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作物学报 ›› 2022, Vol. 48 ›› Issue (5): 1181-1190.doi: 10.3724/SP.J.1006.2022.11042

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

青稞新基因HvMEL1 AGO的克隆和条纹病胁迫下的表达

姚晓华1,2(), 王越1(), 姚有华1,2, 安立昆1,2, 王燕1,2, 吴昆仑1,2,*()   

  1. 1青海大学, 青海西宁 810016
    2青海省农林科学院 / 青海省青稞遗传育种重点实验室 / 国家麦类改良中心青海青稞分中心, 青海西宁 810016
  • 收稿日期:2021-04-09 接受日期:2021-09-01 出版日期:2022-05-12 网络出版日期:2021-10-21
  • 通讯作者: 吴昆仑
  • 作者简介:姚晓华, E-mail: yaoxiaohua009@126.com;
    王越, E-mail: joanwy919@163.com第一联系人：**同等贡献
  • 基金资助:
    国家自然科学基金项目(31960427);国家自然科学基金项目(31660388);青海省科技厅应用基础研究项目(2019-ZJ-7075);国家现代农业产业技术体系建设专项(CARS-05);国家重点研发计划项目资助(2020YFD1001403)

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 Published:2022-05-12 Published online:2021-10-21
  • Contact: WU Kun-Lun
  • 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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摘要：

为筛选与青稞条纹病相关的AGO类基因, 本研究以青稞抗病品种昆仑14号和感病品种1141为材料, 从感病和正常叶片的转录组测序结果中获得一个差异表达的AGO家族新基因, 克隆验证了该基因为青稞HvMEL1 AGO。HvMEL1 AGO基因全长3462 bp, 其中蛋白质编码区(CDS, coding domain sequence)在昆仑14号和1141品种中的一致性为100%, 无内含子, 全长3161 bp, 包含一个3129 bp开放阅读框, 编码1043个氨基酸, 理论等电点为9.33, 预测蛋白分子量为115,865.58 Da。蛋白质序列分析表明, HvMEL1 AGO为亲水性的不稳定酸性蛋白, 具有高度保守的DUF1785、PAZ和PIWI结构域, 属于AGO基因家族成员。进化树分析表明, HvMEL1 AGO与大麦AGO家族中HvAGO12、HvAGO18、HvAGO1D、HvAGO1B在拟南芥AGO家族系统发育树上属于AGO1一类; 与HvAGO12的亲缘关系最近。蛋白质互作预测结果表明, 在水稻中与MEL1作用密切的已知蛋白为DCL类, 分别为DCL1、DCL2A、DCL3A、DCL3B和DCL4。半定量和定量PCR结果表明, 条纹病胁迫下, 抗病品种昆仑14号与感病品种1141的HvMEL1 AGO基因表达量显著下降; 且1141显著高于昆仑14号(P<0.01)。推测青稞HvMEL1 AGO基因在青稞抗条纹病过程中发挥重要作用。本研究为探索HvMEL1 AGO基因在青稞抗条纹病过程中的作用及调控机制奠定基础。

关键词: 青稞, 条纹病, HvMEL1 AGO基因, 基因表达

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

表1

本实验用到的引物序列"

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

图1

HvMEL1 AGO基因PCR扩增图 a: CDS区域扩增图; b: 3'端扩增图; c: 5'端扩增图; M1: DL2000 DNA marker; M2: λ-Hin d III digest; 1、2、3和4分别代表1141未感病、1141感病、昆仑14未感病和昆仑14感病叶片扩增产物; 5和6分别代表3'和5'端扩增产物。"

图2

HvMEL1 AGO基因全序列及推导的氨基酸序列"

图3

HvMEL1 AGO基因结构域预测"

图4

HvMEL1 AGO蛋白磷酸化位点预测"

图5

HvMEL1 AGO蛋白二级结构预测 蓝色: α-螺旋; 红色: 延伸链; 绿色: β转角; 橙色: 无规则卷曲。"

图6

HvMEL1 AGO蛋白三级结构预测"

图7

HvMEL1 AGO与其他禾本科植物MEL1 AGO多序列比对 Hv: 大麦(青稞); Td: 小麦; Bd: 二穗短柄草; Os: 水稻; At: 节节麦; Sb: 高粱; 黑线代表DUF1785结构域; 红线代表PAZ结构域; 绿线代表PiWi结构域。"

图8

HvMEL1 AGO蛋白与大麦和拟南芥AGO系统进化分析"

图9

HvMEL1 AGO蛋白与水稻相关蛋白互作分析 每个节点代表由一个单一的蛋白质编码基因座产生的蛋白质; 空节点: 未知三维结构的蛋白质; 填充节点: 三维结构是已知的或预测的蛋白质; 明亮的蓝色线: 来自策划的数据库; 紫色线: 实验确定的; 绿色线: 基因邻接; 红色线: 基因融合; 浅绿色线: 文本挖掘; 黑色线: 共表达; 浅紫色线: 蛋白质同源。"

图10

HvMEL1 AGO基因荧光定量和半定量 a: 青稞MEL AGO基因的荧光定量检测结果; b: 青稞MEL AGO基因的半定量的检测结果; KL14N代表抗条纹病品种‘昆仑14号’正常叶片; KL14S代表抗条纹病品种‘昆仑14号’感病叶片; 1141N代表抗条纹病品种1141正常叶片; 1141S代表抗条纹病品种1141感病叶片; 不同的大写字母表示差异极显著(P < 0.01)。"

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