马铃薯<i>StPHR1</i>基因克隆与表达特征分析

doi:10.7668/hbnxb.20195260

摘要/Abstract

摘要：

PHR1是平衡植物抗病性与低磷胁迫抗性的关键因子。为研究马铃薯StPHR1基因的性质和功能,探明StPHR1在马铃薯抵抗早疫病菌侵染过程中的作用。以马铃薯为材料,采用PCR技术克隆得到了马铃薯StPHR1基因CDS序列,利用生物信息学软件分析预测StPHR1的结构、理化性质和亲缘关系,随后利用qRT-PCR技术分析StPHR1在早疫病菌侵染马铃薯时和不同激素处理下的表达量,并通过激光共聚焦扫描显微技术进行了蛋白质亚细胞定位分析。结果表明:StPHR1基因CDS全长为1 353 bp,编码450个氨基酸。蛋白分子式为C₂₁₄₇H₃₃₉₉N₅₉₅O₇₁₁S₁₈,分子质量为49.51 ku,理论等电点为5.07,编码亲水性不稳定蛋白,无信号肽,无跨膜结构,二级结构主要由无规则卷曲和α螺旋组成。系统进化树分析发现,StPHR1蛋白与拟南芥的亲缘关系最近;保守结构域分析发现,StPHR1蛋白和其他PHR1一样,在其蛋白C末端同时具有MYB-CC和MYB保守结构域。相对表达量分析发现,StPHR1被茄链格孢和水杨酸显著诱导表达,推测StPHR1可能在茄链格孢侵染马铃薯和水杨酸响应方面起重要作用;亚细胞定位显示,StPHR1蛋白定位于细胞核中。推测StPHR1可能通过其MYB转录因子活性和响应水杨酸参与调控马铃薯对茄链格孢菌的抗性。

关键词: 马铃薯, 早疫病菌, StPHR1, 生物信息学分析, 亚细胞定位

Abstract:

PHR1 is a crucial factor in balancing plant disease resistance and low phosphorus stress resistance.To investigate the nature and function of the StPHR1 gene in potato and to explore the role of StPHR1 in the process of potato resistance to Alternaria solani infection,the CDS sequence of the StPHR1 gene was cloned by PCR technology using potatoes as the research material,and the structural,physicochemical properties,and phylogenetic relationships of StPHR1 were analyzed and predicted using bioinformatics software,then,the expression level of StPHR1 during the infection of potatoes by A.solani and under different hormone treatments was analyzed using qRT-PCR technology,and subcellular localization analysis of the protein was conducted using laser confocal microscopy technology.The results showed that the CDS of the StPHR1 gene was 1 353 bp,encoding 450 amino acids.The protein had a molecular formula of C₂₁₄₇H₃₃₉₉N₅₉₅O₇₁₁S₁₈,a molecular weight of 49.51 ku,and a theoretical isoelectric point of 5.07,encoding a hydrophilic,unstable protein with no signal peptide and no transmembrane structure.Its secondary structure consisted mainly of random coil and α-helix.Phylogenetic tree analysis revealed that the StPHR1 protein was most closely related to Arabidopsis thaliana; conservative domain analysis revealed that the StPHR1 protein,like other PHR1s,possesses both MYB-CC and MYB conserved structural domains at its C-terminus.Relative expression analysis found that StPHR1 was significantly induced by A.solani and salicylic acid,and it was hypothesized that StPHR1 played an important role in A.solani infection of potato and in the response to salicylic acid; and the subcellular localization showed that the StPHR1 protein was localized in the nucleus.It is hypothesized that StPHR1 may regulate potato resistance to A.solani through its MYB transcription factor activity and response to salicylic acid.

Key words: Potato, Alternaria solani, StPHR1, Bioinformatics analysis, Subcellular localization

彭泽驰, 姜海滨, 丁丽丽, 杨志浩, 杨志辉, 朱杰华. 马铃薯StPHR1基因克隆与表达特征分析[J]. 华北农学报, 2025, 40(2): 33-39. doi: 10.7668/hbnxb.20195260.

PENG Zechi, JIANG Haibin, DING Lili, YANG Zhihao, YANG Zhihui, ZHU Jiehua. Cloning and Expression Characteristic Analysis of StPHR1 from Potato[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(2): 33-39. doi: 10.7668/hbnxb.20195260.

http://www.hbnxb.net/CN/Y2025/V40/I2/33

图/表 14

表1 试验所需引物

Tab.1 Primers required for the experiment

引物名称 Primer name	序列(5'—3') Sequence(5'—3')
qStActin7-F	GCTATGTATGTTGCTATTCAG
qStActin7-R	ATCTTCATCAGGTTATCAGTT
qStPHR1-F	TGTTGGTCTTATGTTCTCA
qStPHR1-R	CGATGTCTCACTATATGTTG
StPHR1-F	CG GGATCCATGGAAGCACGCCCTGC
StPHR1-R	GC GTCGACTTCATCCAATTTAGAGCGCTTCA

表2 生物信息学分析数据网址

Tab.2 Bioinformatics analysis data site

功能 Function	网址 Web site
预测氨基酸序列 Predicting amino acid sequences	https://www.ncbi.nlm.nih.gov/orffinder/
预测蛋白分子质量 Predicting protein molecular weight	https://www.detaibio.com/sms2/protein_mw.html
预测理论等电点 Predicting the theoretical isoelectric point (pI)	https://www.detaibio.com/sms2/protein_iep.html
预测亲水性 Predicting hydrophilicity	https://web.expasy.org/protscale/
预测二级结构 Predicting secondary structure	https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl? page=/NPSA/npsa_sopma.html
预测三级结构 Predicting tertiary structure	https://swissmodel.expasy.org/
预测跨膜结构域 Predicting transmembrane structural domains	https://services.healthtech.dtu.dk/services/TMHMM-2.0/
预测信号肽 Predicting signal peptide	https://services.healthtech.dtu.dk/services/SignalP-5.0/

图1 StPHR1蛋白的系统进化分析

Fig.1 Phylogenetic analysis of StPHR1

图2 以马铃薯cDNA为模板扩增StPHR1基因 M.DNA Marker 2000;1.PCR扩增产物。

Fig.2 PCR amplification for StPHR1 gene using potato cDNA as template M.DNA Marker 2000;1.PCR product.

图3 StPHR1蛋白亲水性分析

Fig.3 Hydrophilicity analysis of StPHR1 protein

图4 StPHR1蛋白信号肽预测

Fig.4 Signal peptide prediction of StPHR1

图5 StPHR1蛋白跨膜结构域预测

Fig.5 Transmembrane region prediction of StPHR1

图6 StPHR1蛋白的保守功能域预测分析

Fig.6 Conserved domain analysis of StPHR1

图7 StPHR1蛋白二级结构预测

Fig.7 Secondary structure prediction of StPHR1

图8 StPHR1蛋白三级结构预测

Fig.8 Tertiary structure prediction of StPHR1

图9 StPHR1在茄链格孢侵染5 d高度上调表达不同小写字母表示差异显著(P<0.05)。 t检验。图10同。

Fig.9 StPHR1 is highly up-regulated on 5 d after Alternaria solani infection Different lowercase letters indicate significant differences(P<0.05).Student's t-test.The same as Fig.10.

图10 StPHR1在不同激素处理时的表达模式

Fig.10 Expression pattern of StPHR1 in response to different hormone treatments

图11 亚细胞定位载体pCAMBIA1301-StPHR1-eGFP菌落PCR鉴定 M.DNA Marker 2000;1.阴性对照;2—4.以不同单菌落为模板扩增StPHR1。

Fig.11 PCR identification of pCAMBIA1301-StPHR1-eGFP colonies with subcellular localization vector M.DNA Marker 2000;1.Negative control;2—4.Amplify StPHR1 with different single colonies as the templates.

图12 StPHR1在本氏烟叶片中的亚细胞定位

Fig.12 Subcellular localization of StPHR1 in Nicotiana benthamiana leaves

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马铃薯StPHR1基因克隆与表达特征分析

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