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转基因三倍体毛白杨花粉活力及枯落物外源基因检测

吕威 孙宇涵 张华新 杨庆山 李娟 陈受宜 张万科 李云

吕威, 孙宇涵, 张华新, 杨庆山, 李娟, 陈受宜, 张万科, 李云. 转基因三倍体毛白杨花粉活力及枯落物外源基因检测[J]. 北京林业大学学报, 2019, 41(7): 91-100. doi: 10.13332/j.1000-1522.20190105
引用本文: 吕威, 孙宇涵, 张华新, 杨庆山, 李娟, 陈受宜, 张万科, 李云. 转基因三倍体毛白杨花粉活力及枯落物外源基因检测[J]. 北京林业大学学报, 2019, 41(7): 91-100. doi: 10.13332/j.1000-1522.20190105
Lü Wei, Sun Yuhan, Zhang Huaxin, Yang Qingshan, Li Juan, Chen Shouyi, Zhang Wanke, Li Yun. Pollen vigor and detection of exogenous genes of litter for transgenic triploid Populus tomentosa[J]. Journal of Beijing Forestry University, 2019, 41(7): 91-100. doi: 10.13332/j.1000-1522.20190105
Citation: Lü Wei, Sun Yuhan, Zhang Huaxin, Yang Qingshan, Li Juan, Chen Shouyi, Zhang Wanke, Li Yun. Pollen vigor and detection of exogenous genes of litter for transgenic triploid Populus tomentosa[J]. Journal of Beijing Forestry University, 2019, 41(7): 91-100. doi: 10.13332/j.1000-1522.20190105

转基因三倍体毛白杨花粉活力及枯落物外源基因检测

doi: 10.13332/j.1000-1522.20190105
基金项目: 国家林业局科技发展中心项目(JC-2017-02),北京林业大学重大科研成果培育项目(2017CGP007),北京林业大学重点研发计划培育项目(2016BLPX13),北京林业大学青年教师科学研究中长期项目(2015ZCQ-SW-03)
详细信息
    作者简介:

    吕威。主要研究方向:杨树转基因育种,转基因安全性评价研究。Email:18037935780@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:

    李云,教授,博士生导师。主要研究方向:毛白杨、刺槐、银杏、杉木遗传育种研究。Email:yunli63@163.com 地址:同上

  • 中图分类号: S722.3

Pollen vigor and detection of exogenous genes of litter for transgenic triploid Populus tomentosa

  • 摘要: 目的长期持续的开展转基因林木安全性评价研究,对林木遗传改良育种工作的开展和转基因林木的生物安全性评价具有重要意义。方法对田间种植11年和13年的转AhDREB1基因三倍体毛白杨[(Populus tomentosa × P.bolleana)× P.tomentosa]花粉活力和杂交结实率进行检测。此外,对自然条件下转基因三倍体毛白杨枯落物中外源基因进行检测,并从转基因三倍体毛白杨林下根际土壤中筛选出44株卡那霉素抗性细菌,对其基因组DNA进行PCR检测。结果MTT和TTC染色检测结果表明转基因三倍体毛白杨花粉没有活力,杂交试验结果再次表明转基因三倍体毛白杨花粉不具备可授性和育性,其杂交结实率为0。转基因三倍体毛白杨的枯落物(枝、茎和叶)无论是埋在土里,飘落在地表,飘落在草坪上,其外源基因在3个月后均未能检测到。种植13年的转基因三倍体毛白杨枯落物中的外源基因暂未水平转移到根际土壤可培养细菌基因组中。结论未发现大田种植13年的转基因三倍体毛白杨对周围生态环境造成显著影响。

     

  • 图  1  转基因杨树枯落物外源基因检测试验

    Figure  1.  Detection experiment of exogenous gene in transgenic poplar litter

    图  2  2018年TTC法检测杨树花粉活力

    Figure  2.  Poplar pollen viability tested by TTC staining in 2018

    图  3  2018年MTT法检测杨树花粉活力

    Figure  3.  Poplar pollen viability tested by MTT staining in 2018

    图  4  毛白杨飞絮的果实、败育的种子和银腺杨的种子

    Figure  4.  Fruit of P. tomentosa, abortive seeds and the seeds of P. alba × P. glandulosa

    图  5  转基因三倍体毛白杨枯落物中外源基因的检测

    1 ~ 3:埋在土里的转基因三倍体毛白杨枝、茎和叶;4 ~ 6:飘落在地表的转基因三倍体毛白杨枝、茎和叶;7 ~ 9:飘落在杂草上的转基因三倍体毛白杨枝、茎和叶;CK:阴性对照,CK+:阳性对照,M: 2 000 bp DNA标记。1−3:transgenic hybrid Populus tomentosa branches, stems and leaves buried in the earth;4−6:transgenic hybrid Populus tomentosa branches, stems and leaves falt on the earth’s surface;7−9 transgenic hybrid Populus tomentosa branches, stems and leaves falt on the lawn. CK:negative control,CK+:positive control, M:2 000 bp DNA marker.

    Figure  5.  Detection of exogenous genes in transgenic triploid Populus tomentosa litter

    图  6  转基因杨树林下根际土壤中卡那霉素抗性细菌外源基因和选择标记基因的PCR检测

    1 ~ 44:转基因杨树林下根际土壤中卡那霉素抗性细菌,CK:阴性对照,CK+:阳性对照,M:2 000 bp DNA 标记。1−44:Kanamycin resistant bacteria in the rhizosphere soil of transgenic poplar forest, CK:negative control, CK+:positive control, M: 2 000 bp DNA marker.

    Figure  6.  PCR detection of exogenous and selectable marker genes in kanamycin resistant bacteria in rhizospheric soils of transgenic poplar

    表  1  2016年花粉活力测试结果

    Table  1.   Pollen viability test results in 2016

    品种 VarietyTTC检测 TTC test花粉体外培养检测 Pollen culture in vitro
    转基因三倍体毛白杨 Transgenic triploid P. tomentosa 0 0
    非转基因三倍体毛白杨(受体) Non-transgenic triploid P. tomentosa (receptor) 0 0
    毛新杨 P. tomentosa × P. bolleana 56.7% ± 6.5% 67.4% ± 8.7%
    下载: 导出CSV

    表  2  2018年花粉活力测试结果

    Table  2.   Pollen viability test results in 2018

    品种 VarietyTTC检测 TTC testMTT 检测 MTT test
    转基因三倍体毛白杨 Transgenic triploid P. tomentosa 0 0
    非转基因三倍体毛白杨(受体) Non-transgenic triploid P. tomentosa (receptor) 0 0
    银腺杨 P. alba × P. glandulosa 48.7% ± 7.2% 86.4% ± 5.4%
    下载: 导出CSV

    表  3  2016年转基因三倍体毛白杨杂交试验结果

    Table  3.   Hybridization assay results of transgenic triploid P. tomentosa in 2016

    亲本 Parent授粉小花数量
    Number of flowers
    pollinated
    种子数量
    Number of seeds
    结籽率
    Seed rate/%
    母本 Female    父本 Male  
    毛新杨
    P. tomentosa × P. bolleana
    转基因三倍体毛白杨
    Transgenic triploid P. tomentosa
    1 585 0 0
    毛新杨
    P. tomentosa × P. bolleana
    非转基因三倍体毛白杨(受体)
    Non-transgenic triploid P. tomentosa (receptor)
    1 376 0 0
    毛新杨
    P. tomentosa × P. bolleana
    毛新杨
    P. tomentosa × P. bolleana
    1 104 36 3.26
    下载: 导出CSV

    表  4  2018年转基因三倍体毛白杨杂交试验结果

    Table  4.   Hybridization assay results of transgenic triploid P. tomentosa in 2018

    亲本Parent授粉小花数量
    Number of flowers
    pollinated
    种子数量
    Number of seeds
    结籽率
    Seed rate/%
    母本 Female    父本 Male  
    银腺杨
    P. alba × P. glandulosa
    转基因三倍体毛白杨
    Transgenic triploid P. tomentosa
    1 210 0 0
    银腺杨
    P. alba × P. glandulosa
    非转基因三倍体毛白杨(受体)
    Non-transgenic triploid P. tomentosa (receptor)
    1 085 0 0
    银腺杨
    P. alba × P. glandulosa
    银腺杨
    P. alba × P. glandulosa
    875 128 14.63
    下载: 导出CSV
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  • 收稿日期:  2019-02-28
  • 修回日期:  2019-04-16
  • 网络出版日期:  2019-07-03
  • 刊出日期:  2019-07-01

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