Cloning and expression analysis of the chloroplast fructose-1,6-bisphosphatase gene from Pyropia haitanensis
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Cloning and expression analysis of the chloroplast fructose1,6-bisphosphatase gene from Pyropia haitanensis XIAO Haidong1, CHEN Changsheng1, XU Yan1, JI Dehua1, XIE Chaotian1* 1
Fisheries College, Jimei University, Xiamen 361021, China
Received 6 March 2013; accepted 28 April 2013 ©The Chinese Society of Oceanography and Springer-Verlag Berlin Heidelberg 2014
Abstract 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 5ƍ untranslated region (UTR) of 92 bp, a 3ƍUTR 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. Key words: Pyropia haitanensis, fructose-1,6-bisphosphatase, gene cloning, qRT-PCR, RACE Citation: Xiao Haidong, Chen Changsheng, Xu Yan, Ji Dehua, Xie Chaotian. 2014. Cloning and expression analysis of the chloroplast fructose-1,6-bisphosphatase gene from Pyropia haitanensis. Acta Oceanologica Sinica, 33(4): 92–100, doi: 10.1007/s13131-0140455-0
1 Introduction Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) catalyzes irreversible hydrolysis of fructose-1,6-bisphosphate into fructose-6-phosphate with the concomitant liberation of inorganic phosphate (Marcus et al., 1986). Genetic and kinetic studies have revealed that at least two isozymes of FBPase exist in plants, the one (cFBPase) involved in gluconeogenesis and sucrose synthesis being located in the cytosol (Daie, 1993). The other isoform (cpFBPase) is present in the stroma of the chloroplasts where it is one of the key regulatory components of the reductive pentose-phosphate pathway (Calvin cycle) (Chueca et al., 2002), as it is involved in the regeneration of the acceptor molecule for CO2 fixation, ribulose-l,5-bisphosphate (RuBP). However, the cpFBPase can also involves in the formation of precursors for chloroplastic starch synthesis. That is to say, the product of cpFBPase activity, fructose-6-phosphate, not only serves the Calvin cycle but also represents the metabolite which channels photoassimilates into (transitory) starch biosynthesis in le
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