Welcome to Acta Agronomica Sinica,

Acta Agron Sin ›› 2011, Vol. 37 ›› Issue (09): 1533-1539.doi: 10.3724/SP.J.1006.2011.01533

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Construction and Application of a Reference Plasmid Suitable for Determination of CpTI and cry1A Gene Dosages in Genetically Modified Cottons

SU Chang-Qing1,2,XIE Jia-Jian1,*,SUN Yao1,PENG Yu-Fa1,*   

  1. 1 State Key Laboratory for Biology of Plant Diseases and Insect Pests / Institute of Plant Protection, China Inspection Test Center for Environmental Safety of Transgenic Crops, Ministry of Agriculture / Chinese Academy of Agricultural Sciences, Beijing 100193, China; 2 Department of Life Science, Hengshui College in Hebei, Hengshui 053000, China
  • Received:2011-01-19 Revised:2011-04-27 Online:2011-09-12 Published:2011-06-28
  • Contact: 谢家建, E-mail: jjxie@ippcaas.cn; 彭于发, E-mail: yfpeng@ippcaas.cn

Abstract: In transgenic detection field, a preferable reference material (RM) has been developing with the advantage of easy availability, high purity, low cost and good stability, which is more suitable for detecting multiple target genes. In the present study, we constructed a multi-target reference plasmid named pMD-CCS containing cowpea trypsin inhibitor (CpTI) and Bacillus thuringiensis insecticidal crystal protein (cry1A) and cotton endogenous gene Stearoyl-acyl carrier protein desaturase (Sad1) sequencestargeting the key exogenous gene types of the insect resistant cotton varieties (Gossypium hirsutum L.) in China. The real-time quantitative PCR methods for CpTI and cry1A were established using pMD-CCS as the RM.Thedosagess of CpTI and cry1A from nine insect resistant cotton varieties were determined. The average CpTI dosages were 0.020–0.018 copies/genome and the average cry1A dosages were 1.377–2.136 copies/genome in three insect resistant cotton varieties including Kemian3. The average cry1A dosages were 0.887–2.564 copies/genome in six ones including Ezamian1-F1. The standard deviations (SD) of the quantitative measurement ranged from 0.001–0.049. The above results demonstrated that pMD-CCS could be used as the RM for the quantitative measurement of CpTI and cry1A genes in insect resistant cotton varieties.

Key words: Insect resistant cotton, CpTI gene, cry1A gene, Gene-specific real-time PCR, Reference plasmid

[1]James C. Global status of commercialized biotech/GM crops: ISAAA Briefs. No.42, 2010. ISAAA: Ithaca, NY
[2]Guo S-D(郭三堆), Ni W-C(倪万潮), Xu Q-F(徐琼芳). Expressive carrier with coded insect-killing protein fusion gene, and transfer gene plant. China, 1995, patent number: 95119563.8 (in Chinese)
[3]Guo S-D(郭三堆), Cui H-Z(崔洪志), Xu Q-F(徐琼芳), Ni W-C(倪万潮). Expressive carrier with coded two insect-killing protein fusion genes, and transfer gene plant. China, 1998, Patent number: ZL98102885.3 (in Chinese)
[4]Guo S-D(郭三堆), Cui H-Z(崔洪志), Xia L-Q(夏兰芹), Wu D-L(武东亮), Ni W-C(倪万潮), Zhang Z-L(张震林), Zhang B-L(张宝龙), Xu Y-J(徐英俊). Development of Bivalent Insect-resistant transgenic cotton plants. Sci Agric Sin (中国农业科学), 1999, 32(3): 1–7 (in Chinese with English abstract)
[5]Huang J K, Rozelle S, Pray C, Wang Q F. Plant biotechnology in China. Science, 2002, 295: 674–677
[6]http://www.stee.agri.gov.cn/biosafety/spxx/P020091127591594596689.pdf
[7]Elenis D S, Kalogianni D P, Glynou K, Ioannou P C, Christopoulos T K. Advances in molecular techniques for the detection and quantification of genetically modified organisms. Anal Bioanal Chem, 2008, 392: 347–354
[8]Kuribara H, Shindo Y, Matsuoka T, Takubo K, Futo S, Aoki N, Hirao T, Akiyama H, Goda Y, Toyada M, Hino A. Novel reference molecules for quantification of genetically modified maize and soybean. J AOAC Int, 2002, 85: 1077–1089
[9]Yang L T, Pan A H, Zhang K W, Guo J C, Yin C S, Chen J X, Huang C, Zhang D B. Identification and quantification of three genetically modified insect resistant cotton lines using conventional and Taqman real-time polymerase chain reaction methods. J Agric Food Chem, 2005, 53: 6222–6229
[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]Yang L T, Guo J C, Pan A H, Zhang H B, Zhang K W, Wang Z M, Zhang D B. Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule. J Agric Food Chem, 2007, 55: 15–24
[12]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-speci?c detections of maize NK603 based on 5' and 3' flanking sequences. Food Control, 2010, 21: 927–934
[13]Yang L T, Chen J X, Huang C, Liu Y H, Jia S R, Pan L W, Zhang D B. Validation of a cotton-speci?c gene, sad1, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic cottons. Plant Cell Rep, 2005 24: 237–245
[14]Arumuganathan K, Earle E D. Nuclear DNA content of some important plant species. Plant Mol Biol Rep, 1991, 9: 208–218
[15]Marmiroli N, Maestri E, Gullì M, Malcevschi A, Peano C, Bordoni R, Bellis G D. Methods for detection of GMOs in food and feed. Anal Bioanal Chem, 392: 369–384
[16]Flavell R B. Inactivation of gene expression in plants as a consequence of specific sequence duplication. Proc Natl Acad Sci USA, 1994, 91: 3490–3496
[17]Vaucheret H, Béclin C, Elmayan T, Feuerbach F, Godon C, Morel J B, Mourrain P, Palauqui J C, Vernhetters S. Transgene-induced gene silencing in plants. Plant J, 1998, 16: 651–659
[18]Mason G, Provero P, Vaira A M, Accotto G P. Estimating the number of integrations intransformed plants by quantitative real-time PCR. BMC Biotechnol, 2002, 2: 20
[19]Livak K J, Schmittgen T D. Analysis of relative gene expression data using Real-time quantitative PCR and the 2–ΔΔCT method. Methods, 2001, 25: 402–408
[20]Song P, Cai C Q, Skokut M, Kosegi B D, Petolino J F. Quantitative real-time PCR as a screening tool for estimating transgene copy number in WHISKERS™-derived transgenic maize. Plant Cell Rep, 2002, 20: 948–954
[21]Yang L T, Ding J Y, Zhang C M, Jia J W, Weng H B, Liu W X, Zhang D B. Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR. Plant Cell Rep, 2005, 23: 759–763
[22]Ginzinger D G. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol, 2002, 30: 503–512
[23]Hernandez M, Pla M, Esteve T, Prat S, Puigdomenech P, Ferrando A. A specific real-time quantitative PCR detection system for event MON810 in maize YieldGard based on the 3?-transgene integration sequence. Transgenic Res, 2003, 12: 179–189
[24]Rønning S B, Vaïtilingom M, Berdal K G, Holst-Jensen A. Event specific real-time quantitative PCR for genetically modified Bt11 maize (Zea mays). Eur Food Res Technol, 2003, 216: 347–354
[25]Jiang L X, Yang L T, Rao J, Guo J C, Wang S, Liu J, Lee S H, Zhang D B. Development and in-house validation of the event-speci?c qualitative and quantitative PCR detection methods for genetically modi?ed cotton MON15985. J Sci Food Agric, 2010, 90: 402–408
[1] WU Zheng-Bin; CHEN Peng; YANG Ye-Hua; SHU Yu-Song;XIE Hong-Bin. Evaluation of the Resistance of Different Insect-resistant Cotton Cultivars to the Pink Bollworm [J]. Acta Agron Sin, 2005, 31(01): 53-57.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!