Identification And Characterization Of The Vacuolar H~+-ATPase Subunit H Gene Family And Functional Analysis Of ZmVHA-H2 Gene

The main function of vacuolar H~+-ATPase(V-ATPase)is to translocate H~+ across biological membranes by hydrolyzing ATP,which plays an important role in plant growth and stress response.The V-ATPase H subunit(VHA-H)is essential for assembly into a stable and active enzyme.The response of VHA-H genes to salt stress has been studied in wheat and Suaeda salsa,but it has not been studied in maize.In view of the important role of VHA-H gene in plant growth and stress response,it is very meaningful to carry out research on the function of ZmVHA-H2 gene in maize.The bioinformatics method was used to identify and analyze the VHA-H gene gene family in 11 main crops.ZmVHA-H2 gene was cloned from maize inbred line 478,and analyzed the fouction under stress.The following results were obtained:1.The identification and analysis of VHA-H gene family in 11 major crops.22 VHA-H genes have identified in 11 major crop plants.Further study on the alignments and evolutionary relationships of the amino acid sequences,gene structures of exons and introns,and protein motif and alternative splice variants of these putative genes suggested the N-terminal domain is the major source of protein diversity and presumably the main functional region of species-specific adaptation,while the C-terminal domain is conserved and probably retains the original functions and characteristics of an ancient V-ATPase.The VHA-H gene family in plants showed genetic structure and transcript diversities mainly in the N-terminal domain,which presumably is the main source of functional diversity of these genes.2.The cloning of maize ZmVHA-H2 gene and its promoter.In this research,a V-ATPase H subunit gene,ZmVHA-H2 was cloned and identified from maize inbred 478.The ZmVHA-H2 gene is 1359 bp,encoding 452 amino acids and contains two domains(N-terminal and C-terminal domain).Evolution tree analysis results show that the evolution of VHA-H gene isconsistent with the evolutionary relationship of species evolution,indicating that this gene is evolutionarily conserved.Analyzed the promoter sequence of the ZmVHA-H2 gene,it can be speculated that the ZmVHA-H2 gene has many stress responses elements.3.The expression pattern of ZmVHA-H2 gene under stress.Under low-phosphorus stress,the expression of ZmVHA-H2 gene in leaves and roots was higher than that before stress,and it was especially obvious in roots.When 20% PEG was used to simulate drought stress,in both leaves and roots,the expression of ZmVHA-H2 gene decreased.Under 200 m M Na Cl stress,the expression pattern of ZmVHA-H2 gene generally decreased first and then increased in leaves,while it increased first and then decreased in roots.The experimental results showed that the ZmVHA-H2 gene responsed to low phosphorus,drought and salt stress.4.The phenotype of transgenic ZmVHA-H2 seedling under stress.Salt tolerant assay showed that the germination rate of ZmVHA-H2 transgenic lines were higher than that of WT plant under Na Cl stress.Seed germination of transgenic lines are higher than wild type under salt treatment.Water-retention experiments of transgenic Arabidopsis showed that the water-retention capacity of transgenic seedling was better at four moments of dehydration: 0.5 h,1.5 h,3.5 h,and 24 h.In conclusion,gene family identification contributed to further understanding of the structure,function,and evolution of the VHA-H genes and their important species-specific roles for crop adaptation and improvement.Overexpressed ZmVHA-H2 gene can improve plant stress resistance.This study provide new candidate genes for genetic improvement of crop stress resistance and provide new clues for understanding the resistance mechanism of V-H~+-ATPase.