耐草甘膦转EPSPS/GAT大豆多重PCR检测体系的建立及应用

doi:10.3864/j.issn.0578-1752.2020.20.003

摘要/Abstract

摘要：

【目的】建立一种精准、高效的草甘膦抗性基因G2-EPSPS和GAT的检测方法,为转基因大豆新品系ZH10-6的广泛应用提供技术支持。【方法】根据抗草甘膦大豆ZH10-6和受体中黄10的分子特征,设计大豆内源参考基因（Actin）、外源基因（G2-EPSPS和GAT）以及侧翼序列（G2EPSPS-2/ZH10P2和ZH10P1/GAT-2）的特异性引物,通过PCR扩增测试引物的特异性和适用性。调整引物配比、DNA模板量、dNTP含量、退火温度和延伸温度等,筛选该多重PCR体系的最适扩增条件。将转基因大豆ZH10-6和受体中黄10的基因组DNA按质量比混合,制备成100%、50%、10%、5%、1%、0.5%、0.1%和0的DNA样品,进行灵敏度检测。运用建立的多重PCR体系检测转基因大豆ZH10-6不同地理来源的11份衍生品系,并根据鉴定结果对该体系的应用性进行评价。【结果】建立的多重PCR方法中引物GmActin11 F/R、G2-EPSPS F/R、GAT F/R、ZH10P1/GAT和G2/ZH10P2可分别扩增出转基因大豆ZH10-6大小为126、430、338、810和1 626 bp的特异性目标条带。用该方法扩增受体中黄10时,除了GmActin11 F/R可以扩增出126 bp目标条带,侧翼序列上游引物ZH10P1和下游引物ZH10P2也可以扩增出632 bp目标条带。多重PCR最适扩增体系为DNA模板量100 ng、5 U·μL^-1 Ex Taq 0.2 μL、10×ExTaq Buffer 2.5 μL、2.5 mmol·L^-1 dNTP 2 μL、10 μmol·L^-1引物（GmActin11 F/R 0.4 μL、G2-EPSPS F/R 0.6 μL、GAT F/R 0.4 μL、ZH10P1/GAT 0.6 μL和G2/ZH10P2 0.6 μL）,ddH₂O补足25 μL。多重PCR扩增最适程序为95℃ 5 min;95℃ 30 s,60℃ 30 s,68℃ 1 min 20 s,35个循环;72℃ 12 min。该多重PCR体系灵敏度为0.5%,符合欧盟有关转基因产品标识的要求。该多重PCR方法特异性很强,可以成功检测受体中黄10、转基因大豆ZH10-6及ZH10-6不同地理来源的11个衍生品系。【结论】建立的转EPSPS/GAT大豆多重PCR检测体系具有高通量、特异性强、操作简便和应用广泛的优点,并且能够快速、准确地检测转基因大豆ZH10-6及其衍生品系。

关键词: 转基因大豆, 抗除草剂, 外源基因, 侧翼序列, 多重PCR

Abstract:

【Objective】This study aims to establish an accurate and efficient high-throughput detection method for herbicide-resistant genes of G2-EPSPS and GAT, providing technical support for the application of transgenic soybean ZH10-6.【Method】Based on the molecular characteristics of glyphosate-resistant soybean ZH10-6 and ZH10, specific primers for endogenous reference genes (Actin), exogenous genes (G2-EPSPS and GAT) and flank sequences (G2EPSPS-2/ZH10P2 and ZH10P1/GAT-2) were designed for testing their specificity and applicability by PCR amplification. The optimal PCR amplified condition of the multiplex PCR system were screened by adjusting primer ratio, DNA template amount, dNTP content, annealing temperature and elongation temperature. The different mix of transgenic soybean ZH10-6 and ZH10 were prepared according to the mass ratio and formed DNA samples of 100%, 50%, 10%, 5%, 1%, 0.5%, 0.1% and 0 for sensitivity detection. Eleven derived lines of transgenic soybean ZH10-6 from different geographical sources were detected for evaluation of its application potential.【Result】Primers of GmActin11 F/R, G2-EPSPS F/R, GAT F/R, ZH10P1/GAT and G2/ZH10P2 were amplified the specific target fragments of 126, 430, 338, 810 and 1 626 bp in the multiple PCR method in transgenic soybean ZH10-6. In addition to the 126 bp target fragment amplified by ZH10 with GmActin11 F/R, the 632 bp target fragments were amplified by ZH10P1/ ZH10P2. The optimal system for multiple PCR amplification included 100 ng DNA template, 5 U·μL^-1 Ex Taq 0.2 μL, 10×ExTaq Buffer 2.5 μL, 2.5 mmol·L^-1 dNTP 2 μL, 10 μmol·L^-1 primers(GmActin11 F/R 0.4 μL, G2-EPSPS F/R 0.6 μL, GAT F/R 0.4 μL, ZH10P1/GAT 0.6 μL and G2/ZH10P2 0.6 μL), ddH₂O supplemented 25 μL. The optimal multiplex PCR amplification procedure: 95℃ for 5 min; 95℃ 30 s, 60℃ 30 s, 68℃ 1 min20 s, 35 cycles; 72℃ 12 min. The sensitivity of the multiplex PCR system is 0.5%, which meets the requirements of the European Union for the labeling of transgenic products. The multiplex PCR method is high specificity and detected ZH10, ZH10-6 and eleven ZH10-6 derived lines successfully. 【Conclusion】The multiple PCR system of EPSPS/GAT were established, which had advantage of high throughput, strong specificity, simple operation and wide application. It can detect transgenic soybean ZH10-6 and its derived lines quickly and accurately.

Key words: transgenic soybean, herbicide resistance, foreign genes, flanking sequence, multiplex PCR

文静,郭勇,邱丽娟. 耐草甘膦转EPSPS/GAT大豆多重PCR检测体系的建立及应用[J]. 中国农业科学, 2020, 53(20): 4127-4136.

WEN Jing,GUO Yong,QIU LiJuan. Establishment and Application of Multiple PCR Detection System for Glyphosate-Tolerant Gene EPSPS/GAT in Soybean[J]. Scientia Agricultura Sinica, 2020, 53(20): 4127-4136.

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基因/标记名称 Gene/Marker name	引物名称 Primer name	引物序列 Primer sequence (5′-3′)	产物大小 Fragment size (bp)
内源参考基因Actin Endogenous gene Actin	GmActin11F	ATCTTGACTGAGCGTGGTTATTCC	126
内源参考基因Actin Endogenous gene Actin	GmActin11R	GCTGGTCCTGGCTGTCTCC	126
侧翼序列ZH10P1/GAT-2 Flanking sequence ZH10P1/GAT-2	ZH10P1	TAATAGTAGAATGGGACTGGTGGAT	810
侧翼序列ZH10P1/GAT-2 Flanking sequence ZH10P1/GAT-2	GAT	GCGGACTTGCTTTGGTGTAAT	810
侧翼序列G2EPSPS-2/ZH10P2 Flanking sequence G2EPSPS-2/ZH10P2	G2	CCCGAATCATCAGGCAAACA	1626
侧翼序列G2EPSPS-2/ZH10P2 Flanking sequence G2EPSPS-2/ZH10P2	ZH10P2	AACACATCATAGTATTCTAAAACGCTT	1626
外源基因G2-EPSPS Exogenous gene G2-EPSPS	G2-EPSPS F	CAAATCCATTACCAACCGTGC	430
外源基因G2-EPSPS Exogenous gene G2-EPSPS	G2-EPSPS R	ACCACCATCAATCTCGAAACG	430
外源基因GAT Exogenous gene GAT	GAT F	CTCAGACCAAACCAGCCGATAG	338
外源基因GAT Exogenous gene GAT	GAT R	GTGTCGAATACCTCTCCCTGCTC	338

衍生品系类型 Derived strain type	引物名称 The name of the primers						衍生品系名称 The name of the derivative lines
衍生品系类型 Derived strain type	GmActin11	G2-EPSPS	GAT	ZH10P1/GAT	G2/ZH10P2	ZH10P1/ZH0P2	衍生品系名称 The name of the derivative lines
类型1 Type 1	√					√	NZG700、ZH10
类型2 Type 2	√	√	√	√	√		NZK100、NZK180、NZJ77、NZJ110、SUN100、ZH10-6
类型3 Type 3	√	√	√			√	NZC400
类型4 Type 4	√	√	√				NZK001
类型5 Type 5	√	√	√	√	√	√	NZC100、NZK150、NZM150