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过表达胡杨PeAnn1负调控拟南芥的抗旱性

武霞 张一南 赵楠 张莹 赵瑞 李金克 周晓阳 陈少良

武霞, 张一南, 赵楠, 张莹, 赵瑞, 李金克, 周晓阳, 陈少良. 过表达胡杨PeAnn1负调控拟南芥的抗旱性[J]. 北京林业大学学报, 2020, 42(6): 14-25. doi: 10.12171/j.1000-1522.20200031
引用本文: 武霞, 张一南, 赵楠, 张莹, 赵瑞, 李金克, 周晓阳, 陈少良. 过表达胡杨PeAnn1负调控拟南芥的抗旱性[J]. 北京林业大学学报, 2020, 42(6): 14-25. doi: 10.12171/j.1000-1522.20200031
Wu Xia, Zhang Yinan, Zhao Nan, Zhang Ying, Zhao Rui, Li Jinke, Zhou Xiaoyang, Chen Shaoliang. Overexpression of PeAnn1 from Populus euphratica negatively regulates drought resistance in transgenic Arabidopsis thaliana[J]. Journal of Beijing Forestry University, 2020, 42(6): 14-25. doi: 10.12171/j.1000-1522.20200031
Citation: Wu Xia, Zhang Yinan, Zhao Nan, Zhang Ying, Zhao Rui, Li Jinke, Zhou Xiaoyang, Chen Shaoliang. Overexpression of PeAnn1 from Populus euphratica negatively regulates drought resistance in transgenic Arabidopsis thaliana[J]. Journal of Beijing Forestry University, 2020, 42(6): 14-25. doi: 10.12171/j.1000-1522.20200031

过表达胡杨PeAnn1负调控拟南芥的抗旱性

doi: 10.12171/j.1000-1522.20200031
基金项目: 国家自然科学基金项目(31770643、31570587),北京市自然科学基金项目(6182030、6172024),中央高校基本科研业务费专项(2019ZY25)
详细信息
    作者简介:

    武霞。主要研究方向:树木逆境生理。Email:wuxia58888@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。主要研究方向:林木逆境生理。Email:lschen@bjfu.edu.cn 地址:同上

  • 中图分类号: S718.43; S792.119

Overexpression of PeAnn1 from Populus euphratica negatively regulates drought resistance in transgenic Arabidopsis thaliana

  • 摘要: 目的Annexins是原核生物和真核生物中普遍存在的一大类膜联蛋白家族,能够参与氧化胁迫、热胁迫、干旱胁迫和盐胁迫等许多胁迫响应过程。但在胡杨中,对膜联蛋白家族在抗逆性中的作用情况还缺乏了解。本文研究胡杨Anneixn1在植物耐受渗透胁迫和干旱中的作用。方法本研究对渗透胁迫诱导的PeAnn1基因表达进行检测,对PeAnn1进行相关生物信息学分析,与毛白杨、拟南芥、大豆和水稻膜联蛋白基因家族成员进行序列比对和系统进化树分析,以过表达PeAnn1拟南芥(PeAnn1-OE1和PeAnn1-OE2)、Annexin1突变体(atann1)和野生型拟南芥(WT)为实验材料,利用不同浓度甘露醇处理(0、150、200、250、300 mmol/L)模拟渗透胁迫,并对各株系进行土壤干旱和复水处理,测定了不同处理株系的萌发率、根长、叶绿素含量及荧光参数、过氧化氢含量、抗氧化酶活性和基因表达等指标,分析了不同基因型拟南芥的抗旱性。结果短期渗透胁迫处理诱导了胡杨叶片中PeAnn1基因的上调表达。PeAnn1基因序列与毛白杨PtAnn1相似度最高,与PtAnn1亲缘关系较近。在甘露醇培养基上,过表达PeAnn1拟南芥的生存率和根长生长受到明显的抑制,且随着甘露醇浓度的升高,差异显著(P < 0.05)。土壤干旱8 d后测得的转基因拟南芥的叶绿素SPAD值、PSⅡ最大光量子效率(Fv/Fm)、实际光合量子产量(ΦPSⅡ)和相对电子传递速率(ETR)均显著低于野生型和突变体(P < 0.05)。复水后,PeAnn1转基因拟南芥光合参数的恢复程度也较低。在渗透胁迫下,转基因植株抗氧化物酶SOD、POD、CAT的活性以及编码基因的表达量显著低于野生型和突变体,不能清除过多活性氧,导致氧化伤害。结论以上结果表明,过表达PeAnn1降低了拟南芥对水分逆境的抗性。

     

  • 图  1  甘露醇处理对胡杨PeAnn1基因表达的影响

    胡杨1年生苗木经250 mmol/L甘露醇处理12 h,渗透胁迫处理0、4、8、12 h后采集叶片进行RT-qPCR分析,每个数值均为3次生物样本重复,*表示差异显著,P < 0.05。One-year-old seedlings of P. euphratica were subjected to 250 mmol/L mannitol and stressed for 12 hours. Then leaves were sampled after being treated for 0, 4, 8, 12 hours under osmotic stress and used for RT-qPCR analysis. Each column is the mean of three biological repeats. Asterisk represents significant differences at P < 0.05 level.

    Figure  1.  Effects of mannitol stress on expression of PeAnn1 in Populus euphratica leaves

    图  2  胡杨PeAnn1基因序列比对与系统进化树分析

    A. 胡杨 PeAnn1基因与其他植物Annexin同源基因多重序列比对。B. 不同植物Annexin蛋白的系统进化树分析。胡杨PeAnn1(XM_011018027.1)、毛白杨PtAnn1(JX986594.1)、拟南芥AtAnn1(NM_103274.4)、野生大豆GsANN(GU474544.1)、水稻OsAnn8(LOC_Os09g20330.1)。A, multiple alignment of the deduced gene sequences of PeAnn1 with other Annexins from different plant species. B, phylogenetic tree analysis of Annexin proteins. P. euphratica Anneixn1(XM_011018027.1), P. tomentosa PtAnn1(JX986594.1), Arabidopsis thaliana AtAnn1(NM_103274.4), Glycine soja GsANN(GU474544.1), and Oryza sative OsAnn8(LOC_Os09g20330.1).

    Figure  2.  Multiple alignment of PeAnn1 gene sequences with other Annexins from different plant species and phylogenetic tree analysis of Annexin proteins

    图  3  不同浓度甘露醇对野生型(WT)、Annexin1突变体(atann1)和过表达PeAnn1(PeAnn1-OE1PeAnn1-OE2)拟南芥萌发率的影响

    A. 甘露醇对种子萌发生长的影响(拟南芥播种在含有不同浓度甘露醇的1/2MS培养基上,生长10 d后的照片);B. 种子萌发率分析。不同字母表示在P < 0.05水平上差异显著,下同。A, effects of mannitol stress on seed germination rate (seeds of Arabidopsis thaliana are allowed to germinate on 1/2 MS medium supplemented with different concentrations of mannitol. Representative photographs are taken after 10 days of treatment); B, analysis on seed germination rate. Different letters denote significant differences at P < 0.05 level, the same below.

    Figure  3.  Effects of different concentrations mannitol on seed germination rate of WT, atann1 and PeAnn1-transgenic Arabidopsis thaliana (PeAnn1-OE1, PeAnn1-OE2)

    图  4  不同浓度甘露醇对野生型(WT)、Annexin1突变体(atann1)和过表达PeAnn1(PeAnn1-OE1和PeAnn1-OE2)拟南芥根长生长的影响

    A. 甘露醇对根长生长的影响(拟南芥播种在1/2MS培养基萌发5 d后,移植到含有不同浓度甘露醇的1/2MS培养基垂直生长7 d后的照片), 标尺为1 cm;B. 根系生长分析。A, effects of mannitol stress on root growth (seeds of Arabidopsis thaliana are allowed to germinate on 1/2 MS medium for 5 days, then transplanted to 1/2 MS medium supplemented with different concentrations of mannitol. Representative photographs are taken after 7 days of treatment). Scale bar = 1 cm; B, root growth analysis.

    Figure  4.  Effects of different concentrations mannitol on root growth of WT, atann1 and PeAnn1-transgenicArabidopsis thaliana (PeAnn1-OE1, PeAnn1-OE2)

    图  5  甘露醇胁迫下野生型(WT)、Annexin1突变体(atann1)和过表达PeAnn1(PeAnn1-OE1PeAnn1-OE2)拟南芥根细胞H2O2荧光强度的变化

    甘露醇(250 mmol/L)胁迫下拟南芥根细胞H2O2荧光强度的变化。H2O2 fluorescence intensity variation of Arabidopsis thaliana under mannitol stress (250 mmol/L).

    Figure  5.  Effects of mannitol stress on H2O2 fluorescence intensity in root cells of wild-type (WT), atann1 and transgenic lines(PeAnn1-OE1, PeAnn1-OE2)

    图  6  甘露醇胁迫下野生型(WT)、Annexin1突变体(atann1)和过表达PeAnn1(PeAnn1-OE1和PeAnn1-OE2)拟南芥中SOD酶活性、POD酶活性和CAT酶活性的变化

    A. 甘露醇胁迫下SOD活性;B. 甘露醇胁迫下POD活性;C. 甘露醇胁迫下CAT活性。A, SOD activity under mannitol stress; B, POD activity under mannitol stress; C, CAT activity under mannitol stress.

    Figure  6.  Effects of mannitol stress on the activities of SOD, POD and CAT in wild-type (WT), atann1 and transgenic lines (PeAnn1-OE1, PeAnn1-OE2)

    图  7  甘露醇胁迫对野生型(WT)、Annexin1突变体(atann1)和过表达PeAnn1(PeAnn1-OE1PeAnn1-OE2)拟南芥抗氧化酶基因AtSODAtPODAtCAT表达的影响

    A. 甘露醇胁迫下AtSOD基因表达;B. 甘露醇胁迫下AtPOD基因表达;C. 甘露醇胁迫下AtCAT基因表达;内参基因为AtACTIN2,n = 3。A, AtSOD expression under mannitol stress; B, AtPOD expression under mannitol stress; C, AtCAT expression under mannitol stress; reference gene: AtACTIN2, n = 3.

    Figure  7.  Effects of mannitol stress on relative expression of antioxidant-enzyme genes (AtSOD, AtPOD and AtCAT) in wild-type (WT), atann1 and transgenic lines (PeAnn1-OE1, PeAnn1-OE2)

    图  8  干旱和复水后野生型(WT)、Annexin1突变体(atann1)和过表达PeAnn1(PeAnn1-OE1和PeAnn1-OE2)拟南芥的光合参数变化

    A. 干旱及复水后拟南芥的生长状况;B~D. 正常浇水、干旱8 d、复水3 d拟南芥的PSⅡ最大光量子效率(Fv/Fm)、相对电子传递效率(ETR)、实际光合量子产量(ΦPSⅡ)的变化。A, plant performance of Arabidopsis thaliana after drought stress and rewatering; B−D, maximum photon efficiency of PSⅡ(Fv/Fm), relative electron transfer efficiency(ETR), and actual photosynthetic quantum yield(ΦPSⅡ) of Arabidopsis thaliana under normal watering, 8 days of drought stress and 3 days of rewatering.

    Figure  8.  Changes of photosynthetic parameters in wild-type (WT), atann1, and transgenic lines (PeAnn1-OE1, PeAnn1-OE2) under drought stress and rewatering

    图  9  干旱和复水后野生型(WT)、Annexin1突变体(atann1)和过表达PeAnn1(PeAnn1-OE1和PeAnn1-OE2)拟南芥的叶绿素相对含量

    拟南芥正常浇水、干旱8 d、复水3 d叶绿素SPAD值的变化。Chlorophyll SPAD values of Arabidopsis thaliana under normal watering, 8 days of drought stress and 3 days of rewatering.

    Figure  9.  Changes of relative chlorophyll content in wild-type (WT), atann1 and transgenic plant (PeAnn1-OE1, PeAnn1-OE2) under drought stress and rewatering

    表  1  本文实验中所用到的引物序列

    Table  1.   Gene-specific primer sequences used in this study

    引物名称 Primer name上游引物 Forward primer (5′−3′)下游引物 Reverse primer (5′−3′)
    AtACTIN2GGTAACATTGTGCTCAGTGGTGGAACGACCTTAATCTTCATGCTGC
    AtSODAGGAAACATCACTGTTGGAGATGAGTTTGGTCCAGTAAGAGGAA
    AtCATAGGATCAAACTTTGAGGGGTAGCTTGTGGTTCCTGGAATCTACT
    AtPODCGTGCCCTTCATATTGTTGGGACGCCATCAACAACGAGTC
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  • 收稿日期:  2020-01-21
  • 修回日期:  2020-04-28
  • 网络出版日期:  2020-06-12
  • 刊出日期:  2020-07-01

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