity in bulked stalk tissue sections. Fine analysis of PFP was conducted on specific developmental tissues and single stalks were divided into immature (internodes 1-3), maturing (internodes 4-5) and mature (internodes 7-8) regions. Sucrose analysis was performed using HPLC and PFP activity was determined enzymatically on each tissue type. The analysis of discrete developmental tissues showed specific PFP activity of 60-80 nmol/min/mg protein in young tissue, an amount which falls in the range previously obtained for sugarcane. However there was no significant difference between PFP or sucrose in the transgenic lines when compared with the wild type controls in any of the three developmental tissues examined. Western blotting and densitometric analysis of the blots confirmed the lack of PFP down-regulation in immature tissue in all lines. A final analysis of PFP iv in immature stalk tissue on selected lines was performed using quantitative PCR, which became available near the end of the study. The fold change of each transgenic line indicated that there was a minor increase in PFP confirming the lack of effect of transgenesis. Although evidence for the expression of the PFP-β transgene was seen in the northern blot, no further evidence for transgenesis could be found to support the desired effect of down-regulation of PFP. Characterization of transgenic stalks in this study was hindered by a limited number of lines available for analysis and large variability between replicate samples. Sampling techniques employed in an attempt to make use of existing standard SASRI mill room practices for sugar analysis highlighted the need for a more precise sampling method, specifically when determining the effects of an enzyme manipulation such as PFP. A refined approach has been developed which will assist researchers in the choice of analytical techniques for screening and characterization of potential transgenic lines in the future.
Thesis (MSc (Genetics))--University of Stellenbosch, 2009.
Dissertations -- Plant biotechnology, Theses -- Plant biotechnology