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林业科学 ›› 2020, Vol. 56 ›› Issue (10): 53-62.doi: 10.11707/j.1001-7488.20201006

• 论文与研究报告 • 上一篇    下一篇

3类转基因南林895杨田间试验的安全性评估

孙伟博,魏朝琼,马晓星,魏辉,诸葛强*   

  1. 南京林业大学生物与环境学院 南京 210037
  • 收稿日期:2019-11-14 出版日期:2020-10-25 发布日期:2020-11-26
  • 通讯作者: 诸葛强
  • 基金资助:
    江苏省自然科学基金项目(BK20170929);国家林业局林业科技发展项目(KJZXSA2018004);转基因生物新品种培育重大专项(2018ZX08020002)

Safety Assessment of a Field Trial of Three Types of Transgenic Poplar Nanlin895

Weibo Sun,Zhaoqiong Wei,Xiaoxing Ma,Hui Wei,Qiang Zhuge*   

  1. College of Biology and the Environment, Nanjing Forestry University Nanjing 210037
  • Received:2019-11-14 Online:2020-10-25 Published:2020-11-26
  • Contact: Qiang Zhuge

摘要:

目的: 转基因植物大田种植可能存在风险,本研究从不同外源基因在转基因杨树中的遗传稳定性及向土壤微生物转移的风险、转基因杨树对土壤微生物群落影响以及对于野生植物的化感作用4个方面进行综合分析,通过评估3类转基因南林895杨田间释放的安全性,为转基因林木的安全评估及田间释放提供参考。方法: 将3种编码不同抗性蛋白的外源基因(PeTLP、Cry1Ac、AtGols)分别构建到植物表达载体pGWB9中,采用根癌农杆菌LBA4404侵染南林895杨叶盘进行转化,筛选阳性植株,获得3类转基因南林895杨(TLP抗病系列、Bt抗虫系列、Gols抗逆系列)共13个株系,于温室培养1年,剪取茎段在试验田中进行扦插种植。田间种植采用随机区和裂区设计2种方式,在20个月的试验周期内,记录试验地环境条件及转基因杨树生长状况;定期采集各转基因系列及对照组南林895杨叶片及周围土壤样品,通过PCR技术检测3种外源基因在转基因杨树中的遗传稳定性,并提取土壤微生物总DNA进行3种外源基因的PCR检测,对外源基因向土壤微生物转移的风险进行评估;采用平板计数方法分析土壤微生物群落组成及变化情况,评估转基因杨树对土壤微生物群落的影响;利用"三明治"方法和莴苣种子模拟转基因杨树对于野生植物的化感作用,记录并分析莴苣种子胚根和胚轴的发育水平,评估转基因杨树对于生态多样性的影响。结果: 试验地环境条件相对稳定,未出现极端天气影响杨树生长,转基因南林895杨的生长趋势与对照组(南林895杨)的生长趋势基本一致,3类转基因杨树的高与各自对照组之间的差异未达到显著水平;3种外源基因均稳定存在于转基因南林895杨中,未发现向土壤微生物中转移的现象;土壤微生物数量受气温和降水量影响显著(P < 0.05),转基因杨树对土壤微生物的群落结构和生长未产生显著影响;3种外源基因转化的南林895杨叶片对于莴苣种子胚根和下胚轴的生长并无显著影响。结论: 本研究中Bt系列、TLP系列和Gols系列3类转基因南林895杨在田间试验中无明显的环境危害。

关键词: 转基因杨树, 田间试验, 安全性

Abstract:

Objective: Potential risks may exist in field plantings of the transgenic trees, four aspects were comprehensively investigated, including the genetic stability of different exogenous genes in the transgenic poplars and the risk of transfer of the exogenous genes to soil microorganisms, the impact of transgenic poplar on soil microbial community and allelopathy to wild plants, the safety of the three types of transgenic poplars for field release were evaluated as a case study for the safety assessment and field release of transgenic trees. Method: The three exogenous genes encoding different resistance proteins (PeTLP, Cry1Ac, ATGols) were constructed into plant expression vector pGWB9. Agrobacterium tumefaciens LBA4404 was used to infect the leaf discs of poplar trees of Nanlin895 (Populus deltoides×P. euramericana 'Nanlin895'), and the positive plants were screened. A total of 13 lines of three types of transgenic poplar Nanlin895 (TLP, Bt, Gols) were obtained and cultured in greenhouse for 1 year. The stem segments were cut and planted in the experimental field. Randomized block design and split-plot design were used in the field planting. The environmental conditions and growth status of the transgenic trees were recorded during the 20 month test period. Leaf samples and soil samples surrounding the sample trees of each transgenic lines and control trees were collected regularly. The genetic stability of three exogenous genes in transgenic poplar was detected by PCR technology, and the total DNA of soil microorganism was extracted for PCR detection of the three exogenous genes, to evaluate the risk of exogenous genes transfer to soil microorganisms. Plate counting method was used to analyze the composition and change of soil microbial community, and the influence of transgenic poplars on soil microbial community was evaluated; the allelopathy of transgenic poplars on wild plants was simulated by 'sandwich' method with lettuce seeds, and the development data of radicle and hypocotyl of lettuce seeds were recorded and analyzed in order to evaluate the impact of transgenic poplars on ecological diversity. Result: The environment conditions of the test field were relatively stable, and there was no extreme weather affecting poplar growth. The growth trend of transgenic poplars was basically consistent with that of the control (poplar Nanlin895), and no significant difference of tree height was found between the transgenic trees and their respective control. The exogenous genes in the transgenic trees were stable, and gene transfer from transgenic trees to soil microorganisms was not found. The quantity of soil microorganisms was significantly affected by temperature and precipitation (P < 0.05), and the community structure and growth of soil microorganisms were not significantly affected by the transgenic trees. The leaves of the transgenic trees with three different exogenous genes had no significant effect on root and hypocotyl growth of the lettuce seeds. Conclusion: Bt, TLP and Gols transgenic trees of poplar Nanlin895 had no obvious environmental hazards in the field testing.

Key words: transgenic poplar, field trial, safety

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