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    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
    shu
    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
    shu

    转AtDME1基因‘84K’杨的获得及目的基因诱导表达分析

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

      摘要
    • 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。

       

      Abstract:
      ObjectiveDNA methylation is an important epigenetic modification that plays an essential role in plant growth and development. In this study, the chemically-induced promoter and the Arabidopsis thaliana demethylation transferase gene AtDME1 were introduced into the genome of Populus alba × P. glandulosa ‘84K’, and the expression of AtDME1 was effectively induced by 17-β-estradiol treatment. The expression characteristics of AtDME1 in transgenic poplar plants were investigated. This study laid a foundation for the establishment of poplar methylation-induced variation system and genetic improvement of poplars.
      MethodThe chemically-induced promoter and AtDME1 were transformed into genome of P. alba × P. glandulosa ‘84K’ using Agrobacterium-mediated transformation. Traditional PCR and DNA sequencing were used to identify the transgenic plants in hygromycin resistant plants. The chemical inducer 17-β-estradiol was used to induce expression of AtDME1 in in vitro leaves of a transgenic clone for 0, 3, 6, 12, 24, 48, 96 and 144 hours, and the expression of AtDME1 gene was detected by quantitative real-time PCR (qRT-PCR).
      ResultA total of 224 hygromycin resistant buds were obtained, among them six hygromycin resistant plants were screened. Finally, six transgenic plants were identified by molecular methods, which were named as AD-1−6. Results of qRT-PCR showed that the expression of AtDME1 reached its highest level after 3 hours treatment of 17-β-estradiol, and its expression gradually decreased after12 hour’s treatment.
      ConclusionThe chemical inducer 17-β-estradiol can rapidly and efficiently induce the expression of AtDME1 gene in transgenic poplar, which lays a solid foundation for further study on the mechanism of DME1 gene in the regulation of poplar genome methylation. It lays a solid foundation for the study of the chemical expression characteristics of poplar.

       

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