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

[摘要] Pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP) is an important glycolyticenzyme and catalyses the reversible conversion of fructose-6-phosphate (Fr-6-P) andpyrophosphate (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 F1varieties. The down-regulation of PFP in cultivar NCo310 in a previous study led to an increasein sucrose accumulation and fibre content in immature tissue. Several potential transgenicsugarcane lines from genotypes 88H0019 and N27, transformed with the untranslatable sensesugarcane PFP-β gene, were characterized in this study. Initial screening for transgenesis wasdetermined by slot blot and Southern blot analysis to confirm the presence of the co-transformedselectable marker npt II transgene. Northern blot analysis confirmed expression of the 1.2 kbPFP-β transcript in 7 of 9 lines analyzed. Sugar analysis using standard South AfricanSugarcane Research Institute (SASRI) mill room practices and HPLC was performed on 12month old pot grown stalks divided into immature and mature tissue sections. The analysis ofwild type 88H0019 showed an average sucrose content of 17.84 and 30.76 g sucrose/stalk inimmature and mature tissue, respectively. However, no significant difference between theputative transgenic plant values and wild type controls was seen. PFP specific activity wasdetermined in these tissues using enzymatic assay analysis and although levels obtained inimmature tissue were between 5-18 nmol/min/mg protein, they were less than values previouslyreported in sugarcane. The results indicated that no down-regulation of PFP in immature tissueoccurred when comparing transgenic and wild type plants.A more discrete internodal tissue sampling method was used to overcome the difficulty ofdetecting small changes in PFP enzyme activity in bulked stalk tissue sections. Fine analysis ofPFP was conducted on specific developmental tissues and single stalks were divided intoimmature (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 oneach tissue type. The analysis of discrete developmental tissues showed specific PFP activity of60-80 nmol/min/mg protein in young tissue, an amount which falls in the range previouslyobtained for sugarcane. However there was no significant difference between PFP or sucrose inthe transgenic lines when compared with the wild type controls in any of the threedevelopmental tissues examined. Western blotting and densitometric analysis of the blotsconfirmed the lack of PFP down-regulation in immature tissue in all lines. A final analysis of PFP ivin immature stalk tissue on selected lines was performed using quantitative PCR, which becameavailable near the end of the study. The fold change of each transgenic line indicated that therewas 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, nofurther evidence for transgenesis could be found to support the desired effect of down-regulationof PFP. Characterization of transgenic stalks in this study was hindered by a limited number oflines available for analysis and large variability between replicate samples. Sampling techniquesemployed in an attempt to make use of existing standard SASRI mill room practices for sugaranalysis highlighted the need for a more precise sampling method, specifically when determiningthe effects of an enzyme manipulation such as PFP. A refined approach has been developedwhich will assist researchers in the choice of analytical techniques for screening andcharacterization of potential transgenic lines in the future.

[发布机构] Stellenbosch University