Welcome to Acta Agronomica Sinica,

Acta Agron Sin ›› 2013, Vol. 39 ›› Issue (08): 1501-1506.doi: 10.3724/SP.J.1006.2013.01501

• RESEARCH NOTES • Previous Articles     Next Articles

Construction and Application of a Reference Plasmid Molecule Suitable for phyA2 of Phytase Transgenic Maize

ZHANG Guang-Yuan1,2,SUN Hong-Wei1,LI Fan1,YANG Shu-Ke1,LU Xing-Bo1,*,ZHAO Lei2,*   

  1. 1 Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Shandong Key Laboratory of Plant Virology, Jinan 250100, China; 2 Shandong Normal University, Jinan 250014, China
  • Received:2013-01-21 Revised:2013-04-22 Online:2013-08-12 Published:2013-05-20
  • Contact: 路兴波,E-mail: luxb99@sina.com, Tel: 0531-83179095;赵蕾,E-mail: zhaolei@sdu.edu.cn,Tel: 0531-88177190


Plasmid molecule based reference material has been shown to be a good alternative as the calibrator for genetically modified organisms (GMOs) identification and quantification. In this study, primers and probe for qualitative and quantitative PCR detection for phytase transgenic maize were designed, and a flexible plasmid pPZ containing phyA2 (243 bp) and zSSIIb (178 bp) sequences, was developed. Primers and plasmid were specifically tested through qualitative PCRs. Results showed that the primers and pPZ plasmid were very specific for phytase transgenic maize since there were no other PCR products amplified using different GMOs and primers except the specific main bands. Four different levels of genetically modified (GM) matrix samples (3.0%, 1.0%, 0.5%, and 0.1%) were detected by real-time PCR when plasmid pPZ and phytase transgenic maize genomic DNA were used as quantification standards, respectively. t-test showed the slop, linearity (R2) and quantification of the two standards for phyA2 and zSSIIb were not significantly different. The results indicated that the pPZ plasmid can be used as standard for phytase transgenic maize detection.

Key words: Phytase transgenic maize, Plasmid, Taqman

[1]Zinin N V, Serkina A V, Gelfand M S. Gene cloning, expression and characterization of novel phytase from Obesumbacterium proteus. FEMS Microbiol Lett, 2004, 236: 283–290

[2]Xiong A S, Yao Q H, Peng R H. Isolation, characterization, and molecular cloning of the cDNA encoding a novel phytase from Aspergillus niger 113 and high expression in Pichia pastori. J Biochem Mol Biol, 2004, 37: 282–291

[3]Brinch-Pedersen H, Olesen A, Rasmussen S K, Holm P B. Generation of transgenic wheat (Triticum aestivum L.) for constitutive accumulation of an Aspergillus phytase. Mol Breed, 2000, 6: 195–206

[4]Zhang L H, An L J, Gao X R. Properties of A. ficuum AS3.324 phytase expressed in tobacco. Process Biochem, 2005, 40: 213–216

[5]Ullah A H, Sethumadhavan K, Mullaney E J. Cloned and expressed fungal phyA gene in alfalfa produces a stable phytase. Biochem Biophys Res Commun, 2002, 290: 1343–1348

[6]Ullah A H, Sethumadhavan K, Mullaney E J. Fungal phyA gene expressed in potato leaves produces active and stable phytase. Biochem Biophys Res Commun, 2003, 306: 603–609

[7]Yu C-H(于彩虹), Tian S-T(田少亭), Lu X-B(路兴波), Li F(李凡), Yang S-K(杨淑珂), Sun H-W(孙红炜). Qualitative and quantitative PCR detection of the Phytase (phyA2) transgenic corn. J Agric Biotechnol (农业生物技术学报), 2012, 20(4): 356–361 (in Chinese with English abstract)

[8]Allnutt T R, Chisholm J, Hird H, Oehlschlager S, Henry C M. Plasmid Standards for Real Time PCR and GM Enforcement Testing. 2006, Certification Report

[9]Isabel T, Pieter W, Marc V, Anne M, Erik V B, Guy V D E, Marc D L. Event-specific plasmid standards and real-time PCR methods for transgenic Bt11, Bt176, and GA21 maize and transgenic GT73 Canola. J Agric Food Chem, 2005, 53: 3041–3052

[10]Yang L T, Pan A H, Zhang K W, Yin C S, Qian B J, Chen J X, Huang C, Zhang D B. Qualitative and quantitative PCR methods for event-speci?c detection of genetically modi?ed cotton Mon1445 and Mon531. Transgenic Res, 2005, 14: 817–831

[11]Li X, Shen K L, Yang L T, Wang S, Pan L W, Zhang D B. Applicability of a novel reference molecule suitable for event-specific detections of maize NK603 based on both 5' and 3' flanking sequences. Food Control, 2010, 21: 927–934

[12]Dong L-H(董莲华), Li L(李亮), Wang J(王晶). Plasmid construction and application of genetic modified maize line pNK603. J Agric Biotechnol (农业生物技术学报), 2011, 19(3): 565–570 (in Chinese with English abstract)

[13]Yang L T, Xu S C, Pan A H, Yin C S, Zhang K W, Wang Z Y, Zhou Z G, Zhang D B. Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified MON863 maize based on the 5'-transgene integration sequence. J Agric Food Chem, 2005, 53: 9312–9318

[14]Pan A H, Yang L T, Xu S C, Yin C S, Zhang K W, Wang Z Y, Zhang D B. Event-speci?c qualitative and quantitative PCR detection of MON863 maize based upon the 3'-transgene integration sequence. J Cereal Sci, 2006, 43: 250–257

[15]Lu J (陆姣), Yang R(杨蓉), Huang K-L(黄昆仑), Zhao W-W(赵薇维), Luo Y-B(罗云波), Yuan Y-F(元延芳), Xu W-T(许文涛). Construction of standard reference molecule and its application in event-specific transgenic detection of genetically modified maize 59122. Food Sci (食品科学), 2011, 32(2): 136–140 (in Chinese with English abstract)

[16]Qin W, Cao J J, Zhu S F. Identified and quantitative detection of the genetically modified maize Mon810 components in processed products. Food Sci, 2003, 24: 132–134

[17]Liu J, Guo J C, Zhang H B, Li N, Yang L T, Zhang D B. Development and in-house validation of the event-specific polymerase chain reaction detection methods for genetically modified soybean MON89788 based on the cloned integration flanking sequence. J Agric Food Chem, 2009, 57: 10524–10530

[18]Jae H K, Su Y K, Hyungjae L, Young R K, Hae Y K. An event-specific DNA microarray to identify genetically modified organisms in processed foods. J Agric Food Chem 2010, 58: 6018–6026

[19]Liu D E, Shen J, Yang L T, Zhang D B. Evaluation of the impacts of different nuclear DNA content in the hull, endosperm, and embryo of rice seeds on GM rice quantification. J Agric Food Chem, 2010, 58: 4582–4587

[20]Wu Y H, Wu G, Xiao L, Lu C M. Event-specific qualitative and quantitative PCR detection methods for transgenic rapeseed hybrids MS1 ? RF1 and MS1 ? RF2. J Agric Food Chem, 2007, 55: 8380–8389

[21]Hari K. S, Kae-Kang H, Wang S J, Liu L F, Chang M C. Simultaneous detection of eight genetically modified maize lines using a combination of event and construct specific multiplex-PCR technique. J Agric Food Chem, 2008, 56: 8962–8968

[22]Kuribara H, Shido Y, Matsuoka T, Takubo K, Futo S, Aoki N. Novel reference molecules for quantitation of genetically modified maize and soybean. J AOAC Int, 2002, 85: 1077–1089

[23]Yoshimura T, Kuribara H, Matsuoka T, Kadama T, Iida M, Watanabe T. Applicability of the quantification of genetically modified organisms to foods processed from maize and soy. J Agric Food Chem, 2005, 53: 2052–2059

[24]European Network of GMO Laboratories. Definition of minimum performance requirements for analytical methods of GMO testing. Ispra, Italy: Biotechnology and GMOs Unit-Community Reference Laboratory, Institute for Health and Consumer Protection, Joint Research Center, European Commission, 2008.

[1] ZHANG Guang-Yuan,SUN Hong-Wei,LI Fan,YANG Shu-Ke,LU Xing-Bo,ZHAO Lei. Event-Specific PCR Detection Method of Genetically Modified Maize MIR162 and Its Standardization [J]. Acta Agron Sin, 2013, 39(07): 1141-1147.
[2] LI Jun,LIU Xin,CAO Ying-Long,WU Yu-Hua,LI Jian-Meng,WU Gang,ZHANG Li,LU Chang-Ming. Establishment of Phytase-Specific Qualitative PCR Detection Method and Construction of a Positive Plasmid Molecule [J]. Acta Agron Sin, 2012, 38(04): 639-647.
[3] SU Chang-Qing, XIE Jia-Jian, SUN Xiao, PENG Yu-Fa. Construction and Application of a Reference Plasmid Suitable for Determination of CpTI and cry1A Gene Dosages in Genetically Modified Cottons [J]. Acta Agron Sin, 2011, 37(09): 1533-1539.
[4] WU Ying ; SONG Feng-Sun ; LU Xu-Zhong; ZHAO Wei; YANG Jian-Bo; LI Li ;. Detecting Genetically Modified Soybean by Real-time Quantitative PCR Technique [J]. Acta Agron Sin, 2007, 33(10): 1733-1737.
Full text



No Suggested Reading articles found!