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胡杨bZIP转录因子PebZIP26和PebZIP33基因的克隆及功能分析

张影 练从龙 段卉 路信 夏新莉 尹伟伦

张影, 练从龙, 段卉, 路信, 夏新莉, 尹伟伦. 胡杨bZIP转录因子PebZIP26和PebZIP33基因的克隆及功能分析[J]. 北京林业大学学报, 2017, 39(7): 18-30. doi: 10.13332/j.1000-1522.20170109
引用本文: 张影, 练从龙, 段卉, 路信, 夏新莉, 尹伟伦. 胡杨bZIP转录因子PebZIP26和PebZIP33基因的克隆及功能分析[J]. 北京林业大学学报, 2017, 39(7): 18-30. doi: 10.13332/j.1000-1522.20170109
ZHANG Ying, LIAN Cong-long, DUAN Hui, LU Xin, XIA Xin-li, YIN Wei-lun. Cloning and functional analysis of PebZIP26 and PebZIP33 transcription factors from Populus euphratica[J]. Journal of Beijing Forestry University, 2017, 39(7): 18-30. doi: 10.13332/j.1000-1522.20170109
Citation: ZHANG Ying, LIAN Cong-long, DUAN Hui, LU Xin, XIA Xin-li, YIN Wei-lun. Cloning and functional analysis of PebZIP26 and PebZIP33 transcription factors from Populus euphratica[J]. Journal of Beijing Forestry University, 2017, 39(7): 18-30. doi: 10.13332/j.1000-1522.20170109

胡杨bZIP转录因子PebZIP26和PebZIP33基因的克隆及功能分析

doi: 10.13332/j.1000-1522.20170109
基金项目: 

国家自然科学基金项目 31270656

“十二五”国家科技支撑计划项目 2015BAD07B01

详细信息
    作者简介:

    张影。主要研究方向:植物抗逆分子生物学。Email: zhangying_email@126.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:

    夏新莉,教授,博士生导师。主要研究方向:植物抗逆分子生物学。Email:xiaxl@bjfu.edu.cn  地址:同上

    尹伟伦,教授,博士生导师。主要研究方向:植物生理与生物技术。Email:yinwl@bjfu.edu.cn  地址:同上

  • 中图分类号: S718.43;Q943.2;S792.119

Cloning and functional analysis of PebZIP26 and PebZIP33 transcription factors from Populus euphratica

  • 摘要: bZIP(basic region/leucine zipper motif)是一类在真核生物中分布广泛的超大转录因子家族,参与调节植物的生长发育、衰老、激素调控、能量代谢、病原防御等过程。胡杨是研究抗逆分子机制的模式木本植物,但是关于胡杨的bZIP功能迄今未见研究报道。本研究从胡杨中克隆得到PebZIP26和PebZIP33两个转录因子的cDNA,经分析,分别编码439与371个氨基酸且PebZIP26与PebZIP33基因的表达受干旱、脱水及盐胁迫诱导。构建植物表达载体,利用农杆菌花序侵染法转化拟南芥,获得CaMV35S:PebZIP26和CaMV35S:PebZIP33超表达株系和空载体对照株系。在含有150 mmol/L NaCl及300 mmol/L甘露醇培养基上,PebZIP26和PebZIP33超表达株系根长均长于野生型,叶片嫩绿,无发黄萎蔫现象;另外,对盆土中的幼苗进行21 d的干旱处理和盐处理,超表达PebZIP26及PebZIP33株系耐旱耐盐性较强,表现为胁迫环境中植株营养生长较好,株高显著高于野生型(WT)及空载体对照株系,干旱复水后,植株存活率为61%及48%,显著高于WT的9%及空载体对照的12%。超表达PebZIP26及PebZIP33株系相比于WT和abf1及tga1(拟南芥同源基因ABF1及TGA1突变体)气孔关闭对外源ABA处理更加敏感,且对氧化胁迫的抗性较强。综上所述,胡杨PebZIP26和PebZIP33转录调节因子可能通过调控气孔开度和活性氧水平及根的生长正向调节植物抗旱耐盐性,进一步丰富了木本植物bZIP的基因功能认识,为遗传育种提供基因资源及理论依据。

     

  • 图  1  拟南芥各株系PCR鉴定结果及GUS染色鉴定

    M.标记DNA; WT.野生型; VC.空载体对照;1~8.条带1~8;A.拟南芥PebZIP26的DNA鉴定; B.拟南芥PebZIP33的DNA鉴定; C.拟南芥PebZIP26的GUS鉴定; D.拟南芥PebZIP33的GUS鉴定。

    Figure  1.  PCR and GUS detection of each strain of Arabidopsis thaliana

    M, DNA marker; WT, wide type; VC, vacant carrier; 1-8, line 1-8; A, PCR result of PebZIP26;B, PebZIP33 DNA identification.

    图  2  PebZIP26基因及PebZIP33基因与毛果杨同源基因编码氨基酸序列比对及基因结构图

    A.PebZIP26基因与PtrbZIP26基因的氨基酸序列比对;B.PebZIP33基因与PtrbZIP33基因氨基酸序列比对;C.PebZIP26基因与PebZIP33基因结构图。

    Figure  2.  Comparison of PebZIP26 and PebZIP33 proteins between P. euphratica and P. trichocarpa

    A, comparison of amino acid sequence between gene PebZIP26 and PtrbZIP26; B, comparison of amino acid sequence between gene PebZIP33 and PtrbZIP33; C, gene structure of PebZIP26 and PebZIP33.

    图  3  胡杨PebZIP26和PebZIP33蛋白的系统进化树分析

    Figure  3.  Molecular phylogenetic tree analysis of PebZIP26 and PebZIP33 species

    图  4  不同处理下胡杨PebZIP26和PebZIP33基因在胡杨叶片中的表达

    A.脱水处理下PebZIP26的表达量; B.脱水处理下PebZIP33的表达量; C.不同水势下PebZIP26的表达量; D.不同水势下PebZIP33的表达量;E.200 mmol/L NaCl处理下PebZIP26的表达量;F.200 mmol/L NaCl处理下PebZIP33的表达量;RC.对C组(30%水势)进行复水。不同字母表示在P<0.05水平上差异显著。

    Figure  4.  Expression of PebZIP26 and PebZIP33 in leaf under treatments in Populus euphratica

    A, expression of PebZIP26 under dehydrate treatment; B, expression of PebZIP33 under dehydrate treatment; C, expression of PebZIP26 under different water potentials; D, expression of PebZIP33 under different water potentials; E, expression of PebZIP26 under 200 mmol/L NaCl treatment; F, expression of PebZIP26 under 200 mmol/L NaCl treatment; RC, rewater of C group (30% water potentral); Different letters mean significant difference at P < 0.05 level.

    图  5  3个株系(WT、PebZIP26、PebZIP33)在不同处理下的根长比较

    A~F:不同处理条件下的根长表型;G.不同处理条件下的根长统计。不同字母表示在P<0.05水平上差异显著。

    Figure  5.  Comparison in root length of three lines (WT, PebZIP26, PebZIP33)

    A-F, root length under different treatment conditions; G, statistics of root length under different treatment conditions. Different letters mean significant difference at P < 0.05 level.

    图  6  干旱和盐胁迫下各株系的(野生型、空载体、超表达)株高的比较及复水后存活率

    A.干旱处理21 d,野生型与超表达PebZIP26株系株高对比;B.干旱处理21 d,野生型与超表达PebZIP33株系株高对比;C.盐胁迫处理21 d,野生型与超表达PebZIP26株系株高对比;D.盐胁迫处理21 d,野生型与超表达PebZIP33株系株高对比; E.复水后植株存活率;F.株高统计。

    Figure  6.  Comparison in stem length of four lines (WT, VC, PebZIP26, PebZIP33) and survival rate being rewatered after drought and salt stress

    A, stem length of WT and PebZIP26 after 21days drought treatment; B, stem length of WT and PebZIP33 after 21days drought treatment; C, stem length of WT and PebZIP26 after 21days drought treatment. D, stem length of WT and PebZIP33 after 21days drought treatment. E, survival rate 5 days later being rewatered after drought. F, statistics of stem length.

    图  7  离体叶片失水率以及地上部分干物质含量

    Figure  7.  Water loss rate of cut leaves and dry matter content in Populus euphratica

    图  8  离体叶片脱水处理2 h以及5 μmol/L外源ABA处理2 h时的组织化学染色检测活性氧

    Figure  8.  Histochemical staining assay detection of H2O2 and O2 accumulation with diaminobenzidin followed dedehydrate and 5 μmol/L exogenous ABA treatment

    图  9  5及10 μmol/L外源ABA处理后各株系的气孔开度

    A.外源ABA处理后各株系气孔开度对比图;B.不同处理下各株系气孔开放宽度与长度比例。

    Figure  9.  Stomatal aperture ratio in response to ABA treatment in each strain

    A, comparison of stomatal aperture of each strain after ABA treatment in WT and PebZIP26, PebZIP33 transgenic and corresponding mutant A. thaliana plants; B, the ratio of width and length of stomatal in different treatments.

    图  10  PebZIP33延缓种子的萌发及植株的营养生长

    A.PebZIP33与WT及tga1在150 mmol/L NaCl培养基的发芽趋势;B.PebZIP33与WT及TGA1在300 mmol/L甘露醇培养基的发芽趋势;C.同一时期WT与PebZIP33植株比较。

    Figure  10.  PebZIP33 delay the germination of seed and nutritional growth

    A, germination trend of PebZIP33 and WT and tga1 on 150 mmol/LNaCl medium; B, germination trend of PebZIP33 and WT and tga1 on 300 mmol/L mannitol medium; C, comparison of WT and PebZIP33 on leaf area at the period.

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  • 收稿日期:  2017-03-29
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