In silico cloning and bioinformatic analysis of PEPCK gene in Fusarium oxysporum

Abstract

Phosphoenolpyruvate carboxykinase (PEPCK), a critical gluconeogenic enzyme, catalyzes the first committed step in the diversion of tricarboxylic acid cycle intermediates toward gluconeogenesis. According to the relative conservation of homologous gene, a bioinformatics strategy was applied to clone Fusarium oxysporum phosphoenolpyruvate carboxykinase gene (PEPCK) by blasting search of EST database with homologous gene cDNA of Neurospora crassa and identified. Some characters of the PEPCK that were analyzed and predicted by the tools of bioinformatics in the following aspects include the composition of amino acid sequences, physical and chemical properties, O-glycosylation site, hydrophobicity or hydrophilicity, secondary and tertiary structure of the protein and function. These results showed that the full-length of PEPCK was 1771 bp and it contained a complete ORF (1575 bp), encoded 524 amino acids, which is much conserved in ascomycetes. The calculated molecular weight of PEPCK was 58358.2 Da, theoretical pI of 6.84. It has 20 α-helices, 37 sheets, and 12 glycosylation sites. It was a hydrophilic and stable protein with active site, ATP-binding site, metalbinding site and substrate-binding site.

The pepper phosphoenolpyruvate carboxykinase CaPEPCK1 is involved in plant immunity against bacterial and oomycete pathogens

“…The derived amino acid sequence encoded by CaPEPCK1 is 662 residues in length and is predicted to have a theoretical isoelectric point (pI) of 6.55 and a molecular weight of 73.2 kDa, as determined using the Expasy Compute pI/Mw tool (http://web.expasy.org/ compute_pi/). Domain search analysis of CaPEPCK1 using the SMART server (http://smart.embl-heidelberg.de/) identified a PEPCK_ATP domain at amino acid position 138-609 that is known to be conserved in bacteria, fungi, insects, and plants (Friedlander et al 1996;Bahrami et al 2001;Lee et al 2002;Li et al 2010). Sequence comparison analysis using the BLASTx server (http://blast.ncbi.nlm.…”

In Silicon Cloning and Bioinformatics Analysis of the Raphanus Sativus WUS Gene

“…With more and more EST and genome sequencing data were reported, it would become possible and feasible to isolate and identify the functional genes by in silico cloning. Many successful examples strongly support the fact that in silico cloning is absolutely a feasible tool for gene cloning and presents some advantages, compared to the traditional methods [8]. In this study, the full cDNA of Raphanus sativu WUS was obtained primarily by searching and splicing the EST sequences.…”

Monitoring of Stiffness Estimation for Tomato During Storage Period

“…Study by Khandaker et al [25] showed the similar decreasing trend in fruit physical properties, which continuous decrease in weight an diameter of the fruit as days increase. The physical properties evolution directly was effecting the value of the stiffness of the tomatoes [19], [26].…”