园艺学报 ›› 2021, Vol. 48 ›› Issue (6): 1079-1093.doi: 10.16420/j.issn.0513-353x.2020-0902
马俊杰1, 宋丽娜1, 李乐1, 马晓春1, 靳磊1, 徐伟荣2,3,4,*()
收稿日期:
2021-03-16
修回日期:
2021-05-11
出版日期:
2021-06-25
发布日期:
2021-07-07
通讯作者:
徐伟荣
E-mail:xuwr@nxu.edu.cn
基金资助:
MA Junjie1, SONG Lina1, LI Le1, MA Xiaochun1, JIN Lei1, XU Weirong2,3,4,*()
Received:
2021-03-16
Revised:
2021-05-11
Online:
2021-06-25
Published:
2021-07-07
Contact:
XU Weirong
E-mail:xuwr@nxu.edu.cn
摘要:
对山葡萄(Vitis amurensis)‘左山-1'中的1个类钙调素磷酸酶B亚基蛋白基因VaCBL6进行克隆及功能分析。VaCBL6位于第4条染色体,开放阅读框为777 bp,编码258个氨基酸;结构域分析表明,该蛋白包含4个EF-hand与1个跨膜结构域;蛋白进化树分析表明,VaCBL6与欧洲葡萄VvCBL9和VvCBL6相似性较高,分别为98.84%、94.96%。VaCBL6编码蛋白定位于细胞核与细胞膜。VaCBL6在根与卷须中有较高的表达,且其表达受低温、干旱、NaCl和ABA的诱导。基于酵母体系的转录激活分析表明,VaCBL6无转录激活活性。100 ~ 125 μmol · L -1 NaCl和0.25 ~ 1.25 μmol · L -1 ABA处理均抑制了VaCBL6转基因拟南芥种子的萌发,且转化拟南芥幼苗对NaCl与ABA的敏感性提高。以上结果说明VaCBL6在植物的逆境胁迫应答中具有重要作用。
中图分类号:
马俊杰, 宋丽娜, 李乐, 马晓春, 靳磊, 徐伟荣. 山葡萄VaCBL6参与非生物胁迫和ABA途径的响应[J]. 园艺学报, 2021, 48(6): 1079-1093.
MA Junjie, SONG Lina, LI Le, MA Xiaochun, JIN Lei, XU Weirong. VaCBL6 from Vitis amurensis Involved in Abiotic Stress Response and ABA-mediated Pathway[J]. Acta Horticulturae Sinica, 2021, 48(6): 1079-1093.
引物 Primer | 引物序列(5′-3′) Primer sequence |
---|---|
qPCR-CBL6-F | TGCTGCGACTTCCACAAA |
qPCR-CBL6-R | CTCATACAGTGCCTCCACTTC |
RT-VvActin-F | CTATCCTTCGTCTTGACCTTGCTG |
RT-VvActin-R | AGTGGTGAACATGTAACCCCTCTC |
Semi-CBL6-F | GGAAGAGCTTCAATTGGCGT |
Semi-CBL6-R | ACTGTGGTGATGTCCTTCAAAT |
AtActin2-RT-F | TGAGCAAAGAAATCACAGCACT |
AtActin2-RT-R | CCTGGACCTGCCTCATCATAC |
VaCBL6-ORF-F | ATGAGTTCTTGGCAGGGAACGGCG |
VaCBL6-ORF-R | TCATTCTTCAACCTCAGTATTGAAAACAAAGC |
221-VaCBL6-F | GAGAGAACACGGGGGACTCTAGAATGAGTTCTTGGCAGGGAACGGCG |
221-VaCBL6-R | TTACCCATGGTACCCCGCTCGAGTTCTTCAACCTCAGTATTGAAAACAAAGC |
BD-CBL6-F | atggccatggaggccgaattcATGAGTTCTTGGCAGGGAACG |
BD-CBL6-R | ccgctgcaggtcgacggatccTCATTCTTCAACCTCAGTATTGAAAAC |
Entry-VaCBL6-F | AAAAAAgCAggCTTTgACTTTATgAgTTCTTggCAgggAACg |
Entry-VaCBL6-R | AAAgCTgggTCTAgAgACTTTCCTTCTTCAACCTCAgTATTgA |
表1 各种载体构建及VaCBL6 表达检测引物
Table 1 Primers of vector generation and the expression of VaCBL6
引物 Primer | 引物序列(5′-3′) Primer sequence |
---|---|
qPCR-CBL6-F | TGCTGCGACTTCCACAAA |
qPCR-CBL6-R | CTCATACAGTGCCTCCACTTC |
RT-VvActin-F | CTATCCTTCGTCTTGACCTTGCTG |
RT-VvActin-R | AGTGGTGAACATGTAACCCCTCTC |
Semi-CBL6-F | GGAAGAGCTTCAATTGGCGT |
Semi-CBL6-R | ACTGTGGTGATGTCCTTCAAAT |
AtActin2-RT-F | TGAGCAAAGAAATCACAGCACT |
AtActin2-RT-R | CCTGGACCTGCCTCATCATAC |
VaCBL6-ORF-F | ATGAGTTCTTGGCAGGGAACGGCG |
VaCBL6-ORF-R | TCATTCTTCAACCTCAGTATTGAAAACAAAGC |
221-VaCBL6-F | GAGAGAACACGGGGGACTCTAGAATGAGTTCTTGGCAGGGAACGGCG |
221-VaCBL6-R | TTACCCATGGTACCCCGCTCGAGTTCTTCAACCTCAGTATTGAAAACAAAGC |
BD-CBL6-F | atggccatggaggccgaattcATGAGTTCTTGGCAGGGAACG |
BD-CBL6-R | ccgctgcaggtcgacggatccTCATTCTTCAACCTCAGTATTGAAAAC |
Entry-VaCBL6-F | AAAAAAgCAggCTTTgACTTTATgAgTTCTTggCAgggAACg |
Entry-VaCBL6-R | AAAgCTgggTCTAgAgACTTTCCTTCTTCAACCTCAgTATTgA |
图2 VaCBL6和其他物种CBL氨基酸序列的聚类分析 采用MEGA 7.0的邻接法构建系统进化树。相关分类群聚集在一起的分支旁边显示百分比(%),分支长度以每个点的替换数量来度量。自展值为1 000。
Fig. 2 Phylogenetic relationships of Vitis amurensis VaCBL6 with CBLs from other plant species Protein sequences were aligned to construct a phylogenetic tree using the Neighbor-Joining(NJ)method by MEGA 7.0 software. The percentage of trees with related clusters were displayed next to the branches,and the phylogenetic tree was drawn proportionally with branch lengths measured in the number of substitutions per site. Bootstrap value was 1 000.
图4 山葡萄VaCBL6 在不同组织中的表达特性 *,** 表示VaCBL6的表达量与根(表达量为1)相比差异显著(* α = 0.05,** α = 0.01)。
Fig. 4 Expression pattern of VaCBL6 in various tissues of Vitis amurensis *,** indicated significant differences compared with the expression level of root(expression level is 1)(* α = 0.05,** α = 0.01).
图5 山葡萄在非生物胁迫与ABA处理下叶片中VaCBL6的表达分析 * 和 ** 代表与同期对照相比差异显著(* α = 0.05,** α = 0.01)。
Fig. 5 Expression pattern of VaCBL6 in grape leaves under abiotic stress and ABA treatment * and ** indicated significant differences compared with control groups(* α = 0.05,** α = 0.01).
图8 VaCBL6过表达拟南芥株系的鉴定 A:RNA分离电泳;B:半定量RT-PCR分析;C:qRT-PCR分析。WT:野生型。** α = 0.01。
Fig. 8 Identification of VaCBL6-overexpressing lines in Arabidopsis thaliana A:Agarose gel electrophoresis of RNA;B:Expression of VaCBL6 analyzed by semi quantitative RT-PCR;C:Expression level of VaCBL6 analyzed by qRT-PCR. WT:Wild type. ** α = 0.01.
图9 VaCBL6过量表达拟南芥株系(#1、#2和#6)在含不同浓度NaCl的1/2 MS培养基上的生长情况 WT:野生型;#1,#2和#6:转基因株系。下同。
Fig. 9 Phenotypes of Arabidopsis thaliana overexpressing VaCBL6 lines(#1,#2 and #6)on 1/2 MS medium supplemented with different concentrations of NaCl WT:Wild type;#1,#2 and #6:35S::VaCBL6 transgenic lines. The same below.
图10 VaCBL6过量表达拟南芥株系(#1、#2和 #6)在含不同浓度NaCl的1/2 MS培养基上0 ~ 7 d的萌发率
Fig. 10 Germination rates of Arabidopsis thaliana overexpressing VaCBL6 lines(#1,#2 and #6)under different concentrations of NaCl at 0-7 d
图11 VaCBL6过量表达拟南芥株系(#1、#2和#6)在含不同浓度NaCl的1/2 MS培养基上播种10 d时的绿苗率
Fig. 11 Germination rates of green cotyledon of seedlings of Arabidopsis thaliana overexpressing VaCBL6 lines (#1,#2 and #6)grown for 10 days under different concentrations of NaCl
图12 VaCBL6过表达拟南芥株系(#1、#2和#6)在含不同浓度ABA的1/2 MS培养基上的生长情况
Fig. 12 Phenotypes of Arabidopsis thaliana overexpressing VaCBL6 lines(#1,#2 and #6) on 1/2 MS medium supplemented with different concentrations of ABA
图13 VaCBL6过表达拟南芥株系(#1、#2和#6)在含不同浓度ABA的1/2 MS培养基上0 ~ 7 d的萌发率
Fig. 13 Germination rates of Arabidopsis thaliana overexpressing VaCBL6 lines(#1,#2 and #6) under different concentrations of ABA at 0-7 d
图14 VaCBL6过表达拟南芥株系(#1、#2和#6)在含不同浓度ABA的1/2 MS培养基上播种10 d时的绿苗率
Fig. 14 Germination rates of green cotyledon of seedlings of Arabidopsis thaliana overexpressing VaCBL6 lines (#1,#2 and #6)grown for 10 d under different concentrations of ABA
[1] |
Albrecht V, Ritz O, Linder S, Harter K, Kudla J. 2001. The NAF domain defines a novel protein-protein interaction module conserved in Ca2+ regulated kinases. The EMBO Journal, 20 (5):1051-1063.
doi: 10.1093/emboj/20.5.1051 URL |
[2] |
Batistič O, Waadt R, Steinhorst L, Held K, Kudla J. 2010. CBL mediated targeting of CIPKs facilitates the decoding of calcium signals emanating from distinct cellular stores. The Plant Journal, 61 (2):211-222.
doi: 10.1111/j.1365-313X.2009.04045.x URL |
[3] | Batistič O, Kudla J. 2012. Analysis of calcium signaling pathways in plants. Biochimica et Biophysica Acta(BBA)-General Subjects, 1820 (8):1283-1293. |
[4] |
Carmona M J, Cubas P, Calonje M, Martinez-Zapater J M. 2007. Flowering transition in grapevine(Vitis vinifera L.). Canadian Journal of Botany, 85 (8):701-711.
doi: 10.1139/B07-059 URL |
[5] | Chen Sha-sha, Lan Hai-yan. 2011. Signal transduction pathways in response to salt stress in plants. Plant Physiology Journal, 47 (2):119-128. (in Chinese) |
陈莎莎, 兰海燕. 2011. 植物对盐胁迫响应的信号转导途径. 植物生理学报, 47 (2):119-128. | |
[6] |
Chen X, Gu Z, Xin D, Hao L, Liu C, Huang J, Ma B, Zhang H. 2011. Identification and characterization of putative CIPK genes in maize. Journal of Genetics and Genomics, 38 (2):77-87.
doi: 10.1016/j.jcg.2011.01.005 URL |
[7] |
Clough S J, Bent A F. 1998. Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal, 16 (6):735-743.
doi: 10.1046/j.1365-313x.1998.00343.x URL |
[8] | Dong Lian-hong, Shi Su-juan, Nuruzzaman Manik S. 2015. Advances in research of CBL family in plant. Journal of Nuclear Agricultural Sciences, 29 (5):892-898. (in Chinese) |
董连红, 史素娟, Nuruzzaman Manik S. 2015. 植物CBL基因家族的研究进展. 核农学报, 29 (5):892-898. | |
[9] |
Gu Z, Ma B, Jiang Y, Chen Z, Su X, Zhang H. 2008. Expression analysis of the calcineurin B-like gene family in rice(Oryza sativa L.)under environmental stresses. Gene, 415 (1-2):1-12.
doi: 10.1016/j.gene.2008.02.011 URL |
[10] |
Hepler P K. 2005. Calcium:a central regulator of plant growth and development. The Plant Cell, 17 (8):2142-2155.
doi: 10.1105/tpc.105.032508 URL |
[11] |
Ishitani M, Liu J, Halfter U, Kim C S, Shi W, Zhu J K. 2000. SOS 3 function in plant salt tolerance requires N-myristoylation and calcium binding. The Plant Cell, 12:1667-1677.
doi: 10.1105/tpc.12.9.1667 URL |
[12] |
Kolukisaoglu U, Weinl S, Blazevic D, Batistič O, Kudla J. 2004. Calcium sensors and their interacting protein kinases:genomics of the Arabidopsis and rice CBL-CIPK signaling networks. Plant Physiology, 134 (1):43-58.
doi: 10.1104/pp.103.033068 URL |
[13] | Ling Q, Zeng Q, Wu J, Hu F, Li Q, Qi Y. 2019. Expression analysis of CBL1 and CBL6 genes in sugarcane under abiotic stress. Molecular Plant Breeding, 10 (1):1-10. |
[14] |
Liu J, Zhu J K. 1998. A calcium sensor homolog required for plant salt tolerance. Science, 280:1943-1945.
doi: 10.1126/science.280.5371.1943 URL |
[15] |
Liu P, Duan Y, Liu C, Xue Q, Guo J, Qi T, Kang Z, Guo J. 2018. The calcium sensor TaCBL4 and its interacting protein TaCIPK 5 are required for wheat resistance to stripe rust fungus. Journal of Experimental Botany, 69 (21):4443-4457.
doi: 10.1093/jxb/ery227 URL |
[16] | Mullins M G, Bouquet A, Williams L E. 1992. Biology of the grapevine. UK: Cambridge University Press. |
[17] | Qiu Q S, Guo Y, Dietrich M A, Schumaker K S, Zhu J K. 2002. Regulation of SOS1,a plasma membrane Na+/H+ exchanger in Arabidopsis thaliana,by SOS2 and SOS3. Proceedings of the National Academy of Sciences, 99 (12):8436-8441. |
[18] |
Quan R, Lin H, Mendoza I, Zhang Y, Cao W, Yang Y, Shang M, Chen S, Pardo J M, Guo Y. 2007. SCABP8/CBL10,a putative calcium sensor,interacts with the protein kinase SOS 2 to protect Arabidopsis shoots from salt stress. The Plant Cell, 19 (4):1415-1431.
doi: 10.1105/tpc.106.042291 URL |
[19] |
Sánchez-Barrena M J, Martínez-Ripoll M, Zhu J K, Albert A. 2005. The structure of the Arabidopsis thaliana SOS3:molecular mechanism of sensing calcium for salt stress response. Journal of Molecular Biology, 345 (5):1253-1264.
doi: 10.1016/j.jmb.2004.11.025 URL |
[20] |
Tang R J, Liu H, Yang Y, Yang L, Gao X S, Garcia V J, Luan S, Zhang H X. 2012. Tonoplast calcium sensors CBL2 and CBL 3 control plant growth and ion homeostasis through regulating V-ATPase activity in Arabidopsis. Cell Research, 22 (12):1650-1665.
doi: 10.1038/cr.2012.161 URL |
[21] | Tang R J, Yang Y, Yang L, Liu H, Wang C T, Yu M M, Gao X S, Zhang H X. 2014. Poplar calcineurin B-like proteins PtCBL10A and PtCBL10B regulate shoot salt tolerance through interaction with PtSOS 2 in the vacuolar membrane. Plant Cell & Environment, 37 (3):573-588. |
[22] |
Weinl S, Kudla J. 2009. The CBL-CIPK Ca 2+-decoding signaling network: function and perspectives. New Phytologist, 184 (3):517-528.
doi: 10.1111/nph.2009.184.issue-3 URL |
[23] |
Wu F H, Shen S C, Lee L-Y, Lee S H, Chan M T, Lin C S. 2009. Tape- Arabidopsis Sandwich-a simpler Arabidopsis protoplast isolation method. Plant Methods, 5:16.
doi: 10.1186/1746-4811-5-16 URL |
[24] |
Xu W, Shen W, Ma J, Ya R, Zheng Q, Wu N, Yu Q, Yao W, Zhang N, Zhang J. 2020. Role of an Amur grape CBL-interacting protein kinase VaCIPK02 in drought tolerance by modulating ABA signaling and ROS production. Environmental and Experimental Botany, 172:103999.
doi: 10.1016/j.envexpbot.2020.103999 URL |
[25] |
Yu Y, Xia X, Yin W, Zhang H. 2007. Comparative genomic analysis of CIPK gene family in Arabidopsis and Populus. Plant Growth Regulation, 52 (2):101-110.
doi: 10.1007/s10725-007-9165-3 URL |
[26] | Yu Yi-he, Li Xiu-zhen, Guo Da-long, Yang Ying-jun, Li Gui-rong, Li Xue-qiang, Zhang Guo-hai. 2016. Isolation and expression analysis of calcineurin B-like protein VvCBL4 in grapevines. Journal of Fruit Science, 33 (4):385-392. (in Chinese) |
余义和, 李秀珍, 郭大龙, 杨英军, 李桂荣, 李学强, 张国海. 2016. 葡萄类钙调磷酸酶B亚基蛋白基因 VvCBL4的克隆与表达分析. 果树学报, 33 (4):385-392. | |
[27] |
Zhang H, Yin W, Xia X. 2008. Calcineurin B-Like family in Populus:comparative genome analysis and expression pattern under cold,drought and salt stress treatment. Plant Growth Regulation, 56 (2):129-140.
doi: 10.1007/s10725-008-9293-4 URL |
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