Gene Cloning and Functional Research of Importin IMPα2 in Solanum tuberosum

doi:10.7668/hbnxb.20195990

Abstract

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

CLC Number:

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.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20195990

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

Figures/Tables 5

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.

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(-)

Fig.2 Subcellular localization of StIMPα2

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).

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).

References 26

[11]	Chen C, De Masi R, Lintermann R, Wirthmueller L. Nuclear import of Arabidopsis poly(ADP-ribose) polymerase 2 is mediated by importin-α and a nuclear localization sequence located between the predicted SAP domains[J].Frontiers in Plant Science, 2018,9:1581.doi:10.3389/fpls.2018.01581.
[12]	Chen C, Kim D, Yun H R, Lee Y M, Yogendra B, Bo Z, Kim H E, Min J H, Lee Y S, Rim Y G, Kim H U, Sung S, Heo J B. Nuclear import of LIKE HETEROCHROMATIN PROTEIN1 is redundantly mediated by importins α-1,α-2 and α-3[J]. The Plant Journal, 2020, 103(3):1205-1214.doi:10.1111/tpj.14796. URL
[13]	Wirthmueller L, Roth C, Banfield M J, Wiermer M. Hop-on hop-off:importin-α-guided tours to the nucleus in innate immune signaling[J]. Frontiers in Plant Science, 2013,4:149.doi:10.3389/fpls.2013.00149.
[14]	Palma K, Zhang Y L, Li X. An importin α homolog,MOS6,plays an important role in plant innate immunity[J]. Current Biology, 2005, 15(12):1129-1135.doi:10.1016/j.cub.2005.05.022. URL
[15]	Zeng L P, Gomez Mendez M F, Guo J Z, Jiang J S, Zhang B L, Chen H, Le B, Ke H Y, Dehesh K. Activation of stress-response genes by retrograde signaling-mediated destabilization of nuclear importin IMPα-9 and its interactor TPR2[J]. Molecular Plant, 2024, 17(6):884-899.doi:10.1016/j.molp.2024.04.008. URL
[16]	Chen T T, Peng J, Yin X, Li M J, Xiang G Q, Wang Y J, Lei Y, Xu Y. Importin-αs are required for the nuclear localization and function of the Plasmopara viticola effector PvAVH53[J]. Horticulture Research, 2021,8:46.doi:10.1038/s41438-021-00482-6.
[17]	Wang L N, Zhang K P, Wang Z H, Yang J, Kang G Z, Liu Y, You L Y, Wang X F, Jin H B, Wang D W, Guo T C. Appropriate reduction of importin-α gene expression enhances yellow dwarf disease resistance in common wheat[J]. Plant Biotechnology Journal, 2024, 22(3):572-586.doi:10.1111/pbi.14204. URL
[18]	Xu Y, Liu L, Zhao P, Tong J, Zhong N Q, Zhang H J, Liu N. Genome-wide identification,expression profile and evolution analysis of karyopherin β gene family in Solanum tuberosum group phureja DM1-3 reveals its roles in abiotic stresses[J]. International Journal of Molecular Sciences, 2020, 21(3):931.doi:10.3390/ijms21030931. URL
[19]	Stewart M. Molecular mechanism of the nuclear protein import cycle[J]. Nature Reviews Molecular Cell Biology, 2007, 8(3):195-208.doi:10.1038/nrm2114. pmid: 17287812
[20]	Li Q, Zhang X C, Hu W T, Liang X T, Zhang F, Wang L Z, Liu Z J, Zhong Y. Importin-7 mediates memory consolidation through regulation of nuclear translocation of training-activated MAPK in Drosophila[J]. Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(11):3072-3077.doi:10.1073/pnas.1520401113.
[21]	Volpon L, Culjkovic-Kraljacic B, Osborne M J, Ramteke A, Sun Q X, Niesman A, Chook Y M, Borden K L B. Importin 8 mediates m⁷G cap-sensitive nuclear import of the eukaryotic translation initiation factor eIF4E[J]. Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(19):5263-5268.doi:10.1073/pnas.1524291113. pmid: 27114554
[22]	Liu H H, Xiong F, Duan C Y, Wu Y N, Zhang Y, Li S. Importin β4 mediates nuclear import of GRF-interacting factors to control ovule development in Arabidopsis[J]. Plant Physiology, 2019, 179(3):1080-1092.doi:10.1104/pp.18.01135. URL
[23]	Jiang C J, Shoji K, Matsuki R, Baba A, Inagaki N, Ban H, Iwasaki T, Imamoto N, Yoneda Y, Deng X W, Yamamoto N. Molecular cloning of a novel importin α homologue from rice,by which constitutive photomorphogenic 1 (COP1) nuclear localization signal (NLS)-protein is preferentially nuclear imported[J]. Journal of Biological Chemistry, 2001, 276(12):9322-9329.doi:10.1074/jbc.M006430200. pmid: 11124253
[24]	Lukhovitskaya N I, Cowan G H, Vetukuri R R, Tilsner J, Torrance L, Savenkov E I. Importin-α-mediated nucleolar localization of Potato mop-top virus TRIPLE GENE BLOCK1(TGB1)Protein facilitates virus systemic movement,whereas TGB1 self-interaction is required for cell-to-cell movement in Nicotiana benthamiana[J]. Plant Physiology, 2015, 167(3):738-752.doi:10.1104/pp.114.254938. pmid: 25576325
[25]	Luo Y J, Wang Z J, Ji H T, Fang H, Wang S F, Tian L N, Li X. An Arabidopsis homolog of importin β1 is required for ABA response and drought tolerance[J]. The Plant Journal, 2013, 75(3):377-389.doi:10.1111/tpj.12207. URL
[26]	Wu S J, Wang L C, Yeh C H, Lu C A, Wu S J. Isolation and characterization of the Arabidopsis heat-intolerant 2(hit2)mutant reveal the essential role of the nuclear export receptor EXPORTIN1A (XPO1A) in plant heat tolerance[J]. New Phytologist, 2010, 186(4):833-842.doi:10.1111/j.1469-8137.2010.03225.x.
[1]	Chook Y, Blobel G. Karyopherins and nuclear import[J]. Current Opinion in Structural Biology, 2001, 11(6):703-715.doi:10.1016/S0959-440X(01)00264-0. pmid: 11751052
[2]	Cook A, Bono F, Jinek M, Conti E. Structural biology of nucleocytoplasmic transport[J]. Annual Review of Biochemistry, 2007, 76:647-671.doi:10.1146/annurev.biochem.76.052705.161529. pmid: 17506639
[3]	Tamura K, Hara-Nishimura I. Functional insights of nucleocytoplasmic transport in plants[J]. Frontiers in Plant Science, 2014,5:118.doi:10.3389/fpls.2014.00118.
[4]	Wang W, Ye R Q, Xin Y, Fang X F, Li C L, Shi H Q, Zhou X P, Qi Y J. An importin β protein negatively regulates microRNA activity in Arabidopsis[J]. The Plant Cell, 2011, 23(10):3565-3576.doi:10.1105/tpc.111.091058. pmid: 21984696
[5]	Görlich D, Prehn S, Laskey R A, Hartmann E. Isolation of a protein that is essential for the first step of nuclear protein import[J]. Cell, 1994, 79(5):767-778.doi:10.1016/0092-8674(94)90067-1. pmid: 8001116
[6]	Moroianu J, Blobel G, Radu A. Previously identified protein of uncertain function is karyopherin alpha and together with karyopherin beta docks import substrate at nuclear pore complexes[J]. Proceedings of the National Academy of Sciences of the United States of America, 1995, 92(6):2008-2011.doi:10.1073/pnas.92.6.2008. pmid: 7892216
[7]	Radu A, Blobel G, Moore M S. Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins[J]. Proceedings of the National Academy of Sciences of the United States of America, 1995, 92(5):1769-1773.doi:10.1073/pnas.92.5.1769. pmid: 7878057
[8]	Oka M, Yoneda Y. Importin α:functions as a nuclear transport factor and beyond[J]. Proceedings of the Japan Academy, Series B, 2018, 94(7):259-274.doi:10.2183/pjab.94.018.
[9]	Christie M, Chang C W, Róna G, Smith K M, Stewart A G, Takeda A A S, Fontes M R M, Stewart M, Vértessy B G, Forwood J K, Kobe B. Structural biology and regulation of protein import into the nucleus[J]. Journal of Molecular Biology, 2016, 428(10):2060-2090.doi:10.1016/j.jmb.2015.10.023. URL
[10]	Wirthmueller L, Roth C, Fabro G, Caillaud M C, Rallapalli G, Asai S T, Sklenar J, Jones A M E, Wiermer M, Jones J D G, Banfield M J. Probing formation of cargo/importin-α transport complexes in plant cells using a pathogen effector[J]. The Plant Journal, 2015, 81(1):40-52.doi:10.1111/tpj.12691. pmid: 25284001

Please choose a citation manager

Content to export