Analysis of enzymes involved in starch phosphate metabolism

[摘要] ENGLISH ABSTRACT: This project examined the role of proteins in starch phosphate metabolism. The first partwas aimed at the functional characterization of the SEX4, LSF1 and LSF2 genes in bothplants and bacteria. Constructs were produced to allow for expression of the threeproteins in E. coli with the SEX4 and LSF2 proteins being successfully purified and usedto produce antibodies. Immunoblot analysis indicated that the antibodies recognised therepective proteins in extracts, but it was not clear if they actually recognised the proteinsor the GST tags they were fused to.Virus induced gene silencing constructs were also produced to allow repression ofthese three genes in Nicotiana benthamiana. This resulted in a starch excess phenotypebeing observed in the leaves of silenced plants which is consistent with the known orpresumed roles for the genes. The antibodies produced were not specific enough toconfirm that the respective protein were actually repressed, but it is likely that this wasthe case as plants infiltrated at the same time with a VIGS vector designed to repressphytoene desaturase exhibited a chlorophyll bleaching phenotype. These data confirmthat SEX4 and LSF1 probable play the same role in N. benthamiana as in Arabidopsis,and provide evidence that LSF2 is also necessary for starch degradation.It was also attempted to characterise these proteins with respect to their substrateutilization by setting up a glyco-array experiment. Various potato starches fromgenetically modified plants were subjected to hydrolytic attack by starch degradingenzymes and fractionated by anion exchange chromatography to produce a multitude ofglucans. These will be spotted onto glass filters and probed with the purified proteins tosee if they bind to specific starch breakdown products preferentially. ivThe project also involved investigating the effect the SEX4 protein has on E. coliglycogen contents. SEX4 was expressed in wild type and glgX mutant E. coli strains as ithas been shown that this stops glycogen accumulation in the wild type, but not the glgXmutant. The cells were grown in liquid culture and glycogen contents measured. In liquidcultures SEX4 had no effect on glycogen contents in the wild type, possible because ofproblems with plasmid stability in the strain used.This final part of the project investigated the effect that a gwd mutation has oncarbohydrate metabolism in leaves and fruits of the Micro-tom tomato cultivar. Starchand soluble sugar contents were measured in leaves and ripening fruits. A starch excessphenotype was found in the leaves, but no change in starch contents was determined ineither the placenta or pericarp of the fruit. Soluble sugar contents were reduced in thefruit tissues, although the reason for this in unclear.

[发布机构] Stellenbosch University