我们的网站为什么显示成这样?

可能因为您的浏览器不支持样式,您可以更新您的浏览器到最新版本,以获取对此功能的支持,访问下面的网站,获取关于浏览器的信息:

|Table of Contents|

马尾松PmCBL3基因的克隆及其表达分析(PDF)

《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

Issue:
2017年04期
Page:
30-36
Column:
研究论文
column:
2017-07-31

Article Info:/Info

Title:
Cloning and expression analysis of PmCBL 3 from Pinus massoniana
Article ID:
1000-2006(2017)04-0030-07
Author:
蔡 琼丁贵杰*丁 波张仁波杜明凤
贵州省森林资源与环境研究中心,贵州大学林学院,贵州 贵阳 550025
Author(s):
CAI Qiong DING Guijie*DING Bo ZHANG Renbo DU Mingfeng
Institute for Forest Resources &
Environment of Guizhou, College of Forestry, Guizhou University, Guiyang 550025, China
Keywords:
马尾松 PmCBL3 基因克隆 表达分析
Keywords:
Pinus massoniana PmCBL3 gene cloning expression analysis
Classification number :
S795
DOI:
10.3969/j.issn.1000-2006.2017.04.006
Document Code:
A
Abstract:
【目的】为明确马尾松(Pinus massoniana)CBL基因结构特征及干旱胁迫下的表达特性,探讨该基因在马尾松抗旱调控过程中的生物学功能。【方法】以马尾松优良家系为试材,采用RT-PCR和RACE方法克隆马尾松CBL基因全长cDNA,运用生物信息学方法,分析其蛋白质性质和亲缘关系,利用荧光定量PCR分析干旱胁迫下的基因表达特性。【结果】获得马尾松的一个CBL同源基因,命名为PmCBL3,该基因cDNA序列全长1 035 bp,包括681 bp的完整开放阅读框,编码226个氨基酸。PmCBL3蛋白含4个EF-hand功能域,且相邻EF-hand功能域之间的氨基酸数目非常保守,属于EFh家族蛋白。系统进化分析表明:该蛋白与其他植物CBL同源性达62%~98%,其中与北美云杉(Picea sitchensis)CBL亲缘关系最近。qRT-PCR结果显示,PmCBL3在马尾松根中表达量最高,茎和叶次之; 干旱第15天表达量最大,随干旱胁迫加剧,表达量逐渐降低。【结论】PmCBL3基因属于CBLs家族,与北美云杉亲缘关系最近。该基因响应干旱胁迫,推测其可能参与马尾松干旱逆境的应答调控。
Abstract:
【Objective】CBLs participates in plants response to abiotic stress. The objectives of this study were to investigate biological function of CBL, and to provide a candidate gene and theoretical basis for the drought tolerant breeding for Pinus massoniana, by cloning the CBL from P. massoniana, and analyzing its sequence features, evolutionary relationships and expression characteristics. 【Method】The full-length cDNA of CBL had been isolated from P. massoniana by RT-PCR and RACE, and bioinformatics analysis was conducted to analyze the properties and phylogenetic tree. In addition, the expression profile of CBL was analyzed by qRT-PCR under drought stress.【Result】A full-length cDNA, designated PmCBL3, was obtained. Sequence analysis revealed that PmCBL3 was 1 035 bp in length, including a 681 bp open reading frame that encodes 226 amino acids. PmCBL3 comprised four EF-Hand functional domains, and the distance between the EF-hand motifs was conservative. Evolutionary analysis further revealed that PmCBL3 had the closest relationship with Picea sitchensis, and CBL exhibited 62%-98% homologies with other plants. qRT-PCR results showed that PmCBL3 expression was the highest in the roots, then was in the stems and leaves. PmCBL3 expression was in the peak at 15 d after treatment, but decreased with drought stress aggravation. 【Conclusion】The results indicated that PmCBL3 belonged to the member of CBLs family genes, and had the closest relationship with CBL of P. sitchensis. PmCBL3 responded drought stress, which suggested that it was involved in drought stress regulation

References

[1] BATISITCˇ O, KUDLA J. Plant calcineurin B-like proteins and their interacting protein kinases[J].Biochimica Et Biophysica Acta, 2009, 1793(6):985-992.DOI:10.1016/j.bbamcr.2008.10.006.
[2] REN X L, QI G N, FENG H Q, et al.Calcineurin B-like protein CBL10 directly interacts with AKT1 and modulates K+ homeostasis in Arabidopsis[J].Plant Journal for Cell & Molecular Biology, 2013, 74(2):258-266.DOI: 10.1111/tpj.12123.
[3] ZHANG H C, YIN W L, XIA X L.Calcineurin B-like family in Populus: comparative genome analysis and expression pattern under cold, drought and salt stress treatment[J].Plant Growth Regulation, 2008, 56(2): 129-140.DOI:10.1007/s10725-008-9293-4.
[4] 赵晋锋, 余爱丽, 田岗, 等.谷子CBL基因鉴定及其在干旱、高盐胁迫下的表达分析[J].作物学报, 2013, 39(2): 360-367.DOI: 10.3724/SP.J.1006.2013.00360. ZHAO J F, YU A L, TIAN G, et al.Identification of CBL genes from foxtail millet(Setaria italic[L.] Beauv.)and its expression under drought and salt stresses[J].Acta Agronomica Sinica, 2013, 39(2):360-367.
[5] GU Z M, MA B J, JIANG Y, et al.Expression analysis of the calcineurin B-like gene family in rice(Oryza sativa L.)under environmental stresses[J].Gene, 2008, 415: 1-12.DOI:10.1016/j.gene.2008.02.011.
[6] MAHIJAN S, TUTEJA N. Cold, salinity and drought stresses: an overview[J].Archives of Biochemistry & Biophyssics, 2005, 444(2):139-158.DOI:10.1016/j.abb.2005.10.018.
[7] PANDEY G K, CHEONG Y H, KIM K N, et al.The calcium sensor calcineurin B-like 9 modulates abscisic acid sensitivity and biosynthesis in Arabidopsis[J].Plant Cell, 2004, 16(7):1912-1924.DOI/10.1105/tpc.021311.
[8] 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.DOI: 10.1016/j.cell.2006.06.011.
[9] CHEONG Y H, KIM K N, PANDEY G K, et al.CBL1, a calcium sensor that differentially regulates salt, drought, and cold responses in Arabidopsis[J].Plant Cell, 2003, 15(8): 1833-1845.DOI/10.1105/tpc.012393.
[10] D'ANGELO C, WEINL S, BATISTIC O, et al.Alternative complex formation of the Ca2+-regulated protein kinase CIPK1 controls abscisic acid-dependent and independent stress responses in Arabidopsis[J].Plant Journal for Cell & Molecular Biology, 2006, 48(6): 857-872.DOI: 10.1111/j.1365-313X.2006.02921.x.
[11] 丁志强, 尚桂军, 李娜,等.沙冬青CBL1蛋白纯化及性质研究[J].中国生物工程杂志, 2011, 31(2): 23-29.DOI:10.13523/j.cb.20110205. DING Z Q, SHANG G J, LI N, et al.Purification and characterization of CBL1 from Ammopiptanthus mongolicus[J].China Biotechnology, 2011, 31(2): 23-29.
[12] WANG M Y, GU D, LIU T, et al.Overexpression of a putative maize calcineurin B-like protein in Arabidopsis confers salt tolerance[J].Plant Molecular Biology, 2007, 65(6): 733-746.DOI:10.1007/s11103-007-9238-8.
[13] GAO P, ZHAO P M, WANG J, et al.Co-expression and preferential interaction between two calcineurin B-like proteins and a CBL-interacting protein kinase from cotton[J].Plant Physiology & Biochemistry, 2008, 46(10): 935-940.DOI:10.1016/j.plaphy.2008.05.001.
[14] HWANG Y S, BETHKE P C, CHEONG Y H, et al.A gibberellin-regulated calcineurin B in rice localizes to the tonoplast and is implicated in vacuole function[J].Plant Physiology, 2005, 138(3): 1347-1358.
[15] 丁贵杰, 周志春, 王章荣, 等.马尾松纸浆用材树种培育与利用[M].北京:中国林业出版社, 2006: 111. DING G J, ZHOU Z C, WANG Z R, et al.Cultivation and utilization of pulpwood stand for Pinus massoniana[M].Beijing: China Forestry publishing house, 2006:111.
[16] 蔡琼, 丁贵杰, 文晓鹏.马尾松水通道蛋白PmPIP1 基因克隆及在干旱胁迫下的表达分析[J].浙江农林大学学报, 2016, 33(2): 191-200.DOI:10.11833/j.issn.2095-0756.2016.02.002. CAI Q, DING G J, WEN X P.Cloning of the PmPIP1 gene from Pinus massoniana and its expression with drought stress[J].Journal of Zhejiang A & F University, 2016, 33(2): 191-200
[17] 施积炎, 丁贵杰, 袁小凤.不同家系马尾松苗木水分参数的研究[J].林业科学, 2004, 40(3): 51-55. SHI J Y, DING G J, YUAN X F. Studies on water parameters in Pinus massoniana seedlings of different families[J].Scienita Silvae Sinicae, 2004, 40(3): 51-55.
[18] 施积炎, 丁贵杰, 袁小凤.不同家系马尾松维持水分平衡能力及综合评价[J].上海交通大学学报(农业科学版), 2004, 22(2): 143-148. SHI J Y, DING G J, YUAN X F.Ability to maintain water of Masson pine from different family[J].Journal of Shanghai Jiaotong University(Agricultural Science), 2004, 22(2): 143-148.
[19] 袁小凤, 丁贵杰, 施积炎.土壤干旱胁迫对不同品系马尾松水分利用效率的影响[J].浙江师范大学学报(自然科学版), 2008, 31(3): 328-331. YUAN X F, DING G J, SHI J Y.Effect of soil drought stress on water use efficiency of Masson pine of different species[J].Journal of Zhejiang Normal University(Nat Sci), 2008, 31(3): 328-331.
[20] FAN F H, CUI B W, ZHANG T, et al.The temporal transcriptomic response of Pinus massoniana seedlings to phosphorus deficiency[J].PLoS One, 2014, 9(8): e105068.DOI:10.1371/journal.pone.0105068.
[21] PFAFFL M W.A new mathematical model for relative quantification in real-time RT-PCR[J].Nucleic Acids Research, 2001, 29(9): 2002-2007.
[22] BATISTIC O, KUDLA J.Integration and channeling of calcium signaling through the CBL calcium sensor/CIPK protein kinase network[J].Planta, 2004, 219(6): 915-924.DOI: 10.1007/s00425-004-1333-3.
[23] 付海辉, 辛培尧, 许玉兰, 等.几种经济植物UFGT基因的生物信息学分析[J].基因组学与应用生物学, 2010, 30(1): 92-102. FU H H, XIN P Y, XU Y L, et al.Bioinformatics analysis of UFGT gene from several economic plants[J].Genomics and Applied Biology, 2010, 30(1): 92-102.
[24] TOMINAGA M, HARADA A, KINOSHITA T, et al.Biochemical characterization of calcineurin B-like-interacting protein kinase in Vicia Guard Cells[J].Plant Cell Physiology, 2010, 51(3): 408-421.DOI:10.1093/pcp/pcq006.
[25] 张和臣, 叶楚玉, 夏新莉, 等.逆境条件下植物CBL-CIPK信号途径转导的分子机制[J].分子植物育种, 2009, 7(1): 143-148. ZHANG H C, YE C Y, XIA X L, et al.The mechanism of CBL-CIPK signal transduction under stresses in plants[J].Molecular Plant Breeding, 2009, 7(1): 143-148.
[26] 汤湖斌, 闵康康, 徐玲玲, 等.CBL-CIPKs信号系统的研究进展[J].中国细胞生物学学报, 2015, 37(1): 100-105.DOI: 10.11844/cjcb.2015.01.0129. TANG H B, MIN K K, XU L L, et al.Research progress in CBL-CIPKs signaling system[J].Chinese Journal of Cell Biollgy, 2015, 37(1): 100-105.
[27] BATISTICˇ O, KUDLA J. Plant calcineurin B-like proteins and their interacting protein kinases[J].Biochimica Et Biophysica Acta, 2009, 1793(6): 985-992.DOI:10.1016/j.bbamcr.2008.10.006.
[28] LEWIT-BENTLEY A, RETY S. EF-hand calcium-binding proteins[J].Current Opinion in Structural Biology, 2000, 10(6): 637-643.DOI:10.1016/S0959-440X(00)00142-1.
[29] BATISTIC O, KUDLA J. Integration and channeling of calcium signaling through the CBL calcium sensor/CIPK protein kinase network[J].Planta, 2004, 219(6): 915-924.DOI: 10.1007/s00425-004-1333-3.
[30] CHEONG Y H, KIM K N, PANDEY G K, et al. CBL1, a calcium sensor that differentially regulates salt, drought, and cold response in Arabidopsis[J], Plant Cell, 2003, 15(8): 1833-1845.DOI: 10.1105/tpc.012393.
[31] ZHANG H C, YIN W L, XIA X L. Calcineurin B-Like family in Populus: comparative genome analysis and expression pattern under cold, drought stress treatment[J].Plant Growth Regulation, 2008, 56(2): 129-140.DOI:10.1007/s10725-008-9293-4.
[32] 董凤丽, 刘杰, 黄河, 等.甘菊CBL基因的克隆与表达分析[J].草业科学, 2014, 31(7): 1283-1289.DOI: 10.11829\\j.issn.1001-0629.2013-0684. DONG F L, LIU J, HUANG H, et al.Cloning and expression analysis of CBL genes from Chrysanthemum lavandulifolium[J].Pratacultural Science, 2014, 31(7): 1283-1289.
[33] LI D, SONG S, XIA X L, et al.Two CBL genes from Populus euphratica confer multiple stress tolerance in transgenic triploid white poplar[J].Plant Cell, Tissue and Organ Culture, 2012, 109(3): 477-489.DOI:10.1007/s11240-011-0112-7.
[34] 李慧, 李刚波, 丛郁, 等.杜梨类钙调磷酸酶B亚基蛋白基因PbCBL2的克隆和功能初探[J].园艺学报, 2013, 40(8): 1445-1455. LI H, LI G B, CONG Y, et al. Isolation of a calcineurin B-like protein gene PbCBL2 from Pyrus betulaefolia and preliminary study of gene function[J].Acta Horticulturae Sinica, 2013, 40(8): 1445-1455.
[35] 李慧, 丛郁, 常有宏, 等.杜梨类钙调磷酸酶B亚基蛋白PbCBL10基因的克隆和表达特性研究[J].果树学报, 2012, 28(4): 550-556.DOI:10.13925/j.cnki.gsxb.2012.04.013. LI H, CONG Y, CHANG Y H, et al.Cloning and expression characteristics of a calcineurin B-like protein gene(PbCBL10)from Pyrus betulaefolia[J].Journal of Fruit Science, 2012, 28(4): 550-556.
[36] 韩金龙, 李慧, 丛郁, 等.杜梨CBL1和CBL7基因对非生物逆境的响应[J].果树学报, 2014, 31(4): 529-535.DOI: 10.13925/j.cnki.gsxb.20130515. HAN J L, LI H, CONG Y, et al.Comparison of two CBL genes on stress tolerance functions from Pyrus betulaefolia[J].Journal of Fruit Science, 2014, 31(4): 529-535.
[37] 邓小敏.小麦CBL基因和CIPK基因的克隆及在非生物胁迫中的功能研究[D].武汉: 华中科技大学, 2013. DENG X M. Cloning and functional analysis of wheat CBL and CIPK genes in response to abiotic stresses[D].Wuhan: Huazhong University of Science and Technology, 2013.
[38] 陈晓兵.白芥CBL基因克隆及其在干旱胁迫下的表达分析[D].郑州: 郑州大学, 2015. CHEN X B. CBL gene cloning of Sinapis alba L.and its expression analysis under drought stress[D].Zhengzhou: Zhengzhou University, 2015.
[39] 许园园,蔺经,李晓刚,等.梨CBL基因家族全基因组序列的鉴定及非生物胁迫下的表达分析[J].中国农业科学, 2015, 48(4): 735-747.DOI: 10.3864/j.issn.0578-1752.2015.04.11. XU Y Y, LIN J, LI X G, et al.Identification and expression analysis under abiotic stresses of the CBL gene family in Pear[J].Scientia Agricultura Sinica, 2015, 48(4): 735-747.
[40] CHEONG Y H, SUNG S J, KIM B G, et al.Constitutive overexpression of the calcium sensor CBL5 confers osmotic or drought stress tolerance in Arabidopsis[J].Molecules and Cells, 2010, 29(2): 159-165.DOI:10.1007/s10059-010-0025-z.
[41] CHEN L, REN J, SHI H Y, et al. TdCBL6, a calcineurin B-like gene from wild emmer wheat(Triticum dicoccoides), is involved in response to salt and low-K+ stresses[J].Molecular Breeding, 2015, 35(1): 1-12.DOI: 10.1007/s11032-015-0229-1.
[42] 胡斐, 曾巧英, 凌秋平, 等.转拟南芥AtCBL9和AtCIPK23基因甘蔗对低钾胁迫的响应研究[J].甘蔗糖业, 2015(3): 5-10. HU F, ZENG Q Y, LING Q P, et al.Research on response of transgenic sugarcane carrying AtCBL9 and AtCIPK23 genes under low potassium stress[J].Sugarcane and Canesugar, 2015(3): 5-10.

Memo

Memo:
收稿日期:2016-09-03 修回日期:2016-12-23
基金项目:国家自然科学基金项目(31260183); “十三五”国家科技支撑计划(2015BAD09B0102); 贵州省重大专项项目(黔科合重大专项字[2012]6001号); 贵州省人才基地建设项目(黔人领发[2009]9号)
第一作者:蔡琼(dukecq@sina.com),博士生。*通信作者:丁贵杰(gjdinggzu@126.com),教授。
引文格式:蔡琼,丁贵杰,丁波,等. 马尾松PmCBL3基因的克隆及其表达分析[J]. 南京林业大学学报(自然科学版),2017,41(4):30-36.
Last Update: 1900-01-01