Analysis And Functional Study Of HD-Zip Gene Family In Barley

Barley(Hordeum vulgare L.)is the fourth largest crop in the world after wheat,rice and corn.It is one of the oldest foods and feed crops in the world and has high economic value.The latest physical,genetic,and functional sequence assembly of the barley genome was completed in 2012 and 2016,respectively,and provides important reference materials for future research in crop breeding,improvement,gene function,and evolution.Barley has strong drought resistance,low temperature resistance and salt and alkali resistance,and the regulation of environmental adaptation mechanisms is more complicated.Homeodomainleucine zipper(HD-Zip)proteins belong to the Homeobox(HB)protein superfamily and are a type of plant-specific transcription factors.Previous studies have shown that the HD-Zip transcription factor family is related to plant epidermal development,cuticle development and stress resistance.In this study,using the ‘Morex' genomic data of barley as a reference,the members of the HD-Zip gene family were identified,and the characteristics and evolutionary relationships of the members of the HD-Zip gene family were analyzed through bioinformatics analysis;Analyze the transcriptome data of this family member to determine its tissue expression pattern,and treat barley seedlings under drought,cold,and salt stress.The q RT-PCR experiment was used to study the response rules of barley HD-Zip gene family members under different abiotic stresses.The codon preference of this family was analyzed,and the results of the previous expression pattern analysis were used to select the barley HD-Zip gene that is representative of the family and had a significant stress response.The function of the barley HD-Zip gene was studied using the Arabidopsis genetic transformation system.The results are as follows:(1)A total of 32 non-redundant barley HD-Zip genes were identified,named Hv HD-Zip 1-32,and divided into subfamily I-IV based on the identified and bound DNA sequences,conserved motifs,and protein functions.The Hv HD-Zip family gene coding sequence(CDS)is 594 bp to 2949 bp in length,and the corresponding protein is 198 to 983 amino acids in length.Hv HD-Zip proteins are all hydrophilic and localized in the nucleus.The chromosome distribution showed that the overall distribution of Hv HD-Zip gene was more balanced on 7 chromosomes of barley.Each of the 32 Hv HD-Zip proteins contains corresponding conserved motifs constituting the HD and LZ domains,however,HD-Zip III and HD-Zip IV subfamily members have more complex domains than HD-Zip I and HD-Zip II subfamilies.(2)The results of synteny analysis show that there are 26,231,and 499 genomewide collinear blocks between barley and Arabidopsis,rice,and wheat,which contains 3(9.4%),14(43.8%),and 25(78.1%)HD-Zip gene pairs,respectively.Interestingly,no segmental or tandem duplication events were identified in the Hv HD-Zip gene family from the 1593 gene pairs in genome-wide duplication events.Statistics found that the number of HD-Zip members in barley was significantly less than the number of members reported in the other 13 plant species.(3)Analysis of tissue expression patterns showed that most Hv HD-Zip genes(67.7%)had low expression levels,but their expression was still significantly tissuespecific.Real-time quantitative PCR(q RT-PCR)analysis showed that the stress response patterns of Hv HD-Zip gene were very different under different tissues.The specific performance is that the Hv HD-Zip gene shows high sensitivity to drought stress in barley root tissue,while it shows a significant cold stress response in leaf tissue.Moreover,the stress response of HD-Zip I and HD-Zip IV subfamilies is significantly higher than HD-Zip II and HD-Zip III subfamilies.(4)The barley HD-Zip family gene favored codons ending in C or G.The most frequently used codon was CUG and two optimal codons are AUC and AGG.The GC content of the third base of the triplet codon(GC3)and the effective number of codons(ENC)prove that the codons of this family of genes have a low preference for codons and are mainly affected by mutation pressure during evolution.Cluster analysis showed that HD-Zip gene clustering based on relative synonymous codon usage(RSCU)was no trelated to species evolution.The optimal receptor analysis of Hv HD-Zip IV 5,are presentative gene of barley HD-Zip gene family,found that E.coli is more suitable as a heterologous expression receptor,and rice is more suitableas as genetic transformation model plants for this gene.However,in order to perform heterologous expression with high efficiency,the codon of Hv HD-Zip IV 5 gene needs to be modified and optimized.(5)The CDS sequence of Hv HD-Zip IV 5 gene in barley and Tibet hulless barley was 2388 bp in length and encoded 796 amino acids.Sequence alignment revealed that there were 8 SNP loci between the two species,but did not show spatial structure differences.The q RT-PCR experiment and transcriptome data analysis were used to determine the expression of Hv HD-Zip IV 5 in the roots,stems,leaves,flowers,and seeds of barley and Tibet hulless barley,the expression of this gene in young tissues was higher than that in mature tissues.It has a high level of expression in leaves and has tissue specificity.Hv HD-Zip IV 5 gene showed different response patterns between different species of barley and Tibet hulless barley seedlings under drought,cold and salt stress.This indicates that the gene is involved in the adaptive regulation of plants to different abiotic stresses,and has tissue and species differences.