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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）  2019, Vol. 45 Issue (6): 675-684    DOI: 10.3785/j.issn.1008-9209.2019.04.081
园艺学     
转AM79-EPSPS基因耐草甘膦大豆的获得及功能验证
翁嘉慧1,2(),楼亿圆1,徐京1,何军光1(),张晓丽1,刘永立2()
1.浙江新安化工集团股份有限公司，杭州 311600
2.浙江大学农业与生物技术学院，杭州 310058
Acquisition and functional validation of transgenic AM79-EPSPS glyphosate-resistant soybean (Glycine max L.)
Jiahui WENG1,2(),Yiyuan LOU1,Jing XU1,Junguang HE1(),Xiaoli ZHANG1,Yongli LIU2()
1.Zhejiang Xin’an Chemical Industry Group Co. , Ltd. , Hangzhou 311600, China
2.College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China
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摘要：

以天隆一号大豆为受体材料，通过农杆菌介导的遗传转化方法，从消毒时间、乙酰丁香酮（acetosyringone, AS）添加浓度、草甘膦添加浓度和生根阶段吲哚丁酸（indole-3-butyric acid, IBA）浸没时间等因素出发，探究并建立高效的转AM79-EPSPS基因大豆转化体系，将具有我国自主知识产权的新基因AM79-EPSPS导入大豆，并验证它在转基因大豆中的功能。结果表明：当种子消毒时间为6～8 h，AS添加浓度为200 μmol/L，草甘膦添加浓度为100 μmol/L，IBA浸没时间为30 s时，转化效果最好。通过该转化体系获得了6株转AM79-EPSPS基因耐草甘膦大豆苗，并对其T0、T1、T2代植株进行了蛋白检测及草甘膦喷施实验。结果表明：在各世代转基因植株中均能检测出AM79-EPSPS蛋白，说明AM79-EPSPS基因能够在大豆中正常表达；喷施草甘膦后，非转基因大豆植株全部枯萎，而转AM79-EPSPS基因大豆植株均能正常生长，表现出明显的草甘膦耐受性。
关键词： 转基因大豆;  AM79-EPSPS基因;  草甘膦;  转化体系    
Abstract:

In order to introduce a new gene AM79-EPSPS with independent intellectual property rights into soybean and verify its function in transgenic soybean, an efficient transformation system of AM79-EPSPS gene was established through Agrobacterium-mediated genetic transformation from disinfection time, acetosyringone (AS) concentration, glyphosate concentration and indole-3-butyric acid (IBA) immersion time at the rooting stage with the receptor material of Tianlong No. 1 soybean. It was clear that the transformation effect was the best under the following optimal condition: the disinfection time of 6-8 h, the AS concentration of 200 μmol/L, the glyphosate concentration of 100 μmol/L, and the IBA immersion time of 30 s. Six transgenic AM79-EPSPS glyphosate-resistant soybeans were obtained, and the protein detection and glyphosate spraying experiments were carried out on the T0, T1 and T2 generation plants. The results showed that AM79-EPSPS protein could be detected in all generation plants containing AM79-EPSPS gene, which indicated that AM79-EPSPS gene could be expressed normally in the soybean. After spraying glyphosate, all non-transgenic soybean plants withered, while the transgenic AM79-EPSPS gene soybean plants could normally grow and showed obvious glyphosate tolerance.
Key words: transgenic soybean    AM79-EPSPS gene    glyphosate    conversion system
收稿日期: 2019-04-08 出版日期: 2020-01-20
CLC:  S 565.1  
基金资助: 国家转基因生物新品种培育科技重大专项(2016ZX08003-001)
通讯作者: 何军光,刘永立     E-mail: wengjh5132@126.com;hejunguang121@126.com;liuyongli@zju.edu.cn;hejunguang121@126.com
作者简介: 翁嘉慧（https://orcid.org/0000-0001-6292-8427），E-mail：wengjh5132@126.com
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引用本文:

翁嘉慧,楼亿圆,徐京,何军光,张晓丽,刘永立. 转AM79-EPSPS基因耐草甘膦大豆的获得及功能验证[J]. 浙江大学学报（农业与生命科学版）, 2019, 45(6): 675-684.

Jiahui WENG,Yiyuan LOU,Jing XU,Junguang HE,Xiaoli ZHANG,Yongli LIU. Acquisition and functional validation of transgenic AM79-EPSPS glyphosate-resistant soybean (Glycine max L.). Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(6): 675-684.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2019.04.081    或    http://www.zjujournals.com/agr/CN/Y2019/V45/I6/675

图1  AM79-pC3301载体图谱CaMV 35S promoter：花椰菜花叶病毒35S启动子；SP：豌豆中rbcS叶绿体转运肽；nos terminator：胭脂碱合成酶基因终止子；T-border (right)：农杆菌介导的T-DNA右边界序列；pVS1 sta：pVS1质粒的稳定位点；pVS1 rep：pVS1质粒的复制起始位点；pBR322 bom：pBR322质粒的bom位点，迁移蛋白质mop的作用位点；pBR322 ori：pBR322质粒的复制起始位点；kanamycin (R)：编码AphA-3蛋白，赋予细菌卡那霉素耐受性；T-border (left)：农杆菌介导的T-DNA左边界序列。

指标

Index

消毒时间 Disinfection time/h
0468101224

污染率

Pollution rate/%

99.33±1.15a65.33±10.07b1.33±2.31c0.67±1.15c0.00±0.00c1.33±2.31c0.00±0.00c

萌发率

Germination rate/%

88.67±3.06bc92.67±3.07ab95.33±3.07a93.33±1.15ab82.67±4.16c68.00±7.21d18.00±6.00e
表1  不同消毒时间下大豆种子的污染率和萌发率
图2  不同草甘膦浓度对大豆外植体出芽率和芽伸长率的影响A～F.草甘膦添加浓度分别为0、60、80、100、200、500 μmol/L。

指标

Index

草甘膦浓度 Glyphosate concentration/(μmol/L)
0406080100200500

出芽率

Budding rate/%

95.23±3.15a74.83±4.26b60.53±7.74c54.43±6.21c38.77±7.32d9.50±3.11e3.40±1.21e

芽伸长率

Bud elongation rate/%

93.23±5.13a60.53±4.26b40.13±2.31c33.33±4.22c10.87±4.26d0.00±0.00d0.00±0.00d
表2  不同草甘膦浓度下大豆外植体出芽率和芽伸长率

指标

Index

AS浓度 AS concentration/(μmol/L)
050100150200250300

出芽率

Budding rate/%

30.63±5.40c33.33±3.10bc38.77±2.05ab41.50±3.11a40.10±3.11a29.27±3.15c18.40±4.05d

芽伸长率

Bud elongation rate/%

6.80±1.21cd6.80±3.11cd10.20±2.00abc12.23±4.05ab14.30±4.10a8.17±2.05bcd3.40±1.21d
表3  不同AS浓度下大豆外植体出芽率和芽伸长率

指标

Index

浸没时间 Immersion time/s
03060120180300600

生根率

Rooting rate/%

3.33±5.77d96.67±5.77a93.33±5.77ab86.67±11.55ab83.33±5.77b50.00±10.00c10.00±10.00d
表4  不同IBA浸没时间下再生苗的生根率
图3  转基因大豆再生苗基因组DNA的PCR检测结果M：DL2000 DNA分子质量标志物；A1～A9：转化所得大豆苗； N：阴性受体材料；P：质粒。
图4  T0代阳性植株AM79-EPSPS蛋白表达检测结果P：阳性对照（AM79-EPSPS蛋白标准品）；N：阴性对照（EB004缓冲液）；A2～A3、A6～A9：转基因大豆苗。C：试纸条显色线； T：AM79-EPSPS蛋白显色线。
图5  T0代转AM79-EPSPS基因大豆植株抗性测试结果A.转基因大豆苗；B.阴性对照。

世代

Generation

存活率 Survival rate/%

阴性对照

Negative control

转基因植株

Transgenic plants

T00100
T10100
T20100
表5  T0～T2代转基因大豆植株喷施草甘膦后存活率统计
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