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白桦BpSPL6基因启动子的克隆及表达分析

李豆 苏功博 胡晓晴 宋婷婷 孙庆斌 徐昭 刘雪梅 王宏伟

李豆, 苏功博, 胡晓晴, 宋婷婷, 孙庆斌, 徐昭, 刘雪梅, 王宏伟. 白桦BpSPL6基因启动子的克隆及表达分析[J]. 北京林业大学学报. doi: 10.12171/j.1000-1522.20200174
引用本文: 李豆, 苏功博, 胡晓晴, 宋婷婷, 孙庆斌, 徐昭, 刘雪梅, 王宏伟. 白桦BpSPL6基因启动子的克隆及表达分析[J]. 北京林业大学学报. doi: 10.12171/j.1000-1522.20200174
Li Dou, Su Gongbo, Hu Xiaoqing, Song Tingting, Sun Qingbin, Xu Zhao, Liu Xuemei, Wang Hongwei. Cloning and expression analysis of BpSPL6 promoter from Betula platyphylla[J]. Journal of Beijing Forestry University. doi: 10.12171/j.1000-1522.20200174
Citation: Li Dou, Su Gongbo, Hu Xiaoqing, Song Tingting, Sun Qingbin, Xu Zhao, Liu Xuemei, Wang Hongwei. Cloning and expression analysis of BpSPL6 promoter from Betula platyphylla[J]. Journal of Beijing Forestry University. doi: 10.12171/j.1000-1522.20200174

白桦BpSPL6基因启动子的克隆及表达分析

doi: 10.12171/j.1000-1522.20200174
基金项目: 黑龙江头雁创新团队计划(林木遗传育种创新研究团队),中央高校基本科研业务费专项基金E类项目(2572015EA05)
详细信息
    作者简介:

    李豆。主要研究方向:林木遗传育种。Email:1171057158@qq.com  地址:150040 黑龙江省哈尔滨市香坊区和兴路26号东北林业大学生命科学学院

    责任作者:

    刘雪梅,博士,教授。主要研究方向:林木遗传育种。Email:695898040@qq.com 地址:同上

    王宏伟,博士,副教授。主要研究方向:生物活性物质制备与应用。Email:84970486@qq.com 地址:同上

  • 中图分类号: S718.46

Cloning and expression analysis of BpSPL6 promoter from Betula platyphylla

  • 摘要:   目的  SPL是植物特有的转录因子,参与植物幼年期向成年期的转变、营养生长向生殖生长的转变、花发育、孢子发生、叶片和根发育、逆境响应等多个过程,在植物的生长发育过程中起着非常重要的作用。探究白桦中BpSPL6基因启动子区的顺式作用元件,以及该启动子在正常和胁迫条件下的表达模式,可为进一步研究BpSPL6基因的功能提供参考,也可为了解白桦的抗逆机制提供依据。  方法  以本实验室组培白桦的总DNA为模板,经PCR克隆了BpSPL6基因上游1 703 bp的启动子序列,用PLACE和Plant CARE在线软件分析启动子区的顺式作用元件。构建了BpSPL6基因启动子驱动GUS报告基因的植物表达载体并转化拟南芥,探究其组织表达特性和胁迫条件下的表达模式。  结果  PCR成功克隆了BpSPL6基因上游1 703 bp的启动子序列,对启动子区的顺式作用元件预测发现除了含有核心启动元件TATA-box、CAAT-box外,还包括2种特异组织表达元件(根、花粉),10种激素响应元件(生长素、赤霉素、水杨酸、脱落酸),4种脱水响应元件等。对转基因拟南芥进行GUS染色结果表明,BpSPL6基因启动子驱动的GUS基因在转基因拟南芥中的表达具有时空特异性。在拟南芥的整个发育过程中,BpSPL6基因启动子驱动GUS基因在真叶叶片中表达,但是表达部位不同。随着叶片的生长,首先在叶片的顶端表达,随后扩展到叶片的叶脉并直至整个叶片,并且表达量逐渐升高。同时BpSPL6基因启动子驱动的 GUS 基因在拟南芥营养生长时期的根部都有表达。并且经氯化钠和甘露醇胁迫后其表达量降低。对比两种胁迫,受到氯化钠胁迫后GUS基因的表达量变化更大,说明对氯化钠胁迫的响应更加强烈。  结论  BpSPL6基因可能参与了植物的叶片、根发育以及对盐和干旱胁迫的响应。

     

  • 图  1  BpSPL6基因启动子的克隆

    M. DL-5000 Marker; 1. BpSPL6基因启动子扩增产物。M, DL-5000 Marker; 1, amplified production of BpSPL6 promoter.

    Figure  1.  Cloning of BpSPL6 promoter by PCR

    图  2  BpSPL6基因启动子核苷酸序列多重比对

    1-BpSPL6-promoter. 测序结果;2-BpSPL6-promoter. 白桦基因组数据库中启动子序列;3-BpSPL6-promoter. 欧洲白桦基因组数据库中启动子序列。1-BpSPL6-promoter, sequencing results; 2-BpSPL6-promoter, BpSPL6 promoter sequences in Betula platyphylla genome database; 3-BpSPL6-promoter, BpSPL6 promoter sequences in Betula pendula genome database.

    Figure  2.  Multi-alignment of nucleotide sequences of BpSPL6 promoter

    图  3  白桦BpSPL6基因启动子序列及顺式作用元件分布

    方框及彩色表示预测的顺势作用元件序列;方框下文字表示对应元件名称;图片下部注释了彩色序列对应的元件名称;下划线表示启动子上游未克隆序列;双下划线并加粗表示起始密码子位置。The box and color represent the predicted cis-elements; text under the box represents the corresponding cis-element name; the cis-element names corresponding to the color sequence are annotated at the bottom of the picture; the underline indicates the uncloned sequence upstream of the promoter; the double underline and bold indicate the position of start codon.

    Figure  3.  Promoter sequence and cis-element distribution of BpSPL6 gene promoter for B. platyphylla

    图  4  大肠杆菌重组质粒的菌液PCR检测

    M. DL-5000 Marker;1. 阴性对照(水对照);2 ~ 3. 大肠杆菌转化子。M, DL-5000 Marker; 1, negative control(water control); 2−3, Escherichia coli transformant.

    Figure  4.  PCR result of positive Escherichia coli clone

    图  5  转基因拟南芥的检测

    M. DL-5000 Marker;1. 阳性对照;2 ~ 6. 转基因单株;7. 阴性对照(野生型拟南芥)。M, DL-5000 Marker; 1, positive control; 2−6, transgenic lines; 7, negative control(wild type A. thaliana).

    Figure  5.  PCR detection of transgenic A. thaliana

    图  6  营养生长期的转基因拟南芥GUS染色

    A ~ C. 生长7 d的拟南芥;A. 整株幼苗;B. 真叶;C. 根。D ~ G. 生长10 d的拟南芥;D. 整株幼苗;E. 真叶;F. 下胚轴;G. 根。A−C, A. thaliana seedling grown for 7 days; A, whole seedling; B, euphylla; C, root. D−G, A. thaliana seedling grown for 10 days; D, whole seedling; E:euphylla;F, hypocotyl; G, root.

    Figure  6.  Staining of GUS in transgenic A. thaliana in vegetative phase

    图  7  生殖生长期的转基因拟南芥GUS染色

    A ~ G. 生长25 d的拟南芥;A. 整株幼苗;B. 成熟叶片;C. 幼嫩叶片;D. 幼嫩叶片和花;E. 花;F. 幼果;G. 根。A−G, A. thaliana seedling grown for 25 days; A, whole seedling; B, mature leaf; C, young leaf; D, young leaf and flower; E, flower; F, young fruit; G, root.

    Figure  7.  Staining of GUS in transgenic A. thaliana in reproductive phase

    图  8  不同非生物胁迫处理下转基因拟南芥的GUS染色

    A. 整株幼苗;B. 成熟叶片;C. 幼嫩叶片;D. 根;E. 下胚轴。A, whole seedling; B, mature leaf; C, young leaf; D, root; E, hypocotyl.

    Figure  8.  Staining of GUS in transgenic A. thaliana under different abiotic stress treatments

    表  1  启动子顺式作用元件分析

    Table  1.   Analysis of promoter cis-elements

    元件名称 Element name基序序列 Motif sequence个数 Number生物学功能 Biological function
    TATA-box TATA 6 核心启动元件 Core actuating element
    CAAT-box CAAT 22 启动子增强区保守元件 Conservative element of promoter enhance region
    GT1-motif GGTTAAT 5 光响应元件 Element involved in light responsiveness
    Box 4 ATTAAT 3 光响应元件 Element involved in light responsiveness
    GA-motif ATAGATAA 2 光响应元件 Element involved in light responsiveness
    AT1-motif AATTATTTTTTATT 1 光响应元件 Element involved in light responsiveness
    AuxRR-core GGTCCAT 2 生长素响应元件 Element involved in auxin responsiveness
    TGA-element AACGAC 1 生长素响应元件 Element involved in auxin responsiveness
    CATATGGMSAUR CATATG 4 生长素响应元件 Auxin response element
    NTBBF1ARROLB ACTTTA 4 生长素响应元件 Auxin response element
    GARE2OSREP1 TAACGTA 1 赤霉素响应元件 Gibberellin response element
    WRKY71OS TGAC 12 赤霉素响应元件 Gibberellin response element
    P-box CCTTTTG 1 赤霉素响应元件 Gibberellin response element
    WBOXATNPR1 TTGAC 4 水杨酸响应元件 Element involved in salicylic acid responsiveness
    DPBFCOREDCDC3 ACACNNG 3 脱落酸响应元件 Element involved in abscisic acid responsiveness
    ABRE ACGTG 1 脱落酸响应元件 Element involved in abscisic acid responsiveness
    MYB1AT WAACCA 5 脱水响应元件 Dehydration response element
    MYCCONSENSUSAT CANNTG 12 脱水及寒冷响应元件 Dehydration and cold response element
    ACGTATERD1 ACGT 4 脱水响应元件 Dehydration response element
    MYBCORE CNGTTR 1 脱水响应元件 Dehydration response element
    WBOXNTERF3 TGACY 7 伤害响应元件 Damage response element
    DOFCOREZM AAAG 33 伤害响应元件 Damage response element
    CCAATBOX1 CCAAT 4 热激信号响应元件 Heat shock signal response element
    POLLEN1LELAT52 AGAAA 14 花粉特异表达的顺式作用元件 Cis-acting element for pollen specific expression
    ROOTMOTIFTAPOX1 ATATT 10 与根相关的顺式作用元件 Cis-element element in root
    注:基序序列:N = A/T/G/C,R = A/G,W = A/T,Y = C/T。Notes: motif sequences: N = A/T/G/C, R = A/G, W = A/T, Y = C/T.
    下载: 导出CSV
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  • 收稿日期:  2020-06-05
  • 修回日期:  2020-07-16
  • 网络出版日期:  2021-06-01

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