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作物学报 ›› 2011, Vol. 37 ›› Issue (09): 1533-1539.doi: 10.3724/SP.J.1006.2011.01533

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

一种适合转基因棉CpTI和cry1A基因剂量测定的标准质粒的构建和应用

苏长青1,2，谢家建1,*，孙爻1，彭于发1,*   

  1. 1 植物病虫害生物学国家重点实验室 / 农业部转基因植物环境安全监督检验测试中心(北京) / 中国农业科学院植物保护研究所, 北京100193; 2 衡水学院生命科学系，河北衡水053000
  • 收稿日期:2011-01-19 修回日期:2011-04-27 出版日期:2011-09-12 网络出版日期:2011-06-28
  • 通讯作者: 谢家建, E-mail: jjxie@ippcaas.cn; 彭于发, E-mail: yfpeng@ippcaas.cn
  • 基金资助:

    本研究由国家重点基础研究发展计划(973计划)项目(2007CB109201)和国家转基因新品种培育重大科技专项(2011ZX08012-002, 2011ZX08011-001)资助。

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 Published:2011-09-12 Published online:2011-06-28
  • Contact: 谢家建, E-mail: jjxie@ippcaas.cn; 彭于发, E-mail: yfpeng@ippcaas.cn

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被引次数

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摘要： 在转基因检测领域，标准质粒以容易获得、纯度高、成本低和稳定性好的优点逐渐被广泛应用，适合同时检测多个靶标基因的需求。本研究针对我国抗虫棉(Gossypium hirsutum L.)的主要外源基因类型，构建含有豇豆胰蛋白酶抑制剂基因(CpTI)、苏云金杆菌晶体杀虫蛋白基因(cry1A)和棉花内标准基因硬脂酰-酰基载体蛋白脱饱和酶基因(Sad1)的多靶标质粒pMD-CCS作为标准质粒，建立了CpTI和cry1A基因的实时荧光定量PCR方法。测定了我国9个抗虫棉品种中的CpTI和cry1A基因剂量，显示科棉3号等3个抗虫棉品种中CpTI基因的平均剂量为0.020~0.018拷贝/基因组，cry1A基因的平均剂量为1.377~2.136拷贝/基因组；鄂杂棉1号F1等6个抗虫棉品种中cry1A基因的平均剂量为0.887~2.564拷贝/基因组，定量结果的标准差(SD)范围在0.001~0.149之间。结果说明，pMD-CCS适合作为标准物质用于抗虫棉中CpTI和cry1A基因的定量测定。

关键词: 抗虫棉, CpTI基因, cry1A基因, 基因特异性实时PCR, 标准质粒

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
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