Loquat Pyruvate Kinase Molecular Mechanism Research

The last step of pyruvate kinase catalyzeing glycolysis is an irreversible reaction, which plays animportant role in glycolytic pathway. In this study,the full-length sequence of loquat pyruvate kinasegene was isolated, and analyzed by subcellular localization. To study the function of this gene, weconstructured a binary expression vector, and introduced it into Nicotiana tabacum L. cv. NC89. Themorphological and physiological function of transgenic tobacco were exploreed to explain themolecular mechanism regulated by loquat pyruvate kinase. The main results are as follows:The conserved fragment of interested cDNA was obtained by RT-PCR. The full length of theLoquat pyruvate kinase cDNA was isolated by5’-RACE and3’-RACE, and named as EjPK. The clonecontains1884nucleotides with a5’UTR of161bp, a3’UTR of193bp and a coding region of1530bpcomprising510amino acid. The deduced nucleic acid and amino acid sequences of EjPK showed78%—84%homology with other cytoplasm pyruvate kinase from various species. EjPK belongs to thebranch of the cytoplasm pyruvate kinase, which have high identities with PK from Vitis vinifera,max(L)Merril,and Citrus L.The ORF region of EjPK was ligated to the upstream of GFP in pCAMBIA-1302by CaMV35Spromoter. The recombinant plasmid was transformed into onion epidermal cells by genegun. Theconfocal imaging of GFP fluorescence showed that35S:EjPK-GFP fusion protein is mainly in thecytoplasm, partly in the cell membrane, but have no fluorescent signal in the nucleus.The composition and amount of sugar and organic acid in different developmental stage of white-fleshed cultivar and red-fleshed cultivar were measured by HPLC. The results indicate that sucrose,glucose, fructose and sorbitol are the main sugar in loquat fruit. Fructose and glucose increased with thematurity of the fruit. The fluctuation of sucrose content is slight. The organic acid loquat fruit iscomposed of malic acid, citric acid, quinic acid, alpha ketone glutaric acid, succinic acid andoxaloacetic acid. Almost all of them increase gradually and fall in later period. Real time PCR revealed that EjPK was detected from different organizations (root, stem, leaf,flower, fruits), and decline in the early developmental stage of fruits, then increase at later stage, but fallduring maturation.The EjPK ORF were introduced into tobacco via Agrobacterium-mediated transformation. PCRscreening showed that the target gene were integrated into the tobacco genome. The detection result ofphenotype and physiology showed that overexpression EjPK plants flowered earlier and had variationalcontent of sugar in the early stage. In total, the results of our study provides a theoretical basis toexplore the regulatory function of pyruvate kinase in glycometabolism, and provide a new insight intothe mechanism of fruit quality in loquat.