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.

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.

AMP makes native snake muscle fructose-1,6-bisphosphatase to an alkaline enzyme

A substance in the crude preparation of NADP+ has been found, which activates snake muscle fructose-1,6-bisphosphatase at pH 9.2 and inhibits the enzyme at pH 7.5. After isolation and extensive characterization, the substance has been determined to be AMP. The activation depends on the concentrations of Mg2+ and could be observed only at concentrations above 1 mmol/L. In the presence of AMP, snake muscle fructose-1,6-bisphosphatase resembles an alkaline enzyme. Kinetic studies indicate that AMP and Mg2+ competitively regulate the activity of the enzyme. AMP releases the inhibition of Mg2+ at high concentration at alkaline pH. It has been reported that fructose-1,6-bisphosphatase with a pH optimum in the alkaline region is caused by limited proteolysis. AMP is also able to make fructose-1,6-bisphosphatase to be an alkaline enzyme. This finding indicates that proteolysis may not be the only reason for shift of the optimum pH of fructose-1,6-bisphosphatase to alkaline side and it may imply some significance in physiological regulation.