高粱抗病相关基因<i>SbRPM1</i>的克隆及表达分析

doi:10.7668/hbnxb.20194651

摘要/Abstract

摘要：

为探究RPM1在高粱抗病过程中的作用,以高粱抗黑穗病品种SX44B为材料,采用同源克隆法获得一个高粱SbRPM1基因。生物信息学分析结果显示:该基因的cDNA全长2 802 bp,预测共编码933个氨基酸。该基因编码蛋白的理论分子质量为106.1 ku,等电点为7.11,为亲水性蛋白质;该蛋白无跨膜结构,亚细胞定位在细胞质中。保守结构域分析显示,SbRPM1蛋白含有RX-CC-like、NB-ARC和LRR结构域,属于NLRs受体蛋白家族中的CNL类蛋白。系统发育关系分析表明,SbRPM1蛋白与南荻RPM1蛋白亲缘关系最近。通过实时荧光定量PCR技术检测SbRPM1基因的表达模式,结果表明,该基因在叶和花序中表达量较高,其次是根,在茎中表达量最低。在接种丝黑穗病原菌后24~72 h抗病品种中SbRPM1基因的表达显著上调,表明该基因受病原菌诱导表达,在高粱抗病反应中扮演着重要角色。首次在高粱中克隆得到SbRPM1基因CDS序列,解析了该基因的结构、性质与表达的特征。

关键词: 高粱, SbRPM1基因, 基因克隆, 生物信息学分析, 表达分析

Abstract:

In order to explore the role of RPM1 in sorghum disease resistance,a sorghum SbRPM1 gene was obtained from sorghum smut resistant variety SX44B by homologous cloning method.The bioinformatics analysis results showed that the total length of the cDNA of SbRPM1 gene was 2 802 bp,encoding 933 amino acids,and its protein had a theoretical molecular weight of 106.1 ku and an isoelectric point of 7.11,which was a hydrophilic protein.The SbRPM1 protein had no transmembrane structure,and its subcellular localization was in the cytoplasm.Conservative domain analysis showed that SbRPM1 protein contained RX-CC-like,NB-ARC and LRR domains,and belonged to CNL proteins in the NLRs family.Phylogenetic analysis showed that SbRPM1 protein was most closely related to the RPM1 protein of Miscanthus lutarioriparius.The expression pattern of SbRPM1 gene was detected by Real-time quantitative PCR,and the results showed that the expression of SbRPM1 gene was higher in leaves and inflorescence,followed by roots,and the lowest in stem.The expression of SbRPM1 gene was significantly up-regulated at 24—72 h in disease-resistant varieties after inoculation with Sporisorium reilianum pathogen,suggesting that this gene could be induced by S.reilianum and played an important role in sorghum disease resistance.In this study, the CDS sequence of the SbRPM1 gene was cloned for the first time in sorghum, and the structure, nature and expression of the gene were characterized.

Key words: Sorghum, SbRPM1 gene, Gene clone, Bioinformatics analysis, Expression analysis

王慧珍, 张朝政, 黄一鸣, 黎耀心, 程子洋, 乐超银. 高粱抗病相关基因SbRPM1的克隆及表达分析[J]. 华北农学报, 2024, 39(2): 153-160. doi: 10.7668/hbnxb.20194651.

WANG Huizhen, ZHANG Chaozheng, HUANG Yiming, LI Yaoxin, CHENG Ziyang, YUE Chaoyin. Cloning and Expression Analysis of Disease Resistance Gene SbRPM1 in Sorghum[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(2): 153-160. doi: 10.7668/hbnxb.20194651.

http://www.hbnxb.net/CN/Y2024/V39/I2/153

图/表 8

表1 引物信息

Tab.1 Primer information

引物名称 Primer name	引物序列(5'—3') Primer sequences (5'—3')	功能 Function
SbRPM1-F	AGTGAATTCGAGCTCGGTAC	基因克隆
SbRPM1-R	GCCCGGGATCCGATATCTAG
qRT-SbRPM1-F	CGGTTATGCTGGAGTTAAGA	qRT-PCR
qRT-SbRPM1-R	AACAGCATTACGCAAGTCTA
qRT-SbACTIN-F	CGAAGACATACTGGACGCAACA
qRT-SbACTIN-R	GGGCGGAAAGAATTAGAAGC

图1 SbRPM1基因的PCR扩增产物 M.Magic Ruler DNA Marker;1.SbRPM1片段。

Fig.1 PCR products of SbRPM1 gene M.Magic Ruler DNA Marker;1.SbRPM1 DNA segment.

图2 高粱SbRPM1与其他植物RPM1氨基酸序列的多重比对红色下划线为NB-ARC结构域。

Fig.2 The multiple alignment of SbRPM1 and other plant RPM1 amino acid sequences The red under line is NB-ARC domain.

图3 SbRPM1蛋白及其同源序列的系统发育树分析

Fig.3 Phylogenetic tree analysis of SbRPM1 protein and its homologues

图4 SbRPM1蛋白的理化性质分析及结构预测 A.SbRPM1蛋白的亲/疏水性预测;B.SbRPM1蛋白二级结构预测;C.SbRPM1蛋白的三级结构预测;D.SbRPM1蛋白的保守结构域。

Fig.4 Physicochemical property analysis and structure prediction of SbRPM1 protein A.Prediction of affinity/hydrophobicity of SbRPM1 protein;B.Secondary structure of SbRPM1 protein; C.Tertiary structure of SbRPM1 protein;D.Conserved domain of SbRPM1 protein.

图5 SbRPM1蛋白信号肽、跨膜区及磷酸化位点预测 A.SbRPM1蛋白信号肽预测;B.SbRPM1蛋白跨膜区预测;C.SbRPM1蛋白磷酸化位点预测。

Fig.5 SbRPM1 protein signal peptide,transmembrane region and phosphorylation site prediction A.SbRPM1 protein signal peptide prediction;B.SbRPM1 protein transmembrane region prediction; C.SbRPM1 protein phosphorylation site prediction.

图6 SbRPM1基因在SX44B不同组织中的表达不同小写字母表示不同组织间差异显著(P<0.05)。图7同。

Fig.6 Expression of SbRPM1 gene in different tissues of SX44B Different lowercase letters indicate significant difference in different tissue (P<0.05).The same as Fig.7.

图7 SbRPM1在抗感高粱品种中受丝黑穗病菌诱导的表达特征

Fig.7 Expression of SbRPM1 gene induced by S.reilianumt in resistant and susceptible sorghum varieties

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高粱抗病相关基因SbRPM1的克隆及表达分析

Cloning and Expression Analysis of Disease Resistance Gene SbRPM1 in Sorghum

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高粱抗病相关基因SbRPM1的克隆及表达分析

Cloning and Expression Analysis of Disease Resistance Gene SbRPM1 in Sorghum

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