Genome-wide screening of hexokinase gene family and functional elucidation of HXK2 response to cold stress in Jatropha curcas

Hexokinase, the key rate-limiting enzyme of plant respiration and glycolysis metabolism, has been found to play a vital role in plant sugar sensing and sugar signal transduction. Using Jatropha curcas genome database and bioinformatics method, J. curcas HXK gene family (JcHXK) was identified and its phylogenetic evolution, functional domain, signal peptide at the N-terminal, and expression analysis were conducted. The results showed that a total of 4 HXK genes (JcHXK1, JcHXK2, JcHXK3, and JcHKL1) with 9 exons were systematically identified from J. curcas. JcHXK1, JcHXK3, and JcHKL1 with putative transmembrane domain at the N-terminal belonged to the type of secretory pathway protein, and JcHXK2 contained putative chloroplast targeting peptide. Quantitative real-time PCR (qRT-PCR) analysis revealed that all the four JcHXKs were expressed in different tissues of the leaves, roots, and seeds; however, JcHXK1 and JcHKL1 expression were higher in the roots, whereas JcHXK2 and JcHXK3 showed over-expression in the leaves and seeds, respectively. Furthermore, all the four JcHXKs were up-regulated in the leaves after cold stress at 12 °C; however, only JcHXK3 remarkably demonstrated cold-induced expression in the roots, which reached the highest expression level at 12 h (2.28-fold). According to the cis-acting element analysis results, JcHXK2 contained the most low temperature responsive elements, which was closely related to the cold resistance in J. curcas. A pET-28a-JcHXK2 prokaryotic recombinant expression vector was successfully constructed and a 57.0 kDa protein was obtained, JcHXK2 revealed catalytic activity towards glucose and fructose, with a higher affinity for glucose than fructose. The subcellular localization assays revealed that JcHXK2 was localized in the chloroplast. The results of this study might provide theoretical foundation for further studies on gene cloning and functional verification of HXK family in J. curcas.