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

Abstract

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

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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References 34

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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

Establishment and Application of Multiple PCR Detection System for Glyphosate-Tolerant Gene EPSPS/GAT in Soybean

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