喜旱莲子草AP2/ERF基因家族的鉴定及其在除草剂胁迫下的表达模式

Genome-Wide Identification of AP2/ERF Gene Family in Alternanthera philoxeroides and Its Expression Patterns Under Herbicide Stresses

【背景】喜旱莲子草(Alternanthera philoxeroides)是一种极难防除的恶性入侵杂草,给我国生态环境造成了严重危害。AP2/ERF(APETALA2/ethylene responsive factor)家族是植物中最大的转录因子家族之一,不仅参与植物体内多种信号网络的调控,还在植物响应除草剂胁迫中发挥重要作用。【目的】系统分析喜旱莲子草ApAP2/ERF家族成员的基本特征,揭示其在除草剂胁迫下的表达模式,明确ApAP2/ERF潜在的生物功能,挖掘抗除草剂的潜在靶标基因,为精准、合理地选择除草剂提供依据。【方法】利用本地BLASTp从喜旱莲子草基因组数据库中对AP2/ERF家族成员进行鉴定,运用MEME、ExPASyServer10、Plant-mPLoc、SWISS-MODEL、NCBI SRA数据库和psRNA Target在线网站获取保守基序(Motif)、蛋白理化性质、亚细胞定位、三级结构、转录组、靶向miRNA信息。通过GFF3基因组注释文件获取基因结构信息。利用MEGA 11、TBtools和R语言绘制系统发育树、表达模式热图和miRNA靶向关系图等。采用RT-qPCR法分析ApAP2/ERF家族成员在5种除草剂和不同时间点(0—7 d)处理下的表达模式。【结果】从喜旱莲子草中共鉴定出96个ApERF、9个ApAP2和4个ApRAV,并根据其在基因组中的位置分别命名为ApERF1—ApERF96、ApAP2-1—ApAP2-9和ApRAV1—ApRAV4。鉴定到的ApAP2/ERF均为亲水蛋白,位于细胞核和细胞质中;ApAP2/ERF表达模式受地理条件、水分条件和低钾胁迫的调控。41%草甘膦处理中,ApERF7/74/94在3—7 d内被高度诱导;50%异丙隆处理中,6个ApERF均在特定的时间被高度诱导;10%乙羧氟草醚处理中,ApERF7/13/49/94表达量呈现先升高后下降再上升的趋势;20%氯氟吡氧乙酸处理中,ApERF7/13/49/54/94在0.5 d时被高度诱导;13%噁草酮处理中,ApERF13/49/54/74在短时间内显著下调表达。【结论】鉴定出109个喜旱莲子草AP2/ERF家族成员,位于同一亚组的成员具有相似的motif。ApAP2/ERF的表达受到地理条件、水分条件和低钾胁迫的调控,同时也受除草剂的诱导,推测其通过影响乙烯信号通路以响应除草剂胁迫。

【Background】Alternanthera philoxeroides is a malignant invasive weed that is extremely difficult to control,causing serious harm to ecology and environment in China.The AP2/ERF(APETALA2/ethylene responsive factor)family is one of the largest transcription factor families in plants,which not only participates in the regulation of various signal networks in plants,but also plays an important role in plant response to herbicides.【Objective】The objective of this study is to systematically analyze the basic characteristics of ApAP2/ERF,reveal its expression patterns under herbicide stress,decipher the biological functions of ApAP2/ERF in response to herbicide stress,identify potential target genes for herbicide resistance,and to provide a theoretical basis for accurate and reasonable selection of herbicides.【Method】The AP2/ERF family members were identified from A.philoxeroides genome database using local BLASTp.MEME,ExPASyServer10,Plant-mPLoc,SWISS-MODEL,NCBI SRA database,and psRNA Target online website were used to obtain conserved motif,protein physicochemical property,subcellular localization,tertiary structure,transcriptome,and targeted miRNA information.Gene structure information was obtained from the GFF3 genome annotation file.Phylogenetic tree,expression pattern heatmap,and miRNA target relationship network were constructed using MEGA 11,TBtools,and R software.The expression patterns of AP2/ERF family members in response to five herbicides and at different time points(0-7 d)were analyzed using RT-qPCR.【Result】A total of 96 ApERF,9 ApAP2,and 4 ApRAV genes were identified from A.philoxeroides,and they were named ApERF1 to ApERF96,ApAP2-1 to ApAP2-9,and ApRAV1 to ApRAV4,respectively.The identified ApAP2/ERF proteins are hydrophilic.Subcellular localization prediction revealed that the ApAP2/ERFs are located in the nucleus and cytoplasm.The expression patterns of ApAP2/ERFs were regulated by geographical condition,water condition,and low potassium stress.Under 41%glyphosate treatment,ApERF7/74/94 were highly induced within 3-7 days;under 50%isoproturon treatment,all six ApERFs were highly induced at a specific time;under 10%fluoroglycofen treatment,the expression levels of ApERF7/13/49/94 showed a trend of first increasing,then decreasing,and then increasing;under 20%fluroxypyr treatment,ApERF7/13/49/54/94 were highly induced at 0.5 d;under 13%oxadiazon treatment,ApERF13/49/54/74 were significantly downregulated in expression in a short period of time.【Conclusion】109 ApAP2/ERF family members were identified,and members located in the same group have similar motifs.The expression of ApAP2/ERFs is regulated by geographical condition,water condition,and low potassium stress,and is also induced by herbicides,suggesting that it responds to herbicide stress by influencing the ethylene signaling pathway.