Cloning and expression analysis of the chloroplast fructose-1,6-bisphosphatase gene from Pyropia haitanensis

Fructose-1,6-bisphosphatase（FBPase） is one of the key enzymes in Calvin circle and starch biosynthesis. In this study, the full-length of cpFBPase gene from Pyropia haitanensis was cloned by using rapid amplification of cDNA ends（RACE） technology. The nucleotide sequence of PhcpFBPase consists of 1 400 bp, including a 5′ untranslated region（UTR） of 92 bp, a 3′？UTR of 69 bp, and an open reading frame（ORF） of 1 236 bp, which can be translated into a 412-amino-acid putative peptides with a molecular weight of 44.3 kDa and a theoretical pI of 5.23. Multiple sequence alignment indicated that the protein belonged to the chloroplast FBPase enzyme. Phylogenetic analysis showed that the protein assembled with the cpFBPase of a thermal tolerant unicellular red micro-algae Galdieria sulphuraria. Expression patterns analyzed by qRT-PCR revealed that the expression of PhcpFBPase gene in the thallus phage was 7-fold higher than in the conchocelis phage, which suggested the different mechanisms of inorganic carbon utilization among the different life phages of P. haitanensis. And the different response modes of PhcpFBPase mRNA levels to high temperature and desiccation stress indicated that PhcpFBPase played an important role in responsing to abiotic stress.

Isolation of a 1 195 bp 5′-Flanking Region of Rice Cytosolic Fructose-1,6-bisphosphatase and Analysis of Its Expression in Transgenic Rice

A genomic DNA fragment containing the 5'-upstream sequence and part of the open reading frame corresponding to the cytosolic fructose-1,6-bisphosphatase (cyFBPase) cDNA was isolated by Genome Walking. The 1 195 lip 5'-flanking region which started from the translation initiation ATG codon was fused to reporter gene encoding beta-glucuronidase (GUS) and stably transferred to rice via particle bombardment. Strong GUS activity was detected in leaves and leaf sheaths of transgenic rice, but not in culms and roots. Histochemical localization revealed that GUS expression was exclusively restricted to mesophyll cells in transgenic rice. Our results indicate that the 1 195 bp fragment contains all the cis-elements required for directing mesophyll-specific expression pattern in rice.

Overexpression of Brassica napus cytosolic fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase genes significantly enhanced tobacco growth and biomass

Elevated activities of cytosolic fructose-1,6-bisphosphatase(cyFBPase) and sedoheptulose-1,7-bisphosphatase(SBPase)are associated with higher yields in plants. In this study, the expression levels of the cyFBPase and SBPase genes were increased by overexpressing rape(Brassica napus) cDNA in tobacco(Nicotiana tabacum) plants. The transgenic plants coexpressing cy FBPase and SBPase(TpFS), or expressing single cy FBPase(TpF) or SBPase(TpS) had 1.77-, 1.55-, 1.23-fold cyFBPase and 1.45-, 1.12-, 1.36-fold SBPase activities as compared to the wild-type(WT), respectively. Photosynthesis rates of TpF, TpS and TpFS increased 4, 20 and 25% compared with WT plants. The SBPase and cyFBPase positively regulated each other and functioned synergistically in transgenic tobacco plants. In addition, the sucrose contents of the three transgenic plants were higher than that of WT plants. The starch accumulation of the TpFS and TpS plants was improved by 53 and 37%, but slightly decreased in TpF plants. Moreover, the transgenic tobacco plants harbouring SBPase and/or cyFBPase genes showed improvements in their growth, biomass, dry weight, plant height, stem diameter, leaf size,flower number, and pod weight. In conclusion, co-expression of SBPase and cyFBPase may pave a new way for improving crop yield in agricultural applications.

Status epilepticus due to fructose-1,6-bisphosphatase deficiency caused by FBP1 gene mutation

Introduction:Fructose-1,6-bisphosphatase (FBPase) deficiency is a rare inherited disorder in gluconeogenesis,characterized by hypoglycemia,ketonuria,metabolic acidosis and convulsions.Case presentation:We describe two brothers with FBPase deficiency.The proband developed severe hypoglycemia and progressed to status epilepticus,and the brother showed slightly hypoglycemia with a good prognosis.Whole exome sequencing (WES) identified compound heterozygous variants [c.333+1333+2delinsTC and c.490G>A (p.Gly164Ser)] in fructose-1,6-bisphosphatase 1 gene in the two brothers,which were inherited from the father and the mother,respectively.Conclusion:Genetic analysis provided a solid basis for a definite diagnosis and the determination of precision therapies for the patient.

Vacuole import and degradation pathway: Insights into a specialized autophagy pathway

Glucose deprivation induces the synthesis of pivotagluconeogenic enzymes such as fructose-1,6-bisphos-phatase, malate dehydrogenase, phosphoenolpyruvatecarboxykinase and isocitrate lyase in Saccharomycescerevisiae. However, following glucose replenishment,these gluconeogenic enzymes are inactivated and de-graded. Studies have characterized the mechanismsby which these enzymes are inactivated in response toglucose. The site of degradation of these proteins hasalso been ascertained to be dependent on the dura-tion of starvation. Glucose replenishment of short-termstarved cells results in these proteins being degradedin the proteasome. In contrast, addition of glucose tocells starved for a prolonged period results in theseproteins being degraded in the vacuole. In the vacuoledependent pathway, these proteins are sequestered inspecialized vesicles termed vacuole import and degra-dation (Vid). These vesicles converge with the endo-cytic pathway and deliver their cargo to the vacuolefor degradation. Recent studies have identified thatinternalization, as mediated by actin polymerization, isessential for delivery of cargo proteins to the vacuolefor degradation. In addition, components of the targetof rapamycin complex 1 interact with cargo proteins during glucose starvation. Furthermore, Tor1p dissoci-ates from cargo proteins following glucose replenish-ment. Future studies will be needed to elaborate on the importance of internalization at the plasma membrane and the subsequent import of cargo proteins into Vid vesicles in the vacuole dependent degradation pathway.