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作物学报 ›› 2019, Vol. 45 ›› Issue (9): 1365-1374.doi: 10.3724/SP.J.1006.2019.81087

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大麦蛋白质二硫键异构酶基因家族的鉴定与表达分析

时丽洁1,2,蒋枞璁2,王方梅1,2,杨平2,*(),冯宗云1,*()   

  1. 1 四川农业大学农学院大麦青稞研究中心, 四川成都 611130
    2 中国农业科学院作物科学研究所, 北京 100081
  • 收稿日期:2018-12-11 接受日期:2019-04-15 出版日期:2019-09-12 网络出版日期:2019-04-18
  • 通讯作者: 杨平,冯宗云
  • 作者简介:E-mail: 1013842716@qq.com
  • 基金资助:
    本研究由中国农业科学院基本科研业务费工程(S2018YC01);本研究由中国农业科学院基本科研业务费工程(S2018PY03);国家现代农业产业技术体系(大麦青稞)建设专项资助(CARS-05)

Genome-wide characterization and transcriptional analysis of the protein disulfide isomerase-like genes in barley (Hordeum vulgare)

SHI Li-Jie1,2,JIANG Cong-Cong2,WANG Fang-Mei1,2,YANG Ping2,*(),FENG Zong-Yun1,*()   

  1. 1 Barley Research Center, Colleague of Agronomy, Sichuan Agricultural University, Chengdu 611130, Sichuan, China
    2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2018-12-11 Accepted:2019-04-15 Published:2019-09-12 Published online:2019-04-18
  • Contact: Ping YANG,Zong-Yun FENG
  • Supported by:
    This study was supported by the Fundamental Research Funds for Central Non-Profit of Chinese Academy of Agricultural Sciences(S2018YC01);This study was supported by the Fundamental Research Funds for Central Non-Profit of Chinese Academy of Agricultural Sciences(S2018PY03);the China Agriculture Research System(CARS-05)

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摘要：

蛋白质二硫键异构酶(protein disulfide isomerases, PDIs)是定位于真核生物内质网中的一类蛋白, 具有催化蛋白质二硫键氧化还原反应与协助蛋白质发生构型变化、类似分子伴侣的功能, 参与生长发育、生物或非生物胁迫应答等方面的调控。真核生物中PDI基因家族的分子鉴定已有报道, 但是对大麦PDI基因家族的鉴定分析鲜有报道。本研究利用生物信息学分析手段鉴定了10个大麦PDI-Like基因(HvPDILs), 并分析了相应编码蛋白的结构特征。与其他物种PDILs的系统发育分析发现, 10个大麦PDILs分处在8个不同的系统分支, 与小麦PDILs高度同源。对公共数据库中的组织和时空表达数据分析发现, HvPDILs基因均表达, 且具组织和时空表达特异性。对接种大麦温性花叶病毒(BaMMV)之后的叶片样品进行基因表达水平分析, 发现其中5个HvPDILs基因出现显著的表达差异, 说明其参与病毒感染过程, 但参与方式及作用机制还有待后续研究。

关键词: 大麦, 蛋白质二硫键异构酶, 基因家族, 大麦黄花叶病

Abstract:

Protein disulfide isomerase (PDI) and PDI-like proteins (PDILs) belong to a protein family that are usually located on endoplasmic reticulum and highly conserved across eukaryotes. The main function of PDILs is catalyzing the redox and isomerization of disulfide bonds in protein precursors via the thioredoxin (TRX) domain. PDILs have been also reported as chaperones in folding and unfolding of proteins, thus being important in growth and development, as well as biotic or abiotic stress responses of plant. In this study, we identified by bioinformatics analysis 10 members of barley PDILs (HvPDILs), which were subjected for analyzing the physical location, protein structure and subcellular localization. The phylogenetic analysis revealed eight sub-branches of plant PDILs, and barley PDILs showed a high sequence homology with wheat PDILs. The transcriptional analysis revealed large variation of the transcription abundance of PDILs in different tissues and development stages. Notably, mechanical inoculation of Barley mild mosaic virus (BaMMV) resulted in transcriptional re-programming of several HvPDILs. This result suggests the association between PDILs and BaMMV infection, despite that the functional mechanism remains largely unknown and needs further studies.

Key words: barley, protein disulfide isomerase, gene family, barley yellow mosaic virus disease

表1

大麦PDI基因家族信息"

基因名称
Gene name		 水稻同源基因
Rice orthologous gene		 NCBI登录号
Acc. ID		 基因ID
Gene ID		 染色体位置
Chromosomal location		 cDNA全长
Full length cDNA
HvPDIL1-1 OsPDIL1_1 AK357991.1 HORVU4Hr1G043910.1 4H:350262817-350266613 1542
HvPDIL1-2 OSPDIL1_2 AK368066.1 HORVU2Hr1G076390.6 2H:549859450-549862026 1554
HvPDIL1-3 OSPDIL1_4 AK370108.1 HORVU6Hr1G001700.1 6H:5173471-5182932 1764
HvPDIL1-4 OSPDIL1_5 AK366963.1 HORVU7Hr1G026030.2 7H:44299270-44302170 1635
HvPDIL2-1 OSPDIL2_1 AK249580.1 HORVU1Hr1G022810.1 1H:98741192-98744845 1104
HvPDIL2-2 OSPDIL2_3 AK353639.1 HORVU5Hr1G067570.1 5H:513285275-513290639 1323
HvPDIL5-1 OSPDIL5_1 AK250421.1 HORVU4Hr1G056730.1 4H:476590992-476593244 456
HvPDIL5-2 OSPDIL5_2 AK251979.1 HORVU2Hr1G075810.4 2H:545243929-545244816 1245
HvPDIL5-3 OSPDIL5_3 AK249445.1 HORVU6Hr1G059710.3 6H:395564404-395567220 1257
HvPDIL5-4 OSPDIL5_4 AK251501.1 HORVU2Hr1G046000.3 2H:243330620-243337299 1458

图1

HvPDILs基因的染色体分布 图中6个白色圆柱代表大麦的6条染色体, 圆柱左侧和右侧分别是基因的物理位置(Mb)和基因名称。由于3H染色体上没有PDILs基因, 故未展示。"

图2

大麦PDILs蛋白的结构示意图"

表2

大麦PDILs蛋白特质"

图3

大麦与小麦、水稻、玉米、拟南芥PDILs同源蛋白的系统进化树"

图4

HvPDILs基因的组织表达情况 EMB: 胚胎; ROO1: 幼根; LEA: 苗期地上部分; INF2: 新生穗; NOD: 第3节间分蘖; CAR5: 前期灌浆籽粒; CAR15: 后期灌浆籽粒; ETI: 黄化苗; LEM: 主穗; LOD: 浆片; PAL: 内稃; EPI: 外颖壳; RAC: 穗轴; ROO2: 灌浆期根部; SEN: 衰老叶。"

表3

qPCR引物及扩增片段"

引物名称
Primer name		 引物序列
Sequence (5'-3')		 退火温度
Tm (°C)		 扩增片段大小
Size of
amplicon (bp)		 备注
Remarks
HvUBC-F AAGCAGCCAGAATGTACAGCGAGAAC 63 152 内参基因
Reference gene
HvUBC-R GGTACAGACCAGCAAAGCCAGAAATG
BaMMV-F GGCACTTTGTGTTATGCAATGG 63 138 检测BaMMV
Check the presence of BaMMV
BaMMV-R TGCCACCCTCAGGTAGGATTAG
HvPDIL1_1-F CGTTGCTTGGTTGAAGGATT 60 199 检测基因表达
Check the expression of HvPDIL1-1
HvPDIL1_1-R GATGGGTGCTAGCTTCTTGC
HvPDIL1_2-F CAGACCCGACCAACCATAAG 60 194 检测基因表达
Check the expression of HvPDIL1-2
HvPDIL1_2-R ATACTCGAAGGCACGGTCAG
引物名称
Primer name		 引物序列
Sequence (5'-3')		 退火温度
Tm (°C)		 扩增片段大小
Size of amplicon (bp)		 备注
Remarks
HvPDIL1_3-F GTGTGGCCATTGTCAGTCAC 60 200 检测基因表达
Check the expression of HvPDIL1-3
HvPDIL1_3-R CCCCCTCGAAAGTTATAGGC
HvPDIL1_4-F AGTTGGCCAAGCATTTCAGT 60 198 检测基因表达
Check the expression of HvPDIL1-4
HvPDIL1_4-R CTGCAGCTTCTCCTTGATGA
HvPDIL2_1-F CGGGACTTGGATGACTTTGT 60 200 检测基因表达
Check the expression of HvPDIL2-1
HvPDIL2_1-R AGGACCACTGAGCTTTGCAG
HvPDIL2_2-F CTAGCGCAGATGTGATGGAA 60 198 检测基因表达
Check the expression of HvPDIL2-2
HvPDIL2_2-R GCTTCTCCAGATCAGCTTGC
HvPDIL5_1-F CGAAGAGACCTTCTCCGACA 60 200 检测基因表达
Check the expression of HvPDIL5-1
HvPDIL5_1-R GAATGTCCACCTTTGAGCAGA
HvPDIL5_2-F CCCCGAGTTAGATGAAGCTG 60 199 检测基因表达
Check the expression of HvPDIL5-2
HvPDIL5_2-R AAGATTGCGGACAAGCAAGT
HvPDIL5_3-F GAAACTTCCATGCGGTTGAT 60 199 检测基因表达
Check the expression of HvPDIL5-3
HvPDIL5_3-R CCAAACTCTCCAAACCCTCA
HvPDIL5_4-F CCTGAAATGGATGGCAGAAT 60 200 检测基因表达
Check the expression of HvPDIL5-4
HvPDIL5_4-R TCCATAGCCGCGACTAAACT

图5

病毒接种后叶片中HvPDILs基因的相对表达分析 差异显著性统计方法为t检验(*P < 0.05; **P < 0.01), 误差棒表示标准误差。"

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