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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (5): 1181-1190.doi: 10.3724/SP.J.1006.2022.11042

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Isolation and expression of a new gene HvMEL1 AGO in Tibetan hulless barley under leaf stripe stress

YAO Xiao-Hua1,2(), WANG Yue1(), YAO You-Hua1,2, AN Li-Kun1,2, WANG Yan1,2, WU Kun-Lun1,2,*()   

  1. 1Qinghai University, Xining 810016, Qinghai, China
    2Qinghai Academy of Agriculture and Forestry Academy / Qinghai Key Laboratory of Hulless Barley Genetics and Breeding / Qinghai Subcenter of National Hulless Barley Improvement, Xining 810016, Qinghai, China
  • Received:2021-04-09 Accepted:2021-09-01 Online:2022-05-12 Published:2021-10-21
  • Contact: WU Kun-Lun E-mail:yaoxiaohua009@126.com;wklqaaf@163.com
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    National Natural Science Foundation of China(31960427);National Natural Science Foundation of China(31660388);Project of Qinghai Science and Technology Department(2019-ZJ-7075);Agriculture Research System of China(CARS-05);National Key Research and Development Program of China(2020YFD1001403)

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Abstract:

To explore AGO genes related to leaf stripe in Tibetan hulless barley (BLS), we obtained a differentially expressed HvMEL1 AGO gene from the transcriptional sequencing in normal and diseased leaves from resistant Tibetan hulless barley variety Kunlun 14 and the susceptible variety 1141. The HvMEL1 AGO gene was 3462 bp in length, of which the CDS (coding domain sequence) was 100% consistent in Kunlun 14 and 1141 varieties. The length of HvMEL1 AGO gene was 3161 bp without intron, contained a 3129 bp open reading frame, encoded 1043 amino acids, had a theoretical isoelectric point of 9.33, and has a predicted molecular weight of 115,865.58 Da. Protein sequence analysis showed that HvMEL1 AGO was a hydrophilic unstable acidic protein with highly conserved structural domains of DUF1785, PAZ and PIWI, belonging to AGO gene family. Phylogenetic tree analysis showed that HvMEL1 AGO belonged to the AGO1 class in Arabidopsis AGO family phylogenetic tree with HvAGO12, HvAGO18, HvAGO1D and HvAGO1B in barley AGO family, and were closely related to HvAGO12. The predicted protein interactions showed that the known proteins that acted closely with MEL1 in rice were the DCL classes, DCL1, DCL2A, DCL3A, DCL3B and DCL4, respectively. Semi-quantitative and quantitative PCR results indicated that the relative expression levels of HvMEL1 AGO gene were significantly down-regulated in 1141 and Kunlun 14 under BLS. We hypothesized that HvMEL1 AGO gene played an important role in the streak resistance mechanism of barley. This study lays the foundation for exploring the role and regulatory mechanism of HvMEL1 AGO in the process of BLS resistance.

Key words: hulless barley, leaf stripe, HvMEL1 AGO gene, relative expression level

Table 1

The primers used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5°‒3°)		 用途
Purpose
HvMEL1 AGO-F CTCTCTCTCTCTCTCTCTCTTGCCA 基因克隆Gene cloning
HvMEL1 AGO-R TACTCTAAGACACTACGAGCAACAG 基因克隆Gene cloning
HvMEL1 AGO-SF AAGGTTTGGAGTGCTGGAGAGG 表达引物Primers for Real-time PCR
HvMEL1 AGO-SR CCGTGACAGGCTTGAAGAAGGA 表达引物Primers for Real-time PCR
TC139057 F GAAGGATGAGCAAAAGGCCCT 内参引物Control primer
TC139057 R GGCAGGCAGACTCATTTCTTCC 内参引物Control primer
MEL1 AGO-3'RACE1 GGCCAGAGATCACCAAGTACAGAG 基因克隆Gene cloning
MEL1 AGO-3'RACE2 CTTGTCTCTGCTCAACCACACAGG 基因克隆Gene cloning
MEL1 AGO-5'RACE1 AGTGGTTGGCGCGGATCATCACCT 基因克隆Gene cloning
MEL1 AGO-5'RACE2 CGGTCTCGGTGACGAAGAGCTTCT 基因克隆Gene cloning

Fig. 1

Amplification products of HvMEL1 AGO gene a: amplification products of the CDS region; b: amplification product of the 3' end; c: amplification product of the 5' end; M1: DL2000 DNA marker; M2: λ-Hin d III digest; 1, 2, 3, and 4 represent of 1141 not susceptible, 1141 susceptible, KL14 not susceptible and KL14 susceptible, respectively; 5 and 6 represent 3' and 5' end amplification products, respectively."

Fig. 2

Full sequence of HvMEL1 AGO gene and deduced amino acid sequence"

Fig. 3

Domain prediction of HvMEL1 AGO gene"

Fig. 4

Prediction of phosphorylation sites of HvMEL1 AGO"

Fig. 5

Secondary structure prediction of HvMEL1 AGO protein Blue: α-helix; Red: extended chain; Green: β-turn; Orange: random coil."

Fig. 6

Tertiary structure prediction of HvMEL1 AGO protein"

Fig. 7

Multiple alignment of HvMEL1 AGO and MEL1 AGO in other Gramineae plants Hv: Hordeum vulgare; Td: Triticum dicoccoides; Bd: Brachypodium distachyon; Os: Oryza sativa; At: Aegilops tauschii; Sb: Sorghum bicolor; Black line: DUF1785 domain; Red line: PAZ domain; Green line: PiWi domain."

Fig. 8

Phylogenetic tree analysis of the HvMEL1 AGO and AGO in barley and Arabidopsis"

Fig. 9

Interaction analysis of HvMEL1 AGO protein with rice related proteins Each node represents all the proteins produced by a single, protein-coding gene locus; empty nodes: proteins of unknown 3D structure; filled nodes: some 3D structure is known or predicted; bright blue lines: from curated databases; purple lines: experimentally determined; green lines: gene neighborhood; red lines: gene fusions; light green lines: textmining; black lines: co-expression; light purple lines: protein homology."

Fig. 10

qPCR and RT-PCR of HvMEL1 AGO gene a: qPCR of HvMEL1 AGO gene; b: RT-PCR of HvMEL1 AGO gene; KL14N indicates for Kunlun14 without BLS; KL14S indicates for Kunlun14 with BLS; 1141N indicates 1141 without BLS; 1141S indicates 1141 with BLS; different capital letters are extremely significantly different at 0.01 level."

[1] 强小林, 迟德钊, 冯继林. 青藏高原区域青稞生产与发展现状. 西藏科技, 2008, 33(3):11-17.
Qiang X L, Chi D Z, Feng J L. Current status of hulless barley production and development in the Tibetan Plateau region. Tibet Sci Technol, 2008, 33(3):11-17 (in Chinese).
[2] 原红军, 曾兴权, 王玉林, 徐齐君, 韦泽秀, 尼玛扎西. 青稞法尼基转移酶β亚基编码基因HbERA1的克隆及表达分析. 麦类作物学报, 2014, 34:1465-1470.
Yuan H J, Zeng X Q, Wang Y L, Xu Q J, Wei Z X, Mimazhaxi. Cloning and characterization of beta subunit of protein farnesyl transferase HbERA1 in Tibetan hulless barley (Hordeum vulgare subsp. vulgare). J Triticeae Crops, 2014, 34:1465-1470 (in Chinese with English abstract).
[3] Gatti A, Rizza F, Delogu G, Terzi V, Porta-puglia A, Vannacci G. Physiological and biochemical variability in a population of Drechslera graminea. J Genet Breed, 1992, 46:179-186.
[4] International Barley Sequencing Consortium (IBSC). A physical, genetic and functional sequence assembly of the barley genome. Nature, 2012, 491:711-716.
doi: 10.1038/nature11543
[5] Arru L, Francia E, Pecchioni N. Isolate-specific QTLs of resistance to leaf stripe (Pyrenophora graminea) in the ‘Steptoe’ × ‘Morex’ spring barley cross. Theor Appl Genet, 2003, 106:668-675.
pmid: 12595996
[6] 郑果, 王春明, 洪流, 王生荣. 7种杀菌剂对大麦条纹病的防治效果. 草原与草坪, 2011, 31(6):65-68.
Zheng G, Wang C M, Hong L, Wang S R. Control effect of 7 fungicides on barley stripe disease. Grassl Turf, 2011, 31(6):65-68 (in Chinese with English abstract).
[7] 王建. 青稞条纹病的发生与防治. 江西农业, 2018, 11(12):25.
Wang J. Incidence and control of barley streak disease. Jiangxi Agric, 2018, 11(12):25 (in Chinese).
[8] Yan J H, Yao Q, Guo Q Y, Chen H M, Hou L, Xu S C. Control effect of four seed coatings on barley leaf stripe caused by drechslera gramine. Plant Prot, 2016, 42:233-236.
[9] Arru L, Niks RE, Lindhout P, Valé G, Francia E, Pecchioni N. Genomic regions determining resistance to leaf stripe (Pyrenophora graminea) in barley. Genome, 2002, 45:460-466.
pmid: 12033613
[10] Biselli C, Urso S, Bernardo L, Tondelli A, Tacconi G, Martino V, Grando S, Valè G. Identification and mapping of the leaf stripe resistance gene Rdg1a in Hordeum spontaneum. Theor Appl Genet, 2010, 120:1207-1218.
doi: 10.1007/s00122-009-1248-2 pmid: 20041226
[11] Haegi A, Bonardi V, Dall’Aglio E, Glissant D, Tumino G, Collins N C, Bulgarelli D, Infantino A, Stanca A M, Delledonne M, Valè G. Histological and molecular analysis of Rdg2a barley resistance to leaf stripe. Mol Plant Pathol, 2008, 9:463-478.
doi: 10.1111/mpp.2008.9.issue-4
[12] Bulgarelli D, Biselli C, Collins N C, Consonni G, Stanca A M, Schulze-Lefert P, Valè G. The CC-NB-LRR-Type Rdg2a resistance gene confers immunity to the seed-borne barley leaf stripe pathogen in the absence of hypersensitive cell death. PLoS One, 2010, 5:e12599.
[13] 姚晓华, 王越, 安立昆, 姚有华, 杨雪, 白羿雄, 吴昆仑. 青稞HvtAGO1基因的克隆及其在条纹病胁迫下的表达. 西北植物学报, 2021, 41:20-28.
Yao X H, Wang Y, An L K, Yao Y H, Yang X, Bai Y X, Wu K L. Identification and expression analysis of HvtAGO1 gene in response to barley leaf stripe in Tibetan hulless barley. Acta Bot Boreali-Occident Sin, 2021, 41:20-28 (in Chinese with English abstract).
[14] 杨雪, 姚晓华, 安立昆, 姚有华, 白羿雄, 吴昆仑. 青稞NBS-LRR类基因HvtRGA的克隆与条纹病胁迫表达分析. 西北植物学报, 2020, 40:1655-1662.
Yang X, Yao X H, An L K, Yao Y H, Bai Y X, Wu K L. Isolation and expression analysis of NBS-LRR HvtRGA gene in hulless barley under stripe disease stress. Acta Bot Boreali-Occident Sin, 2020, 40:1655-1662 (in Chinese with English abstract).
[15] 吴宽然, 杨建明, 朱靖环, 金婷. 大麦条纹病抗性及防治研究进展. 浙江农业学报, 2013, 25:903-907.
Wu K R, Yang J M, Zhu J H, Jin T. Advances of research on control of barley leaf stripe disease. Acta Agric Zhejiangensis, 2013, 25:903-907 (in Chinese with English abstract).
[16] Inal B, Türktas M, Eren H, Ilhan E, Okay S, Atak M, Erayman M, Unver T. Genome-wide fungal stress responsive miRNA expression in wheat. Planta, 2014, 240:1287-1298.
doi: 10.1007/s00425-014-2153-8
[17] Bohmert K, Camus I, Bellini C, Bouchez D, Caboche M, Benning C. AGO1 defines a novel locus of Arabidopsis controlling leaf development. EMBO J, 1998, 17:170-180.
pmid: 9427751
[18] Hervé V, Edward N. AGO1 homeostasis involves differential production of 21-nt and 22-nt miR168 Species by MIR168a and MIR168b. PLoS One, 2009, 4:e6442.
[19] Komiya R, Ohyanagi H, Niihama M, Watanabe T, Nakano M, Kurata N, Nonomura K I. Rice germline-specific HvMEL1 AGO protein binds to phasiRNAs generated from more than 700 lincRNAs. Plant J, 2014, 78:385-397.
doi: 10.1111/tpj.12483
[20] Tucker M R, Okada T, Hu Y, Scholefield A, Taylor J M, Koltunow A M G. Somatic small RNA pathways promote the mitotic events of megagametogenesis during female reproductive development in Arabidopsis. Development, 2012, 139:1399.
doi: 10.1242/dev.075390
[21] Xu R R, Liu C Y, Li N, Zhang S Z. Global identification and expression analysis of stress-responsive genes of the Argonaute family in apple. Mol Genet Genomics, 2016, 291:2015-2030.
doi: 10.1007/s00438-016-1236-6
[22] Luo M, Peng H, Gao J, Pan G T, Zhang Z M. Identification and functional analysis of miRNAs in response to banded leaf and sheath blight in Zea mays. Chin J Biochem Mol Biol, 2012, 28:1122-1132.
[23] 黄赳. NRTs基因的克隆及其功能研究. 中国农业科学院硕士学位论文, 北京, 2021.
Huang J. Cloning and Functional Study of NRTs Gene. MS Thesis of Chinese Academy of Agricultural Sciences, Beijing, China, 2021.
[24] 王越, 姚晓华, 吴昆仑, 白羿雄, 魏晓星. 青稞HVA1和blt4.9基因对模拟水分胁迫的响应差异及其在抗旱育种中的应用. 麦类作物学报, 2019, 39:666-674.
Wang Y, Yao X H, Wu K L, Bai Y X, Wei X X. Difference of HVA1 and blt4.9 Gene expression patterns under simulated drought stress and the potention application in drought tolerance breeding in hulless barley. J Triticeae Crops, 2019, 39:666-674 (in Chinese with English abstract).
[25] Pfaffl M W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res, 2001, 29:e45.
[26] Agustín S, Lucas D, Abelardo V, Manuel T, Francisco T, Marcela D. Genome-wide analysis of AGO, DCL and RDR gene families reveals RNA-directed DNA methylation is involved in fruit abscission in Citrus sinensis. BMC Plant Biol, 2019, 19:401.
doi: 10.1186/s12870-019-1998-1
[27] Song J J, Joshua-Tor L. Argonaute and RNA-getting into the groove. Curr Opin Struct Biol, 2006, 16:5-11.
doi: 10.1016/j.sbi.2006.01.010
[28] Wang Y, Juranek S, Li H, Sheng G, Tuschl T, Patel D J. Structure of an Argonaute silencing complex with a seed-containing guide DNA and target RNA duplex. Nature, 2008, 456:921-926.
doi: 10.1038/nature07666
[29] Zeng X Q, Xu T, Ling Z H, Wang Y L, Li X F, Xu S Q, Xu Q J, Zha S, Qimei W M, Basang Y Z, Dunzhu J B, Yu M Z, Yuan H J, Nyima T. An improved high-quality genome assembly and annotation of Tibetan hulless barley. Sci Data, 2020, 7:139.
doi: 10.1038/s41597-020-0480-0
[30] Ta K N, Sabot F, Adam H, Vigouroux Y, Mita S D, Ghesquière A, Do N V, Gantet P, Jouannic S. miR2118-triggered phased siRNAs are differentially expressed during the panicle development of wild and domesticated African rice species. Rice, 2016, 9:10.
doi: 10.1186/s12284-016-0082-9 pmid: 26969003
[31] Vaucheret H. Plant ARGONAUTES. Trends Plant Sci, 2008, 13:350-358.
doi: 10.1016/j.tplants.2008.04.007 pmid: 18508405
[32] Fernández-Nohales P, Domenech M J, Martínez de Alba A E, Micol J L, Ponce M R, Madueño F. AGO1 controls Arabidopsis inflorescence architecture possibly by regulating TFL1 expression. Ann Botlondon, 2014, 114:1471-1481.
[33] Thiébeauld O, Charvin M, Rastogi M S, Yang F, Pontier D, Pouzet C, Bapaume L, Li G, Deslandes L, Lagrange T, Alfano J R, Navarro L. A bacterial GW-effector targets Arabidopsis AGO1 to promote pathogenicity and induces Effector-triggered immunity by disrupting AGO1 homeostasis. BioRxiv, 2017, doi: 10.1101/215590.
doi: 10.1101/215590
[34] Zhang J, Zhang H, Srivastava A K, Pan Y, Bai J, Fang J, Shi H, Zhu J K. Knock-down of rice microrna166 confers drought resistance by causing leaf rolling and altering stem xylem development. Plant Physiol, 2018, 176:2082-2094.
doi: 10.1104/pp.17.01432
[35] Vaucheret H, Vazquez F, Crété P, Bartel D P. The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev, 2004, 18:1187-1197.
doi: 10.1101/gad.1201404
[36] Sheng K P, Gullerova M. Noncanonical functions of microrna pathway enzymes—drosha, dgcr8, dicer and ago proteins. FEBS Lett, 2018, 592:992-1004.
[37] Kim V N. Sorting out small RNAs. Cell, 2008, 133:25-26.
doi: 10.1016/j.cell.2008.03.015
[38] Cui D L, Meng J Y, Ren X Y, Yue J J, Fu H Y, Huang M T, Zhang Q Q, Gao S J. Genome-wide identification and characterization of DCL, AGO and RDR gene families in. Sci Rep, 2020, 10:13202.
doi: 10.1038/s41598-020-70061-7
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