Identification Of Hexokinase Gene Family In Glycine Max And Functional Research Of GmHXK2 Under Salt Stress

Hexokinase(HXK)is the key enzyme of glucose glycolysis in plants,which plays an important role in sensing sugar signal and responding to abiotic stress.HXK has been widely studied in many species,but the detailed information of HXK gene family in Glycine max has not been reported yet.In this study,HXK gene family members in Glycine max were systematically analyzed by bioinformatics method,and q RT-PCR was performed to explore the expression pattern of GmHXKs under salt and drought stress.By cloning the GmHXK2 gene of Glycine max,expression vector was constructed,and then Wild-type Arabidopsis and homozygous mutants(athkl1,athxk2 and athxk3)were transformed to obtain transgenic lines to further verify the function of the GmHXK2 gene.The results are as follows:1.Based on the genomic information of Glycine max,17 HXK members were identified.The length of the DNA varied from 1058 to 8549 bp and CDS were 471 to1812 bp in length,encoding 156 to 504 amino acids and corresponding to molecular weights ranging from 17.42 to 55.40 k Da.It were predicted that most GmHXKs were acidic(p I<7).HXK members in Glycine max were predicted to be located in the chloroplast and mitochondria,and six of them had chloroplast turning peptide(CTP).There were 20 chromosomes in Glycine max,and GmHXKs genes were distributed on9 chromosomes.Multiple sequence alignment analysis showed that most GmHXKs were multidomain proteins,which contained two phosphate sites(I and II),two connect sites(I and II),one α-helix site,one adenosine binding site,and one sugar binding site.HXK members from Arabidopsis thaliana,Solanum lycopersicum and Glycine max were divided into three categories by phylogenetic tree.The gene structure and conserved motifs of most GmHXKs were similar,composed of 9 exons and 8 introns,and contained 10 conserved motifs.In addition,the promoters of GmHXKs contained cis-elements involved in plant growth and development,phytohormone response elements such as gibberellin,auxin,abscisic acid and son on,and abioc stress response elements such as salt,drought,cold and so on.2.q RT-PCR was performed to analyze the expression pattern of HXK genes in Glycine max under drought and salt stress.Under salt stress,the expression of GmHXK10,GmHXK11 and GmHXK16 were significantly up-regulated in the roots,and GmHXK12 was significantly expressed in the leaves,while the expression of GmHXK4 and GmHXK7 were significantly down-regulated.GmHXK14-17 genes was significantly expressed in the leaves responsing to drought stress,and the expression of most GmHXK genes were significantly up-regulated in the roots after 2 h of drought treatment.3.GmHXK2(Gene ID:GLYMA＿09G144600)was 2033 bp in length,encoded296 aa,had a relative molecular weight of 32.92 k Da,and contained 9 extrons and 8introns.GmHXK2 gene was constructed into plant expression vector p MDC83 after being cloned,and then transformed into Arabidopsis thaliana WT and homozygous mutants athkl1,athxk2 and athxk3.4.The GmHXK2 transgenic plants were identified to confirm that GmHXK2 gene was successfully transferred into Arabidopsis and expressed the target protein.The subcellular localization was observed by laser confocal microscope,and it was found that GmHXK2 was located on vacuole membrane and cell membrane.In addition,the hexokinase activity of transgenic plants was significantly higher than that of untransformed plants,which indicating that GmHXK2 had hexokinase activity.5.WT,mutants and transgenic plants were treated with salt and glucose.It was found that the growth of GmHXK2 transgenic plants was significantly better than that of untransformed Arabidopsis under salt stress,and the root length,dry and fresh weight,chlorophyll and proline contents increased,while malondialdehyde content decreased,which indicated that the heterologous expression of GmHXK2 gene promoted the growth of plants and enhanced the salt tolerance of plants.Compared with seedlings under salt treatment,the growth of Arabidopsis plants treated with salt and 100 m M glucose was better,and the root length,dry and fresh weight,chlorophyll and proline contents increased,while malondialdehyde content decreased,indicating that 100 m M exogenous glucose could alleviate the inhibition of salt stress on plant growth.In this study,the physicochemical characteristics,chromosome location,multiple sequence alignment,phylogenetic relationship,gene structure and conserved motifs,cis-acting elements and expression patterns of HXK members under salt and drought stress were systematically analyzed,and the characteristics of GmHXKs were revealed.The experiments of GmHXK2 transgenic Arabidopsis under salt stress proved that the expression of GmHXK2 could promote the growth of plants,and enhanced the salt tolerance of plants,which provided theoretical basis for the breeding of stress resistant crop in saline alkali areas.