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林业科学 ›› 2021, Vol. 57 ›› Issue (11): 85-93.doi: 10.11707/j.1001-7488.20211109

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转多基因107杨对目标害虫的抗性检测

韩昆瑾,郭娟娟,吕梓晴,李玉言,王世杰,杨敏生,王进茂*   

  1. 河北农业大学林学院 河北省林木种质资源与森林保护重点实验室 保定 071001
  • 收稿日期:2020-12-09 出版日期:2021-11-25 发布日期:2022-01-12
  • 通讯作者: 王进茂
  • 基金资助:
    国家林业和草原局科技发展中心项目(KJZXZH202001);转基因生物新品种培育重大专项(2018ZX08020002)

Detection of Resistance of Multi-Gene Transgenic Populus×euramericana 'Neva' to Target Pests

Kunjin Han,Juanjuan Guo,Ziqing Lü,Yuyan Li,Shijie Wang,Minsheng Yang,Jinmao Wang*   

  1. Key Laboratory of Germplasm Resources of Forest Tree and Forest Protection of Hebei College of Forestry, Hebei Agricultural University Baoding 071001
  • Received:2020-12-09 Online:2021-11-25 Published:2022-01-12
  • Contact: Jinmao Wang

摘要:

目的: 探索转多基因107杨外源基因是否稳定存在且表达,筛选对鳞翅目和鞘翅目害虫具双抗性的转基因株系,为培育转多基因抗虫杨树新品种提供有价值的参考。方法: 对107杨转Cry1Ac-Cry3A-BADH基因的3个株系(ECAA1-3)和转Cry1Ac-Cry3A-NTHK1基因的3个株系(ECAB1-3)的2年生田间苗进行PCR检测、毒蛋白含量检测和抗虫性检测。结果: PCR凝胶电泳检测证明目的基因仍在转多基因107杨的基因组中;转多基因107杨Bt毒蛋白检测证明,8月份各转基因株系均含有Cry1Ac和Cry3A毒蛋白,Cry1Ac毒蛋白含量为3.60~20.76 ng·g-1,Cry3A毒蛋白含量为3 479.66~7 230.29 ng·g-1。室内饲虫试验证明,转多基因107杨具有双抗性,对美国白蛾和柳蓝叶甲幼虫均表现出抗性,其中株系ECAB1对美国白蛾的致死率最高,为100%,株系ECAA1、ECAA2和ECAA3对美国白蛾1龄幼虫具有明显抑制其生长发育的作用;除转基因株系ECAA2外,其他株系均表现为对柳蓝叶甲高抗虫性,对1龄幼虫的致死率均达到100%,对1、2龄混龄幼虫的致死率均在85%~100%之间,对成虫致死率在66%以上。结论: 转多基因107杨各株系的外源基因仍存在且稳定表达,其抗虫性与非转基因对照相比均有显著提高,但不同转基因株系间差异显著,株系ECAA3和ECAB1对美国白蛾和柳蓝叶甲幼虫均具有较高抗虫性。

关键词: 107杨, Bt基因, 多基因转化, 基因表达, 抗虫性

Abstract:

Objective: The purpose of this study was to explore the stability and expression of exogenous gene in transgenic poplar 107(Populus×euramericana 'Neva') and to screen transgenic lines with double resistance to Lepidoptera and Coleoptera pests, providing valuable reference for breeding new varieties of transgenic poplar with multiple genes resistant to pests. Method: PCR, toxin protein content detection and insect resistance detection were performed in 2-year-old field seedlings of three lines (ECAA1-3) transformed with Cry1Ac-Cry3A-BADH genes and three lines (ECAB1-3) transformed with Cry1Ac-Cry3A-NTHK1 genes. Result: PCR gel electrophoresis test proved that the target gene was still in the genome of transgenic poplar 107. The detection of Bt toxin of transgenic poplar 107 showed that the 2-year-old transgenic lines in August all contained Cry1Ac and Cry3A toxin, but the content of Cry3A toxin was relatively high. The toxic protein content of Cry1Ac was 3.60-20.76 ng·g-1, and the toxic protein content of Cry3A was 3 479.66-7 230.29 ng·g-1. Indoor insect feeding test showed that the transgenic poplar 107 had resistance to both Hyphantria cunea and Plagiodera versicolora larvae, among which ECAB1 had the highest lethal rate to H. cunea (100%), and ECAA1, ECAA2 and ECAA3 significantly inhibited the growth and development of H. cunea larvae of the first instar. With the exception of transgenic line ECAA2, all the other lines showed high insect resistance to P. versicolora, with the lethal rate of 100% for the first instar larvae, 85%-100% for the first and second instar mixed larvae, and over 66% for adults. Conclusion: The exogenous genes of transgenic poplar 107 were still present and stably expressed, and their insect resistance was significantly improved compared with that of non-transgenic controls. However, there were significant differences among different transgenic lines, the strains ECAA3 and ECAB1 showed high insect resistance to the larvae of H. cunea and P. versicolora.

Key words: Populus×euramericana 'Neva', Bt gene, polygenic transformation, gene expression, insect resistance

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