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作物学报 ›› 2023, Vol. 49 ›› Issue (7): 1818-1828.doi: 10.3724/SP.J.1006.2023.23048

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

转基因玉米ND207转化事件特异性定性PCR检测方法及其标准化

常丽娟1(), 梁晋刚2,*(), 宋君1, 刘文娟1, 付成平3, 代晓航1, 王东1, 魏超1, 熊梅1   

  1. 1四川省农业科学院农业质量标准与检测技术研究所, 四川成都 610066
    2农业农村部科技发展中心, 北京 100176
    3四川省农业科学院遥感与数字农业研究所, 四川成都 610066
  • 收稿日期:2022-06-16 接受日期:2022-11-25 出版日期:2023-07-12 网络出版日期:2022-12-30
  • 通讯作者: *梁晋刚, E-mail: liangjingang@agri.gov.cn
  • 作者简介:E-mail: 277989595@126.com
  • 基金资助:
    本研究由2020年农业国家标准和行业标准制修订项目计划(农质标函(2020)128号);四川省自然科学基金(面上) 项目(2022NSFSC0140);四川省农业科学院“1+9”揭榜挂帅科技攻关项目——生物安全前沿技术资助(1+9KJGG006)

Event-specific PCR detection method of transgenic maize ND207 and its standardization

CHANG Li-Juan1(), LIANG Jing-Gang2,*(), SONG Jun1, LIU Wen-Juan1, FU Cheng-Ping3, DAI Xiao-Hang1, WANG Dong1, WEI Chao1, XIONG Mei1   

  1. 1Institute of Quality Standard and Testing Technology Research, Sichuan Academy of Agricultural Sciences, Chengdu 610066, Sichuan, China
    2Development Center of Science and Technology, MARA, Beijing 100176
    3Institute of Remote Sensing and Digital Agriculture, Sichuan Academy of Agricultural Sciences, Chengdu 610066, Sichuan, China
  • Received:2022-06-16 Accepted:2022-11-25 Published:2023-07-12 Published online:2022-12-30
  • Contact: *E-mail: liangjingang@agri.gov.cn
  • Supported by:
    The Project of Agricultural National and Industrial Standards in 2020(农质标函(2020)128号);The Natural Science Foundation of Sichuan Province(2022NSFSC0140);The Fund of “1+9” Science and Technology Project of Sichuan Academy of Agricultural Sciences——Advanced Technology for Biosafety(1+9KJGG006)

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摘要：

转基因玉米ND207是中国农业大学研发的转mCry1Ab和mCry2Ab基因的抗虫玉米, 本研究的目的是建立ND207转化事件特异性定性PCR检测方法。根据研发者提供的引物进行PCR扩增, PCR产物测序分析, 获得了5'端旁侧序列262 bp, 包括109 bp的载体序列和153 bp的玉米基因组序列, 3'端旁侧序列316 bp, 包括76 bp的载体序列和240 bp的玉米基因组序列。针对两端旁侧序列设计14条引物, 组成25对引物组合进行引物筛选, 选中3'端一对最佳引物优化PCR反应体系及反应条件, 建立ND207的转化事件特异性定性PCR检测方法, PCR产物片段大小为166 bp。经过测试, 该方法的检出限为0.1%, 相当于20个拷贝的ND207特异分子片段。国内8家转基因生物安全检测机构对本方法进行了特异性测试、检出限测试和再现性测试, 循环验证报告显示: 该方法符合国家标准方法的各项要求, 可在检测行业推广应用。ND207转化事件特异性定性PCR检测方法的建立, 为我国ND207及其衍生品系的安全监管提供技术支撑。

关键词: 转基因玉米, ND207, 旁侧序列, 转化事件特异性, 定性PCR

Abstract:

Transgenic maize ND207 is an insect-resistant maize with mCry1Ab and mCry2Ab genes developed by China Agricultural University. The objective of this study is to develop the event-specific PCR detection method for ND207. The PCR amplification was performed according to the primers provided by the ND207 developer. The sequence of insertion site of ND207 was sequenced and obtained 262 bp of the 5'-flanking sequence, including 109 bp vector sequence and 153 bp maize genome sequence, 316 bp of the 3'-flanking sequence, including 76 bp vector sequence and 240 bp maize genome sequence. Fourteen primers were designed at both ends to form 25 primer pairs. The best primer pair at the 3' end was selected to optimize the PCR reaction system and reaction condition. The event-specific qualitative PCR detection method of ND207 was established and the PCR product size was 166 bp. After testing, the results showed that the detection limit of this method was 0.1%, equivalent to 20 copies of ND207 specific molecule fragment. Eight GMO safety testing institutions in China tested specificity, detection limit, reproducibility of the method, and the circular verification report revealed that the method met the requirement of the national standard method, which could be promoted and applied in the testing industry. The established event-specific qualitative PCR detection method of ND207 provides the technical support for the safety supervision of ND207 and its derivatives in China.

Key words: transgenic maize, ND207, flanking sequence, event-specific, qualitative PCR

图1

转基因玉米ND207外源插入序列图谱"

表1

转基因玉米ND207 PCR扩增引物"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 片段大小
Amplicon size (bp)		 引物位置
Primer position
ND207 5F 5'-CGGTCGATGAACGTGAACAAG-3' 254 5'-端旁侧序列
5'-flanking sequence
ND207 5R 5'-CAGTACATTAAAAACGTCCGCAA-3'
ND207 3F 5'-GTTTTTATGATTAGAGTCCCGCAAT-3' 303 3'-端旁侧序列
3'-flanking sequence
ND207 3R 5'-CAGGATGGGCTTCATGTACTCC-3'

表2

PCR反应引物序列信息表"

名称
Primer name		 引物序列
Primer sequence (5'-3')		 引物位置
Primer position
ND207 5'-F1
ND207-5'-F2
ND207-5'-F3		 5'-ATGAACGTGAACAAGGTGAAGC-3'
5'-CGATGAACGTGAACAAGGTGAA-3'
5'-AGGTGAAGCTCTACGACGC-3'		 5'-端旁侧序列
5'-flanking sequence
ND207-5'-R1 5'-CCGCAATGTGTTATTAAGTTGTCTA-3'
ND207-5'-R2 5'-AACGTCCGCAATGTGTTATTAAGT-3'
ND207-5'-R3 5'-ATGTGTTATTAAGTTGTCTAAGCGT-3'
ND207-3'-F1 5'-TGATTAGAGTCCCGCAATTATACAT-3' 3'-端旁侧序列
3'-flanking sequence
ND207-3'-F2 5'-CGATAGAAAACAAAATATAGCGCG-3'
ND207-3'-F3 5'-AACAAAATATAGCGCGCAATC-3'
ND207-3'-F4 5'-TGATTAGAGTCCCGCAATTATAC-3'
ND207-3'-R1 5'-AGGTCCTCCCGGAACGC-3'
ND207-3'-R2 5'-GACGACGGCGGGAAGCT-3'
ND207-3'-R3 5'-GTGCGCCGACGAGACG-3'
ND207-3'-R4 5'-CGTGGACGGCCTTCATAG-3'

表3

PCR反应引物组合信息表"

编号
Number		 正向引物
Forward primer		 反向引物
Reverse primer		 片段大小
Amplication size (bp)		 引物位置
Position
5'-1 ND207-5'-F1 ND207-5'-R1 235 5'-端旁侧序列
5'-flanking sequence
5'-2 ND207-5'-F1 ND207-5'-R2 240
5'-3 ND207-5'-F1 ND207-5'-R3 230
5'-4 ND207-5'-F2 ND207-5'-R1 237
5'-5 ND207-5'-F2 ND207-5'-R2 242
5'-6 ND207-5'-F2 ND207-5'-R3 232
5'-7 ND207-5'-F3 ND207-5'-R1 222
5'-8 ND207-5'-F3 ND207-5'-R2 227
5'-9 ND207-5'-F3 ND207-5'-R3 217
3'-1 ND207-3'-F1 ND207-3'-R1 278 3'-端旁侧序列
3'-flanking sequence
3'-2 ND207-3'-F2 ND207-3'-R1 245
3'-3 ND207-3'-F3 ND207-3'-R1 237
3'-4 ND207-3'-F4 ND207-3'-R1 278
3'-5 ND207-3'-F1 ND207-3'-R2 257
3'-6 ND207-3'-F2 ND207-3'-R2 224
3'-7 ND207-3'-F3 ND207-3'-R2 216
3'-8 ND207-3'-F4 ND207-3'-R2 257
3'-9 ND207-3'-F1 ND207-3'-R3 219
3'-10 ND207-3'-F2 ND207-3'-R3 186
3'-11 ND207-3'-F3 ND207-3'-R3 178
3'-12 ND207-3'-F4 ND207-3'-R3 219
3'-13 ND207-3'-F1 ND207-3'-R4 166
3'-14 ND207-3'-F2 ND207-3'-R4 133
3'-15 ND207-3'-F3 ND207-3'-R4 125
3'-16 ND207-3'-F4 ND207-3'-R4 166

图2

转基因玉米ND207两端旁侧序列与玉米基因组序列比对图 图中黑色背景表示相比较序列的碱基相同, 蓝色背景表示相比较序列的碱基不同。"

图3

ND207引物筛选"

图4

引物特异性筛选 1: 阳性对照; 2: 阴性对照; 3: 空白对照; 4: 转基因玉米混样; 5: 转基因水稻混样; 6: 转基因油菜混样; 7: 转基因大豆混样; 8: 转基因棉花混样。"

图5

引物灵敏度筛选"

图6

PCR反应的退火温度和引物浓度测试"

表4

PCR反应体系"

试剂
Reagent		 终浓度
Final concentration		 体积
Volume
水 Water —
10×PCR缓冲液 10×PCR buffer solution 1× 2.5 µL
25 mmol L-1氯化镁溶液 25 mmol L-1 MgCl solution 1.5 mmol L-1 1.5 µL
dNTPs混合溶液(各2.5 mmol L-1) dNTPs mixture (each 2.5 mmol L-1) 0.2 mmol L-1 2.0 µL
正向引物10 µmol L-1 ND207-F Forward primer 10 µmol L-1 ND207-F 0.4 µmol L-1 1.0 µL
反向引物10 µmol L-1 ND207-R Reverse primer 10 µmol L-1 ND207-R 0.4 µmol L-1 1.0 µL
Taq DNA聚合酶 Taq DNA polyase 0.025 U µL-1 —
25 mg L-1 DNA模板 25 mg L-1 DNA templet 2.0 mg L-1 2.0 µL
总体积 Total volume 25.0 µL

图7

方法的灵敏度测试"

图8

方法的检出限测试"

图9

方法的再现性测试"

表5

ND207的转化事件特异性定性PCR检测方法循环验证结果"

样品名称
Sample name		 定性PCR检测结果
Detection results of qualitative PCR
阳性对照Positive control + + + + + +
阴性对照Negative control - - - - - -
1% ND207玉米 1% transgenic maize ND207 + + + + + +
其他转基因玉米混合样Transgenic corn mixture samples - - - - - -
转基因大豆混合样Transgenic soybean mixture samples - - - - - -
转基因水稻混合样Transgenic rice mixture samples - - - - - -
转基因棉花混合样Transgenic cotton mixture samples - - - - - -
转基因油菜混合样Transgenic rape mixture samples - - - - - -
非转基因玉米混合样Non-transgenic maize mixture samples - - - - - -
0.1% ND207玉米 0.1% transgenic maize ND207 + + + + + +
0.05% ND207玉米 0.05% transgenic maize ND207 - - - - - -
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