文静^,1,2, 郭勇², 邱丽娟^1,21东北农业大学农学院,哈尔滨150030
²中国农业科学院作物科学研究所/国家农作物基因资源与遗传改良重大科学工程/农业部作物种质资源 与生物技术重点开放实验室,北京100081

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

WEN Jing^,1,2, GUO Yong², QIU LiJuan^{1,2 1}College of Agriculture, Northeast Agricultural University, Harbin 150030
²Institute of Crop Sciences, Chinese Academy of Agricultural Sciences/The National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI)/Key Laboratory of Germplasm & Biotechnology (MOA), Beijing 100081

责任编辑: 李莉
收稿日期:2020-01-3接受日期:2020-03-7网络出版日期:2020-10-16

基金资助:

抗除草剂转基因大豆新品种培育.2016ZX08004001

Received:2020-01-3Accepted:2020-03-7Online:2020-10-16
作者简介 About authors
文静,E-mail: 496064503@qq.com。





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.
Keywords：transgenic soybean;herbicide resistance;foreign genes;flanking sequence;multiplex PCR


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本文引用格式
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 doi:10.3864/j.issn.0578-1752.2020.20.003
WEN Jing, GUO Yong, QIU LiJuan. Establishment and Application of Multiple PCR Detection System for Glyphosate-Tolerant Gene EPSPS/GAT in Soybean[J]. Scientia Acricultura Sinica, 2020, 53(20): 4127-4136 doi:10.3864/j.issn.0578-1752.2020.20.003

0 引言

【研究意义】自1994年第一个商业化的转基因植物（FlavrSavr tomato）被批准上市以来,植物基因工程技术在现代农业中得到了广泛的应用^[1]。目前已有24个国家/地区种植了转基因作物,42个国家/地区进口转基因作物用于粮食、饲料和加工。全球转基因作物的种植面积从1996年的170万hm²增加到2018年的1.917亿hm²,增长了113倍,而转基因大豆作为种植面积最大的转基因作物,种植面积达到全球转基因作物种植面积的50%^[2]。随着转基因作物产业化和商业化程度的不断扩大,全球对转基因食品安全、环境风险和伦理问题的争议加剧^[3],越来越多的国家和地区要求对转基因食品和成分进行标识^[4]。部分国家要求转基因生物（genetically modified organism,GMO）含量需要低于一定阈值水平的限制。例如,欧盟将非转基因背景下的转基因物质的阈值设定为0.9%^[5]。因此,标准化的转基因成分检测和鉴定方法是转基因发展和商业化的关键步骤,也在转基因生物的安全评估和风险管理中发挥了重要作用^[6]。现有的转基因检测方法大多基于聚合酶链反应（polymerase chain reaction,PCR）,并由于其具有通用性、敏感性、特异性和高通量的特点,可用于复杂转基因成分分析,包括筛查外源调控元件和目的基因,并对特定的转基因成分进行鉴定^[7]。多重PCR技术是在同一个PCR反应体系中加入多对特异性引物,实现对多个靶标的扩增,更能节省转基因生物检测中时间和成本^[8,9]。【前人研究进展】吕山花等^[10]应用PCR检测方法,以大豆凝集素（lectin）基因为内参,检测了4个抗草甘膦转基因大豆品种及草甘膦敏感大豆品种豫豆12的外源CaMV35S启动子、NOS终止子和CP4-EPSPS成分。目前,中国允许进口的转基因大豆有16种（具体包括A2704-12、CV127、MON87701、MON87769、GTS40-3-2、MON87708、305423、A5547-127、FG72、MON89788、SYHT0H2、MON87701×MON89788、DAS-44406-6、305423× GTS40-3-2、MON87705和356043,其中MON87701× MON89788和305423×GTS40-3-2为复合性状转基因大豆）,农业部针对这些转基因大豆品系发布的检测方法以核酸检测为主,但是其方法均为单重PCR检测。针对转基因大豆建立系统的、高通量的由“公共引物”介导的多重PCR转基因大豆检测体系,可大幅降低检测成本、提高检测效率,为转基因安全评价提供新的检测技术和方法^[11]。多重PCR检测体系并不是多个单重PCR体系检测体系的简单叠加,它需要对PCR体系和程序进行反复试验、优化和验证,保证多重PCR具有足够的特异性、灵敏性和适用性,最终实现多个目标片段的同时扩增,因此,与普通PCR相比,难度更大^[12]。近年来,国内外有诸多****对其进行研究,并建立了许多关于通用元件、基因特异性和转化事件特异性的多重PCR检测体系^[8,13-16]。尹全等^[17]以抗草甘膦大豆GTS40-3-2的通用元件作为检测参数,建立了大豆内源基因lectin、CaMV35S启动子、NOS终止子和CP4-EPSPS的四重PCR检测体系。AO等^[18]根据外源基因的特异性建立了适用于转基因大豆、玉米、水稻中CP4-EPSPS、Cry1A(b)、BAR、PAT检测的多重PCR方法。KIM等^[19]针对RRS、GTS40-3-2、A2704-12、DP356043-5、MON89788、A5547-127和DP305423-1 6个商业化转基因大豆特异性事件,建立了包含内参基因的七重PCR检测体系。付伟等^[20]以转基因大豆DAS44406-6品系为研究对象,利用Multiplex PCR技术与其他技术相结合,建立了多重实时荧光PCR检测体系,检测灵敏度为0.01%。【本研究切入点】目前,大多数转基因大豆的多重PCR技术都是针对通用元件和基因特异性进行检测,或者针对多个商业化特异性转化事件进行筛选,而单一转化事件特异性鉴定鲜见报道^[21]。在转基因生物检测中,转化事件特异性检测的目标片段是外源基因和宿主DNA之间的连接区域,故比单纯基因特异性检测和通用元件检测具有更高的特异性^[22,23]。【拟解决的关键问题】本研究基于转基因成分检测技术及要求,以转基因大豆ZH10-6为研究对象,针对内参基因、外源基因和侧翼序列设计5对特异性引物,构建了转EPSPS/GAT大豆ZH10-6多重PCR反应体系,并用本研究建立的多重PCR检测体系检测了ZH10-6的11个衍生品系,为中国抗草甘膦转基因大豆新品种目标基因鉴定提供信息和方法。

1 材料与方法

1.1 试验材料

转基因大豆ZH10-6、受体中黄10及11个ZH10-6衍生品系均为中国农业科学院作物科学研究所大豆基因资源挖掘与利用实验室保存样品。

1.2 样品基因组DNA的提取

参照植物基因组提取试剂盒说明书提取样品DNA,用紫外分光光度计测量DNA样品质量及浓度,并用1×TE缓冲液将DNA稀释至50 ng·μL^-1。

1.3 引物的设计及筛选

使用软件Primer Premier 5.0进行引物设计,设计时遵从多重PCR检测引物设计与筛选的原则,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（表1）。将各个检测基因的上、下游引物使用灭菌的超纯水稀释为10 μmol·L^-1。

Table 1
表1
表1多重PCR标记及其相关信息
Table 1The markers for multiplex PCR and their related information

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
	GmActin11R	GCTGGTCCTGGCTGTCTCC
侧翼序列ZH10P1/GAT-2 Flanking sequence ZH10P1/GAT-2	ZH10P1	TAATAGTAGAATGGGACTGGTGGAT	810
	GAT	GCGGACTTGCTTTGGTGTAAT
侧翼序列G2EPSPS-2/ZH10P2 Flanking sequence G2EPSPS-2/ZH10P2	G2	CCCGAATCATCAGGCAAACA	1626
	ZH10P2	AACACATCATAGTATTCTAAAACGCTT
外源基因G2-EPSPS Exogenous gene G2-EPSPS	G2-EPSPS F	CAAATCCATTACCAACCGTGC	430
	G2-EPSPS R	ACCACCATCAATCTCGAAACG
外源基因GAT Exogenous gene GAT	GAT F	CTCAGACCAAACCAGCCGATAG	338
	GAT R	GTGTCGAATACCTCTCCCTGCTC

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1.4 引物特异性和适用性检测

单重PCR反应体系为25 μL,包括DNA 2 μL（50 ng·μL^-1）、Ex Taq酶0.2 μL、10×ExTaq Buffer 2.5 μL、dNTP 2 μL、引物（10 μmol·L^-1）0.6 μL和ddH₂O 17.7 μL。

多重PCR反应体系为25 μL,包括DNA 2 μL（50 ng·μL^-1）、Ex Taq酶 0.2 μL、10×ExTaq Buffer 2.5 μL、dNTP 2 μL、引物（10 μmol·L^-1,GmActin11 F/R 0.6 μL、G2-EPSPS F/R 0.6 μL、GAT F/R 0.6 μL、ZH10P1/GAT 0.6 μL、G2/ZH10P2 0.6 μL）和ddH₂O 15.3 μL。

单重和多重PCR反应程序为95℃ 5 min;95℃ 30 s,58℃ 30 s,72℃ 1 min20 s 35个循环;72℃ 8 min;4℃保存。

用1%琼脂糖凝胶电泳检测多重PCR产物,并利用凝胶成像系统观察。

1.5 多重PCR反应体系和程序的优化

以转基因大豆ZH10-6和受体中黄10的DNA为模板,将5对引物的用量设置为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,DNA模板量设置为100、150、200和250 ng 4个梯度,退火温度设置为55℃、58℃和60℃,延伸温度设置为68℃和72℃ 2个梯度,dNTP含量为2、3、4和5 μL 4个梯度,分别进行多重PCR扩增,筛选出五重PCR体系的最优反应条件。

1.6 多重PCR灵敏度检测

为了验证多重PCR体系灵敏性,将相同浓度的转基因大豆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。使用1.5建立的多重PCR体系及程序进行多重PCR方法灵敏度检测。

1.7 多重PCR体系的应用

用建立的多重PCR体系对转基因大豆ZH10-6衍生品系进行检测,包括东北地区和黄淮地区材料,进一步验证多重PCR体系的实用性。

2 结果

2.1 样品DNA检测

使用NanoDrop 2000/2000c超微量分光光度计测定样品DNA浓度和质量,经测定,样品OD₂₆₀/OD₂₃₀比值均为2.0—2.2,OD₂₆₀/OD₂₈₀比值均为1.8—2.0,说明提取的样品DNA质量较好。用1%琼脂糖凝胶电泳对DNA样品进行检测,仅1号样品第三条带有降解,该样品不在后续试验中使用,其余样品几乎无降解,符合多重PCR试验要求（图1）。将样品DNA浓度稀释至50 ng·μL^-1,备用。

图1

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图11%琼脂糖凝胶电泳DNA检测结果

M:2K Plus II;1:中黄10;2:ZH10-6
Fig. 1DNA detection results of 1% agarose gel electrophoresis

M: 2K Plus II; 1: ZH10; 2: ZH10-6

2.2 引物特异性和适用性检测

以转基因大豆ZH10-6和受体中黄10的DNA为模板进行5对引物特异性和适用性检测。单引物扩增时,5对引物均可在目的位置处观察到特异性条带,未观察到明显的非特异性条带。5对引物同时扩增时,转基因大豆ZH10-6可以扩增出多条特异性条带,但个别条带相对微弱（图2）,表明这5对引物可以用于多重PCR区别ZH10-6和受体中黄10,但是需要进一步调整扩增条件。

图2

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图2单基因特异性检测结果

M:2K Plus II;1:中黄10;2:ZH10-6;GmActin11、G2-EPSPS、GAT、ZH10P1/GAT、G2/ZH10P2为PCR扩增引物,5 Pairs of Primers是GmActin11、G2-EPSPS、GAT、ZH10P1/GAT、G2/ZH10P2等量混合
Fig. 2Single gene specific test results

M: 2K Plus II; 1: ZH10; 2: ZH10-6; GmActin11, G2-EPSPSP, GAT, ZH10P1/GAT and G2/ZH10P2 were PCR amplification primers, and 5 Pairs of Primers were equal combinations of GmActin11, G2-EPSPSP, GAT, ZH10P1/GAT and G2/ZH10P2

2.3 多重PCR检测体系的建立

多重PCR体系中,将5对引物用量均设置为0.6 μL时,部分条带不能被扩增（图2）。可以看出在多重PCR中,引物存在强弱之分,需要减少相对较强引物GmActin11 F/R和GAT F/R的用量。以条带亮度作为参考,最终将5对引物的用量设置为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。将DNA模板量、退火温度、延伸温度和dNTP含量均设置为不同梯度,进行同时扩增（图3）,筛选多重PCR最适温度和体系。

图3

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图3梯度多重PCR检测结果

M:2K Plus II;1:中黄10;2:ZH10-6
Fig. 3Results of gradient multiple PCR

M: 2K Plus II; 1: ZH10; 2: ZH10-6


通过不同体系检测（图4）,多重PCR扩增最适体系为:DNA 2—4 μL、Ex Taq 0.2 μL、10×ExTaq Buffer 2.5 μL、dNTP 2 μL、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 min20 s,35个循环;72℃ 12 min;4℃保存。最终扩增产物长度为内参基因126 bp、外源基因G2-EPSPS 430 bp、外源基因GAT 338 bp、侧翼序列ZH10P1/GAT-2 810 bp、侧翼序列G2EPSPS-2/ZH10P2 1 626 bp。其中,受体中黄10除了可以扩增出Actin内参序列以外,还可以扩增出ZH10-6 2对侧翼序列的上游引物ZH10P1和下游引物ZH10P2的632 bp片段,而在ZH10-6中,由于插入外源基因过大,此段序列无法被扩增出。因此,受体中黄10可以被扩增出2条带,而转基因材料ZH10-6可以被扩增出5条带,此体系可以用于鉴定ZH10-6和受体中黄10。

图4

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图4单重及多重PCR检测ZH10-6及中黄10

M:2K Plus II;1:中黄10;2:ZH10-6;GmActin11、G2-EPSPS、GAT、ZH10P1/GAT、G2/ZH10P2为PCR扩增引物,5 Pairs of Primers是GmActin11、G2-EPSPS、GAT、ZH10P1/GAT、G2/ZH10P2等量混合
Fig. 4Single and multiple PCR detection of ZH10-6 and ZH10

M: 2K Plus II; 1: ZH10; 2: ZH10-6; GmActin11, G2-EPSPSP, GAT, ZH10P1/GAT and G2/ZH10P2 were PCR amplification primers, and 5 Pairs of Primers were equal combinations of GmActin11, G2-EPSPSP, GAT, ZH10P1/GAT and G2/ZH10P2

2.4 多重PCR体系灵敏度检测

将模板DNA稀释至设定浓度,通过多重PCR灵敏度试验（图5）,随着受体中黄10质量分数的上升,特异性片段632 bp的条带逐渐变亮,而ZH10-6的5条带则出现不同程度的减弱,特别是外源基因G2-EPSPS的430 bp条带和侧翼序列G2EPSPS-2/ ZH10P2为1 626 bp条带。因为在转基因大豆ZH10-6和非转基因大豆中黄10混合后,相对于原有的多重PCR体系中增添了一重PCR反应。灵敏度是非转基因样品中转基因成分的最低检测限,虽然图5转外源基因G2-EPSPS的430 bp条带和侧翼序列G2EPSPS-2/ ZH10P2为1 626 bp条带相比于其他条带较弱,但是仍可以看出条带所在位置。因此,只要样品中含有0.5%以上的ZH10-6转基因成分,利用此体系都能检测出目标条带。说明建立的多重PCR体系灵敏度可达0.5%,符合欧盟有关转基因产品标识的要求。

图5

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图5多重PCR灵敏度检测

M:2K Plus II;1—7:ZH10-6的质量分数分别为100%、50%、10%、5%、1%、0.5%和0.1%;8:中黄10
Fig. 5Results of multiple PCR sensitivity detection

M: 2K Plus II; 1-7: The mass fraction of ZH10-6: 100%, 50%, 10%, 5%, 1%, 0.5%, 0.1%; 8: ZH10

2.5 多重PCR体系的应用

以转基因大豆ZH10-6衍生品系的DNA为模板,将DNA浓度稀释成50 ng·μL^-1。用建立的多重PCR体系待测大豆进行多重扩增,根据不同的条带类型,判断ZH10-6衍生品系中外源基因和侧翼序列情况（图6）。主要检测到5种带型（表2）,类型1出现与受体中黄10相同带型,认为是未发生杂交的野生型;类型2出现与ZH10-6相同带型;类型3和类型4分别以NZC400和NZK001为例,认为是其他转化事件;第5类带型包含ZH10-6和受体ZH10的全部6条带型,认为是转基因杂合材料。在11份ZH10-6衍生品系中,NZC100和NZC400为黄淮材料,剩余为东北材料。用单重PCR对该扩增结果进行验证,与多重PCR鉴定结果完全一致,证明建立的多重PCR体系可以鉴定转基因大豆ZH10-6和受体中黄10,也可以鉴定不同地区的ZH10-6衍生品系,应用性较为广泛。

图6

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图6ZH10-6衍生品系检测

M:2K Plus II;1:NZK001;2:NZK100;3:NZK150;4: NZK180;5:NZC100;6:NZC400;7:NZJ77;8:NZJ110;9:SUN100;10:NZM150;11:NZG700;12:ZH10;13:ZH10-6
Fig. 6Test results of ZH10-6 derivative lines



Table 2
表2
表2ZH10-6衍生品系类型
Table 2The derived lines of ZH10-6

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
	GmActin11	G2-EPSPS	GAT	ZH10P1/GAT	G2/ZH10P2	ZH10P1/ZH0P2
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

√:多重PCR方法成功扩增出ZH10-6衍生品系的引物类型
√: The primers of the ZH10-6 derived lines were successfully amplified by multiple PCR

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3 讨论

PCR检测技术是转基因作物及产品中外源基因检测的重要手段^[24,25],相比于普通PCR,多重PCR只需一个反应和一次电泳就可同时检出多个目标片段,可极大地节约试剂和时间^[26,27],目前,已被广泛应用到医学及生物等领域^[28]。在研究过程中发现,不同引物序列的多重PCR在实际应用中会受各项参数直接影响。

3.1 多重PCR体系中引物选择的直接依据

本研究中选择特异性强、引物二聚体少的5对引物进行扩增,其中包括起质量控制作用的内源基因,以避免转基因检测的假阴性结果出现。多重PCR扩增时,各个引物的扩增并非均一的,本研究通过5对引物等量加入时的条带亮度对引物进行强弱判断,选择各基因扩增效率相对一致的引物浓度配比条件建立了多重PCR体系。引物设计是多重PCR反应成败的关键,需要遵循PCR引物设计通用准则,即单条引物长度一般为18—25个碱基,且4种碱基的比例要适当,G+C含量一般为40%—60%。同时,选择的引物必须高度特异,彼此之间无同源性,且连续互补的碱基不超过4个,避免出现交叉错配等现象^[29]。引物之间TM值尽可能相差较小,可以实现在同一退火温度下的扩增。

3.2 不同反应体系和条件对多重PCR体系建立的影响

在建立多重PCR反应体系时,反应体系和条件是关键因素^[30,31]。反应体系主要包括DNA模板量、dNTP含量等。本研究将DNA模板量从100 ng增加至250 ng时,扩增效果没有明显变化,说明DNA模板量对多重PCR体系扩增影响较小。同时发现在多重PCR扩增中,dNTP含量可以同单重PCR扩增设置一致,dNTP含量过高反而会抑制反应。

反应条件是另一个关键因素,主要包括退火温度和延伸温度。一般情况下,可以依据引物的TM值减少5℃直接选择退火温度,但有时结果与预期并不一致。较为简单的方法就是设置54℃为初始退火温度,如果多重PCR扩增产生非特异性条带,需适当提高引物退火温度,反之则降低引物退火温度^[32]。退火温度应在Tm值允许范围内选择较高的温度,以确保PCR反应的特异性。但在这个过程中会出现目标片段扩增变弱的现象,可以通过延长延伸时间来消除。同时适当降低延伸温度有利于目标条带的产生。本研究中设置了55℃、58℃和60℃ 3个退火温度,68℃和72℃ 2个延伸温度,最终在退火温度为60℃,延伸温度为68℃时,多重PCR体系扩增效果最好。

3.3 目标片段的选择对多重PCR体系灵敏度的影响

在多重PCR目标片段的选择时,目标片段必须具有高度特异性,避免目标片段之间的竞争性扩增,才能实现高效灵敏的扩增反应,建议PCR产物片段大小在200 bp以内时,片段间隔大于30 bp;产物在500 bp以上时,片段间隔要大于70 bp^[33]。此外,各个目的片段长度应差异明显,以便于鉴别,但最大和最小片段最好相差不超过400 bp,否则会导致多重PCR体系灵敏度降低,且体系建立的难度较大。目前转基因大豆多重PCR技术中,董立明等^[34]选择商品化早、种植面积大的5种转基因大豆品系305423、MON89788、CV127、GTS40-3-2、356043及大豆内源基因Lectin为研究对象,选用相应的国家标准和文献中的引物,建立了能同时检测5种转基因大豆品系和大豆内源基因的六重PCR检测体系,检测最大片段和最小片段相差400 bp,该体系能够从0.1%及以上含量的阳性对照样品中检测出全部6种靶标成分。本研究检测的目标片段是外源基因和宿主DNA之间的连接区域,具有更高的特异性,扩增最大目标片段与最小片段之间高达1 500 bp,但灵敏度也能达到0.5%,完全符合欧盟有关转基因标识的要求。同时,该多重PCR体系在ZH10-6和受体中黄10混合体系中,实现了六重PCR扩增。

4 结论

通过设计内参基因、外源基因和侧翼序列引物并对反应体系和条件进行优化,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。

参考文献 View Option 原文顺序
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被引期刊影响因子

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Abstract

This paper presents an overview of GMO testing methodologies and how these have evolved and may evolve in the next decade. Challenges and limitations for the application of the test methods as well as to the interpretation of results produced with the methods are highlighted and discussed, bearing in mind the various interests and competences of the involved stakeholders. To better understand the suitability and limitations of detection methodologies the evolution of transformation processes for creation of GMOs is briefly reviewed.

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BACKGROUND: There are about 80 biotech crop events that have been approved by safety assessment in Korea. They have been controlled by genetically modified organism (GMO) and livingmodified organism (LMO) labeling systems. The DNA-based detection method has been used as an efficient scientific management tool. Recently, the multiplex polymerase chain reaction (PCR) and DNA chip have been developed as simultaneous detection methods for several biotech crops' events.
RESULTS: The event-specific multiplex PCR method was developed to detect five biotech maize events: MIR604, Event 3272, LY 038, MON 88017 and DAS-59122-7. The specificity was confirmed and the sensitivity was 0.5%. The screening DNA chip was developed from four endogenous genes of soybean, maize, cotton and canola respectively along with two regulatory elements and seven genes: P35S, tNOS, pat, bar, epsps1, epsps2, pmi, cry1Ac and cry3B. The specificity was confirmed and the sensitivity was 0.5% for four crops' 12 events: one soybean, six maize, three cotton and two canola events.
CONCLUSION: The multiplex PCR and DNA chip can be available for screening, gene-specific and event-specific analysis of biotech crops as efficient detection methods by saving on workload and time. (C) 2014 Society of Chemical Industry

[15] GUO J C, CHEN L L, LIU X, GAO Y, ZHANG D B, YANG L T. A multiplex degenerate PCR analytical approach targeting to eight genes for screening GMOs
Food Chemistry, 2012,132(3):1566-1573.
DOI:10.1016/j.foodchem.2011.11.096URL
Currently, the detection methods with lower cost and higher throughput are the major trend in screening genetically modified (GM) food or feed before specific identification. In this study, we developed a quadruplex degenerate PCR screening approach for more than 90 approved GMO events. This assay is consisted of four PCR systems targeting on nine DNA sequences from eight trait genes widely introduced into GMOs, such as CP4-EPSPS derived from Acetobacterium tumefaciens sp. strain CP4, phosphinothricin acetyltransferase gene derived from Streptomyces hygroscopicus (bar) and Streptomyces viridochromogenes (pat), and Cry1Ab, Cry1Ac, Cry1A(b/c), mCry3A, and Cry3Bb1 derived from Bacillus thuringiensis. The quadruplex degenerate PCR assay offers high specificity and sensitivity with the absolute limit of detection (LOD) of approximate 80 target copies. Furthermore, the applicability of the quadruplex PCR assay was confirmed by screening either several artificially prepared samples or samples of Grain Inspection, Packers and Stockyards Administration (GIPSA) proficiency program. (C) 2011 Elsevier Ltd.

[16] JAMES D, SCHMIDT A M, WALL E, GREEN M, MASRI S. Reliable detection and identification of genetically modified maize, soybean, and canola by multiplex PCR analysis
Journal of Agricultural and Food Chemistry, 2003,51(20):5829-5834.
DOI:10.1021/jf0341159URLPMID:13129280 [本文引用: 1]
Multiplex PCR procedures were developed for simultaneously detecting multiple target sequences in genetically modified (GM) soybean (Roundup Ready), maize (event 176, Bt11, Mon810, T14/25), and canola (GT73, HCN92/28, MS8/RF3, Oxy 235). Internal control targets (invertase gene in corn, lectin and beta-actin genes in soybean, and cruciferin gene in canola) were included as appropriate to assess the efficiency of all reactions, thereby eliminating any false negatives. Primer combinations that allowed the identification of specific lines were used. In one system of identification, simultaneous amplification profiling (SAP), rather than target specific detection, was used for the identification of four GM maize lines. SAP is simple and has the potential to identify both approved and nonapproved GM lines. The template concentration was identified as a critical factor affecting efficient multiplex PCRs. In canola, 75 ng of DNA template was more effective than 50 ng of DNA for the simultaneous amplification of all targets in a reaction volume of 25 microL. Reliable identification of GM canola was achieved at a DNA concentration of 3 ng/microL, and at 0.1% for GM soybean, indicating high levels of sensitivity. Nonspecific amplification was utilized in this study as a tool for specific and reliable identification of one line of GM maize. The primer cry1A 4-3' (antisense primer) recognizes two sites on the DNA template extracted from GM transgenic maize containing event 176 (European corn borer resistant), resulting in the amplification of products of 152 bp (expected) and 485 bp (unexpected). The latter fragment was sequenced and confirmed to be Cry1A specific. The systems described herein represent simple, accurate, and sensitive GMO detection methods in which only one reaction is necessary to detect multiple GM target sequences that can be reliably used for the identification of specific lines of GMOs.

[17] 尹全, 李忆, 宋君, 王东, 张富丽, 刘文娟, 常丽娟, 刘勇. 多重PCR检测抗草甘膦大豆GTS40-3-2
大豆科学, 2016,35(4):666-671.
[本文引用: 1]

YIN Q, LI Y, SONG J, WANG D, ZHANG F L, LIU W J, CHANG L J, LIU Y. Multiplex PCR for detection of roundup-ready soybean GTS40-3-2
Soybean Science, 2016,35(4):666-671. (in Chinese)
[本文引用: 1]

[18] AO J, LI Q, GAO X, YU Y, LI L, ZHANG M. A multiplex nested PCR assay for the simultaneous detection of genetically modified soybean, maize and rice in highly processed products
Food Control, 2011,22(10):1617-1623.
DOI:10.1016/j.foodcont.2011.03.018URL [本文引用: 1]
The use of genetically modified organisms (GMOs) as food products becomes more and more widespread. The European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or food ingredients. Thus, analytical methods for detection of the GMOs are necessary in order to verify compliance with labeling requirements. There are few effective screening methods for highly processed GM (genetically modified) products. Four genes (CP4-EPSPS, Cry1A(b), BAR, and, PAT) are common exogenous genes used in commercialized transgenic soybean, maize, and rice. In the present study, a multiplex nested polymerase chain reaction (PCR) method was developed to simultaneously detect the four exogenous genes and one endogenous gene in two runs. We tested eleven representative highly processed products samples (soya lecithin, soya protein powder, chocolate beverage, infant rice cereal, soybean refine oil, soybean salad oil, maize oil, maize protein powder, maize starch, maize jam) using the developed method, and amplicons of endogenous gene and transgenic fragments were obtained from all the processed products except for soybean refined oil, soybean salad oil and maize oil, and the sensitivity was 0.005%. These results indicate that multiplex nested PCR is appropriate for qualitative detection of transgenic soybean, maize and rice in highly processed products except for refined oil. (C) 2011 Elsevier Ltd.

[19] KIM J H, JEONG D, KIM Y R, KWON Y K, RHEE G S, ZHANG D B, KIM H Y. Development of a multiplex PCR method for testing six GM soybean events
Food Control, 2013,31(2):366-371.
DOI:10.1016/j.foodcont.2012.10.015URL [本文引用: 1]
Genetically modified (GM) soybean and derived products make up a large part of the biotech-derived food and feed market. As more GM soybean varieties have been approved for commercialization, labeling requirement by South Korea and other countries needs the technical testing methods. This paper reports the development of a multiplex PCR method for identifying six commercialized GM soybean events using the event-specific fragment. Event specific primers targeting Roundup Ready Soybean (RRS, GTS40-3-2), A2704-12, DP356043-5, MON89788, A5547-127, and DP305423-1 were designed, and a multiplex PCR assay consisting of six event-specific fragments and one endogenous lectin fragment was developed. The specificity of the event-specific PCR method was confirmed using 20 GM events of maize, soybean, cotton, and canola. The limit of detection (LOD) for each event in the multiplex PCR is approximately 0.05%. Intra-lab validation by two different operators,confirmed the specificity and LOD of this multiplex PCR method. The method was used to test 30 soybean-derived foods from South Korean and US markets, and results revealed three varieties of GM soybean (RRS, A2704-12, and MON89788) in 19 of the 30 food samples tested. This work provides an efficient and cost-effective approach for event-specific analysis of six commercialized GM soybean varieties and related processed foods in Korea. (c) 2012 Elsevier Ltd.

[20] 付伟, 任娇, 魏霜, 袁俊杰, 周广彪, 吴希阳, 朱水芳, 刘中勇. 转基因大豆DAS44406-6多重荧光定量PCR检测方法的建立
食品工业科技, 2017,38(4):63-67.
[本文引用: 1]

FU W, REN J, WEI S, YUAN J J, ZHOU G B, WU X Y, ZHU S F, LIU Z Y. Multiplex real-time PCR for the detection of genetically modified soybean DAS44406-6
Science and Technology of Food Industry, 2017,38(4):63-67. (in Chinese)
[本文引用: 1]

[21] 鲁军, 李刚, 赵建宁, 杨殿林, 修伟明. 5种转基因油菜转化体特异性多重PCR检测方法
生物安全学报, 2017,26(3):244-250.
[本文引用: 1]

LU J, LI G, ZHAO J N, YANG D L, XIU W M. Event-specific multiplex PCR detection method for five genetically modified canola lines
Entomological Journal of East China, 2017,26(3):244-250. (in Chinese)
[本文引用: 1]

[22] JENSEN A H. Testing for genetically modified organisms (GMOs): Past, present and future perspectives
Biotechnology Advances, 2009,27(6):1071-1082.
DOI:10.1016/j.biotechadv.2009.05.025URL [本文引用: 1]

Abstract

This paper presents an overview of GMO testing methodologies and how these have evolved and may evolve in the next decade. Challenges and limitations for the application of the test methods as well as to the interpretation of results produced with the methods are highlighted and discussed, bearing in mind the various interests and competences of the involved stakeholders. To better understand the suitability and limitations of detection methodologies the evolution of transformation processes for creation of GMOs is briefly reviewed.

[23] YANG L T, GUO J C, PAN A H, ZHANG H B, ZHANG K W, WANG Z M, ZHANG D. Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule
Journal of Agricultural and Food Chemistry, 2007,55(1):15-24.
URLPMID:17199308 [本文引用: 1]

[24] LI Y J, LI J, WU Y H, CAO Y L, LI J, ZHU L, LI X F, HUANG S M, WU G. Successful detection of foreign inserts in transgenic rice TT51-1 (BT63) by RNA-sequencing combined with PCR
Journal of the Science of Food and Agriculture, 2016,97(5):1634-1639.
DOI:10.1002/jsfa.7913URLPMID:27436567 [本文引用: 1]
BACKGROUND: As event-specific sequence information for most unauthorised genetically modified organisms (GMOs) is currently still unavailable, detecting unauthorised GMOs remains challenging. Here, we used insect-resistant rice TT51-1 as an example to develop a novel approach via detecting GMOs by RNA-seq (sequencing) and PCR. RNA-seq of TT51-1 generated 4.8 million (M) 21-nt cDNA tags. Alignment to the Oryza sativa subsp. japonica reference genome revealed 24 098 unmapped tags. Foreign tags from the nopaline synthetic enzyme gene (NOS) terminator and insect-resistant genes were then identified by searching against the NCBI VecScreen and NT databases. RESULTS: To further isolate foreign DNA sequences, putative NOS terminator and insect-resistant gene tags were combined and used directly as primer pairs for long-range PCR, producing a 5016-bp fragment. The inserted DNA sequence of TT51-1 has been submitted to a database, and thus, similarity analysis using the database could identify a test sample. CONCLUSION: The novel approach has a great potential for application to the detection and identification of unauthorised GMOs in food and feed products. (c) 2016 Society of Chemical Industry.

[25] RANDHAWA G J, CHHABRA R, SINGH M. Decaplex and real-time PCR based detection of MON531 and MON15985 Bt cotton events
Journal of Agricultural & Food Chemistry, 2010,58(18):9875-9881.
DOI:10.1021/jf100466nURLPMID:20687600 [本文引用: 1]
The genetically modified (GM) Bt crops expressing delta-endotoxins from Bacillus thuringiensis provide protection against a wide range of lepidopteron insect pests throughout the growing season of the plant. Bt cotton is the only commercialized crop in India that is planted on an area of 7.6 million hectares. With the increase in development and commercialization of transgenic crops, it is necessary to develop appropriate qualitative and quantitative methods for detection of different transgenic events. The present study reports on the development of a decaplex polymerase chain reaction (PCR) assay for simultaneous detection of transgene sequences, specific transgene constructs, and endogenous stearoyl acyl desaturase (Sad1) gene in two events of Bt cotton, i.e., MON531 and MON15985. The decaplex PCR assay is an efficient tool to identify and discriminate the two major commercialized events of Bt cotton, i.e., MON531 and MON15985, in India. Real-time PCR assays were also developed for quantification of cry1Ac and cry2Ab genes being employed in these two events. The quantitative method was developed using seven serial dilutions containing different levels of Bt cotton DNA mixed with a non-Bt counterpart ranging from 0.01 to 100%. The results revealed that the biases from the true value and the relative standard deviations were all within the range of +/-20%. The limit of quantification (LOQ) of the developed real-time PCR method has also been established up to 0.01%.

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Journal of Virological Methods, 2005,129(1):47-55.
URLPMID:15951030 [本文引用: 1]

[27] YAN W. Multiplex PCR primer design for simultaneous detection of multiple pathogens
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URLPMID:25697653 [本文引用: 1]

[28] 赵红庆, 苑锡铜, 黄留玉. 多重PCR技术在病原检测中的应用
生物技术通讯, 2007,18(5):863-865.
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ZHAO H Q, YUAN X T, HUANG L Y. Application of multiplex PCR in detection of pathogens
Letters in Biotechnology, 2007,18(5):863-865. (in Chinese)
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[29] 曹洪志, 颜其贵, 郭万柱, 樊汶樵, 肖雪, 李成贤. 多重PCR技术在动物疫病诊断中的应用
中国动物检疫, 2007,24(1):45-47.
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CAO H Z, YAN Q G, GUO W Z, FAN W Q, XIAO X, LI C X. Application of multiplex PCR in the diagnosis of animal diseases
China Journal of Animal Quarantine, 2007,24(1):45-47. (in Chinese)
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[30] 刘志杰, 李如举, 曾智勇, 周莉, 汤德元, 李谦, 肖超能. 多重PCR反应的影响因素及其优化
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[33] 陈贞, 芦春斌, 杨梦婕, 马骏, 白卫斌, 吴希阳. 多重PCR检测转基因菜籽粕中的转基因成分
植物检疫, 2011,25(3):35-38.
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CHEN Z, LU C B, YANG M J, MA J, BAI W B, WU X Y. Multiplex PCR for detection of genetically modified components of rapeseed meal
Plant Quarantine, 2011,25(3):35-38. (in Chinese)
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[34] 董立明, 李葱葱, 邢珍娟, 邵改革, 夏蔚, 闫伟, 李飞武. 利用多重PCR技术快速检测五个转基因大豆品系
大豆科学, 2016,35(6):1002-1007.
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DONG L M, LI C C, XING Z J, SHAO G G, XIA W, YAN W, LI F W. Rapid detection of five genetically modified soybean lines by multiplex PCR method
Soybean Science, 2016,35(6):1002-1007. (in Chinese)
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