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胡杨PeREM1.3过表达提高烟草耐盐性的机制

张会龙 武霞 尧俊 赵楠 赵瑞 李金克 沈昕 陈少良

张会龙, 武霞, 尧俊, 赵楠, 赵瑞, 李金克, 沈昕, 陈少良. 胡杨PeREM1.3过表达提高烟草耐盐性的机制[J]. 北京林业大学学报, 2019, 41(1): 1-9. doi: 10.13332/j.1000-1522.20180338
引用本文: 张会龙, 武霞, 尧俊, 赵楠, 赵瑞, 李金克, 沈昕, 陈少良. 胡杨PeREM1.3过表达提高烟草耐盐性的机制[J]. 北京林业大学学报, 2019, 41(1): 1-9. doi: 10.13332/j.1000-1522.20180338
Zhang Huilong, Wu Xia, Yao Jun, Zhao Nan, Zhao Rui, Li Jinke, Shen Xin, Chen Shaoliang. Overexpression mechanism of PeREM1.3 from Populus euphratica enhancing salt tolerance in transgenic tobacco[J]. Journal of Beijing Forestry University, 2019, 41(1): 1-9. doi: 10.13332/j.1000-1522.20180338
Citation: Zhang Huilong, Wu Xia, Yao Jun, Zhao Nan, Zhao Rui, Li Jinke, Shen Xin, Chen Shaoliang. Overexpression mechanism of PeREM1.3 from Populus euphratica enhancing salt tolerance in transgenic tobacco[J]. Journal of Beijing Forestry University, 2019, 41(1): 1-9. doi: 10.13332/j.1000-1522.20180338

胡杨PeREM1.3过表达提高烟草耐盐性的机制

doi: 10.13332/j.1000-1522.20180338
基金项目: 

高等学校学科创新引智计划项目 111Project

中央高校基本科研业务费专项 2017ZY07

国家自然科学基金项目 31570587

国家自然科学基金项目 31770643

北京市自然科学基金项目 6182030

教育部科学技术研究(科学技术类)项目 113013A

北京市自然科学基金项目 6172024

高等学校学科创新引智计划项目 B130007

详细信息
    作者简介:

    张会龙,博士生。主要研究方向:树木逆境生理。Email: hlzhang1988@sina.cn  地址:100083北京市海淀区清华东路35号北京林业大学生物科学与技术学院

    责任作者:

    沈昕,教授。主要方研究向:分子生物学。Email: xinshen77@126.com  地址:同上

    陈少良,博士,教授。主要方研究向:树木逆境生理。Email:lschen@bjfu.edu.cn  地址:同上

  • 中图分类号: S718.43;S792.119;Q943.2

Overexpression mechanism of PeREM1.3 from Populus euphratica enhancing salt tolerance in transgenic tobacco

  • 摘要: 目的盐害作为一类非生物胁迫严重危害了农作物的生存以及产量。Remorin作为一类植物特有的蛋白质在植物适应环境过程中具有重要功能。本研究克隆了胡杨remorin蛋白PeREM1.3的编码基因PeREM1.3,并研究PeREM1.3基因在植物耐盐性中的作用。方法笔者将基因构件35S::PeREM1.3转入模式植物烟草中, 在盐胁迫条件下,通过生理生化的方法对表达PeREM1.3的转基因烟草进行基因功能的分析。结果研究显示PeREM1.3蛋白定位于细胞质膜上,其编码基因PeREM1.3的开放阅读框(ORF)长600 bp,编码199个氨基酸。胡杨中PeREM1.3能够响应盐胁迫和渗透胁迫表达上调。结果表明,在烟草中过表达PeREM1.3明显地提高了耐盐性。过表达PeREM1.3的烟草转基因株系中抗氧化物酶如SOD、POD和CAT活性显著提高,降低了活性氧水平,调控活性氧平衡。另外植物抗逆相关基因SOS1、HAKNHA1、VAG1和PMA4的转录水平显著增高,调控K+/Na+平衡。结论这些结果说明PeREM1.3蛋白通过维持植物的活性氧平衡和K+/Na+平衡来提高植物的耐盐性。

     

  • 图  1  不同胁迫处理下胡杨PeREM1.3基因在胡杨叶片中的表达量变化

    A.NaCl处理下PeREM1.3的表达量;B.甘露醇处理下PeREM1.3的表达量;不同字母表示在P<0.05水平上差异显著。

    Figure  1.  Expression changes of PeREM1.3 in leaf under different stress treatments in Populus euphratica

    A.Expression of PeREM1.3 under NaCl treatment; B. Expression of PeREM1.3 under mannitol treatment; different letters mean significant difference at P < 0.05 level.

    图  2  胡杨PeREM1.3序列分析

    A.胡杨PeREM1.3氨基酸序列与其他物种REM多重系列比对; B.胡杨PeREM1.3的系统进化树分析。Pe.胡杨; Pt.毛果杨; At.拟南芥。

    Figure  2.  Analysis of the amino acid sequence encoded by PeREM1.3

    A, multiple amino acid sequence alignment of PeREM1.3 with other REM from different plant species; B, phylogenetic tree analysis of PeREM proteins; Pe, Populus euphratica; Pt, Populus trichocarpa; At, Arabidopsis thaliana.

    图  3  PeREM1.3的亚细胞定位

    PeREM1.3-GFP.GFP荧光场;FM4-64.膜染料;Merged.叠加场。

    Figure  3.  Subcellular location of PeREM1.3

    PeREM1.3-GFP, GFP field; FM4-64, plasma membrane dye; Merged, merged field.

    图  4  转基因烟草各株系荧光定量和半定量RT-PCR检测PeREM1.3

    PeREM1.3基因在野生型(WT)、转空载体(VC)和转基因烟草(OE1、OE6、OE7、OE9、OE11、OE16、OE18、OE22)中的表达量,EF1α为内参基因。A为荧光定量PCR分析;B为半定量PCR分析。不同字母表示在P<0. 05水平上差异显著。下同。

    Figure  4.  Quantitative reverse transcription PCR and semi-quantitative reverse transcription PCR analysis of each line in tobacco plants

    Expression level of PeREM1.3 of wild-type (WT), vector control (VC) and PeREM1.3-transgenic tobacco (OE1, OE6, OE7, OE9, OE11, OE16, OE18, OE22) plants, EF1α was used as the internal control. A, real-time PCR analysis. B, semi-quantitative sRT-PCR analysis. Different letters mean significant difference at P < 0.05 level. The same below.

    图  5  烟草在含有不同浓度NaCl培养基上的根长生长情况

    A. NaCl对根长生长的影响; B.根系生长的定量分析;烟草播种在普通1/2MS培养基上,垂直生长7 d后,移植到含有0、150、175 mmol/L NaCl培养基上,生长5 d后统计根长生长情况并拍照,不同字母表示在P<0.05水平上差异显著。

    Figure  5.  Root length of tobacco on MS medium supplemented with different NaCl concentrations

    A, effect of NaCl on root length; B, quantitative analysis of root growth; seeds of each line were sowed on 1/2 MS medium for 7 days, then transferred to new MS medium with 0, 150, 175 mmol/L NaCl, after 5 days, root length was measured and photographed. Different letters mean significant difference at P < 0.05 level.

    图  6  盐处理后烟草中SOD酶活性(A)、POD酶活性(B)和CAT酶活性(C)变化

    A.盐处理下SOD酶活性;B.盐处理下POD酶活性;C.盐处理下CAT酶活性。不同字母表示在P<0.05水平上差异显著。

    Figure  6.  Activity of SOD (A), POD (B) and CAT (C) in tobacco plants after salt treatment

    A, activity of SOD under NaCl treatment; B, activity of POD under NaCl treatment; C, activity of CAT under NaCl treatment; different letters mean significant difference at P < 0.05 level.

    图  7  盐胁迫对PeREM1.3转基因烟草抗逆相关基因SOS1、HAKNHA1、VAG1和PMA4表达的影响

    内参基因:EF1α,n=3。不同字母表示在P<0.05水平上差异显著。

    Figure  7.  Effects of NaCl on relative expression of stress- resistance related genes in transgenic tobacco plants

    Reference gene: EF1α, n=3. Different letters mean significant difference at P < 0. 05 level.

    表  1  本研究中使用的引物序列以及名字

    Table  1.   Gene-specific primer sequences and names used in this work

    引物名称
    Primer name
    上游引物
    Forward primer(5′-3′)
    下游引物
    Reverse primer(5′- 3′)
    HAKATCCACACCGAGCTTGTTTCAGGATGGGTCCAATTCTTCCCACCAAGA
    SOS1GCGTGCTTATTTCCACCTTTTGTTTGATGACGGCTCCCCAGT
    PMA4TTTCCCGAGCACAAGTATGAGGTAACCTCCAAGAACAACAC
    NHA1CCTTATGCTTGTCGGTGCTTTCGCCCATTGTGCTTCCCTTTC
    VAG1GGCACGTAACCACAGTGAAGAGAAGCAGCCATGCCTAGTC
    EF1αGCTGTGAGGGACATGCGTCAAAGTAGTAGATATCGCGAGTACCACCA
    PeActinATTGGCCTTGGGGTTAAGAGCACACTGGAGTGATGGTTGG
    PeREM1.3-0029GFPGCTGCAGCATGGCAGAGGAGGAGCCAAAGCCCGGGTAAAAAAATTCCAAGAAGCTTC
    PeREM1.3-2300CGGGATCCATGGCAGAGGAGGAGCCAAAGGCGTCGACCTATAAAAAAATTCCAAGAAGCTT
    PeREM1.3GTCCAAAGCAGAAAACAAAGCTCACACGTTTAGCTTCGATAATCGCC
    PeREM1.3-FulllengthATGGCAGAGGAGGAGCCAAAGCTATAAAAAAATTCCAAGAAGCTT
    下载: 导出CSV
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  • 收稿日期:  2018-10-19
  • 修回日期:  2018-11-29
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