马铃薯核运输蛋白IMPα2基因克隆及功能分析

doi:10.7668/hbnxb.20195990

摘要/Abstract

摘要：

为探究马铃薯中StIMPα的功能,以马铃薯克星一号为材料,通过PCR技术成功克隆了StIMPα2基因。生物信息学分析结果表明,该基因的编码区(CDS)全长为1 590 bp,其编码的蛋白含有IMPα家族的典型结构域。系统进化树分析显示,StIMPα2与拟南芥中的AtIMPα-1和AtIMPα-2亲缘关系最近,暗示其功能的保守性。此外,对StIMPα2启动子序列的分析发现,其区域内含有多种与生物及非生物胁迫响应相关的顺式作用元件。为进一步明确其细胞内定位,构建了StIMPα2-GFP融合表达载体,并通过农杆菌介导的瞬时转化法在本氏烟草叶片中进行表达。激光共聚焦显微镜观察结果显示,GFP荧光信号特异性地富集于细胞核,证实StIMPα2是一个定位于细胞核的蛋白。表达模式分析发现,StIMPα2的表达受低温、高盐、干旱等非生物胁迫以BTH的显著诱导。在功能验证方面,通过农杆菌浸润法在本氏烟草中过表达StIMPα2,并接种马铃薯致病疫霉。病理学表型分析显示,与对照相比,过表达StIMPα2的烟草叶片其病斑面积显著减小;同时,通过实时荧光定量PCR检测致病疫霉生物量,也证实了StIMPα2过表达植株中的致病疫霉生物量显著降低。综上所述,StIMPα2是一个受多种生物与非生物胁迫诱导表达的核定位蛋白,并通过正调控植物免疫反应来增强对致病疫霉的抗性。

关键词: 马铃薯, StIMPα2, 克隆, 致病疫霉, 抗性

Abstract:

To investigate the function of StIMPα in potato, this study used the potato cultivar Kexing No.1 as material and successfully cloned the StIMPα2 gene via PCR. Bioinformatic analysis revealed that the coding sequence (CDS) of StIMPα2 had a length of 1 590 bp, encoding a protein containing the typical domains of the IMPα family. Phylogenetic tree analysis indicated that StIMPα2 was most closely related to AtIMPα-1 and AtIMPα-2 from Arabidopsis thaliana, suggesting functional conservation. Furthermore, analysis of the StIMPα2 promoter region identified multiple cis-acting elements associated with responses to biotic and abiotic stresses. To determine its subcellular localization, a StIMPα2-GFP fusion expression vector was constructed and transiently expressed in leaves of Nicotiana benthamiana via Agrobacterium-mediated transformation. Confocal laser scanning microscopy showed that the GFP fluorescence signal was specifically enriched in the nucleus, confirming that StIMPα2 is a nuclear-localized protein. Expression pattern analysis demonstrated that StIMPα2 expression was significantly induced by abiotic stresses such as low temperature, high salinity, and drought, as well as by BTH (benzothiadiazole). For functional validation, StIMPα2 was overexpressed in N. benthamiana via Agrobacterium infiltration, followed by inoculation with Phytophthora infestans. Pathological phenotype analysis showed that compared with the control, the lesion area on leaves overexpressing StIMPα2 was significantly reduced. Meanwhile, Quantitative Real-time PCR detection of P. infestans biomass confirmed a significant decrease in pathogen biomass in StIMPα2-overexpressing plants. In conclusion, these results indicate that StIMPα2 is a nuclear-localized protein induced by various biotic and abiotic stresses, and it enhances resistance to P. infestans by positively regulating plant immune responses.

Key words: Solanum tuberosum, StIMPα2, Cloning, Phytophthora infestans, Resistance

中图分类号:

张一帆, 林睿, 周娜, 胡安琪, 白薇. 马铃薯核运输蛋白IMPα2基因克隆及功能分析[J]. 华北农学报, 2025, 40(6): 71-76. doi: 10.7668/hbnxb.20195990.

ZHANG Yifan, LIN Rui, ZHOU Na, HU Anqi, BAI Wei. Gene Cloning and Functional Research of Importin IMPα2 in Solanum tuberosum[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(6): 71-76. doi: 10.7668/hbnxb.20195990.

http://www.hbnxb.net/CN/Y2025/V40/I6/71

图/表 5

图1 StIMPα2的克隆及编码蛋白的生物信息学分析 A.StIMPα2克隆电泳图(M.TaKaRa DL5000 Marker);B.StIMPα2蛋白保守结构域预测;C.StIMPα2的系统进化分析。

Fig.1 Cloning of stIMPα2 and bioinformatics analysis of its coding protein A.PCR results of StIMPα2(M.TaKaRa DL5000 Marker);B.Protein conserved domain prediction of StIMPα2;C.Phylogenetic analysis of StIMPα2.

表1 StIMPα2启动子区域顺式响应元件分析

Tab.1 Analysis of cis-acting regulatory elements in the promoter region of StIMPα2

元件名称 Element name	序列 Sequence	元件数量/个 Element quantity	功能 Function	所在位置/bp Position
MRE	AACCTAA	1	光响应元件	532(-)
GT1-motif	GGTTAAT	1	光响应元件	535(+)
G-Box	CACGTT	2	光响应元件	9(-);451(+)
Myb	TAACTG	2	防卫和胁迫响应元件	1 272(+);1 739(+)
MYC	CATTTG	4	干旱、低温和ABA响应元件	132(-);573(-);867(-);1 970(+)
DRE core	GCCGAC	1	高盐和低温响应元件	877(+)
CGTCA-motif	CGTCA	1	茉莉酸响应元件	219(+)
TCA-element	CCATCTTTTT	1	水杨酸响应元件	1 984(-)
ABRE	ACGTG	2	脱落酸响应元件	10(+);451(-)
ARE	AAACCA	2	生长素元件	1 003(-);1 939(-)

图2 StIMPα2的亚细胞定位

Fig.2 Subcellular localization of StIMPα2

图3 低温胁迫、盐胁迫、干旱胁迫和BTH诱导后 StIMPα2的相对表达量不同小写字母表示不同时间点差异显著(P<0.05)。

Fig.3 The relative expression level of StIMPα2 induced by low temperature stress,salt stress,drought stress and BTH induction Different lowercase letters indicate significant differences at different time points (P<0.05).

图4 过表达StIMPα2增强本氏烟草对致病疫霉的抗性 A.本氏烟草中StIMPα2相对表达量;B.本氏烟草叶片病斑展示结果;C.本氏烟草叶片病斑面积统计;D.本氏烟草叶片中致病疫霉相对生物量。EV.转化pRI 101-AN的空载体,OE-StIMPα2.转化StIMPα2-pRI 101-AN的过表达载体。不同小写字母表示不同叶片种类差异显著(P<0.05)。

Fig.4 Overexpression of StIMPα2 enhances resistance to Phytophthora infestans in Nicotiana benthamiana A.Relative expression levels of StIMPα2 in Nicotiana benthamiana;B.Disease lesion phenotypes in Nicotiana benthamiana leaves;C.Statistical analysis of lesion areas in Nicotiana benthamiana leaves;D.Relative biomass of Phytophthora infestans in leaves.EV.Empty vector pRI 101-AN,OE-StIMPα2.Overexpression vector StIMPα2-pRI 101-AN.Different lowercase letters indicate significant differences among leaf types (P<0.05).

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