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过表达胡杨PeRIN4基因拟南芥提高质膜H+-ATPase活性和耐盐性

张一南 王洋 张会龙 尧俊 邓佳音 赵瑞 沈昕 陈少良

张一南, 王洋, 张会龙, 尧俊, 邓佳音, 赵瑞, 沈昕, 陈少良. 过表达胡杨PeRIN4基因拟南芥提高质膜H+-ATPase活性和耐盐性[J]. 北京林业大学学报, 2017, 39(11): 1-8. doi: 10.13332/j.1000-1522.20170124
引用本文: 张一南, 王洋, 张会龙, 尧俊, 邓佳音, 赵瑞, 沈昕, 陈少良. 过表达胡杨PeRIN4基因拟南芥提高质膜H+-ATPase活性和耐盐性[J]. 北京林业大学学报, 2017, 39(11): 1-8. doi: 10.13332/j.1000-1522.20170124
ZHANG Yi-nan, WANG Yang, ZHANG Hui-long, YAO Jun, DENG Jia-yin, ZHAO Rui, SHEN Xin, CHEN Shao-liang. Over-expression of PeRIN4 enhanced salinity tolerance through up-regulation of PM H+-ATPase in Arabidopsis thaliana[J]. Journal of Beijing Forestry University, 2017, 39(11): 1-8. doi: 10.13332/j.1000-1522.20170124
Citation: ZHANG Yi-nan, WANG Yang, ZHANG Hui-long, YAO Jun, DENG Jia-yin, ZHAO Rui, SHEN Xin, CHEN Shao-liang. Over-expression of PeRIN4 enhanced salinity tolerance through up-regulation of PM H+-ATPase in Arabidopsis thaliana[J]. Journal of Beijing Forestry University, 2017, 39(11): 1-8. doi: 10.13332/j.1000-1522.20170124

过表达胡杨PeRIN4基因拟南芥提高质膜H+-ATPase活性和耐盐性

doi: 10.13332/j.1000-1522.20170124
基金项目: 

国家自然科学基金项目 31770643

高等学校学科创新引智计划项目 111Project

教育部创新团队发展计划项目 IRT13047

高等学校学科创新引智计划项目 B130007

教育部科学技术研究(科学技术类)项目 113013A

国家自然科学基金项目 31570587

北京市自然科学基金项目 6172024

详细信息
    作者简介:

    张一南,博士生。主要研究方向:树木逆境生理。Email: xhzyu007@163.com  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:

    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

Over-expression of PeRIN4 enhanced salinity tolerance through up-regulation of PM H+-ATPase in Arabidopsis thaliana

  • 摘要: 本文克隆了RIN4(RPM1-interacting protein 4)在胡杨中的同源基因PeRIN4,并在拟南芥中进行过表达,通过研究转基因株系的耐盐表型、质膜H+-ATPsae活性及H+ 、Na+、K+等的动态离子流,揭示了PeRIN4基因在植物响应和适应盐胁迫环境中的作用。利用定位载体pGreen0029-PeRIN4-GFP瞬时转化拟南芥叶肉细胞原生质体的方法,对胡杨PeRIN4蛋白进行亚细胞定位,发现该蛋白定位在细胞的胞质中。耐盐表型实验结果显示,在100 mmol/L NaCl处理下,拟南芥PeRIN4过表达株系(OE1和OE8)的生存率和根长均明显高于野生型(WT)和转空载体拟南芥(VC),说明PeRIN4基因能够提高拟南芥的耐盐性。与WT和VC相比,拟南芥PeRIN4过表达株系质膜H+-ATPsae的活性较高。动态离子流数据显示,在盐胁迫下,PeRIN4过表达株系外排H+和Na+ 离子的能力强于野生型和转空载体拟南芥,然而K+的外流却弱于WT和VC。因此,PeRIN4蛋白具有调节质膜H+-ATPsae活性的功能。拟南芥质膜H+-ATPsae活性的提高主要有两方面的作用:一是可以增强H+泵的质子动力势,驱动Na+/H+逆向转运蛋白,提高Na+外排的能力;二是抑制质膜的去极化,减少K+离子通过去极化激活的外向型K+通道(DA-KORCs)和非选择性阳离子通道(DA-NSCCs)外流,维持了K+/Na+平衡,从而提高PeRIN4转基因拟南芥的耐盐性。

     

  • 图  1  胡杨PeRIN4序列分析

    A.胡杨RIN4氨基酸序列与其他物种RIN4多重系列比对。B.不同物种RIN4同源蛋白的系统进化树分析。Pe.胡杨;Pt.毛果杨;At.拟南芥;Hv.大麦;Mt.苜蓿;Zm.玉蜀黍。

    Figure  1.  Bioinformatic analysis of amino acid sequence encoded by PeRIN4

    A, Multiple amino acid sequence alignment of PeRIN4 with other RIN4s from different plant species. B, Phylogenetic tree analysis of RIN4 proteins. Pe, Populus euphratica; Pt, Populus trichocarpa; At, Arabidopsis thaliana; Hv, Hordeum vulgare; Mt, Medicago truncatula; Zm, Zea mays.

    图  2  拟南芥各株系半定量反转录PCR

    PeRIN4基因在野生型(WT)、转空载体(VC)和转基因(OE1、OE2、OE7、OE8、OE11)拟南芥中的表达量,Atactin2为内参基因。

    Figure  2.  Semi-quantitative reverse transcription PCR analysis of PeRIN4 in arabidopsis

    Expression level of PeRIN4 in wild-type (WT), vector control (VC) and PeRIN4-transgenic arabidopsis (OE1, OE2, OE7, OE8, OE11), Atactin2 was used as the internal control.

    图  3  PeRIN4的亚细胞定位

    pGreen0029为不含PeRIN4基因的空载体。GFP:绿色荧光蛋白;Chlorophyll:叶绿体自发荧光;Merged:叠加场;Bright:明场。

    Figure  3.  Subcellular location of PeRIN4

    pGreen0029 is vector control. GFP: green fluorescent protein; Chlorophyll: auto fluorescence; Merged: merged field; Bright: bright field. Scale bar=250 μm.

    图  4  NaCl对拟南芥生存率和根长生长的影响

    A. NaCl对拟南芥生长、生存率的影响;B.拟南芥生存率的统计分析;拟南芥直接播种在含有0或100 mmol/L NaCl培养基上,生长10 d后统计生存率;C. NaCl对根长生长的影响; D.根长生长的统计分析;拟南芥播种在普通MS培养基上,垂直生长7 d后,移植到含有0或100 mmol/L NaCl培养基上,生长3 d后统计根长生长情况(选取90株进行测定)。每个实验重复3次,每个数值均为3次重复的平均值,误差线代表平均标准差。不同小写字母表示差异显著,P<0.05。下同。标尺为1 cm。

    Figure  4.  Effects of NaCl on survival rate and root length of arabidopsis grown on MS medium

    A. Effect of NaCl on plant growth and survival rate; B. Quantitative analysis of survival rate; seeds of each line were sowed on MS medium supplemented with or without 100 mmol/L NaCl. After 10 days of salt treatment, survival rate was measured. C. Effect of NaCl on root length; D. Quantitative analysis of root length (with 90 plants); seeds of each line were sowed on MS medium for 7 days, then transferred to new MS medium supplemented with or without NaCl. After 3 days of salt treatment, root length was measured. Each column is the mean of three independent experiments and error bars represent the standard error of the mean. Different letters represent significant differences at P < 0.05 level. The same below. Scale bar=1 cm.

    图  5  NaCl对拟南芥根尖H+、Na+和K+离子流的影响

    A.拟南芥盐处理根尖瞬时H+离子流;B.拟南芥根尖稳态Na+离子流;C.拟南芥根尖稳态K+离子流。每个实验重复3次,每个数值均为3次重复的平均值,误差线代表平均标准差。

    Figure  5.  Effects of NaCl on H+, Na+ and K+ fluxes in arabidopsis root tips

    A. Transient H+ flux kinetics in arabidopsis root tips; B. Steady Na+ fluxes in arabidopsis root tips; C. Steady Na+ fluxes in arabidopsis root tips. Each column is the mean of three independent experiments and error bars represent the standard error of the mean. Scale bar=1 cm.

    图  6  拟南芥质膜H+-ATPase水解活性分析

    每个实验重复3次,每个数值均为3次重复的平均值,误差线代表平均标准差。

    Figure  6.  Hydrolytic activity of PM H+-ATPase in arabidopsis

    Each column is the mean of three independent experiments and error bars represent the standard error of the mean.

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  • 收稿日期:  2017-04-10
  • 修回日期:  2017-05-05
  • 刊出日期:  2017-11-01

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