红三叶响应淹水胁迫的相关通路及差异表达基因分析_尚盼盼.pdf

第 32 卷第 4 期Vol.32，No.4112-1282023 年 4 月草业学报ACTA PRATACULTURAE SINICA尚盼盼，曾兵，屈明好，等.红三叶响应淹水胁迫的相关通路及差异表达基因分析.草业学报，2023，32（4）：112128.SHANG Pan-pan，ZENG Bing，QU Ming-hao，et al.Analysis of metabolic pathways and differentially expressed genes of Trifolium pratense respondingto waterlogging stress.Acta Prataculturae Sinica，2023，32（4）：112128.红三叶响应淹水胁迫的相关通路及差异表达基因分析尚盼盼1，曾兵1，屈明好1，李明阳1，杨兴云1，郑玉倩1，沈秉娜1，毕磊1，杨成1，曾兵1，2*（1.西南大学动物科学技术学院，重庆 402460；2.草食动物科学重庆市重点实验室，重庆 400715）摘要：淹水胁迫是影响植物生长发育和分布的重要非生物胁迫，对植物淹水胁迫的研究是解决近年来极端强降水天气下植物生产管理的关键。红三叶作为优质豆科牧草，耐淹性较差，长期水淹会导致烂根死亡。为研究红三叶淹水胁迫下的分子响应机理，本研究通过 Illumina高通量测序平台，以耐涝型品种“红龙”淹水胁迫下 0、8 和 24 h 三个时间点的幼苗叶片为材料进行转录组测序，将测序数据与参考基因组比对后进行差异表达基因（DEGs）分析和功能注释。结果显示，与对照 0 h相比，“红龙”在淹水胁迫 8 h后，有 5065个 DEGs，其中，上调表达基因 2442个，下调表达基因 2623个；在淹水胁迫 24 h后，有 9022个 DEGs，其中，上调表达基因 4279个，下调表达基因 4743个。基因本体数据库富集结果显示，DEGs 主要富集于代谢过程、细胞过程、生物调节、细胞、催化活性等条目；东京基因与基因组数据库富集结果显示，DEGs显著富集于植物激素信号调节、植物-病原互作、碳代谢和乙醛酸及二羧酸代谢等通路中，其中乙醛酸及二羧酸代谢通路中过氧化氢酶和甲酸脱氢酶等抗氧化酶相关基因高表达；并且发现差异表达的 AP2/ERF、WRKY、bHLH、NAC、bZIP 等重要转录因子在红三叶响应淹水胁迫中也发挥重要作用。最后利用qRT-PCR对 DEGs进行表达模式的分析验证，发现其与 RNA-Seq结果一致，证实了测序结果的准确性。本研究根据转录组信息对 DEGs展开功能注释、代谢通路、转录因子等方面的分析研究，初步了解红三叶对淹水胁迫的分子响应机理，为后续候选基因功能挖掘提供了基础数据和理论支撑。关键词：红三叶；淹水胁迫；转录组测序；代谢通路；差异表达基因Analysis of metabolic pathways and differentially expressed genes of Trifoliumpratense responding to waterlogging stressSHANG Pan-pan1，ZENG Bing1，QU Ming-hao1，LI Ming-yang1，YANG Xing-yun1，ZHENG Yu-qian1，SHEN Bing-na1，BI Lei1，YANG Cheng1，ZENG Bing1，2*1.College of Animal Science and Technology，Southwest University，Chongqing 402460，China；2.Chongqing Key Laboratory ofHerbivore Science，Chongqing 400715，ChinaAbstract：Waterlogging stress is an important abiotic stress that affects plant growth，development and distribution，and research on plant waterlogging stress is key to addressing plant production management under extreme heavyrainfall in recent years.Trifolium pratense is a high-quality legume forage，but is poorly tolerant to waterlogging，and long-term waterlogging can lead to root rot and death.In order to investigate the molecular response mechanismof T.pratense under waterlogging stress，the transcriptome was sequenced from seedling leaves of the waterlogging-tolerant variety“Hong Long”at 0，8 and 24 h using an Illumina high-throughput sequencing platform，and theDOI：10.11686/cyxb2022139http：/收稿日期：2022-03-30；改回日期：2022-05-13基金项目：中央高校基本科研业务费项目荣昌专项（XDJK2020RC001），重庆市现代山地特色高效农业产业技术体系（草食牲畜）2023和西南大学大学生创新训练项目（X202210635042；X202210635203）资助。作者简介：尚盼盼（1996-），女，甘肃定西人，在读硕士。E-mail：1916001864 通信作者 Corresponding author.E-mail：第 32 卷第 4 期草业学报 2023 年sequenced data were compared with the reference genome for differentially expressed genes（DEGs）and functionalannotation.The results showed that，compared with the control 0 h，“Hong Long”had 5065 DEGs after 8 h ofwaterlogging stress，among which 2442 genes were up-regulated and 2623 genes were down-regulated；After 24 h ofwaterlogging stress，there were 9022 DEGs，among which 4279 genes were up-regulated and 4743 genes weredown-regulated.The gene ontology（GO）enrichment results showed that DEGs were mainly enriched in terms ofmetabolic process，cellular process，biological regulation，cell and catalytic activity；The kyoto encyclopedia ofgenes and genomes（KEGG）enrichment results showed that DEGs were significantly enriched in pathways such asplant hormone signal transduction，plant-pathogen interaction，carbon metabolism and glyoxylate and dicarboxylatemetabolism，in which antioxidant enzymes such as peroxidase and formic dehydrogenase were highly expressed in theglyoxylate and dicarboxylate metabolism pathways.This study also found that the differentially expressed AP2/ERF，WRKY，bHLH，NAC，bZIP and other important transcription factors also played important roles in theresponse of T.pratense to waterlogging stress.Finally，the expression pattern of DEGs was verified by qRT-PCRand found to be consistent with the RNA-Seq results，confirming the accuracy of the sequencing results.In thisstudy，functional annotation，metabolic pathways and transcription factors of DEGs were analyzed based on thetranscriptome information，which provided a preliminary understanding of the molecular response mechanisms of T.pratense to waterlogging stress and provided basic data and theoretical direction for the subsequent functional miningof candidate genes.Key words：Trifolium pratense；waterlogging stress；transcriptomic sequencing；metabolic pathways；differentiallyexpressed genes红三叶（Trifolium pratense）是豆科三叶草属多年生草本植物，又名红车轴草、红荷兰翘摇，喜温暖湿润气候，耐旱性较差，耐湿性良好，适合在年降水量较充沛的地方生长，但红三叶不耐淹，长期水淹会烂根死亡。其营养价值和药用价值高，富含黄酮类物质、蛋白质、氨基酸等成分1-3。具有适口性好、易消化、再生速度快、鲜草产量高等特点。因此，在国内外被广泛种植和利用2。红三叶中含有黄酮和异黄酮类物质，有研究表明，岷山红三叶青干草中异黄酮含量约为大豆（Glycine max）籽粒中异黄酮含量的 10倍4-5，是妇女保健产品和药用产品中异黄酮类物质提取的重要原料6。作为优质豆科牧草，红三叶在土壤肥力建设和种植制度中发挥多重效益，并且在青贮时对蛋白质的利用率明显高于紫花苜蓿（Medicago sativa），作为牧草的物种多样性对不同的气候和耕作系统也起重要作用7，是英国、美国、新西兰等地重要的栽培牧草之一，在我国西南、西北、华中、新疆等地也均有分布8。淹水胁迫是植物遭受的主要非生物胁迫之一，与其他非生物胁迫共同决定了植物的特定结构和分布9-10。近年来我国多地受强降水的影响，2021年河南省、重庆市等地暴发的持续性强降水天气最大时降水量高达 201.9mm，长期的涝害严重破坏植物的自然分布规律，直接影响生物多样性11，并且会对作物生长和粮食安全产生毁灭性的影响12。而淹水胁迫的