Construction and Application of Plant Expression Vector Containing NLS-GFP Fusion Gene
LUAN Hong-Ying1,2, CHEN Ai-E3, WANG Hui-Jie1,2, LIU Jing1,2, WANG Hong-Yang1,2,*, LI Can-Hui1,2,*
1 Yunnan Key Laboratory of Potato Biology, Yunnan Normal University, Kunming 650500, China; 2 Key Lab of Potato Biology in Universities of Yunnan Province, Yunnan Normal University, Kunming 650500, China; 3 Teaching Affairs Department, Yunnan Normal University, Kunming 650500, China
Abstract：Plant cells are highly compartmentalized into distinct subcellular structures, which requires proper assembly of relevant protein players in the right place at the right time. The nucleus is the control center of the cell, which entails protein expression and trafficking in a highly coordinated manner. Nuclear localization signal (NLS) promotes localization of proteins into nucleus that plays an important role in studying the cell biological functions of proteins. Based on the limitations of current NLS plant expression vectors, this study has obtained a new pRI101-NLS-GFP plant expression vector by fusing NLS with GFP based on the seamless cloning. To determine the subcellular localization of GFP, pRI101-NLS-GFP was transiently expressed in Nicotiana benthamiana. NLS-GFP fusion protein accumulated strongly in the nucleus, while the fluorescence of GFP spreaded the whole cell. In the meantime, the RXLR (arginine-x-leucine-arginine) effector gene PITG_04314 (PITG: Phytophthora infestans T30-4 gene, GenBank No. XM_002905913) was used to further investigate whether the vector could successfully introduce protein into the nucleus. Previous studies have shown that PITG_04314 localizes to the nucleus with additional cytoplasmic background and functions in thenucleus. To further confirm the localization, PITG_04314 was fused with pRI101-NLS-GFP and pRI101-NLS. Consistent with previous results, GFP-PITG_04314 was localized to the nucleus with cytoplasmic background, whereas NLS-GFP-PITG 04314 accumulated strongly in the nucleus. Transient overexpression GFP-PITG_04314 and NLS-GFP-PITG_04314 could both promote the Phytophthorainfestans colonization, indicated that PITG_04314 functions in the nucleus. Taken together, this study showed that NLS-GFP plant expression vector based on seamless cloning technology could promote the target protein into the nucleus, suggesting it could be widely used in the study of biological function of pathogen proteins.
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