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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."

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