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

刚毛柽柳SAIR6长链非编码RNA耐盐功能初探

许欣 卢惠君 王玉成 王智博 及晓宇

许欣, 卢惠君, 王玉成, 王智博, 及晓宇. 刚毛柽柳SAIR6长链非编码RNA耐盐功能初探[J]. 北京林业大学学报, 2021, 43(3): 36-43. doi: 10.12171/j.1000-1522.20200235
引用本文: 许欣, 卢惠君, 王玉成, 王智博, 及晓宇. 刚毛柽柳SAIR6长链非编码RNA耐盐功能初探[J]. 北京林业大学学报, 2021, 43(3): 36-43. doi: 10.12171/j.1000-1522.20200235
Xu Xin, Lu Huijun, Wang Yucheng, Wang Zhibo, Ji Xiaoyu. Salt stress tolerance analysis of SAIR6 long non-coding RNA in Tamarix hispida[J]. Journal of Beijing Forestry University, 2021, 43(3): 36-43. doi: 10.12171/j.1000-1522.20200235
Citation: Xu Xin, Lu Huijun, Wang Yucheng, Wang Zhibo, Ji Xiaoyu. Salt stress tolerance analysis of SAIR6 long non-coding RNA in Tamarix hispida[J]. Journal of Beijing Forestry University, 2021, 43(3): 36-43. doi: 10.12171/j.1000-1522.20200235

刚毛柽柳SAIR6长链非编码RNA耐盐功能初探

doi: 10.12171/j.1000-1522.20200235
基金项目: 黑龙江省科学基金项目(QC2018017)
详细信息
    作者简介:

    许欣。主要研究方向:林木逆境生理与分子生物学。Email:1056296957@qq.com 地址:150040黑龙江省哈尔滨市香坊区和兴路26号东北林业大学林木遗传育种国家重点实验室

    责任作者:

    及晓宇,博士,副教授。主要研究方向:林木逆境生理与分子生物学。Email:jixy0219@163.com 地址:同上

  • 中图分类号: S718.43;S793.5

Salt stress tolerance analysis of SAIR6 long non-coding RNA in Tamarix hispida

  • 摘要:   目的  长链非编码RNA(lncRNA)是一类长度大于200个核苷酸、无蛋白质编码能力或编码能力极低的转录本,已有研究表明,lncRNA是植物胁迫反应中关键的调控因子之一。本研究拟对刚毛柽柳SAIR6长链非编码RNA是否具有提高刚毛柽柳的耐盐能力进行分析,为阐明刚毛柽柳lncRNA响应盐胁迫的分子调控机制奠定基础,进一步丰富木本植物lncRNA响应逆境胁迫分子机制的研究。  方法  本研究从盐胁迫下刚毛柽柳转录组中筛选出一条差异表达的lncRNA-224223.1,将其命名为ThSAIR6。利用实时荧光定量PCR(qRT-PCR)技术分析盐胁迫下刚毛柽柳叶组织中ThSAIR6的表达模式,初步鉴定其是否响应盐胁迫。为了进一步分析ThSAIR6的抗逆功能,构建其植物过表达载体(pROKII-ThSAIR6),利用农杆菌介导的高效遗传瞬时转化体系,获得ThSAIR6瞬时过表达及对照刚毛柽柳植株。在盐胁迫下分别对ThSAIR6瞬时过表达及对照植株的耐盐相关生理指标进行测定,判断ThSAIR6是否能提高刚毛柽柳的耐盐能力。  结果  qRT-PCR结果表明,在盐胁迫24 h后,ThSAIR6在刚毛柽柳植株中的表达量显著上升(P < 0.05),说明该lncRNA能响应盐胁迫。抗逆生理指标的测定结果表明,ThSAIR6在刚毛柽柳中过表达能够使H2O2$ {\rm{O}}_{\rm{2}}^{ {-} {\text{•}}} $的含量显著降低(P < 0.05),使POD、SOD的酶活性显著增强(P < 0.05);同时降低刚毛柽柳组织中的死亡细胞数量、电解质渗透率及失水率。  结论  刚毛柽柳SAIR6长链非编码RNA能响应盐胁迫;在盐胁迫下,ThSAIR6过表达显著减轻了植物组织细胞的受损程度,增强了POD、SOD酶活性,降低了植株内H2O2$ {\rm{O}}_{\rm{2}}^{ {-} {\text{•}}} $的含量,提高了活性氧(ROS)清除能力,有效提高了刚毛柽柳的耐盐能力。

     

  • 图  1  长链非编码RNA ThSAIR6序列

    Figure  1.  ThSAIR6 sequence of long non-coding RNA

    图  2  ThSAIR6克隆及载体构建PCR检测结果

    A. 植物过表达载体pROKⅡ-ThSAIR6构建图谱; B. ThSAIR6克隆结果; C. ThSAIR6 大肠杆菌菌液 PCR 结果,1为 阳性对照, 8为 阴性对照; D. ThSAIR6农杆菌菌液PCR结果; M为 Trans 2k DNA Marker。A, the map of plant overexpression vector pROKⅡ-ThSAIR6; B, the PCR of ThSAIR6; C, identification of recombinant plasmid ThSAIR6 by PCR, in picture C, 1 means positive control, 8 means negative control; D, Agrobacterium suspension containing recombinant plasmid ThSAIR6 by PCR; M, Trans 2k DNA Marker.

    Figure  2.  PCR testing results of vector construction and ThSAIR6 cloning

    图  3  盐胁迫下刚毛柽柳ThSAIR6的表达模式分析

    Figure  3.  Analysis of expression pattern of ThSAIR6 in Tamarix hispida under salt stress

    图  4  盐胁迫下ThSAIR6在瞬时过表达及对照刚毛柽柳植株中的表达情况

         *P < 0.05;下同。*P < 0.05; The same below.

    Figure  4.  Expression level of ThSAIR6 in transient overexpression (OE) and control of Tamarix hispida under salt stress

    图  5  盐胁迫下对照及瞬时过表达植株电解质渗透率、失水率分析

    Figure  5.  Electrolyte leakage and water loss rate analyses of control and overexpressing plants under salt stress

    图  6  盐胁迫下对照及瞬时过表达植株ROS水平及POD、SOD活性分析

    Figure  6.  Analysis of ROS accumulation and the activities of POD and SOD in OE and control plants under salt stress

    表  1  引物序列

    Table  1.   Primer sequences

    用途 Application引物 Primer引物序列(5′—3′) Primer sequence (5′−3′)
    q-ThSAIR6-F GCCTGGTTGGTTTATCTG
    q-ThSAIR6-R TCCCACTACCCTACCTTAT
    实时荧光定量PCR
    Quantitative real-time PCR
    Actin-F AAACAATGGCTGATGCTG
    Actin-R ACAATACCGTGCTCAATAGG
    β-tubulin-F GGAAGCCATAGAAAGACC
    β-tubulin-R CAACAAATGTGGGATGCT
    基因克隆
    Gene cloning
    ThSAIR6-F-infu GACTCTAGAGGATCCCCGAAAGATGTTGAGGCGGAC
    ThSAIR6-R-infu GGAAATTCGAGCTCGGTACCCGTATCAGTACGGGTCCAATAC
    载体验证
    Vector verification
    pROKⅡ-F ATGTGATATCTCCACTGACGT
    pROKⅡ-R ATCGCAAGACCGGCAACAGGA
    下载: 导出CSV
  • [1] Alcazar R, Marco F, Cuevas J C, et al. Involvement of polyamines in plant response to abiotic stress[J]. Biotechnology Letters, 2006, 28(23): 1867−1876. doi: 10.1007/s10529-006-9179-3
    [2] Lopez-Perez L, Martinez-Ballesta M C, Maurel C, et al. Changes in plasma membrane lipids, aquaporins and proton pump of broccoli roots, as an adaptation mechanism to salinity[J]. Phytochemistry, 2009, 70(4): 492−500. doi: 10.1016/j.phytochem.2009.01.014
    [3] Yamaguchi A, Abe M. Regulation of reproductive development by non-coding RNA in Arabidopsis: to flower or not to flower[J]. Journal of Plant Research, 2012, 125(6): 693−704. doi: 10.1007/s10265-012-0513-7
    [4] Bardou F, Merchan F, Ariel F, et al. Dual RNAs in plants[J]. Biochimie, 2011, 93(11): 1950−1954. doi: 10.1016/j.biochi.2011.07.028
    [5] Wierzbicki A T. The role of long non-coding RNA in transcriptional gene silencing[J]. Current Opinion in Plant Biology, 2012, 15(5): 517−522. doi: 10.1016/j.pbi.2012.08.008
    [6] Chen L L, Carmichael G G. Decoding the function of nuclear long non-coding RNAs[J]. Current Opinion in Cell Biology, 2010, 22(3): 357−364. doi: 10.1016/j.ceb.2010.03.003
    [7] Bai Y, Dai X, Harrison A P, et al. RNA regulatory networks in animals and plants: a long noncoding RNA perspective[J]. Briefings in Functional Genomics, 2015, 14(2): 91−101. doi: 10.1093/bfgp/elu017
    [8] Begcy K, Dresselhaus T. Epigenetic responses to abiotic stresses during reproductive development in cereals[J]. Plant Reproduction, 2018, 31(4): 343−355. doi: 10.1007/s00497-018-0343-4
    [9] Liu J, Jung C, Xu J, et al. Genome-wide analysis uncovers regulation of long intergenic noncoding RNAs in Arabidopsis[J]. The Plant Cell, 2012, 24(11): 4333−4345. doi: 10.1105/tpc.112.102855
    [10] Qin T, Zhao H, Cui P, et al. A nucleus-localized long non-coding RNA enhances drought and salt stress tolerance[J]. Plant Physiology, 2017, 175(3): 1321−1336. doi: 10.1104/pp.17.00574
    [11] Li S, Yu X, Lei N, et al. Genome-wide identification and functional prediction of cold and/or drought-responsive lncRNAs in cassava[J/OL]. Scientific Reports, 2017, 7: 45981 (2017−04−07) [2018−12−11]. https://www.nature.com/articles/srep45981.
    [12] Karlik E, Gozukirmizi N. Expression analysis of lncRNA AK370814 involved in the barley vitamin B6 salvage pathway under salinity[J]. Molecular Biology Reports, 2018, 45(6): 1597−1609. doi: 10.1007/s11033-018-4289-2
    [13] Livak K, Schmittgen T. Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆Ct Method[J]. Methods, 2001, 25(4): 402−408. doi: 10.1006/meth.2001.1262
    [14] Ji X, Zheng L, Liu Y, et al. A transient transformation system for the functional characterization of genes involved in stress response[J]. Plant Molecular Biology Reporter, 2014, 32(3): 732−739. doi: 10.1007/s11105-013-0683-z
    [15] 王关林, 方宏荺. 植物基因工程实验技术指南[M]. 2版. 北京: 科学出版社, 2016.

    Wang G L, Fang H J. Laboratory guide for plant genetic engineering[M]. 2nd ed. Beijing: Science Press, 2016.
    [16] Zhang X, Wang L, Meng H, et al. Maize ABP9 enhances tolerance to multiple stresses in transgenic Arabidopsis by modulating ABA signaling and cellular levels of reactive oxygen species[J]. Plant Molecular Biology, 2011, 75(4−5): 365−378. doi: 10.1007/s11103-011-9732-x
    [17] Kim M, Ahn J W, Jin U H, et al. Activation of the programmed cell death pathway by inhibition of proteasome function in plants[J]. Journal of Biological Chemistry, 2003, 278: 19406−19415. doi: 10.1074/jbc.M210539200
    [18] 毋若楠, 王红, 杨成成, 等. 拟南芥lncRNA-At5NC056820过表达载体构建及其转基因植株的抗旱性研究[J]. 西北植物学报, 2017, 37(10):22−27.

    Wu R N, Wang H, Yang C C, et al. Construction of lncRNA-At5NC056820 Overexpression vector in Arabidopsis thaliana and study on drought resistance of transgenic plants[J]. Acta Botanica Boreali-Occidentalia Sinica, 2017, 37(10): 22−27.
    [19] Wu J, Liu C, Liu Z, et al. Pol III-dependent cabbage BoNR8 long ncRNA affects seed germination and growth in Arabidopsis[J]. Plant & Cell Physiology, 2019, 60(2): 421−435.
    [20] Yan Q, Wu F, Yan Z, et al. Differential co-expression networks of long non-coding RNAs and mRNAs in Cleistogenes songorica under water stress and during recovery[J/OL]. BMC Plant Biology, 2019, 19(1): 23 (2019−01−11)[2019−06−23]. https://doi.org/10.1186/s12870-018-1626-5.
    [21] Gai Y P, Yuan S S, Zhao Y N, et al. A novel LncRNA, MuLnc1, associated with environmental stress in mulberry (Morus multicaulis) [J/OL]. Frontiers in Plant Science, 2018, 9: 669 (2018−05−29) [2019−04−21]. https://doi.org/10.3389/fpls.2018.00669.
    [22] 卢惠君, 李子义, 梁瀚予,等. 刚毛柽柳NAC24基因的表达及抗逆功能分析[J]. 林业科学, 2019, 55(3):57−66.

    Lu H J, Li Z Y, Liang H Y, et al. Expression and stress tolerance analysis of NAC24 from Tamarix hispida[J]. Scientia Silvae Sinicae, 2019, 55(3): 57−66.
    [23] He Z, Li Z, Lu H, et al. The NAC protein from Tamarix hispida, ThNAC7, confers salt and osmotic stress tolerance by increasing reactive oxygen species scavenging capability[J]. Plants, 2019, 8(7): 221. doi: 10.3390/plants8070221
  • 加载中
图(6) / 表(1)
计量
  • 文章访问数:  433
  • HTML全文浏览量:  162
  • PDF下载量:  36
  • 被引次数: 0
出版历程
  • 收稿日期:  2020-08-10
  • 修回日期:  2020-09-14
  • 网络出版日期:  2021-03-20
  • 刊出日期:  2021-04-16

目录

    /

    返回文章
    返回