Identification Of HXK Gene Family In Zea Mays And Functional Analysis Of ZmHXK6?ZmHXK7 Genes To Improve Salt Tolerance In Arabidopsis Thaliana

Hexokinase(HXK)is a promoter enzyme of glucose involved in glycolysis,which not only regulates plant respiration,but also plays an important role in the induction and transmission of sugar signals.In this study,the members of maize HXK gene family were identified by bioinformatics method,and the tissue differential expression of this gene family was analyzed under salt and drought stress.The maize Zm HXK6 and Zm HXK7 genes were cloned to construct expression vectors,and the mutant Arabidopsis thaliana athxk3 was transformed by dips method to obtain homozygous transgenic plants.to study the function of Zm HXK6 and Zm HXK7 genes in the process of stress resistance.The main results are as follows:(1)Based on the online database,we identified 10 members of maize HXK gene family.Chromosomal location prediction showed that these members were distributed on 4 of the 10 chromosomes of maize.Subcellular location prediction predicted that maize HXK family members were located in cytoplasm or mitochondria.Phylogenetic analysis showed that maize HXK family could be divided into four subgroups.Multiple sequence alignment showed that most maize HXK proteins had phosphoric acid sites and sugar binding domains.The gene structure and promoter prediction analysis of maize HXK gene family showed that the members of maize HXK gene family were relatively conservative in gene structure and most of them contained 9 exons.The promoter regions of HXK gene family members in maize have multiple stress response elements of hormones?growth and abiotic factors.Real-time quantitative PCR analysis showed that Zm HXK5,Zm HXK6,Zm HXK7 and Zm HXK8 genes were mostly upregulated in maize leaves and roots under salt and drought stress,suggesting that maize HXK gene family may be involved in the response to salt and drought stress.(2)The target genes Zm HXK6(Zm00001D043511?T002)and Zm HXK7(Zm00001D037689?T002)were cloned,and their CDS sequences were 1221 bp and841 bp,respectively.The molecular weights were 44.6 KDa and 30.1 KDa,respectively.After homozygous mutants athxk3(Salk?022188C)and athxk5(Salk?080584C)were identified,they were screened for salt tolerance and the mutant athxk3 was selected as the transgenic receptor plant.The recombinant expression vector of the target gene was constructed by Gateway technology.After screening and identification,homozygous transgenic Arabidopsis thaliana was obtained: athxk3 35S::Zm HXK6 and athxk335S::Zm HXK7.(3)We used molecular biology techniques to identify homozygous transgenic Arabidopsis thaliana at DNA,transcription and protein expression levels;Subcellular localization showed that the target gene proteins were localized in the cytoplasm.Transgenic Zm HXK6 and Zm HXK7 genes significantly increased the hexokinase(HK)activity of Arabidopsis thaliana mutants.(4)Transgenic Zm HXK6 and Zm HXK7 could restore the mutant's plant height to different degrees.athxk3 35S::Zm HXK6 and athxk3 35S:: Zm HXK7 were 15.1% and51.4% higher than athxk3,respectively.The growth of branches and pods was also more lush than that of athxk3.These results indicated that Zm HXK6 and Zm HXK7 were involved in regulating the growth and development of Arabidopsis thaliana to different degrees.(5)Under different salt concentrations,the dry and fresh weight,lateral root number and chlorophyll content of transgenic Arabidopsis thaliana with Zm HXK6 and Zm HXK7 genes were higher than those of mutant Arabidopsis thaliana,while the content of proline increased and the content of malondialdehyde decreased,indicating that transgenic Arabidopsis thaliana was less susceptible to salt toxicity than mutant Arabidopsis.In other words,transgenic Zm HXK6 and Zm HXK7 genes can improve the salt tolerance of Arabidopsis mutants.(6)Under the treatment of salt and glucose,the application of glucose alone promoted the growth of Arabidopsis thaliana.Compared with the treatment of salt alone,the exogenous application of glucose increased the dry and fresh weight of plants under salt stress,increased the content of proline,decreased the content of malondialdehyde,and alleviated the damage of salt stress to Arabidopsis thaliana,the recovery effect of transgenic Arabidopsis thaliana was better than that of mutant.In conclusion,Zm HXK6 and Zm HXK7 genes are involved in regulating the growth and development of Arabidopsis thaliana and improving salt tolerance;the application of exogenous glucose can alleviate the damage of salt stress to Arabidopsis thaliana and promote plant growth.