枇杷HD-ZipⅠ转录因子家族全基因组鉴定及表达模式分析

Genome-wide identification and expression pattern analysis of HD-ZipⅠtranscription factor family in loquat

【目的】同源结构域-亮氨酸拉链(HD-Zip)转录因子参与多种植物的非生物胁迫响应过程。然而,在枇杷中,HD-ZipⅠ基因家族尚未被鉴定。【方法】利用生物信息学方法对全基因组范围内枇杷HD-ZipⅠ基因家族成员进行鉴定和综合分析,通过qRT-PCR法分析基因家族成员在枇杷不同组织和干旱胁迫下的表达特征。【结果】在枇杷基因组中筛选出20个HD-ZipⅠ家族成员。共线性分析结果发现了3对串联复制序列和22对片段复制序列,表明串联复制和片段复制可能促进了枇杷HD-ZipⅠ基因家族的扩张。蛋白序列比对分析表明,所有的HD-ZipⅠ家族成员均具有高度保守的HD和Zip结构域。系统发育分析表明,枇杷HD-ZipⅠ家族可以分为7个分支。HD-ZipⅠ各基因在枇杷不同组织中的表达模式有所差异。启动子序列分析表明,HD-ZipⅠ家族成员的启动子上含有多个与干旱胁迫和胁迫相关激素信号响应的顺式作用元件。干旱处理能够诱导EjHB9、EjHB10、EjHB17、EjHB18和EjHB20在叶片中的表达显著上调,预示着这些基因参与枇杷对干旱胁迫的响应。【结论】鉴定出5个受干旱胁迫显著诱导表达的枇杷HD-ZipⅠ基因,为进一步研究HD-ZipⅠ基因在响应枇杷干旱胁迫中的分子功能提供理论依据。

【Objective】Homologous structural domain-leucine zip(HD-Zip)transcription factors are involved in a variety of plant abiotic stress response processes.However,the HD-ZipⅠgene family has not been identified in loquat.【Methods】A genome-wide identification and analysis of the loquat HDZipⅠtranscription factor were carried out using bioinformatic methods for identification.The expression patterns of HD-ZipⅠfamily members in different tissues and by various drought treatments were examined by qPCR.【Results】A total of 20 putative loquat HD-ZipⅠfamily members were identified by searching the Big Seven Stars loquat genome database.The HD-ZipⅠmembers were further named EjHB1-EjHB20 according to their positions on 10 different chromosomes.We performed covariance analysis within the loquat genome and found 25 duplicate gene pairs in the HD-ZipⅠfamily,including3 tandem duplicate gene pairs and 22 fragment duplicate gene pairs.The nucleotide sequence identity of the HD-ZipⅠduplicate pairs ranged from 42.04%to 93.71%,and the Ka/Ks ratios ranged from 0.08to 0.43.To further investigate the phylogenetic relationships among HD-ZipⅠfamily members in different plant species,phylogenetic trees were constructed for HD-ZipⅠprotein sequences in loquat,apple,Arabidopsis thaliana and rice.The HD-ZipⅠproteins were classified into nine clades,namelyα,β1,β2,γ,δ,ε,φ1,φ2 andζ.Among them,theφ1 andζclades contained only the family members of Arabidopsis thaliana and rice,respectively,and were not clustered with the HD-ZipⅠgenes of apple and loquat.The members of the loquat HD-ZipⅠclades clustered closer to the apple homologues and further away from the rice homologues.In addition,loquat,apple,Arabidopsis and rice had the most members in theαclade,followed by theγclade.Multiple sequence comparison of 20 loquat HD-ZipⅠproteins using DNAMAN software revealed that all HD-ZipⅠproteins had HD and Zip conserved structural domains.To further investigate the relationship between loquat HD-ZipⅠproteins,we constructed a phylogenetic tree of all loquat HD-ZipⅠprotein sequences and analysed their gene structures and motifs.Similar to the results of the phylogenetic analysis described above,the loquat HD-ZipⅠgene family was divided into seven clades:α,β1,β2,γ,δ,εandφ2.Since the intron-exon structure of genes played a crucial role in the evolution of multigene families,we examined the intron-exon structures of 20 loquat HD-ZipⅠgenes to better understand their structural evolution.Theγclade members had one intron,theβ1 clade members had three introns,and the other branch members contained two introns.Combined with phylogenetic analysis,we found that genes in the same branch had similar intron-exon structures,whereas the intron-exon structures of different branches differed.To gain insight into the differences and functions of the loquat HD-ZipⅠprotein,we used the MEME programme to identify its motifs.We identified 10motifs ranging from 20 to 50 residues in length.All predicted motifs were identified only once in each HD-ZipⅠprotein.Except for motif 1,which was present in all HD-ZipⅠproteins,the remaining nine motifs were only present in certain branches.Tissue expression analysis showed that HD-ZipⅠwas found in loquat roots,stems,leaves,flowers and fruits.The results showed that EjHB3,EjHB6,EjHB8,EjHB15 and EjHB20 were mainly expressed in leaves,and EjHB9,EjHB16 and EjHB18 were mainly expressed in roots.Most members had high expression levels in stems and low expression levels in fruits.In addition,EjHB11,EjHB12 and EjHB13 were expressed at higher levels in flowers than in other tissues,while other members were also generally expressed at lower levels in flowers.Cis-acting element analysis revealed that most HD-ZipⅠpromoters contained ABRE elements,which were normally involved in ABA-related responses.And HD-ZipⅠpromoters contained drought-inducible elements(MBS),as well as defence and stress-responsive elements(TC-rich repeats).In addition,there were a number of cis-elements associated with stress response and stress-related hormone signalling,such as MYB,MYC,SA and MeJA.The HD-ZipⅠfamily contained cis-acting elements associated with drought stress.To identify the role of HD-ZipⅠin the regulation of drought tolerance in loquat,we analysed the expression of 20 HD-ZipⅠgenes under drought stress.It was shown that the expression levels of EjHB9,EjHB10,EjHB17 and EjHB18 in theγ-clade and EjHB20 in theε-branch significantly increased after drought treatment,whereas EjHB2 and EjHB19 in theβ2-branch were significantly downregulated by drought.【Conclusion】In this study,20 members of the HD-ZipⅠtranscription factor family were identified from the complete loquat protein sequence,and promoter prediction analysis indicated that they responded to drought stress.Expression analysis after drought treatment also confirmed that loquat HD-ZipⅠtranscription factors may play an important role in drought stress response.The present study may provide a reference for the future analysis of the mechanism of loquat HD-ZipⅠgenes and the development of drought-resistant breeding in loquat.