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AtDME1基因‘84K’杨的获得及目的基因诱导表达分析

吴晓娟 鲁俊倩 常英英 钟姗辰 苏晓华 张冰玉

吴晓娟, 鲁俊倩, 常英英, 钟姗辰, 苏晓华, 张冰玉. 转AtDME1基因‘84K’杨的获得及目的基因诱导表达分析[J]. 北京林业大学学报, 2020, 42(6): 26-32. doi: 10.12171/j.1000-1522.20190068
引用本文: 吴晓娟, 鲁俊倩, 常英英, 钟姗辰, 苏晓华, 张冰玉. 转AtDME1基因‘84K’杨的获得及目的基因诱导表达分析[J]. 北京林业大学学报, 2020, 42(6): 26-32. doi: 10.12171/j.1000-1522.20190068
Wu Xiaojuan, Lu Junqian, Chang Yingying, Zhong Shanchen, Su Xiaohua, Zhang Bingyu. Genetic transformation of Populus alba × P. glandulosa ‘84K’ with AtDME1 and its chemical-inducible expression analysis[J]. Journal of Beijing Forestry University, 2020, 42(6): 26-32. doi: 10.12171/j.1000-1522.20190068
Citation: Wu Xiaojuan, Lu Junqian, Chang Yingying, Zhong Shanchen, Su Xiaohua, Zhang Bingyu. Genetic transformation of Populus alba × P. glandulosa ‘84K’ with AtDME1 and its chemical-inducible expression analysis[J]. Journal of Beijing Forestry University, 2020, 42(6): 26-32. doi: 10.12171/j.1000-1522.20190068

AtDME1基因‘84K’杨的获得及目的基因诱导表达分析

doi: 10.12171/j.1000-1522.20190068
基金项目: 中央级公益性科研院所基本科研业务费专项重点项目子项目“杨树工业用材新型品种选育研究”(CAFYBB2017ZA001-3)
详细信息
    作者简介:

    吴晓娟。主要研究方向:林木遗传育种。Email:yy992@foxmail.com 地址:100091 北京市海淀区青龙桥街道中国林业科学研究院林业研究所

    责任作者:

    张冰玉,博士,研究员。主要研究方向:林木遗传育种。Email:byzhang@caf.ac.cn 地址:同上

Genetic transformation of Populus alba × P. glandulosa ‘84K’ with AtDME1 and its chemical-inducible expression analysis

  • 摘要: 目的DNA甲基化是一种重要的表观遗传标记,在植物生长发育、环境响应等过程中发挥重要作用。本研究将拟南芥去甲基化基因AtDME1导‘84K’杨基因组中,通过化学诱导表达实验,研究AtDME1基因在转基因杨树中的诱导表达特性,为建立杨树甲基化诱导变异体系,进而实现杨树品种改良等奠定良好基础。方法以白杨派优良品种84K杨无菌苗叶片为受体材料,采用农杆菌介导法将化学诱导启动子与AtDME1基因导入‘84K’杨;经过潮霉素筛选、常规PCR检测及DNA测序等方法对抗性植株进行鉴定。通过化学诱导剂17-β-雌二醇对随机挑选的1个转基因株系离体叶片进行诱导处理,处理时间为0、3、6、12、24、48、96、144 h,采用qRT-PCR检测处理叶片中外源基因AtDME1的表达量变化。结果本研究中,潮霉素分化筛选共获得了抗性芽224个,经生根筛选获得6株抗性植株,经分子检测证实这6株抗性植株均为转AtDME1基因植株,扩繁后分别标号为转基因AD-1 ~ 6。qRT-PCR检测结果显示,化学诱导剂17-β-雌二醇处理3 h时,目的基因AtDME1的表达量基本达到最大值,效果持续至12 h后逐渐减弱。结论化学诱导剂17-β-雌二醇能迅速有效地调控转基因杨树中AtDME1基因的表达,为进一步研究DME1基因在杨树基因组甲基化调控方面的作用机制奠定基础,也为杨树化学诱导表达特性的研究做好了铺垫。

     

  • 图  1  植物表达载体pER8-DME1载体结构图

    Figure  1.  Structure of plant expression vector pER8-DME1

    图  2  抗性芽及抗性植株的获得

    A. 分化筛选30 d叶盘;B. 生根筛选15 d植株。A, resistant buds obtained by screening for 30 days on hygromycin selection medium; B, resistant plants obtained by screening for 15 days on hygromycin rooting medium.

    Figure  2.  Hygromycin resistant buds and resistant plants of AtDME1 transgenic P. alba × P. glandulosa ‘84K’

    图  3  DME1基因抗性植株的潮霉素抗性基因PCR检测

    M. DL2000 Marker;1. 阴性对照;2. 阳性对照;3 ~ 8. 转DME1基因抗性植株。下同。M, DL2000 Marker;1, negative control;2, positive control;3−8, hygromycin resistant plants. The same below.

    Figure  3.  PCR detection of HPT gene in AtDME1 transgenic P. alba × P. glandulosa ‘84K’ plants

    图  4  抗性植株DME1基因PCR检测

    Figure  4.  PCR detection of HPT and AtDME1 gene in AtDME1 transgenic P. alba × P. glandulosa ‘84K’ plants

    图  5  不同时间处理下转基因植株AtDME1诱导表达量

    Figure  5.  Expression level of AtDME1 in leaves of transgenic P. alba × P. glandulosa ‘84K’ induced by different time treatments of 17-β-estradiol

    表  1  常规PCR检测相关引物序列

    Table  1.   Primer sequences of PCR

    引物名称
    Primer name
    检测基因
    Detecting gene
    扩增产物长度
    Amplification product length/bp
    引物序列 Primer sequence
    HPHPT843F: 5′-TAAATAGCTGCGCCGATGGT-3′
    R: 5′-GGTTTCCACTATCGGCGAGT-3′
    HDHPT ~ AtDME11 102F: 5′-CTGTCGGGCGTACACAAATC-3′
    R: 5′-GTGCATGTGATCCCGCTAAG-3′
    下载: 导出CSV

    表  2  Real-time PCR反应相关引物序列

    Table  2.   Relative primer sequences of Real-time PCR reaction

    基因
    Gene
    扩增产物长度
    Amplification product length/bp
    引物序列
    Primer sequence
    Actin195F: 5′- AAACTGTAATGGTCCTCCCTCCG -3′
    R: 5′- GCATCATCACAATCACTCTCCGA -3′
    DME1163F: 5′- GGGATTCTGCAACACTGGTG -3′
    R: 5′- AGAAGTCACCTCACTCCTGC -3′
    下载: 导出CSV
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出版历程
  • 收稿日期:  2019-01-25
  • 修回日期:  2019-03-22
  • 网络出版日期:  2020-05-30
  • 刊出日期:  2020-07-01

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