Standardization of PCR and sequencing-based methods for the detection of the presence of

CP4 EPSPS

gene in

Zea mays

Carlos Humberto Chamone Cangussu

, Léia Cardoso

, Alessandra Rejane Ericsson de Oliveira Xavier

*, Josiane dos Santos

Luiz Felipe Lopes Campos

, Murilo Malveira Brandão

, Denys Cunha Fonseca Garcia

, Alexandre Moisés Ericsson de Oliveira

Evandrei Santos Rossi

, Mauro Aparecido de Sousa Xavier10

DOI: https://doi.org/10.35699/2447-6218.2020.19990

Abstract

With the rise in planting of transgenic cultivars in Brazil as well as worldwide, the demand for legal regulations has increased.

The transgenic event MON88017 is often found in maize cultivars marketed in Brazil. The event contains the CP4 EPSPS and

cry3Bb1 genes, which encode tolerance to the herbicide glyphosate and resistance to caterpillars, respectively. Globally,

methodologies to track transgenic events are mandatory. The objective of this study was to standardize a method based on

qualitative PCR and sequencing for detection of the CP4 EPSPS gene in Zea mays. DNA was extracted from three

commercial strains of transgenic maize, containing the MON88017 event, as well as conventional maize. Primers were

designed for partial detection of the zein and CP4 EPSPS genes. PCR reactions were performed for detection of partial regions

of CP4 EPSPS and Zein (as endogenous marker of Z. mays) genes. The three transgenic maize inbred lines tested positive for

Zein and CP4 EPSPS, and the two conventional strains tested negative for CP4 EPSPS and positive for the Zein gene. To

confirm the presence of the genic regions, PCR products were sequenced and showed 100% identity with sequences of Zein

and CP4 EPSPS genes deposited in GenBank. Thus, the results of this study suggest the applicability of an ‘in-house’ method

for the qualitative detection of CP4 EPSPS in genetically modified maize cultivars.

Keywords:

Genetically modified organisms. MON88017. Detection. Maize

1State University of Montes Claros, Montes Claros, MG, Brazil.

https://orcid.org/0000-0002-4487-6130.

2State University of Montes Claros, Montes Claros, MG, Brazil.

https://orcid.org/0000--0002-4605-6160.

3State University of Montes Claros, Montes Claros, MG, Brazil.

https://orcid.org/0000-0001-8558-4196.

4State University of Montes Claros, Montes Claros, MG, Brazil.

https://orcid.org/0000-0002-9406-5918.

5State University of Montes Claros, Montes Claros, MG, Brazil.

https://orcid.org/0000-0002-0622-9805.

6State University of Montes Claros, Montes Claros, Minas Gerais, Brazil.

https://orcid.org/000-003-1238-1042.

7State University of Montes Claros, Montes Claros, Minas Gerais, Brazil.

https://orcid.org/0000-0003-1513-9390.

8Federal University of Uberlândia, Uberlândia, Minas Gerais, Brazil.

https://orcid.org/0000-0001-8162-9174.

9Shull Seeds Ltda, Ribeirão Preto, São Paulo, Brazil.

https://orcid.org/0000-0003-1697-4048.

10State University of Montes Claros, Montes Claros, MG, Brazil.

https://orcid.org/0000-0002-0512-1616.

*Corresponding author: ericsson_aerc@yahoo.com.br

Recebido para publicação em 11 de abril de 2020. Aceito para publicação em 30 de abril de 2020.

e-ISSN: 2447-6218 /

ISSN: 2447-6218. Atribuição CC BY.

CADERNO DE CIÊNCIAS AGRÁRIAS

Agrarian Sciences Journal

Cangussu, C. H. C. et al.

Padronização de método baseado em PCR e sequenciamento para detecção da presença do

gene CP4 EPSPS

em Zea mays

Resumo

O plantio de cultivares transgênicas no Brasil e no mundo tem crescido e demandado regulamentação legal. O evento

transgênico MON8807 é frequentemente encontrado em cultivares de milho comercializadas no Brasil. Esse evento contém

em sua construção os genes CP4 EPSPS e Cry3Bb1 que codificam, respectivamente, a tolerância ao herbicida glifosato e

resistência a lagartas. Metodologias que permitam o rastreamento de eventos transgênicos são globalmente mandatórias. O

objetivo deste trabalho foi padronizar uma metodologia baseada em PCR qualitativo e sequenciamento para detecção do gene

CP4 EPSPS em Zea mays. Para tal linhagens comerciais de milho transgênico contendo o evento MON88017 e milho

convencional foram submetidas ao procedimento de extração de DNA. Foram desenhados oligonucleotídeos para detecção

parcial dos genes zeína e CP4 EPSPS. A reação de PCR para detecção das regiões parciais dos genes CP4 EPSPS e zeína (como

marcador endógeno da espécie Z. mays) foi então realizada. As três linhagens transgênicas de milho testaram positivo para os

genes Zeína e CP4 EPSPS, bem como as duas linhagens convencionais testaram negativo para CP4 EPSPS e positivas somente

para o gene Zeína. A confirmação da presença das regiões gênicas, os produtos de PCR foram sequenciados e apresentaram

100% de identidade com sequência dos genes Zeína e CP4 EPSPS depositados no GenBank. Os resultados deste trabalho

sugerem a aplicabilidade de um método “in house” para detecção qualitativa do gene CP4 EPSPS em cultivares de milho

geneticamente modificado.

Palavras-chave:

Organismos Geneticamente Modificados. MON8807. Detecção. Milho.

Introduction

MON88017 contains two important expression proteins in the

genome: CP4 EPSPS (CP4 5-5-enolpyruvylshikimate-

-3-phosphate) that originated from Agrobacterium tume-

faciens and Cry3Bb1 from Bacillus thuringiensis (ISAAA,

2018). The event presents a genetic construction formed

by a

regulating region, P-E35S (constitutive promoter of the

CaMV 35S gene), a coding region for the CP4 EPSPS

protein, and a terminator region, 3’IN (nopaline synthase)

nig et al., 2004; Miaw, 2014). As the genes are present

the same segment of DNA and occur in a single locus in the

genome, it provided improved efficiency in plant

reproduction and allowed the use of molecular markers to

identify transgenic genes (Conceição et al., 2006).

Maize (Zea mays) makes out the largest volume of

cereal production in the world. Between the years

2018 and

2019, approximately 17,255.6 million hectares

of maize were

planted globally (CONAB, 2019). A large variety is

observed among maize cultivars, for example, in the harvest

of 2016/17 approximately 315 cultivars of corn, of which

214 were transgenic and 101 were conventional, were

observed (Pereira-Filho et al., 2016; Tabima-Cubillos et al.,

2016). In Brazil, the planting of transgenic cultivars has

become very common since the

release of commercial strains.

Since 2009 the country has

been the second-largest producer

of transgenic grains in

the world. In 2017, there were

approximately 50.2 million

hectares of maize cultivated, and

31% of the total land area was used (Cardoso et al., 2018;

ISAAA, 2018).

As soon as they are released for planting and

marketing, all transgenic events must be liable to be de-

tected. In 2003, the Executive Power through the decree no.

4.680/2003, demanded the inclusion of information on the

labels of the packed products in bulk or in natura if it

contained more than 1% GMOs (Brasil, 2003).

Biotechnology has significantly improved maize

cultivars, mainly in relation to agronomic characteristics,

such

as providing tolerance to aerial and root pest car- riers as

well as tolerance to herbicides, in particular to glyphosate

(Fraiture et al., 2015). The genetic construc- tion used to

produce a Genetically Modified Organism (GMO) consists

of three regions: a promoter region, an

encoding gene, and a

terminator region (Wu et al., 2012). The MON88017 event,

obtained through the insertion of

the CP4 EPSPS and

Cry3Bb1 genes was the first product

developed and approved

by the company, Monsanto (now

Bayer), whose 2015/16

season produced 12 cultivars (Cruz et al., 2015).

Compliance with the legislation that regulates

the

commercialization of food and ingredients containing

transgenic components is totally dependent on the sen-

sitivity and reliability of the detection and quantification

methods (Conceição et al., 2006). GMOs are characterized

the presence of one or more segments of exogenous

DNA,

which may or may not provide the expression of new

proteins.

Thus, GMO detection is focused on exogenous DNA

sequences (via PCR-based methods) or transgenic proteins

(via immunoassays, ELISA, and Western blot analysis)

(König et al., 2004).

In Brazil, commercialization of the MON88017

event was approved by the National Technical Commis- sion

on Biosafety (CTNBio) in 2010 for planting as well as

human and animal consumption (ISAAA, 2018). As a bearer

of the Roundup Ready Technology (VT PRO3TM),

Qualitative and quantitative PCR detection me-

thods, which use DNA to monitor genetically modified

crops and their derivatives, are widely used mainly for

the

characteristics or stability of DNA. Protein expression

Cad. . Ciên. Agrá., 12, 1-9. https://doi.org/10.35699/2447-6218.2020.19990

Standardization of PCR and sequencing-based methods for the detection of the presence of

CP4 EPSPS

gene in

Zea mays

may vary depending on the age, variety, environmental

conditions, or even the industrial processes used for the

manufacturing of oils, syrups, and other products

(Cardarelli et al., 2005; Forte et al., 2005; Holst-Jensen et

al., 2003).

and the mixture was stored at -20°C for 24 hours. After

hours, the microtubes were centrifuged at 14000 rpm for 10

minutes and the pellet was washed with 700 µL of 70%

ethanol. The pellet containing the nucleic acids was

then

resuspended in 20 µL of TE buffer (10 mL Tris-HCl 10 mM

pH 8.0, 2 mL EDTA 1 mM) and 1 µL of RNase A 20 mg/mL

and stored at -20°C. The nucleic acids were quantified by

1.0% agarose gel electrophoresis and used in PCR reactions.

Real-time quantitative PCR can be used to detect or

quantify GMO fractions in food products. On the other hand,

qualitative PCR is required for large-scale screening,

when

large numbers of negative samples are expected, and it is

also used for GMO identification (Gašparič et al., 2010).

According to Heide et al. (2008), high levels

of GMOs in

samples produced signals of greater strength

than low levels of

GMOs. The objective of this study was to standardize a

method based on qualitative PCR and sequencing for the

detection of the CP4 EPSPS gene in

Z. mays.

Design of genetic markers for

Zein

and

CP4 EPSPS

genetic regions

Two pairs of PCR-specific primers were used:

(1) Zeo-F/Zeo-R for detection of the Zein maize gene as

described by Cardarelli et al. (2005) with modifications

(Table 1). The sequences of the primers were confirmed with

the sequence of the Zein gene (Genbank accession no.

M23537.1); (2) CP4F/CP4R for detection of the CP4 EPSPS

gene (transgenic event MON88017) as described by Wu et

al. (2012) with modifications (Table 1). The primer

sequences were confirmed with the sequence of

the CP4

EPSPS gene (Genbank accession no. AB209952.1). All primers

were synthesized by GenOne Biotechnologies

(Rio de Janeiro,

Brazil).

Materials and Methods

Obtaining leaf tissue

Three commercial transgenic maize cultivars with

the

presence of the event MON88017 were used: dkb

230PRO3, dkb 240PRO3, and dkb 177 PRO3, and two

conventional maize cultivars: SS7088 and SS8021, kindly

provided by Shull Seeds Ltd. The seeds were planted in a

substrate Bioplant (Bioplant Blender Agricola Ltd.),

kept at

ambient conditions of light and temperature, and

watered

daily. After 10 days, the seedlings were used as a source of

leaf tissue for DNA extractions.

Qualitative PCR for detection of Zein and CP4 EPSPS

genes

The presence of the Zein (an endogenous mar- ker

of Z. mays) and CP4 EPSPS genes (a marker for the

transgenic event MON88017) was verified with the pri- mers

listed in Table 1. The reactions were carried out in a

mixture containing 2x GoTaq® Green Master Mix (Promega

Corporation, USA), 1.5 M MgCl2, 10 µM of

each primer,

and 50 ng of DNA with a total reaction volume of 50 µl. The

amplification conditions

for both

Zein and CP4 EPSPS genes

were as follows: an initial cycle

of denaturation at 95°C for 2

minutes, followed by 35 cycles of denaturation at 95°C for

2 minutes, annealing temperature at 60°C for 50 seconds,

extension at 72°C for 1 minute, and final extension for 10

minutes. The

amplicons were visualized on a 1.5% agarose

gel, stained

with ethidium bromide and documented.

DNA extraction

The DNA of the transgenic and conventional cul-

tivars was extracted from leaf tissue according to the me- thod

of Doyle and Doyle (1990) with some modifications.

Fifty

milligrams of leaves of each cultivar were weighed,

transferred to a crucible containing 0.2 g of autoclaved sand,

and macerated with a pistil. During maceration, 2 mL of

5% cetyltrimethylammonium bromide (CTAB) buffer, 2

µL/mL of 2-mercaptoethanol, 100 mM TrisHCl (pH 8.0), 1

mg/mL proteinase K, 20 mM EDTA, 100 mM

polyvinylpyrrolidone, and 1.4 M NaCl were added. One

milliliters of the macerate obtained were transferred to 2 mL

microtubes and vortexed for 10 seconds, followed by

incubation in a water bath at 70°C for 1 hour. Every

minutes the microtubes were removed from the water

bath and

agitated again with a vortex mixer to induce rupturing of

the cell walls. For DNA extraction, 700 µL of

chloroform:isoamyl alcohol (24:1) was added to the mixture

and homogenized in the vortex mixer for 10 minutes. Then

the samples were centrifuged at 14000 rpm for 10 minutes

and the supernatant was transferred to a new microtube. To

each microtube, 55 µL of 10% CTAB (10% CTAB, 1.4 M

NaCl) and 700 µL chlorofor- m:isoamyl alcohol (24:1) was

added and homogenized with a vortex mixer for 10 minutes.

Then the samples were centrifuged at 14000 rpm for 10

minutes, 700 µL of ice cold isopropanol was added to the

supernatant,

Sequencing of PCR products of Zein and CP4 EPSPS

genes

The PCR products (amplicons of 325 bp and 610

corresponding to Zein and CP4 EPSPS, respectively) were

sequenced by the Sanger method (Ludwig Biotech Ltd., RS,

Brazil - ACTGene Análises Moleculares Ltd.) with the

primers described in Table 1. In order to ob- tain the

amplified sequence, the sequences were aligned with Clustal

Omega software (European Bioinformatics Institute -

https://www.ebi.ac.uk/Tools/msa/clustalo/) and differences

were visualized on electropherograms (Chromas v.2.6.5 -

www.technelysium.com.au) and cor- rected. After obtaining

the amplified sequence, it was used to perform BLASTn

searches (https://blast.ncbi.

Cad. . Ciên. Agrá., 12, 1-9. https://doi.org/10.35699/2447-6218.2020.19990

Cangussu, C. H. C. et al.

nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch). After the

blast searches, the obtained sequences were aligned with

BLASTn sequences with Clustal Omega software

(European Bioinformatics Institute (https://www.ebi.

ac.uk/Tools/msa/clustalo/).

Table 1 – Sequences and information related to the primers for partial detection of Zein and CP4 EPSPS genes in strains of Zea

mays

Gene

Position

Primer

Sequence (5’- 3’)

Amplicon Size (PB)

Reference

Zein

Zeo-F

TGCATTGTTCGCTCTCCTA

107 - 432

325

This study

(EU952871.1)

Zeo-R

CP4-F

GTCGCAGTGACATTGTGGC

CCTCGTCGGGGTCTACGA

CP4 EPSPS

(KX640115.1)

2270 - 2880

610

This study

CP4-R

CAGCGTGGAGGAGCGAA

Results and Discussion

chosen for the design of the primers. The PCR reaction

for

Zein gene detection proved to be positive for the three

analyzed

DNA samples of transgenic Z. mays, since the expected

fragment of 325 bp was amplified (Figure 1). An amplicon

of the same size was also obtained for the two DNA samples

of non-transgenic Z. mays (data not shown).

For this study, commercial transgenic maize seeds that

are sold at local markets were purchased in two cities

in the

north of Minas Gerais State, Brazil. Conventional maize

seeds were kindly donated by the company, Se- mentes

Shull Ltd.

The rationale for obtaining DNA samples from

leaf tissue was based on previous experience of our re-

search group that tested different protocols that used seeds

and distinct DNA extraction methods for Z. mays and

Glicine max (Campos et al., 2018). In this study, the

DNA

samples of seedlings of five different maize cultivars

extracted

with the modified methodology of Doyle and

Doyle (1990)

presented the characteristic profile of bands

with satisfactory

quality to proceed with PCR analyses.

PCR-based screening methods aimed at detecting

genetic elements commonly used in genetically modified

plants are important tools for the detection of food, feed,

and

transgenic seeds samples. The CP4 EPSPS gene is

present

in a large number of transgenic plants, including

Z. mays (Anklam et al., 2002; Matsuoka et al., 2002; Heck

et al., 2005; Marmiroli et al., 2008; Vidal et al., 2015).

In order to establish an ‘in-house’ method for the

detection of the CP4 EPSPS gene in transgenic maize, the

primers CP4-F and CP4-R were designed (Table 1). The

nucleotide sequence corresponding to the CP4 EPSPS gene

was

obtained from GenBank (access no. KX640115.1).

The

regions that comprise the nucleotides 2270 and 2880

of the

CP4 EPSPS gene were chosen for the design of the primers.

The PCR reaction performed for detection of the CP4

EPSPS gene among the three transgenic maize

samples, was

positive, once the expected fragment of 610

bp was amplified

and there was no amplification for the two conventional

maize samples (Figure 1).

Difficulties in the reproducibility of protocols for

DNA extraction from plant species that hinder molecular

studies that directly depend on the quality of extraction of

nucleic acids were discussed in the literature. The presence

of polysaccharides, phenols, and secondary

compounds, is

the main challenge faced in DNA isolation and purification due

to the inhibition of Taq polymerase

activity in PCR reactions

(Mazza and Bittencourt, 2000; Vieira et al., 2010; Sika et al.,

2015; Abdel-Latif and Osman, 2017).

To evaluate the quality of the extracted DNA and

to genetically confirm the Z. mays species among analyzed

samples, the Zeo-R and Zeo-F primers were designed

(Table 1).

Several studies have described the PCR method as

fast and safe for the identification of transgenic maize through

the use of specific primers (Hernández et al., 2003; James

et al., 2003; Germini et al., 2004; Onishi et al., 2005; Kim

et al., 2006). According to Michelini et al. (2008) and Zhang

and Guo (2011), PCR is the most widely used technique

for CP4 EPSPS gene detection in maize cultivars, owing to

its high sensitivity, genetic stability, and presence in

different cultivars.

The Zein gene is described in the literature as a

marker of Z. mays. The full or partial gene regions have been

used for the construction of primers for genotypic

confirmation of Z. mays (Meyer, 1999; Höhne et al., 2002;

Matsuoka et al., 2002; Yamaguchi et al., 2003; Cardarelli

al., 2005; Nascimento et al., 2010; Dinon et al., 2011;

Wu et

al., 2012). In this study, partial region primers were

designed

for the Zein gene based on the gene sequence

deposited in

Genbank (accession number EU952871). The

regions

comprising the nucleotides 107 and 439 were

According to Holst-Jensen et al. (2012) and Broe- ders

et al. (2012), the use of this method to identify diffe-

rent types

of maize with primers specific for CP4 EPSPS, Bar,

Cry1A(b), and/or Pat genes is of great importance,

Cad. . Ciên. Agrá., 12, 1-9. https://doi.org/10.35699/2447-6218.2020.19990

Standardization of PCR and sequencing-based methods for the detection of the presence of

CP4 EPSPS

gene in

Zea mays

because these genes are present in a variety of cultivars of

maize and soya worldwide.

Figure 1 – Detection of Zein and CP4 EPSPS genes by the PCR method on 1.5% agarose gel.

1 1

2000

1500

1000

700

600

500

400

300

200

100

gene

cp4-epsps

610pb

gene zeina

325pb

M: marker of molecular mass 100pb (Ludwig Biotecnologia). The Zein gene amplicon of 325 pb amplified in samples 1 to 3: Line 1: dkb230PRO3, Line 2: dkb

240PRO3 and Line3 dkb 177PRO3. The CP4 EPSPS gene amplicon of 610pb amplified in samples 4 to 7: Line 4: dkb230PRO3, Line 5: dkb 240PRO3, Line

6: dkb 177PRO3 and Line 7: dkb177PRO3 (positive control). Line 8: SS7088 and Line 9: SS8021, two conventional cultivars did not present the CP4 EPSPS

amplicon as expected. Line 10: sterile water (negative control).

Milavec et al. (2014) showed that using pairs of

primers for regulatory regions or inserted genes are more

important for obtaining an effective transgenic screening

assay. Matsuoka et al. (2002) developed a method using

multiple pairs of DNA primers (including genic regions,

promoters or terminators) to effectively identify transgenic

events.

transgenic samples analyzed here, we decided to sub- mit

the PCR products from one of the three cultivars of

transgenic Z. mays (dkb 177 PRO3) for sequencing of the

amplified region. The rationale for this is based on the

objective to establish a robust and reliable methodology for

transgenic screening in Z. mays cultivars.

Database alignment analysis of the sequenced 325

PCR product corresponding to the partial Zein gene

(dkb

177 PRO3) proved to be identical (100% identity) to a cDNA

sequence of Zein described in Genbank accession

number

EU952871.1 (Figure 2).

James et al. (2003) were able to detect transgenic

soybeans with multiplex PCR analysis using three pairs of

primers specific for the CP4 EPSPS gene, 35S promoter, and

NOS terminator, along with two pairs of primers specific

for soy targeting soy lecithin and -actin genes.

The

procedure was able to distinguish the non-transgenic

soybeans

from transgenic soybeans with certainty.

Similarly, database alignment analysis of the

sequenced PCR product of 610 bp corresponding to the

partial gene CP4 EPSPS (CP4 EPSPS dkb 177 PRO3)

proved to be identical (100% identity) to a sequence of a

transgenic cultivar of Z. mays lineage transgenic culti- var

NK603 containing the CP4 EPSPS gene described in

Genbank (KX640115.1) (Figure 3).

Forte et al. (2005) developed a molecular scree-

ning

method through multiplex PCR and amplification of

specific

sequences of soybeans or the 35S promoter and NOS

terminator for the detection of transgenic events.

Nikolić et

al. (2008) investigated triplex PCR methods for the

identification of the lecithin and Zein genes, through the

amplification of the 35S promoter and NOS terminator for the

detection of genetically modified soya and maize,

further

illustrating the effectiveness of the methodology.

The availability of gene sequences of crops of

agronomic value, along with the advancement of genetic

sequencing, has made it possible to predict and isolate

regulatory regions of any region inserted into a genome,

facilitating the screening of transgenic events (Schmutz et

al., 2010; Tran and Mochida, 2010).

Although PCR amplification of the Zein and CP4

EPSPS gene regions was successfully achieved in the

Cad. . Ciên. Agrá., 12, 1-9. https://doi.org/10.35699/2447-6218.2020.19990

Cangussu, C. H. C. et al.

Figure 2 – Alignment of the sequence obtained from the amplicon of 325 bp of the partial Zein gene of the lineage of

transgenic maize dkb 177 PRO3 (Zein dkb 177PRO3). The asterisks indicate the identical nucleotides between

Zein dkb 177PRO3 and the cDNA of Zein of Zea mays Genbank accession number EU952871.1.

Zein dkb 177PRO3

EU952871.1

AAATAGGACCTGCTAGATCAATCGCAGTCCATCGGCCTCAGTCGCACATATCTACTATAC

Zein dkb 177PRO3

EU952871.1

TGCATTGTTCGCT

120

TATACTCTAGGAAGCAAGGACACCACCGCCATGGCAGCCAAGATGCTTGCATTGTTCGCT

*************

Zein dkb 177PRO3

EU952871.1

CTCCTAGCTCTTTGTGCAAGCGCCACTAGTGCGACCCATATTCCAGGGCACTTGCCACCA

************************************************************

180

Zein dkb 177PRO3

EU952871.1

GTCATGCCATTGGGTACCATGAACCCATGCATGCAGTACTGCATGATGCAACAGGGGCTT

************************************************************

133

240

Zein dkb 177PRO3

EU952871.1

GCCAGCTTGATGGCGTGTCCGTCCCTGATGCTGCAGCAACTGTTGGCCTTACCGCTTCAG

************************************************************

193

300

Zein dkb 177PRO3

EU952871.1

ACGATGCCAGTGATGATGCCACAGATGATGACGCCTAACATGATGTCACCATTGATGATG

************************************************************

253

360

Zein dkb 177PRO3

EU952871.1

CCGAGCATGATGTCACCAATGGTCTTGCCGAGCATGATGTCGCAAATGATGATGCCACAA

************************************************************

313

420

Zein dkb 177PRO3

EU952871.1

TGTCACTGCGAC

325

480

TGTCACTGCGACGCCGTCTCGCAGATTATGCTGCAACAGTAGTTACCATTCATGTTCAAC

************

Zein dkb 177PRO3

EU952871.1

325

540

CCAATGGCCATGACGATTCCACCCATGTTCTTACAGCAACCCTTTGTTGGTGCTGCATTC

Zein dkb 177PRO3

EU952871.1

325

600

TAGATAGAAATATTTGTGTTGTACCGAATAATGAGTTGACATGCCATCGCGTGTGACTCA

Zein dkb 177PRO3

EU952871.1

325

646

TTATTAACAATAAAACAAGTTTCCTCTTAAAAAAAAAAAAAAAAAA

Conclusion

Acknowledgements

In this study we designed and tested primers

specific to detect zein and CP4 EPSPS genes in Zea mays

based on literature and sequences available in Genbank.

The

qualitative “in house” PCR and sequenced amplicons showed

to be a reliable method for tracking the presence

of CP4

EPSPS transgenic gene marker in Zea mays due to its

robustness, sensitivity, and specificity. Further studies

should

include the gene marker frequency, others markers

used in Z.

mays and others cultivars as soya and cotton as well as the

development of qualitative and quantita- tive methodologies

to confirm the presence of them in transgenic crops or

transgenic food.

To Brazilian National Council for Scientific and

Technological Development (CNPq), Research Supporting

Foundation of Minas Gerais State (FAPEMIG) and Post-

graduate Program in Biotechnology of State University of

Montes Claros (Unimontes).We would like to thank

Editage

(www.editage.com) for English language editing.

Authors’ contributions

A.R.E.O.X. and M.A.S.X. conceived designed the

study. C.H.C.C., J.S., L.C., L.F.L.C., M.N.B., D.C.F.G.

carried out the experiments. C.H.C.C., A.M.E.O., E.S.R.,

A.R.E.O.X. and M.A.S.X analyzed the data. C.H.C.C.,

A.R.E.O.X. and M.A.S.X. wrote the manuscript. All authors

read and approved the final manuscript.

Cad. . Ciên. Agrá., 12, 1-9. https://doi.org/10.35699/2447-6218.2020.19990

Standardization of PCR and sequencing-based methods for the detection of the presence of

CP4 EPSPS

gene in

Zea mays

Figure 3 – Alignment of the sequence obtained from the amplicon of 610 bp of partial CP4 EPSPS gene of the lineage of

transgenic maize dkb 177PRO3 (CP4 EPSPS dkb 177PRO3). The asterisks indicate the identical nucleotides

between CP4 EPSPS dkb 177PRO3 and transgenic cultivar of Zea mays L. line NK603 containing the CP4 EPSPS

gene described in Genbank accession number KX640115.1.

CP4-EPSPS

KX640115.1

dkb 177PRO3

CCTCGTCGGG

2280

GCGCCGCTCGATTTCGGCAATGCCGCCACGGGCTGCCGCCTGACGATGGGCCTCGTCGGG

**********

CP4-EPSPS

KX640115.1

177PRO3

GTCTACGATTTCGACAGCACCTTCATCGGCGACGCCTCGCTCACAAAGCGCCCGATGGGC

************************************************************

2340

CP4-EPSPS

KX640115.1

dkb

177PRO3

CGCGTGTTGAACCCGCTGCGCGAAATGGGCGTGCAGGTGAAATCGGAAGACGGTGACCGT

************************************************************

130

2400

CP4-EPSPS

KX640115.1

dkb

177PRO3

CTTCCCGTTACCTTGCGCGGGCCGAAGACGCCGACGCCGATCACCTACCGCGTGCCGATG

************************************************************

190

2460

CP4-EPSPS

KX640115.1

dkb

177PRO3

GCCTCCGCACAGGTGAAGTCCGCCGTGCTGCTCGCCGGCCTCAACACGCCCGGCATCACG

************************************************************

250

2520

CP4-EPSPS

KX640115.1

dkb

177PRO3

ACGGTCATCGAGCCGATCATGACGCGCGATCATACGGAAAAGATGCTGCAGGGCTTTGGC

************************************************************

310

2580

CP4-EPSPS

KX640115.1

dkb

177PRO3

GCCAACCTTACCGTCGAGACGGATGCGGACGGCGTGCGCACCATCCGCCTGGAAGGCCGC

************************************************************

370

2640

CP4-EPSPS

KX640115.1

dkb

177PRO3

GGCAAGCTCACCGGCCAAGTCATCGACGTGCCGGGCGACCCGTCCTCGACGGCCTTCCCG

************************************************************

430

2700

CP4-EPSPS

KX640115.1

dkb

177PRO3

CTGGTTGCGGCCCTGCTTGTTCCGGGCTCCGACGTCACCATCCTCAACGTGCTGATGAAC

************************************************************

490

2760

CP4-EPSPS

KX640115.1

dkb

177PRO3

CCCACCCGCACCGGCCTCATCCTGACGCTGCAGGAAATGGGCGCCGACATCGAAGTCATC

************************************************************

550

2820

CP4-EPSPS

KX640115.1

dkb

177PRO3

AACCCGCGCCTTGCCGGCGGCGAAGACGTGGCGGACCTGCGCGTTCGCTCCTCCACGCTG

************************************************************

610

2880

CP4-EPSPS

KX640115.1

dkb

177PRO3

610

2940

AAGGGCGTCACGGTGCCGGAAGACCGCGCGCCTTCGATGATCGACGAATATCCGATTCTC

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