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

doi:10.3864/j.issn.0578-1752.2023.20.008

摘要/Abstract

摘要：

【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。

关键词: 喜旱莲子草, 生物信息学, 转录因子, 除草剂, 表达模式, AP2/ERF基因家族

Abstract:

【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.

Key words: Alternanthera philoxeroides, bioinformatics, transcription factor, herbicide, expression pattern, AP2/ERF gene family

韩晓文, 韩硕, 胡义锋, 王梦如, 陈中义, 朱永兴, 尹军良. 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[J]. 中国农业科学, 2023, 56(20): 4021-4034.

HAN XiaoWen, HAN Shuo, HU YiFeng, WANG MengRu, CHEN ZhongYi, ZHU YongXing, YIN JunLiang. Genome-Wide Identification of AP2/ERF Gene Family in Alternanthera philoxeroides and Its Expression Patterns Under Herbicide Stresses[J]. Scientia Agricultura Sinica, 2023, 56(20): 4021-4034.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2023.20.008

https://www.chinaagrisci.com/CN/Y2023/V56/I20/4021

图/表 9

表1

表2

图1

图2

图3

图4

图5

图6

图7

参考文献 36

[1]	孙思昂, 邓梓妍, 熊佳瑶, 李少斌, 杜何为, 杜小龙. 空心莲子草生物及生态防治研究进展. 南方农业, 2022, 16(9): 164-167.
	SUN S A, DENG Z Y, XIONG J Y, LI S B, DU H W, DU X L. Research progress on biological and ecological control of Alternanthera philoxeroides. South China Agriculture, 2022, 16(9): 164-167. (in Chinese)
[2]	蒋昕晨. 空心莲子草转录组de novo拼接、不定根膨大相关基因挖掘及作用机制解析[D]. 荆州: 长江大学, 2022.
	JIANG X C. De novo assembling of Alternanthera philoxeroides transcriptome, mining of genes related to adventitious root expansion and analysis of their mechanism[D]. Jingzhou: Yangtze University, 2022. (in Chinese)
[3]	娄远来, 邓渊钰, 沈纪冬, 李亚浩. 我国空心莲子草的研究现状. 江苏农业科学, 2002(4): 46-48.
	LOU Y L, DENG Y Y, SHEN J D, LI Y H. Progress of study on Alternantliera philoxeroides Griseb in China. Jiangsu Agricultural Sciences, 2002(4): 46-48. (in Chinese)
[4]	JOFUKU K D, OMIDYAR P K, GEE Z, OKAMURO J K.Control of seed mass and seed yield by the floral homeotic gene APETALA2. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(8): 3117-3122.
[5]	OKAMURO J K, CASTER B, VILLARROEL R, VAN MONTAGU M, JOFUKU K D. The AP 2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 1997, 94(13): 7076-7081.
[6]	ZHAO Y, MA R Y, XU D L, BI H H, XIA Z L, PENG H R. Genome-wide identification and analysis of the AP2 transcription factor gene family in wheat (Triticum aestivum L.). Frontiers in Plant Science, 2019, 10: 1286. doi: 10.3389/fpls.2019.01286
[7]	SAKUMA Y, LIU Q, DUBOUZET J G, ABE H, SHINOZAKI K, YAMAGUCHI-SHINOZAKI K. DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochemical and Biophysical Research Communications, 2002, 290(3): 998-1009. doi: 10.1006/bbrc.2001.6299
[8]	GIL-MONREAL M, GIUNTOLI B, ZABALZA A, LICAUSI F, ROYUELA M. ERF-VII transcription factors induce ethanol fermentation in response to amino acid biosynthesis-inhibiting herbicides. Journal of Experimental Botany, 2019, 70(20): 5839-5851. doi: 10.1093/jxb/erz355
[9]	YANG X, ZHANG Z C, GU T, DONG M C, PENG Q, BAI L Y, LI Y F. Quantitative proteomics reveals ecological fitness cost of multi-herbicide resistant barnyardgrass (Echinochloa crus-galli L.). Journal of Proteomics, 2017, 150: 160-169. doi: 10.1016/j.jprot.2016.09.009
[10]	VALLEJO-REYNA M A, SANTAMARÍA J M, RODRÍGUEZ- ZAPATA L C, HERRERA-VALENCIA V A, PERAZA-ECHEVERRIA S. Identification of novel ERF transcription factor genes in papaya and analysis of their expression in different tissues and in response to the plant defense inducer benzothiadiazole (BTH). Physiological and Molecular Plant Pathology, 2015, 91: 141-151.
[11]	WANG L Y, WANG R L, LEI W, WU J Y, LI C Y, SHI H S, MENG L J, YUAN F, ZHOU Q Y, CUI C. Transcriptome analysis reveals gene responses to herbicide, tribenuron methyl, in Brassica napus L. during seed germination. BMC Genomics, 2021, 22(1): 299. doi: 10.1186/s12864-021-07614-1
[12]	NAKANO T, SUZUKI K, FUJIMURA T, SHINSHI H. Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiology, 2006, 140(2): 411-432. doi: 10.1104/pp.105.073783
[13]	SHU Y, LIU Y, ZHANG J, SONG L, GUO C. Genome-wide analysis of the AP2/ERF superfamily genes and their responses to abiotic stress in Medicago truncatula. Frontiers in Plant Science, 2015, 6: 1247.
[14]	XING H T, JIANG Y S, ZOU Y, LONG X L, WU X L, REN Y, LI Y, LI H L. Genome-wide investigation of the AP2/ERF gene family in ginger: Evolution and expression profiling during development and abiotic stresses. BMC Plant Biology, 2021, 21(1): 561. doi: 10.1186/s12870-021-03329-3 pmid: 34823471
[15]	SHARONI A M, NURUZZAMAN M, SATOH K, SHIMIZU T, KONDOH H, SASAYA T, CHOI I-R, OMURA T, KIKUCHI S. Gene structures, classification and expression models of the AP2/EREBP transcription factor family in rice. Plant and Cell Physiology, 2011, 52(2): 344-360. doi: 10.1093/pcp/pcq196 pmid: 21169347
[16]	ZHANG J, LIAO J Y, LING Q Q, XI Y, QIAN Y X. Genome-wide identification and expression profiling analysis of maize AP2/ERF superfamily genes reveal essential roles in abiotic stress tolerance. BMC Genomics, 2022, 23(1): 125. doi: 10.1186/s12864-022-08345-7 pmid: 35151253
[17]	ZHU Y X, JIANG X C, HAN X W, HAN S, CHEN Z Y, YIN J L, LIU Y Q. Characterization the coding and non-coding RNA components in the transcriptome of invasion weed Alternanthera philoxeroides. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 2021, 49(1): 12242. doi: 10.15835/nbha49112242
[18]	YIN J, WANG L, ZHAO J, LI Y, ZHU Y. Genome-wide characterization of the C₂H₂ zinc-finger genes in Cucumis sativus and functional analyses of four CsZFPs in response to stresses. BMC Plant Biology, 2020, 20(1): 359. doi: 10.1186/s12870-020-02575-1
[19]	杨蕾, 蒋昕晨, 杨杰, 夏雨晨, 陈中义, 马东方, 朱永兴, 尹军良. 空心莲子草MTP基因家族的鉴定、特征及表达分析. 草业科学, 2020, 37(8): 1516-1527.
	YANG L, JIANG X C, YANG J, XIA Y C, CHEN Z Y, MA D F, ZHU Y X, YIN J L. Identification, characterization, and expression analysis of metal tolerance protein (MTP) genes in Alternanthera philoxeroides. Pratacultural Science, 2020, 37(8): 1516-1527. (in Chinese)
[20]	TRAPNELL C, ROBERTS A, GOFF L, PERTEA G, KIM D, KELLEY D R, PIMENTEL H, SALZBERG S L, RINN J L, PACHTER L. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nature Protocols, 2012, 7(3): 562-578. doi: 10.1038/nprot.2012.016 pmid: 22383036
[21]	YIN J L, HOU L, JIANG X C, YANG J, HE Y, ZHOU X K, ZHU X M, GONG A D, ZHU Y X, CHEN Z Y. Identification and validation of reference genes for quantitative real-time PCR studies in alligatorweed (Alternanthera philoxeroides). Weed Science, 2021, 69(4): 404-411. doi: 10.1017/wsc.2021.32
[22]	YIN J L, LIU M Y, MA D F, WU J W, LI S L, ZHU Y X, HAN B. Identification of circular RNAs and their targets during tomato fruit ripening. Postharvest Biology and Technology, 2018, 136: 90-98. doi: 10.1016/j.postharvbio.2017.10.013
[23]	王洁, 吴晓宇, 杨柳, 段巧红, 黄家保. 大白菜ACA基因家族的全基因组鉴定与表达分析. 中国农业科学, 2021, 54(22): 4851-4868. doi: 10.3864/j.issn.0578-1752.2021.22.012.
	WANG J, WU X Y, YANG L, DUAN Q H, HUANG J B. Genome-wide identification and expression analysis of ACA gene family in Brassica rapa. Scientia Agricultura Sinica, 2021, 54(22): 4851-4868. doi: 10.3864/j.issn.0578-1752.2021.22.012. (in Chinese)
[24]	兰孟焦, 肖满秋, 潘皓, 卢凌志, 侯隆英, 葛瑞华, 吴问胜.AP2/ERF转录因子参与植物次生代谢和逆境胁迫响应的研究进展. 植物遗传资源学报, 2023, doi: 10.13430/j.cnki.pngr.20230322001.
	LAN M J, XIAO M Q, PAN H, LU L Z, HOU L Y, GE R H, WU W S.Research progress of AP2/ERF transcription factors participating in plant secondary metabolism and stress response. Journal of Plant Genetic Resources, 2023, doi: 10.13430/j.cnki.pngr.20230322001. (in Chinese)
[25]	SHARMA M K, KUMAR R, SOLANKE A U, SHARMA R, TYAGI A K, SHARMA A K. Identification, phylogeny, and transcript profiling of ERF family genes during development and abiotic stress treatments in tomato. Molecular Genetics and Genomics, 2010, 284(6): 455-475. doi: 10.1007/s00438-010-0580-1 pmid: 20922546
[26]	ZHANG H N, PAN X L, LIU S H, LIN W Q, LI Y H, ZHANG X M. Genome-wide analysis of AP2/ERF transcription factors in pineapple reveals functional divergence during flowering induction mediated by ethylene and floral organ development. Genomics, 2021, 113(2): 474-489. doi: 10.1016/j.ygeno.2020.10.040 pmid: 33359830
[27]	赵冰雪, 聂功平, 张小惠, 黄清俊. 芝麻AP2/ERF基因家族生物信息学鉴定与分析. 应用技术学报, 2022, 22(4): 399-412.
	ZHAO B X, NIE G P, ZHANG X H, HUANG Q J. Bioinformatics identification and analysis of AP2/ERF gene family in sesame. Journal of Technology, 2022, 22(4): 399-412. (in Chinese)
[28]	卞云迪, 张驰, 王雪晴, 杨晨晓, 王光钰, 刘晓颖, 王颖, 方芳, 王振英.小麦AP2/ERF转录因子家族生物信息学分析. 天津师范大学学报 (自然科学版), 2022, 42(4): 39-45.
	BIAN Y D, ZHANG C, WANG X Q, YANG C X, WANG G Y, LIU X Y, WANG Y, FANG F, WANG Z Y.Bioinformatics analysis of TaAP2/ERF transcription factor family in wheat. Journal of Tianjin Normal University (Natural Science Edition), 2022, 42(4): 39-45. (in Chinese)
[29]	陈珂, 张君, 刘嘉斐, 杨洁冰, 李蒙鑫, 陈吉宝, 杨树琼. 绿豆AP2/ERF转录因子家族的生物信息学鉴定与特征分析. 分子植物育种, 2020, 18(20): 6605-6617.
	CHEN K, ZHANG J, LIU J F, YANG J B, LI M X, CHEN J B, YANG S Q. Bioinformatics identification and characteristic analysis of AP2/ERF transcription factor in mung bean. Molecular Plant Breeding, 2020, 18(20): 6605-6617. (in Chinese)
[30]	杨金荣, 崔婉宁, 张瑜, 伯晨, 晁秋杰, 朱艳芳, 段永波, 薛涛, 张爱民. 基于转录组数据的半夏AP2/ERF基因家族鉴定及逆境响应分析. 中国实验方剂学杂志, 2023, 29(5): 176-184.
	YANG J R, CUI W N, ZHANG Y, BO C, CHAO Q J, ZHU Y F, DUAN Y B, XUE T, ZHANG A M. Identification and expression analysis of AP2/ERF family in stress responses of Pinellia ternata based on transcriptome data. Chinese Journal of Experimental Traditional Medical Formulae, 2023, 29(5): 176-184. (in Chinese)
[31]	马芮, 李瑞雪, 郑煜聆, 李永强. 蓝莓AP2/ERF基因家族鉴定及其在休眠解除过程中的表达分析研究. 浙江农业科学, 2022, 63(12): 2854-2864.
	MA R, LI R X, ZHENG Y L, LI Y Q. Identification of blueberry AP2/ERF gene family and analysis of its expression during dormancy release. Journal of Zhejiang Agricultural Sciences, 2022, 63(12): 2854-2864. (in Chinese)
[32]	RITONGA F N, NGATIA J N, WANG Y, KHOSO M A, FAROOQ U, CHEN S. AP2/ERF, an important cold stress-related transcription factor family in plants: A review. Physiology and Molecular Biology of Plants, 2021, 27(9): 1953-1968. doi: 10.1007/s12298-021-01061-8 pmid: 34616115
[33]	ZENG Q Y, LING Q P, FAN L N, LI Y, HU F, CHEN J W, HUANG Z R, DENG H H, LI Q W, QI Y W. Transcriptome profiling of sugarcane roots in response to low potassium stress. PLoS ONE, 2015, 10(5): e0126306. doi: 10.1371/journal.pone.0126306
[34]	KIM M J, RUZICKA D, SHIN R, SCHACHTMAN D P. The Arabidopsis AP2/ERF transcription factor RAP2.11 modulates plant response to low-potassium conditions. Molecular Plant, 2012, 5(5): 1042-1057. doi: 10.1093/mp/sss003
[35]	DOĞRAMACı M, ANDERSON J V, CHAO W S, FOLEY M E. Foliar application of glyphosate affects molecular mechanisms in underground adventitious buds of leafy spurge (Euphorbia esula) and alters their vegetative growth patterns. Weed Science, 2014, 62(2): 217-229. doi: 10.1614/WS-D-13-00156.1
[36]	YUAN L B, MA G Y, GENG Y L, LIU X M, WANG H, LI J, SONG S S, PAN W L, HUN Z Y. Seed dressing with mefenpyr-diethyl as a safener for mesosulfuron-methyl application in wheat: The evaluation and mechanisms. PLoS ONE, 2021, 16(8): e0256884. doi: 10.1371/journal.pone.0256884

除草剂 <BOLD>H</BOLD>erbicide	施液量 Application amount (g·m^-2)	生产商 Manufacturer	剂型 Dosage form
41%草甘膦Glyphosate	0.08	江苏快达农化股份有限公司 Jiangsu Kuaida Agrochemical Co., Ltd	水剂 Aqueous agent
50%异丙隆 Isoproturon	0.05	美丰农业科技（上海）有限公司 Meifeng Agricultural Technology (Shanghai) Co., Ltd	可湿性粉剂 Wettable powder
13%噁草酮 Oxadiazon	0.04	大连越达农药化工有限公司 Dalian Yueda Pesticide Chemical Co., Ltd	乳油 Emulsifiable concentrate
10%乙羧氟草醚 Fluoroglycofen	0.008	潍坊鸿汇化工有限公司 Weifang Honghui Chemical Co., Ltd	乳油 Emulsifiable concentrate
20%氯氟吡氧乙酸 Fluroxypyr	0.01	山东绿霸化工股份有限公司 Shandong Lvba Chemical Co., Ltd	乳油 Emulsifiable concentrate

基因<BOLD>G</BOLD>ene	正向引物Forward primer (5′-3′)	反向引物Reverse primer (5′-3′)
Tubby	CGGTCTAGCCGAAGATTCCA	CGCTTGGTGAAGGCAGACATT
ApERF7	ACCCCAAGCCCAACAACC	TCCTCACCGAACACGTCTCC
ApERF13	TCCTCACCGAACACGTCTCC	CAAACGACATTCACCCAAGAGT
ApERF49	CTCTCCTGCTTCTGTGCTCG	GCCCGATTTCACTACATTGG
ApERF54	CCAAAGTGTTCCCACCCAA	CCCCACCTTCACCTCCAC
ApERF74	GCAACATTAACTGCCAAGCTTC	GGCCCAATTTCATTAGTAGAGCT
ApERF94	TTCAGAGTACCCAAAGAAACGTG	AAATGTCCCGAGCCAAATTC