Study On Bioinformatics Analysis And Functional Verification Of Loquat Sugar Metabolism Of The Key Enzyme HXK Gene Families

Hexokinase which is one of the vital enzymes in sugar metabolism process is the first step involved in glycolysis reaction of irreversible enzymes and also has the function of the catalytic hexose phosphorylation. Based on RNA- seq sequencing of loquat hexokinase bioinformatics analysis, this study transfered the isolated cloned loquat HXK to tobacco, and then made a preliminary validation of the functions of loquat hexokinase in a manner of physiological phenotypic analysis and determination of sugar content of transgenic tobacco plant.The main outcome was as follows.Taking use of the equipment such as NCBI, Ex PASy, and so forth,to analyze the bioinformatics information of RNA – seq ascribed to its unique sequence. The sequential analysis indicates all of its7 EjHXK are to comprise a complete open reading frame, encoding 497 amino acids; Protein analysis showed that molecular mass of protein designed by them is roughly 53 kDa, together with not only its pI ranged from 6.1 to 6.8, but 1 ~ 2 period of transmembrane region, which suggested it is belong to the stability of the hydrophilic membrane protein called zero-signal peptide; Secondary structure mainly consists of both alpha helix and curl at random while there are multiple polypeptide chain cysteine residues site. Conservative area of protein structure forecast contains nucleotide binding site and homologous dimer interface where rich protein phosphorylation protein binding sites and functional sites attach, the prediction analysis reveals both can regulate the function and structure of the protein by means of protein phosphorylation, protein glycosylation as well as acetoxylation.The investigation employing the instruments such as DNAMAN, MEGA5.0 analysis of Ej HXK sequence homologous gene families illustrated that the homology among them is up 95% to 98% consistented with other family members’ similarity and homology. It is higher and and more conservative compared with hexokinase protein sequence homology of woody plant in particular. from the standpoint of phylogenetic relationship analysis, as far as hexokinase concerned there is no obvious boundaries between monocotyledon and dicotyledon clustering distribution,nevertheless EjHXK has higher homology than that in apple,Chinese white pear and cacao tree.The determination of the T1 generation whose EjHXK were obtained through the method of agrobacterium mediated genetic transformation of tobacco transgenic plants of transgenic plants uncovers that excessive expression EjHXK gene resulted in early flowering 12 to 15 days when compared with wild types of tobacco plants.Using HPLC to determine the sugar content of EjHXK transgenic tobacco leaves in different periods,which discovered that the content of glucose and fructose in the transgenic tobacco plants and wild type increased with their growth, but the decreased after the appearance of plant peak,also suggested the trend of the content of glucose was low and the trend of that was unconspicuous.Compared with wid type tobacco, the content of glucose and fructose showed downtrend at a rate of 0.5~1.0mg/mL in different periods.The peak of the total content of glucose,fructose and sucrose which was flowering stage appeared in 75 d and 90 d respectively in tobacco with EjHXK gene and wild type tobacco,which showed the similar trend with phenotype observation.Thus verified loquat hexokinase was capable of catalyzing the phosphorylation of hexoses, involved in regulation of glycometabolism, and promoted plant growth and development.This experiment through the gene and protein sequence analysis and genetic transformation research gives preliminary identification of the loquat EjHXK gene’s functionality, it lays a foundation of further study on loquat glycometabolism as well.