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| Cloning of VaCBL01 Gene from Vitis amurensis and Analysis of Its Interaction with VaCIPKs Proteins |
| SHEN Wei1,2, YAO Wen-Kong1, ZHENG Qiao-Ling1,2, ZHANG Ning-Bo1,2, XU Wei-Rong1,2,3* |
1 College of Agronomy, Ningxia University, Yinchuan 750021, China;
2 Engineering Research Center of Grape and Wine, Ministry of Education, Ningxia University / Ningxia Institute of Grape &Wine / Ningxia Engineering and Technology Research Center of Grape and Wine, Yinchuan 750021, China;
3 Key Laboratory of Modern Molecular Breeding for Dominant and Special Crops in Ningxia, Yinchuan 750021, China |
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Abstract Calcineurin B-like proteins (CBLs) represent a family of newly emerging plant-specific Ca2+ sensors, which could selectively interplay with their respective kinase effectors (CBL-interacting protein kinases, CIPKs), thus playing critical roles in response to signal transduction of various abiotic stresses. To identify the functional characteristics of the CBLs gene involved in stress response in Vitis plants, the cold-tolerant species of Vitis amurensis 'Zuoshan-1' was used as the material to isolate one calcineurin B protein family member VaCBL01 by homologous cloning. Analysis of its sequence and structure indicated that the ORF of VaCBL01 gene (GenBank No. MH921999) was 642 bp, encoding 213 amino acids, with four EF-conserved domains, and a myristoylation site at the N-terminus. Chromosome location prediction indicated that VaCBL01 could be mapped to locus of 5 571 989~5 581 623 in chromosome 2. Sequence alignment revealed that VaCBL01 shared high sequence homology with Vitis vinifera VvCBL01 (100%), followed by tomato (Solanum lycopersicum) SlCBL01 (95.3%), Arabidopsis AtCBL01 (95.3%) and PtCBL01 (94.8%). Phylogenetic analysis of CBL01 gene families across a variety of species suggested VaCBL01 protein had the closest evolutionary relationship with proteins clustered in GroupⅡ, including V. vinifera, tomato, Arabidopsis and Populus trichocarpa. Furthermore, the expression levels of VaCBL01 genes either in different tissue and organs or in response to abscisic acid (ABA) and cold stress were examined by qRT-PCR. Expression of VaCBL01 was found to be ubiquitous in all tissues in V. amurensis, but its expression was relatively high in tendril. VaCBL01 gene was found to respond to ABA and cold stress, suggesting that the grapevine CBL-CIPK network may be a point of convergence for several different signaling pathways. Also, a comparison of interaction patterns of VaCBL01 and 19 VaCIPKs was performed using yeast two-hybrid system. The results showed that none of the 19 VaCIPKs had autotoxicity and self-activation in yeast cells. VaCBL01 exhibited a significant interaction only with a subset of 12 CIPKs (VaCIPK01, 02, 04, 06, 09, 10, 11, 12, 13,16, 17 and 18). For 5 additional CIPKs (VaCIPK03, 07, 15, 19 and 20), a weak tendency of complex formation was observed, whereas 2 CIPKs (VaCIPK08 and 14) did not show any affinity toward AtCBL01. The results would provide an important foundation for further functional dissection of the CBL-CIPK signaling network in grapevine.
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Received: 19 November 2018
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Corresponding Authors:
xuwr@nxu.edu.cn
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