龙眼Aux/IAA基因家族全基因组鉴定及表达分析

Genome-wide characterization and expression analyses of the auxin/in-dole3-acetic acid(Aux/IAA)gene family in Longan(Dimocarpus longan Lour.)

【目的】Aux/IAA作为重要的生长素原初响应因子参与到植物的各个生长发育阶段。为了解Aux/IAA家族基因在龙眼花芽分化过程中的作用,对DlIAA基因家族进行全基因组鉴定和分析,并研究其在外源激素处理下的表达模式。【方法】利用生物信息学方法对Aux/IAA基因家族进行全基因组鉴定与分析,基于RNA-seq数据库分析DlIAA基因家族的表达,利用qRT-PCR探究DlIAA基因家族在龙眼花芽分化过程中和外源IAA处理下的表达情况。【结果】利用龙眼参考基因组数据,鉴定出18个DlIAA基因,依据龙眼、苹果、拟南芥和番茄之间的系统发育关系,可将进化树分为4大类7个亚族。结果表明,DlIAA基因家族与苹果具有较高的同源性,而与拟南芥、番茄的亲缘关系较远。基因结构和结构域中同一类群的不同DlIAA基因家族成员具有较高的相似度。启动子中顺式元件的分布表明,DlIAA基因表达不仅受生长素(AuxREs、TGA-element和TGA-box)的调节,还受其他激素和生长环境的调节。DlIAA基因家族在龙眼9个不同组织中表现出不同的表达模式,且部分DlIAA基因为组织特异性基因。通过对不同品种龙眼花芽的3个RNAseq数据库筛选发现,有5个候选基因(D1IAA2、D1IAA4、D1IAA7、D1IAA15和D1IAA17)均显示出较低的表达量,表明其功能与龙眼花芽分化可能存在一定的关系。进一步的qRT-PCR分析表明,DlIAA2和DlIAA17可能在开花诱导中起作用。DlIAA对外源IAA的反应结果表明,5个候选基因在100μmol·L^(-1) IAA处理下被不同程度地诱导表达,且在500μmol·L^(-1)浓度下均显著上调。【结论】转录组数据显示DlIAA基因参与了龙眼花芽分化过程,DlIAA2和DlIAA17可能在开花诱导中起作用。

【Objective】As an important auxin primary response factor,Aux/IAA is involved in all stages of plant growth and development.Auxin sensing and signal transduction are synergistic effects of various components,in which auxin/indole-3-acetic acid(Aux/IAA)protein plays a key role.Despite their physiological importance have identified in various woody plants,the functions of Aux/IAA genes in longan are still not clear.The purpose of this study was to identify Aux/IAA family genes in longan and analyze their sequences and expression,so as to understand the roles of Aux/IAA family genes in longan especially during flower bud differentiation.【Methods】Aux/IAA gene was identified from the longan genome using Arabidopsis Aux/IAA gene as reference,and the domain of the Aux/IAA gene family was further confirmed by the Hidden Markov Model(HMM).With the help of ExPASy,MEGA 6.0,DNA-MAN,Cytoscape tools and MEME,PlantCARE,STRING and iTOL online website,the bioinformatics of the gene family was analyzed.Their nucleotide sequence characteristics(including the chromosomal location,homology identification,gene structure,the promoter cis element)and the characteristics of the encoding protein(including physical and chemical properties of protein,conservative structure domain and motif,Protein-Protein Interaction network and phylogenetic analysis)were studied using the software.The expression of Aux/IAA gene was analyzed based on RNA-Seq database.Quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was used to study the expression of Aux/IAA gene during the process of flower bud differentiation and treated with exogenous IAA in longan.【Results】18 Aux/IAA genes in longan were identified using reference genomic data.Chromosomal location analysis showed that 18 Aux/IAA genes were concentrated in the scaffold range from number 1 to 3101.18 Aux/IAA genes were named DlIAA1-DlIAA18 according to their locations.The physicochemical properties of Aux/IAA(DlIAA)proteins in longan were that the isoelectric point pI ranged from 5.17(D1IAA4)to 9.31(D1IAA17),and the molecular weight ranged from 19562.08 Da(DlIAA14)to 55478.99 Da(DlIAA17).Except DlIAA5,the other DlIAA proteins were unstable hydrophilic proteins.According to the phylogenetic relationship,evolutionary tree was divided into four classes and nine sub-tribes,in which longan had high homologous with the apple,while had distant relatives with Arabidopsis thaliana.Genetic structure and multiple comparison analysis indicated that longan Aux/IAA gene families had more than one conservative amino acid sites,and contained different numbers of exons(ranged from 2 to 6).Among different branches,introns and exons varied in length.DlIAA-2,-8,-12,-18 and DlIAA-1,-2,-3,-4,-8,-14 lacked the conserved motif of DomainⅠ(motif5),DlIAA-1,-2,-3,-4,-8 and-14 lacked the conserved motif of DomainⅣ-2(motif4).A lot of cis-elements were found by the analysis of DlIAA promoters,including cis-elements related to flowering induction,such as low temperature response motif,circadian rhythm control base sequence motif and light response.The motifs associated with hormone response also were found,such as auxin,methyl jasmonic acid,gibberellin,abscisic acid and salicylic acid.The presence of these cis-regulatory elements indicated that hormones had potential regulatory effects on Aux/IAA genes.In the Protein-Protein Interaction network,a total of 20 hinge genes and 72 network lines were identified,and all the other DlIAA proteins exhibited 70%high reliability except DlIAA-2,-3,-4,-6,-9,-12,-13 and-14,which suggested that proteins of the same gene family would also have parallel functions.FPKM values of 18 DlIAAs genes in 9 different tissues of longan showed that some of the DlIAA genes were tissue-specific,nearly half of DlIAAs(DlIAA-3,-5,-7,-8,-11,-13,-14)had low expression in each tissue,while the differences of DlIAA-7,-8,-13 among tissues were the least.Through the analysis of three transcriptome data of the flower buds in different longan cultivars,five genes(DlIAA-2,-4,-7,-15,-17)were found to have low expression levels during the flower bud differentiation of common longan cultivars and in the grafting newly-sprouting buds of Sijimi.Further qRT-PCR analysis showed that the expression levels of DlIAA2 and DlIAA17 were significantly decreased in longan buds in November and December,which was the phase of physiological differentiation of longan flowering,suggesting that these genes might play a role in flowering induction.Treated with exogenous IAA,the expression levels of DlIAA-7,-15 were significantly suppressed under the 100μmol·L^(-1) IAA treatment,while the expression levels of all five candidate genes increased significantly under 500μmol·L^(-1) IAA concentration.【Conclusion】We identified 18 Aux/IAA members from longan genome,and found that DlIAA2 and DlIAA17 might play a key role in longan flower bud differentiation.Our results would provide comprehensive information on the genetic structure,phylogenetic relationships and expression profiles of Aux/IAA families in longan,and offer a new insight into the complexity of IAAs regulation at transcriptional level.It may be helpful to develop breeding strategies to promote the development of flowering in longan.