烟草NtCBL1基因的克隆、表达载体构建及表达分析

doi:10.7525/j.issn.1673-5102.2017.03.009

植物研究 ›› 2017, Vol. 37 ›› Issue (3): 387-394.doi: 10.7525/j.issn.1673-5102.2017.03.009

烟草NtCBL1基因的克隆、表达载体构建及表达分析

张雪薇, 刘仑, 鲁黎明, 李立芹

四川农业大学农学院, 成都 611130

收稿日期:2016-12-08 出版日期:2017-05-15 发布日期:2017-06-03
通讯作者: 李立芹,E-mail:liliqin88@163.com E-mail:liliqin88@163.com
作者简介:张雪薇(1993-),女,硕士研究生,主要从事烟草分子生物学研究。
基金资助:
植物生理学与生物化学国家重点实验室开放课题（SKLPPBKF1505，SKLPPBKF1506）

Cloning,Construction of Expression Vector and Expression Analysis of NtCBL1 in Nicotiana tabacum

ZHANG Xue-Wei, LIU Lun, LU Li-Ming, LI Li-Qin

College of Agronomy, Sichuan Agricultural University, Chengdu 611130

Received:2016-12-08 Online:2017-05-15 Published:2017-06-03
Supported by:
The Foundation of State Key Laboratory of Plant Physiology and Biochemistry,China(SKLPPBKF1505,SKLPPBKF1506)

摘要/Abstract

摘要： CBL是一类Ca²⁺感受蛋白，在植物适应或抵制逆境胁迫的过程中发挥重要的作用。从烟草品种K326中克隆到了一个CBL1的同源基因，该序列包含了一个642 bp的开放阅读框，编码一个由213个氨基酸残基组成的蛋白，预测分子量为24.5 kDa，等电点为5.03。同源性分析结果显示，该基因与林烟草CBL1、甜辣椒CBL1等具有较高的同源性，故命名为NtCBL1。生物信息学分析表明，NtCBL1具有CBL家族保守的EF-hand钙结合结构域。组织表达分析发现该基因在成熟期的根、茎、叶、花中均有表达，在根中的表达量最高。逆境胁迫实验表明，该基因表达受低钾、高盐、干旱、ABA和低温诱导调控，参与烟草生物与非生物逆境胁迫的响应。并成功构建了NtCBL1-pBI121过表达载体。研究结果为解析NtCBL1在响应逆境胁迫的功能奠定一定理论基础。

关键词: 烟草, NtCBL1, 克隆, 序列分析, 表达

Abstract: CBL is a kind of Ca²⁺ sensor, which plays a pivotal role in adaptation or resistance to stress in plants. A CBL1 homologous gene was cloned from the tobacco cultivar K326, which contained a 642 bp ORF encoding 213 amino acid. The predictedmolecular weight was 24.5 kDa and the isoelectric point(pI) was 5.03. By the homology analysis, the gene had high homology with calcineurin B-like protein 1 of the Nicotiana sylvestris(NsCBL1) and Arabidopsis thaliana(AtCBL1), so named as NtCBL1. By bioinformatics analysis, NtCBL1 had the conserved EF-hand calcium binding domain of the CBL family. Expression patterns showed that the gene was expressed in roots, stems, leaves and flowers in mature stage, and the highest expression level in roots.Expression patterns under adversity stress indicated that the gene expression was induced by low-potassium, high-salt,anddrought, ABA and low-temperature, and participated in the response to biological and abiotic stress in Nicotiana tabacum. The research constructed NtCBL1-pBI121 overexpression vector.The results will provide some basis for the function analysis of NtCBL1 in response to stress.

Key words: tobacco, NtCBL1, cloning, sequence analysis, expression

中图分类号:

S572

张雪薇, 刘仑, 鲁黎明, 李立芹. 烟草NtCBL1基因的克隆、表达载体构建及表达分析[J]. 植物研究, 2017, 37(3): 387-394.

ZHANG Xue-Wei, LIU Lun, LU Li-Ming, LI Li-Qin. Cloning,Construction of Expression Vector and Expression Analysis of NtCBL1 in Nicotiana tabacum[J]. Bulletin of Botanical Research, 2017, 37(3): 387-394.

参考文献

1. 司丛丛,刘好宝,曲平治. 烟草钾离子通道及转基因烟草抗逆性的研究进展[J]. 中国农学通报,2010,26(2):45-49.Si C C,Liu H B,Qu P Z. Research advances in potassium ion channel of tobacco and stress resistance of transgenic tobacco[J]. Chinese Agricultural Science Bulletin,2010,26(2):45-49.
2. 杨铁钊,鲁黎明,王瑞华. 生物技术在我国烟草研究领域的应用[J]. 中国烟草学报,2006,12(3):39-45.Yang T Z,Lu L M,Wang R H. Application of biotechnology in tobacco research in China[J]. Acta Tabacaria Sinica,2006,12(3):39-45.
3. Hepler P K. Calcium:a central regulator of plant growth and development[J]. The Plant Cell,2005,17(8):2142-2155.
4. Luan S,Kudla J,Rodriguez-concepcion M,et al. Calmodulins and calcineurin B-like proteins:calcium sensorsfor specific signal response coupling in plants[J]. The Plant Cell,2002,14(S1):S389-S400.
5. Kolukisaoglu U,Weinl S,Blazevic D,et al. Calcium sensors and their interacting protein kinases:genomics of the Arabidopsis and rice CBL-CIPK signaling networks[J]. Plant Physiology,2004,134(1):43-58.
6. Xu J,Li H D,Chen L Q,et al. A protein kinase,interacting with two calcineurin B-like proteins,regulates K⁺ transporter AKT1 in Arabidopsis[J]. Cell,2006,125(7):1347-1360.
7. Cheong Y H,Kim K N,Pandey G K,et al. CBL:a calciumsensor that differentially regulates salt,drought,and cold responses in Arabidopsis[J]. Plant Cell,2003,15(8):1833-1845.
8. Zhou Y L,Cheng Y,Yang Y Q,et al. Overexpression of SpCBL6,a calcineurin B-like protein of Stipa purpurea,enhanced cold tolerance and reduced drought tolerancein transgenic Arabidopsis[J]. Molecular Biology Reports,2016,43(9):957-966.
9. Kanwar P,Sanyal S K,Tokas I,et al. Comprehensive structural,interaction and expression analysis of CBL and CIPK complement during abiotic stresses and development in rice[J]. Cell Calcium,2014,56(2):81-95.
10. Wang M Y,Gu D,Liut S,et al. Overexpression of a putative maize calcineurin B-like protein in Arabidopsis confers salttolerance[J]. Plant Molecular Biology,2007,65(6):733-746.
11. Tang R J,Yang Y,Yang L Y,et al. Poplar calcineurin B-like proteins PtCBL10A and PtCBL10B regulate shoot salt tolerance through interaction with PtSOS₂ in the vacuolar membrane[J]. Plant,Cell & Environment,2014,37(3):573-588.
12. Zhang H F,Yang B,Liu W Z,et al. Identification and characterization of CBL and CIPK gene families incanola(Brassica napus L.)[J]. BMC Plant Biology,2014,14(1):8.
13. Huertas R,Olías R,Eljakaoui Z,et al. Overexpression of SlSOS2(SlCIPK24) confers salt tolerance to transgenic tomato[J]. Plant,Cell &Environment,2012,35(8):1467-1482.
14. Lewit-bentley A,Réty S. EF-hand calcium-binding proteins[J]. Current Opinion in Structural Biology,2000,10(6):637-643.
15. Towler D A,Adams S P,Eubanks S R,et al. Myristoyl CoA:protein N-myristoyltransferase activities from ratliver and yeast possess overlapping yet distinct peptide substrate specificities[J]. Journal of BiologicalChemistry,1988,263(4):1784-1790.
16. Batisti O,Sorek N,Schültke S,et al. Dual Fatty Acyl modification determines the localization and plasma membrane targeting of CBL/CIPK Ca²⁺ signaling complexes in Arabidopsis[J]. The Plant Cell,2008,20(5):1346-1362.
17. 吴广宇,李国婧,王瑞刚,等. 野大麦HbCBL1和HbCBL2结构与亚细胞定位分析[J]. 华北农学报,2011,26(4):1-7.Wu G Y,Li G J,Wang R G,et al. Analyses for protein structure and subcellular localization of HbCBL1and HbCBL2 from Hordeum brevisubulatum[J]. Acta Agriculturae Boreali-Sinica,2011,26(4):1-7.
18. Zhang H C,Lü F L,Han X,et al. The calcium sensor PeCBL1,interacting with PeCIPK24/25 and PeCIPK26,regulates Na⁺/K⁺ homeostasisin Populus euphratica[J]. Plant Cell Reports,2013,32(5):611-621.
19. Li J,Jiang M M,Ren L,et al. Identification and characterization of CBL and CIPK gene families in eggplant(Solanum melongena L.)[J]. Molecular Genetics and Genomics,2016,291(4):1769-1781.
20. Li J,Long Y,Qi G N,et al. The Os-AKT1 channel is critical for K⁺ uptake in Rice roots and is modulated by the Rice CBL1-CIPK23 complex[J]. The Plant Cell,2014,26(8):3387-3402.
21. Xu L,Zhang D Y,Xu Z L,et al. Comparative expression analysis of Calcineurin B-like family gene CBL10A between salt-tolerant and salt-sensitive cultivars in B. oleracea[J]. Science of the Total Environment,2016,571:1-10.
22. Chen L,Ren F,Zhou L,et al. The Brassica napus Calcineurin B-Like1/CBL-interactingproteinkinase6(CBL1/CIPK6)componentis involved in theplant response to abiotic stress and ABA signalling[J]. Journal of Experimental Botany,2012,63(17):6211-6222.
23. Huang C L,Ding S,Zhang H,et al. CIPK7 is involved in cold response by interacting with CBL1 in Arabidopsis thaliana[J]. Plant Science,2011,181(1):57-64.
24. 张和臣. 逆境条件下胡杨CBL-CIPK信号途径转导的分子机制研究[D]. 北京:北京林业大学,2010:47-49.Zhang H C. The molecular mechanism of CBL-CIPK signal transduction pathway under abiotic stess in Populus euphratica[D]. Beijing:Beijing Forestry University,2010:47-49.

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烟草NtCBL1基因的克隆、表达载体构建及表达分析

Cloning,Construction of Expression Vector and Expression Analysis of NtCBL1 in Nicotiana tabacum

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