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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2012, Vol. 38 Issue (6): 647-654    DOI: 10.3785/j.issn.1008-9209.2011.10.241
生物科学与技术     
  抗除草剂转基因水稻和大豆快速准确检测技术研究
马林1， 周练1， 周正剑1， 唐桂香2， 沈志成2， 寿惠霞1
  （1.浙江大学 生命科学学院，浙江 杭州310058；2.浙江大学 农业与生物技术学院，浙江 杭州310058）
Establishment of methods for identifying rapidly and precisely transgenic rice and soybean containing herbicide-resistant gene bar and EPSPS
MA Lin1, ZHOU Lian1, ZHOU Zheng-jian1, TANG Gui-xiang2, SHEN Zhi-cheng2, SHOU Hui-xia1*
(1. College of Life Sciences, Zhejiang University, Hangzhou 310058, China; 2. College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China)
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摘要： 以实验室培育的转基因水稻和大豆为研究材料，对含草铵膦乙酰转移酶基因(phosphinothricin acetyl transferase gene, bar) 和不与草甘膦结合的突变型5-烯醇丙酮莽草酸-3-磷酸合酶编码基因 (5-enolpyruvylshi- kimate-3-phosate synthase gene, EPSPS) 2种不同除草剂筛选标记的转基因植株进行叶片喷涂、叶片离体平板培养、种子萌发试验，以建立快速、准确的转基因鉴定方法，并探索可有效区分转化和非转化植株鉴定用的筛选剂剂量. 结果表明：这3种方法都能快速、简便、准确地鉴定相应的转基因植株；水稻和大豆叶片对农药吸收具有一定的差异，用300或135 mg/L草铵膦可有效区分是否转入bar基因，用1 %或0.25 %农达喷施可区分是否转入EPSPS基因；用含有50 mg/L草甘膦的平板培养离体大豆叶片可以方便地区分出转基因株系；种子萌发对除草剂剂量十分敏感，用远低于10 mg/L的2种除草剂即可有效区分阳性与阴性种子. 本研究建立的快速鉴定转基因植株的方法在转基因研究材料的鉴定及转基因安全评估中具有重要意义.
Abstract:   Genetic engineering is an important tool in plant functional genomics research and crop improvement applications. During the production of transgenic plants, the regenerated plantlets (T0) through transformation process will often mix with false-positive plants. In addition, the propagation of T0 plants will generate a certain percentage of non-transgenic progenies through the median-segregation of the transgene. Rapid identification of the transformed transgenic plants from the non-transgenic plants is an essential step in the production of transgenic plants. The commonly used method for identifying the existence of transgene in plants is through the polymerase chain reaction (PCR), which uses a pair of the target gene-specific primers to amplify the target DNA fragment from the genomic DNA samples of the tested plants. The use of PCR methods in transgenic plant screening is prone to cross contamination, is time consuming, and is considerably expensive. Thus, the development of methods for identifying transgenic plants in a robust, precise manner is of great importance. In this study, we assessed different methods for identifying rapidly and precisely transgenic rice and soybean plants containing herbicide resistant genes, including phosphinothricin acetyl transferase gene (bar) and modified 5-enolpyruvylshikimate-3-phosphate synthase gene (EPSPS). The methods used for the research included leaf-painting of herbicides, leaf-incubation on the solid medium containing herbicides, and seed germination in presence of herbicides. For these experiments optimum concentrations of herbicides were determined. Results showed that all tested methods could be used for rapid screening of transgenic plants. Application of 300 mg/L Liberty® could easily distinguish the bar-containing rice plants from the non-transgenic ones (Fig. 1), while Liberty® at 135 mg/L was sufficient for the identification of transgenic soybean plants (Fig. 2). Similarly, EPSPS-containing rice and soybean plants were determined by leaf-painting of 1 % and 0.25 % roundup (active gradient of glyphosate was 41 %), respectively (Fig. 3, 4). Solid medium with 50 mg/L glyphosate was suitable to distinguish leaf samples of soybean plants containing EPSPS gene (Fig. 5). Seed germination was very sensitive to selective agents. The germination of the non-transgenic seeds was completely prohibited in water solution containing either of the herbicides at the concentration of 10 mg/L (Fig. 6-9). In conclusion, we establish three methods that can rapidly and precisely identify transgenic rice and soybean plants containing herbicide-resistant gene bar and EPSPS. Our results can be used in both transgenic research and safety assessment of transgenic crop production.
出版日期: 2012-11-20
基金资助:

国家转基因重大专项资助项目(2009ZX08010-013B)
通讯作者: 寿惠霞， Tel: +86-571-88206146；E-mail: huixia@zju.edu.cn   
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引用本文:

马林1， 周练1， 周正剑1， 唐桂香2， 沈志成2， 寿惠霞1.   抗除草剂转基因水稻和大豆快速准确检测技术研究[J]. 浙江大学学报(农业与生命科学版), 2012, 38(6): 647-654.

MA Lin1, ZHOU Lian1, ZHOU Zheng-jian1, TANG Gui-xiang2, SHEN Zhi-cheng2, SHOU Hui-xia1*. Establishment of methods for identifying rapidly and precisely transgenic rice and soybean containing herbicide-resistant gene bar and EPSPS. , 2012, 38(6): 647-654.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2011.10.241    或    http://www.zjujournals.com/agr/CN/Y2012/V38/I6/647

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