高级检索

留言板

尊敬的读者、作者、审稿人, 关于本刊的投稿、审稿、编辑和出版的任何问题, 您可以本页添加留言。我们将尽快给您答复。谢谢您的支持!

姓名
邮箱
手机号码
标题
留言内容
验证码

过表达细叶百合LpNAC6基因增强烟草的耐盐性

刘彬 曹尚杰 王营 崔颖 岳桦 张彦妮

刘彬, 曹尚杰, 王营, 崔颖, 岳桦, 张彦妮. 过表达细叶百合LpNAC6基因增强烟草的耐盐性[J]. 北京林业大学学报, 2020, 42(4): 69-79. doi: 10.12171/j.1000-1522.20190342
引用本文: 刘彬, 曹尚杰, 王营, 崔颖, 岳桦, 张彦妮. 过表达细叶百合LpNAC6基因增强烟草的耐盐性[J]. 北京林业大学学报, 2020, 42(4): 69-79. doi: 10.12171/j.1000-1522.20190342
Liu Bin, Cao Shangjie, Wang Ying, Cui Ying, Yue Hua, Zhang Yanni. Overexpression of LpNAC6 gene in Lilium pumilum enhancing salt tolerance in transgenic tobacco[J]. Journal of Beijing Forestry University, 2020, 42(4): 69-79. doi: 10.12171/j.1000-1522.20190342
Citation: Liu Bin, Cao Shangjie, Wang Ying, Cui Ying, Yue Hua, Zhang Yanni. Overexpression of LpNAC6 gene in Lilium pumilum enhancing salt tolerance in transgenic tobacco[J]. Journal of Beijing Forestry University, 2020, 42(4): 69-79. doi: 10.12171/j.1000-1522.20190342

过表达细叶百合LpNAC6基因增强烟草的耐盐性

doi: 10.12171/j.1000-1522.20190342
基金项目: 中央高校基本科研业务费专项资金项目(2572019BK04),黑龙江省自然科学基金项目(LH2019C004)
详细信息
    作者简介:

    刘彬。主要研究方向:园林植物繁殖栽培及育种。Email:276410256@qq.com 地址:150040 黑龙江省哈尔滨市香坊区和兴路26号东北林业大学园林学院

    责任作者:

    张彦妮,副教授,博士生导师。主要研究方向:园林植物繁殖栽培及育种。Email:ynzhang808@126.com 地址:同上

  • 中图分类号: S682.29

Overexpression of LpNAC6 gene in Lilium pumilum enhancing salt tolerance in transgenic tobacco

  • 摘要: 目的NAC转录因子是一类具有多种生物功能的新型转录因子,在植物抗逆响应中发挥着重要作用。本文旨在克隆并研究细叶百合LpNAC6基因在逆境胁迫下的表达模式,探究其在烟草中响应盐胁迫的功能。方法本研究采用同源克隆技术克隆得到细叶百合LpNAC6基因,利用生物信息学软件对LpNAC6基因进行分析;通过基因枪法对LpNAC6蛋白进行亚细胞定位;利用实时荧光定量PCR的方法分析LpNAC6基因在不同非生物胁迫和不同组织中的表达模式;构建植物表达载体pBI121-LpNAC6-GFP转化烟草,通过对转基因烟草进行盐胁迫处理验证LpNAC6基因的功能。结果LpNAC6基因长909 bp,编码302个氨基酸,存在一个高度保守的NAM结构域,属于NAC基因家族。LpNAC6为不稳定亲水性蛋白,无信号肽和跨膜结构域,有5个糖基化位点和20个磷酸化位点,亚细胞定位在细胞核。LpNAC6基因与黄褐棉的NAC转录因子进化关系最近。细叶百合中LpNAC6基因对ABA、干旱、低温及盐胁迫均有响应。盐胁迫下,过表达LpNAC6基因的转基因烟草其SOD、POD、CAT的活性和叶绿素、脯氨酸、可溶性蛋白的含量均显著高于野生型。结论细叶百合LpNAC6基因能够响应ABA、干旱、低温、盐胁迫等非生物胁迫,其过表达能够提高转基因烟草在盐胁迫下的代谢活力和抗氧化酶活性,从而增强烟草耐盐性。

     

  • 图  1  LpNAC6的核苷酸与氨基酸序列

                 下划线部分表示NAM结构域。The underlined part represents the NAM domain.

    Figure  1.  Nucleotide and amino acid sequences of LpNAC6

    图  2  LpNAC6的亲水/疏水性及信号肽分析

    A. LpNAC6的亲水/疏水性分析;B. LpNAC6的信号肽分析。C-score. 剪切位置分值;S-score. 信号肽分值;Y-score. 综合剪切位置分值。A,hydrophilicity / hydrophobicity analysis of LpNAC6;B,signal peptide analysis of LpNAC6. C-score,the score of the original cut site;S-score,the score of signal peptide;Y-score,the score of the integrated cut site.

    Figure  2.  Hydrophilicity / hydrophobicity and signal peptide analysis of LpNAC6

    图  3  LpNAC6蛋白的跨膜结构域及二级结构预测

    A. LpNAC6跨膜结构域预测;B. LpNAC6二级结构预测。A,prediction of transmembrane domains of LpNAC6;B,prediction of secondary structure of LpNAC6.

    Figure  3.  Prediction of transmembrane domains andsecondary structure of LpNAC6

    图  4  细叶百合LpNAC6与其他物种NAC蛋白的多序列比对

    TYJ30417.1. 黄褐棉;XP_017700768.1. 海枣;OWM84829.1. 石榴;XP_020261897.1. 石刁柏;XP_010907545.3. 油棕;RZS25466.1. 阿比西尼亚红脉蕉;THU44006.1. 野蕉;XP_015940149.1. 蔓花生;XP_024963988.1. 洋蓟;XP_020083042.1. 凤梨;XP_020703744.1. 铁皮石斛。TYJ30417.1,Gossypium mustelinum;XP_017700768.1,Phoenix dactylifera;OWM84829.1,Punica granatum;XP_020261897.1,Asparagus officinalis;XP_010907545.3,Elaeis guineensis;RZS25466.1,Ensete ventricosum;THU44006.1,Musa balbisiana;XP_015940149.1,Arachis duranensis;XP_024963988.1,Cynara cardunculus var. scolymus;XP_020083042.1,Ananas comosus;XP_020703744.1,Dendrobium catenatum.

    Figure  4.  Multiple alignment of LpNAC6 for L. pumilum with the protein sequences of NAC from other plant species

    图  5  LpNAC6蛋白的系统进化树分析

    细叶百合. Lilium pumilum;海枣. Phoenix dactylifera;油棕. Elaeis guineensis;凤梨. Ananas comosus;阿比西尼亚红脉蕉. Ensete ventricosum;野蕉. Musa balbisiana;铁皮石斛. Dendrobium catenatum;黄褐棉. Gossypium mustelinum;石刁柏. Asparagus officinalis;石榴. Punica granatum;蔓花生. Arachis duranensis;洋蓟. Cynara cardunculus var. scolymus

    Figure  5.  Phylogenetic tree analysis of LpNAC6 protein

    图  6  LpNAC6基因在不同组织和非生物胁迫下的表达模式

    A. 150 μmol/L ABA胁迫;B. 20% PEG6000胁迫;C. 2 ℃低温胁迫;D. 200 mmol/L NaCl胁迫。不同小写字母表示在P < 0.05水平上差异显著。下同。A, 150 μmol/L ABA stress;B, 20% PEG6000 stress;C, 2 ℃ low temperature stress;D, 200 mmol/L NaCl stress. Different lowercase letters mean significant difference at P < 0.05 level. The same below.

    Figure  6.  Expression patterns of LpNAC6 in different tissues and under different abiotic stresses

    图  7  LpNAC6蛋白的亚细胞定位

    A. pBI121-GFP蛋白定位;B. pBI121-LpNAC6-GFP融合蛋白定位。A, subcellular localization of pBI121-GFP protein;B, subcellular localization of pBI121-LpNAC6-GFP fusion protein.

    Figure  7.  Subcellular localization of LpNAC6 protein

    图  8  转基因烟草T0代植株PCR鉴定

    A. 鉴定转基因烟草T0代植株DNA;B. 鉴定转基因烟草T0代植株cDNA。M. DL2000 Marker;1. 水对照;2. 野生型对照;3 ~ 8. 转基因阳性植株;9. pBI121- LpNAC6-GFP农杆菌菌液对照。A,DNA identification of transgenic tobacco T0 generation plants;B,cDNA identification of transgenic tobacco T0 generation plants. M,DL2000 Marker;1,water control;2,wide-type control;3−8,positive transgenic plants;9,pBI121- LpNAC6-GFP Agrobacterium solution control.

    Figure  8.  Identification of transgenic tobacco T0 generation plants by PCR

    图  9  转基因烟草种子和幼苗的耐盐性分析

    A. WT、Tr-2和Tr-3烟草种子分布示意图;B. WT、Tr-2和Tr-3烟草种子在1/2MS培养基中的萌发情况;C. WT、Tr-2和Tr-3烟草种子在含150 mmol/L NaCl的1/2MS培养基中的萌发情况;D. WT、Tr-2和Tr-3烟草在150 mmol/L NaCl胁迫下的生根情况;E. 野生型烟草和转基因烟草种子在150 mmol/L NaCl胁迫下的发芽率;F. 野生型烟草和转基因烟草幼苗在150 mmol/L NaCl胁迫下的相对根长。A,distribution of WT,Tr-2 and Tr-3 tobacco seeds;B,germination condition of WT,Tr-2 and Tr-3 tobacco seeds in 1/2MS medium;C,germination condition of WT,Tr-2 and Tr-3 tobacco seeds in 1/2MS medium containing 150 mmol/L NaCl;D,rooting condition of WT,Tr-2 and Tr-3 tobacco under 150 mmol/L NaCl stress;E,germination rate of wild type tobacco and transgenic tobacco seeds under 150 mmol/L NaCl stress;F,relative root length of wild type tobacco and transgenic tobacco seedlings under 150 mmol/L NaCl stress.

    Figure  9.  Analysis of salt tolerance of transgenic tobacco seeds and seedlings

    图  10  盐胁迫下转基因株系和野生型烟草的氧化酶活性变化

    Figure  10.  Changes of oxidase activity of transgenic lines and wild-type tobacco under salt stress

    图  11  盐胁迫下转基因株系和野生型烟草叶绿素、脯氨酸和可溶性蛋白质的含量变化

    Figure  11.  Changes of chlorophyll,proline and soluble protein content of transgenic lines and wild-type tobacco under salt stress

  • [1] Riechmann J L, Heard J, Martin G, et al. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes[J]. Science, 2000, 290: 2105−2110. doi: 10.1126/science.290.5499.2105
    [2] Nuruzzaman M, Manimekalai R, Sharoni A M, et al. Genome-wide analysis of NAC transcription factor family in rice[J]. Gene, 2010, 465(1−2): 30−44. doi: 10.1016/j.gene.2010.06.008
    [3] Pinheiro G L, Marques C S, Costa M D B L, et al. Complete inventory of soybean NAC transcription factors: sequence conservation and expression analysis uncover their distinct roles in stress response[J]. Gene, 2009, 444(1−2): 10−23. doi: 10.1016/j.gene.2009.05.012
    [4] Rushton P J, Bokowiec M T, Laudeman T W, et al. TOBFAC: the database of tobacco transcription factors[J/OL]. BMC Bioinformatics, 2008, 9(1): 53 [2019−07−15]. https://doi.org/10.1186/1471-2105-9-53.
    [5] Zhang J, Huang G Q, Zou D, et al. The cotton(Gossypium hirsutum)NAC transcription factor(FSN1)as a positive regulator participates in controlling secondary cell wall biosynthesis and modification of fibers[J]. New Phytologist, 2018, 217(2): 625−640. doi: 10.1111/nph.14864
    [6] Huang Q J, Wang Y, Li B, et al. TaNAC29, a NAC transcription factor from wheat, enhances salt and drought tolerance in transgenic Arabidopsis[J/OL]. BMC Plant Biology, 2015, 15(1): 268 [2019−07−15]. https://doi.org/10.1186/s12870-015-0644-9.
    [7] Tak H, Negi S, Ganapathi T R. Banana NAC transcription factor MusaNAC042 is positively associated with drought and salinity tolerance[J]. Protoplasma, 2017, 254(2): 803−816. doi: 10.1007/s00709-016-0991-x
    [8] Nakashima K, Tran L S P, Van Nguyen D, et al. Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice[J]. The Plant Journal, 2007, 51(4): 617−630. doi: 10.1111/j.1365-313X.2007.03168.x
    [9] 侯佳, 李发虎, 李龙梅, 等. 野生山丹组织培养体系优化与品种改良研究进展[J]. 中国园艺文摘, 2017, 33(10):67−69, 76. doi: 10.3969/j.issn.1672-0873.2017.10.025

    Hou J, Li F H, Li L M, et al. Advances in tissue culture techniques optimization and variety improvement of wild Lilium pumilum DC.[J]. Chinese Horticulture Abstracts, 2017, 33(10): 67−69, 76. doi: 10.3969/j.issn.1672-0873.2017.10.025
    [10] De Clercq I, Vermeirssen V, Van Aken O, et al. The membrane-bound NAC transcription factor ANAC013 functions in mitochondrial retrograde regulation of the oxidative stress response in Arabidopsis[J]. The Plant Cell, 2013, 25(9): 3472−3490. doi: 10.1105/tpc.113.117168
    [11] 刘锴栋, 袁长春, 黎海利, 等. 番荔枝GA20氧化酶基因的克隆与表达分析[J]. 植物生理学报, 2015, 51(10):1697−1705.

    Liu K D, Yuan C C, Li H L, et al. Cloning and expression analysis of GA20-Oxidase gene from sugar apple (Annona squamosa)[J]. Plant Physiology Journal, 2015, 51(10): 1697−1705.
    [12] Kamiuchi Y, Yamamoto K, Furutani M, et al. The CUC1 and CUC2 genes promote carpel margin meristem formation during Arabidopsis gynoecium development[J/OL]. Frontiers in Plant Science, 2014, 5: 165 [2019−07−15]. https://doi.org/10.3389/fpls.2014.00165.
    [13] 康桂娟, 曾日中, 聂智毅, 等. 巴西橡胶树NAC转录因子HbNAC1基因的克隆及生物信息学分析[J]. 中国农学通报, 2012, 28(34):1−11. doi: 10.3969/j.issn.1000-6850.2012.34.001

    Kang G J, Zeng R Z, Nie Z Y, et al. Cloning and bioinformatics analysis of a NAC transcription factor HbNAC1 from Hevea brasiliensis[J]. Chinese Agricultural Science Bulletin, 2012, 28(34): 1−11. doi: 10.3969/j.issn.1000-6850.2012.34.001
    [14] Furuta K M, Yadav S R, Lehesranta S, et al. Arabidopsis NAC45/86 direct sieve element morphogenesis culminating in enucleation[J]. Science, 2014, 345: 933−937. doi: 10.1126/science.1253736
    [15] Kim S G, Lee S, Seo P J, et al. Genome-scale screening and molecular characterization of membrane-bound transcription factors in Arabidopsis and rice[J]. Genomics, 2010, 95(1): 56−65. doi: 10.1016/j.ygeno.2009.09.003
    [16] Kim S G, Lee A K, Yoon H K, et al. A mem-brane-bound NAC transcription factor NTL8 regulates gibberellic acid-mediated salt signaling in Arabidopsis seed germination[J]. The Plant Journal: for Cell and Molecular Biology, 2008, 55(1): 77−88. doi: 10.1111/j.1365-313X.2008.03493.x
    [17] 樊金娟, 阮燕晔. 植物生理学实验教程[M]. 北京: 中国农业大学出版社, 2015.

    Fan J J, Ruan Y Y. Experimental course of plant physiology[M]. Beijing: China Agricultural University Press, 2015.
    [18] 刘萍, 李明军. 植物生理学实验[M]. 北京: 科学出版社, 2016.

    Liu P, Li M J. Experiments of plant physiology[M]. Beijing: Science Press, 2016.
    [19] 华智锐, 李小玲. 水杨酸浸种对小麦品种‘商麦5226’盐胁迫的缓解效应[J]. 西北农业学报, 2015, 24(9):29−35. doi: 10.7606/j.issn.1004-1389.2015.09.005

    Hua Z R, Li X L. Mitigative effect of seed-soaking by salicylic acid on wheatcultiver of ‘Shangmai 5226’ under salt stress[J]. Acta Agriculturae Boreali-occidentalis Sinica, 2015, 24(9): 29−35. doi: 10.7606/j.issn.1004-1389.2015.09.005
    [20] Zhong R Q, Lee C H, Ye Z H. Functional characterization of poplar wood-associated NAC domain transcription factors[J]. Journal of Plant Physiology, 2010, 152(2): 1044−1055. doi: 10.1104/pp.109.148270
    [21] Olsen A N, Ernst H A, Leggio L L, et al. NAC transcription factors: structurally distinct, functionally diverse[J]. Trends in Plant Science, 2005, 10(2): 79−87. doi: 10.1016/j.tplants.2004.12.010
    [22] Webster D E, Thomas M C. Post-translational modification of plant-made foreign proteins, glycosylation and beyond[J]. Biotech Advances, 2011, 30(2): 410−418.
    [23] 段奥其, 冯凯, 刘洁霞, 等. 芹菜NAC转录因子基因AgNAC1的克隆及其对非生物胁迫的响应[J]. 园艺学报, 2018, 45(6):1125−1135.

    Duan A Q, Feng K, Liu J X, et al. Cloning and response to abiotic stress of NAC transcription gene AgNAC1 in Apium graveolens[J]. Acta Horticulturae Sinica, 2018, 45(6): 1125−1135.
    [24] 樊蕾, 高志英. 番茄SlNAC71基因克隆及表达分析[J]. 分子植物育种, 2018, 16(13):4172−4175.

    Fan L, Gao Z Y. Cloning and expression analysis of SlNAC71 gene in tomato[J]. Molecular Plant Breeding, 2018, 16(13): 4172−4175.
    [25] 张晓菲, 路信, 段卉, 等. 胡杨NAC转录因子PeNAC045基因的克隆及功能分析[J]. 北京林业大学学报, 2015, 37(6):1−10.

    Zhang X F, Lu X, Duan H, et al. Cloning and functional analysis of PeNAC045 from Populus euphratica[J]. Journal of Beijing Forestry University, 2015, 37(6): 1−10.
    [26] 袁义杭, 张鹤华, 游韩莉, 等. 青杄PwNAC42基因的克隆及表达模式分析[J]. 生物技术通报, 2018, 34(3):113−120.

    Yuan Y H, Zhang H H, You H L, et al. Cloning and expression analysis of PwNAC42 in Picea wilsonii[J]. Biotechnology Bulletin, 2018, 34(3): 113−120.
    [27] 王燕飞, 红格日其其格, 王光霞, 等. 中间锦鸡儿CiATAF1基因的亚细胞定位及表达分析[J]. 华北农学报, 2019, 34(3):23−30. doi: 10.7668/hbnxb.201751243

    Wang Y F, Honggeriqiqige, Wang G X, et al. Subcellular localization and expression analysis of CiATAF1 gene in Caragana intermedia[J]. Acta Agriculturae Boreali-Sinica, 2019, 34(3): 23−30. doi: 10.7668/hbnxb.201751243
    [28] Zheng X N, Chen B, Lu G J, et al. Overexpression of a NAC transcription factor enhances rice drought and salt tolerance[J]. Biochemical and Biophysical Research Communications, 2008, 379(4): 985−989.
    [29] 姜红岩, 张蕊, 滕珂, 等. 日本结缕草ZjNAC2基因的克隆、亚细胞定位及表达分析[J]. 草业科学, 2019, 36(6):1553−1562.

    Jiang H Y, Zhang R, Teng K, et al. Molecular cloning, subcellular localization analysis, and expression characterization of ZjNAC2 from Zoysia japonica[J]. Pratacultural Science, 2019, 36(6): 1553−1562.
    [30] Zhao J H, Li M Z, Gu D C, et al. Involvement of rice histone deacetylase HDA705 in seed germination and in response to ABA and abiotic stresses[J]. Biochemical and Biophysica Research Communications, 2016, 470(2): 439−444. doi: 10.1016/j.bbrc.2016.01.016
    [31] Yong Y B, Zhang Y, Lyu Y M. A stress-responsive NAC transcription factor from tiger lily (LlNAC2) interacts with LlDREB1 and LlZHFD4 and enhances various abiotic stress tolerance in Arabidopsis[J/OL]. International Journal of Molecular Sciences, 2019, 20(13): 3225 [2019−07−15]. https://doi.org/10.3390/ijms20133225.
    [32] Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction[J]. Annual Review of Plant Biology, 2004, 55: 373−399. doi: 10.1146/annurev.arplant.55.031903.141701
    [33] Farhangi-Abriz S, Torabian S. Antioxidant enzyme and osmotic adjustment changes in bean seedlings as affected by biochar under salt stress[J]. Ecotoxicology and Environmental Safety, 2017, 137: 64−70. doi: 10.1016/j.ecoenv.2016.11.029
    [34] Zhao X, Yang X W, Pei S Q, et al. The Miscanthus NAC transcription factor MlNAC9 enhances abiotic stress tolerance in transgenic Arabidopsis[J]. Gene, 2016, 586(1): 158−169. doi: 10.1016/j.gene.2016.04.028
    [35] Ma X J, Zhang B, Liu C J, et al. Expression of a populus histone deacetylase gene 84KHDA903 in tobacco enhances drought tolerance[J]. Plant Science, 2017, 265: 1−11. doi: 10.1016/j.plantsci.2017.09.008
    [36] Yu X W, Liu Y M, Wang S, et al. CarNAC4, a NAC-type chickpea transcription factor conferring enhanced drought and salt stress tolerances in Arabidopsis[J]. Plant Cell Reports, 2016, 35(3): 613−627. doi: 10.1007/s00299-015-1907-5
  • 加载中
图(11)
计量
  • 文章访问数:  1418
  • HTML全文浏览量:  556
  • PDF下载量:  26
  • 被引次数: 0
出版历程
  • 收稿日期:  2019-08-27
  • 修回日期:  2019-12-23
  • 网络出版日期:  2020-04-11
  • 刊出日期:  2020-04-27

目录

    /

    返回文章
    返回