Construction and Application of Reference Plasmids for Detection of Transgenic Insect-resistant Crops

doi:10.19586/j.2095-2341.2022.0163

Current Biotechnology ›› 2023, Vol. 13 ›› Issue (1): 83-89.DOI: 10.19586/j.2095-2341.2022.0163

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Construction and Application of Reference Plasmids for Detection of Transgenic Insect-resistant Crops

Haoqian WANG¹(), Yan HUANG²^,³, Yuting SHI⁴, Pengyu ZHU²^,⁵, Yuzhou XIE², Chunmeng HUANG², Xiaoyu LU⁶, Wei FU²^,⁵()

^1.Development Center for Science and Technology，Ministry of Agriculture and Rural Affairs，Beijing 100176，China
^2.Chinese Academy of Inspection and Quarantine，Beijing 100176，China
^3.Beijing Health Center Acadamy，Beijing 101149，China
^4.China Association for Standardization，Beijing 100048，China
^5.Chinese Academy of Inspection and Quarantine Center for Biosafety，Hainan Sanya 572025，China
^6.Animal and Plant Inspection and Quarantine Technology Centre，Guangdong Shenzhen 518038，China

Received:2022-09-29 Accepted:2022-11-07 Online:2023-01-25 Published:2023-02-07
Contact: Wei FU

转基因抗虫作物鉴定质粒标准分子的构建与应用

王颢潜¹(), 黄炎²^,³, 石雨婷⁴, 朱鹏宇²^,⁵, 谢宇宙², 黄春蒙², 卢小雨⁶, 付伟²^,⁵()

^1.农业农村部科技发展中心，北京 100176
^2.中国检验检疫科学研究院，北京 100176
^3.北京卫生职业学院，北京 101149
^4.中国标准化协会，北京 100048
^5.三亚中国检科院生物安全中心，海南三亚 572025
^6.深圳海关动植物检验检疫技术中心，广东深圳 518038

通讯作者: 付伟
作者简介:王颢潜 E-mail: 648368941@qq.com；
基金资助:
海南省重大科技计划项目(ZDKJ202002);中国检科院基本科研业务费(2020JK016)

Abstract

Abstract:

The Bacillus thuringiensis gene is a universal foreign functional gene in transgenic insect-resistant crops. It exists in most insect-resistant transgenic crops. However， the lack of Bt gene detection standard materials limits the development of the detection on crops with transgenic insect-resistant Bt genes in China. In order to make up for the lack of traditional matrix standard materials， this experiment first cloned the three commonly used Bt foreign genes of Cry1Ab， Cry1Ac and Cry3A into the pUC57 plasmid， and carried out the sequence and amplification function of the plasmid by means of sequencing， enzyme digestion and qPCR. Validation， and then test the amplification efficiency and actual application conditions to evaluate the applicability of the genetically modified detection. The results showed that the sequencing results of the plasmid prepared in this study are in complete agreement with the target sequence， and the results of enzyme digestion， qPCR amplification results and amplification efficiency are all in line with expectations. The application in the specific detection of Cry1Ab， Cry1Ac and Cry3A gene events is in line with the positive control. The requirements indicated that the positive plasmid reference molecules prepared in this study can be used as positive reference materials for the specific detection of transgenic Cry1Ab， Cry1Ac and Cry3A gene qPCR events.

Key words: transgene, Bt insect resistance gene, reference plasmids, gene-specific detection, qPCR

摘要：

苏云金芽胞杆菌基因是转基因抗虫作物中通用的外源功能基因，在绝大多数抗虫转基因作物中均有存在，然而Bt基因检测标准样品的缺乏却限制了我国转Bt基因抗虫作物检测工作的发展。为了弥补传统基体标准样品的缺失，首先将Cry1Ab、Cry1Ac、Cry3A 3种常用Bt外源基因克隆到pUC57质粒上，通过测序、酶切和qPCR等技术对质粒的序列和扩增功能进行了验证，然后对扩增效率和实际应用情况加以测试，评价其转基因检测的适用性，构建了质粒标准分子。结果显示，制备的质粒标准分子测序结果与靶标序列完全符合，酶切结果、qPCR扩增结果和扩增效率等均符合预期，在Cry1Ab、Cry1Ac、Cry3A基因特异性检测中的应用符合阳性对照要求，表明制备的阳性质粒标准分子能够作为转Cry1Ab、Cry1Ac、Cry3A基因qPCR基因特异性检测的阳性标准样品。

关键词: 转基因, Bt抗虫基因, 质粒标准分子, 基因特异性检测, qPCR

CLC Number:

S476.2

Haoqian WANG, Yan HUANG, Yuting SHI, Pengyu ZHU, Yuzhou XIE, Chunmeng HUANG, Xiaoyu LU, Wei FU. Construction and Application of Reference Plasmids for Detection of Transgenic Insect-resistant Crops[J]. Current Biotechnology, 2023, 13(1): 83-89.

王颢潜, 黄炎, 石雨婷, 朱鹏宇, 谢宇宙, 黄春蒙, 卢小雨, 付伟. 转基因抗虫作物鉴定质粒标准分子的构建与应用[J]. 生物技术进展, 2023, 13(1): 83-89.

Figures/Tables 6

References 20

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试剂	体积	终浓度
2×TaqMan Universal Master Mix	10 μL	1×
上游引物（10 μmol·L^-1）	1 μL	0.5 μmol·L^-1
下游引物（10 μmol·L^-1）	1 μL	0.5 μmol·L^-1
探针（10 μmol·L^-1）	0.5 μL	0.25 μmol·L^-1
质粒DNA模板	2 μL	50~250 ng
去离子水	补足至20 μL

试剂	体积	终浓度
2×TaqMan Universal Master Mix	10 μL	1×
上游引物（10 μmol·L^-1）	1 μL	0.5 μmol·L^-1
下游引物（10 μmol·L^-1）	1 μL	0.5 μmol·L^-1
探针（10 μmol·L^-1）	0.5 μL	0.25 μmol·L^-1
质粒DNA模板	2 μL	50~250 ng
去离子水	补足至20 μL

基因	标准	引物/探针	序列（5'→3'）	产物大小/bp
Cry1Ab	SN/T 1204—2016	正向引物	5'-CGCGACTGGATCAGGTACA-3'	75
		反向引物	5'-TGGGGAACAGGCTCACGA-3'
		探针	FAM-CCGCCGCGAGCTGACCCTGACCGTG-BHQ
Cry3A	SN/T 1204—2016	正向引物	5'-TCCGGTTACGAGGTTCTT-3'	86
		反向引物	5'-CCATAGATTTGAGCGTCCTT-3'
		探针	FAM-ACCTATGCTCAAGCTGCCAACACCC-BHQ
Cry1Ac	SN/T 1204—2016	正向引物	5'-CGGAAATGCGTATTCAATTCAA-3'	71
		反向引物	5'-TTCTGGACTGCGAACAATGG-3'
		探针	FAM-ACATGAACAGCGCCTTGACCACAGC-BHQ

基因	标准	引物/探针	序列（5'→3'）	产物大小/bp
Cry1Ab	SN/T 1204—2016	正向引物	5'-CGCGACTGGATCAGGTACA-3'	75
		反向引物	5'-TGGGGAACAGGCTCACGA-3'
		探针	FAM-CCGCCGCGAGCTGACCCTGACCGTG-BHQ
Cry3A	SN/T 1204—2016	正向引物	5'-TCCGGTTACGAGGTTCTT-3'	86
		反向引物	5'-CCATAGATTTGAGCGTCCTT-3'
		探针	FAM-ACCTATGCTCAAGCTGCCAACACCC-BHQ
Cry1Ac	SN/T 1204—2016	正向引物	5'-CGGAAATGCGTATTCAATTCAA-3'	71
		反向引物	5'-TTCTGGACTGCGAACAATGG-3'
		探针	FAM-ACATGAACAGCGCCTTGACCACAGC-BHQ

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Construction and Application of Reference Plasmids for Detection of Transgenic Insect-resistant Crops

转基因抗虫作物鉴定质粒标准分子的构建与应用

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