Abstract
A transgenic maize event ZD12-6 expressing a Bacillus thuringiensis (Bt) fusion protein Cry1Ab/Cry2Aj and a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein G10 was characterized and evaluated. Southern blot analysis indicated that ZD12-6 is a single copy integration event. The insert site was determined to be at chromosome 1 by border sequence analysis. Expression analyses of Bt fusion protein Cry1Ab/Cry2Aj and the EPSPS protein G10 suggested that they are both expressed stably in different generations. Insect bioassays demonstrated that the transgenic plants are highly resistant to Asian corn borer (Ostrinia furnacalis), cotton boll worm (Helicoverpa armigera), and armyworm (Mythimna separata). This study suggested that ZD12-6 has the potential to be developed into a commercial transgenic line.
中文概要
目的
研究抗虫抗草甘膦玉米转化体ZD12-6 外源基因的分子特征,对ZD12-6的抗虫和抗草甘膦性状 进行综合评价,为ZD12-6产业化提供基础信息。
创新点
抗虫抗草甘膦转基因玉米ZD12-6是聚合了双抗 虫基因和抗草甘膦基因的转化体,多基因聚合有利于后期品种转育和多性状叠加。对该转化体的 分子特征信息分析和功能评价是评判其产业化价值的重要依据。
方法
利用DNA印迹法(Southern blot)研究外源基因插入拷贝数;利用高效热不对称交错聚合酶链反应(hiTAIL-PCR)方法对外源基因的插入位点进行定位;通过蛋白质印迹法(Western blot)对外源蛋白进行定性分析;利用酶联免疫吸附测定(ELISA)分析外源蛋白表达量;通过生物测定对抗虫和草甘膦的抗性水平进行评估。
结论
抗虫抗草甘膦玉米转化体ZD12-6中外源基因单 拷贝插入,插入位点位于玉米1号染色体。ZD12-6对玉米上的主要害虫具有良好抗性,同时对草甘膦具有较强耐受性,具有产业化推广潜力
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References
Carrillo L, Martinez M, Ramessar K, et al., 2011. Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteineproteases. Plant Cell Rep, 30(1):101–112. https://doi.org/10.1007/s00299-010-0948-z
Cattaneo MG, Yafuso C, Schmidt C, et al., 2006. Farm-scale evaluation of the impacts of transgenic cotton on biodiversity, pesticide use, and yield. Proc Natl Acad Sci USA, 103(20):7571–7576. https://doi.org/10.1073/pnas.0508312103
Chang X, Liu GG, He KL, et al., 2013. Efficacy evaluation of two transgenic maize events expressing fused proteins to Cry1Ab-susceptible and-resistant Ostrinia furnacalis (Lepidoptera: Crambidae). J Econ Entomol, 106(6):2548–2556. https://doi.org/10.1603/EC13100
Chen H, Tang W, Xu CG, et al., 2005. Transgenic indica rice plants harboring a synthetic cry2A* gene of Bacillus thuringiensis exhibit enhanced resistance against lepidopteran rice pests. Theor Appl Genet, 111(7):1330–1337. https://doi.org/10.1007/s00122-005-0062-8
Chen Y, Tian JC, Shen ZC, et al., 2010. Transgenic rice plants expressing a fused protein of Cry1Ab/Vip3H has resistance to rice stem borers under laboratory and field conditions. J Econ Entomol, 103(4):1444–1453. https://doi.org/10.1603/EC10014
Coll A, Nadal A, Collado R, et al., 2009. Gene expression profiles of MON810 and comparable non-GM maize varieties cultured in the field are more similar than are those of conventional lines. Transgenic Res, 18(5):801–808. https://doi.org/10.1007/s11248-009-9266-z
Du DX, Geng CJ, Zhang XB, et al., 2014. Transgenic maize lines expressing a cry1C* gene are resistant to insect pests. Plant Mol Biol Rep, 32(2):549–557. https://doi.org/10.1007/s11105-013-0663-3
Gouffon C, van Vliet A, van Rie J, et al., 2011. Binding sites for Bacillus thuringiensis Cry2Ae toxin on heliothine brush border membrane vesicles are not shared with Cry1A, Cry1F, or Vip3A toxin. Appl Environ Microbiol, 77(10):3182–3188. https://doi.org/10.1128/AEM.02791-10
Gould F, 1998. Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology. Annu Rev Entomol, 43:701–726. https://doi.org/10.1146/annurev.ento.43.1.701
He KL, Wang ZY, Zhou DR, et al., 2000. Methodologies and criterions for evaluating maize resistance to Asian maize borer. J Shenyang Agric Univ, 31(5):439–443 (in Chinese). https://doi.org/10.3969/j.issn.1000-1700.2000.05.009
He KL, Wang ZY, Wen LP, et al., 2004. Transgenic maize evaluated for resistance to the Asian corn borer (Lepidoptera: Pyralidae). Chin Agric Sci Bull, 20(6):240–242, 246 (in Chinese). https://doi.org/10.3969/j.issn.1000-6850.2004.06.077
Hernández-Rodríguez CS,van Vliet A, Bautsoens N, et al., 2008. Specific binding of Bacillus thuringiensis Cry2A insecticidal proteins to a common site in the midgut of Helicoverpa species. Appl Environ Microbiol, 74(24):7654–7659. https://doi.org/10.1128/AEM.01373-08
Hernández-Rodríguez CS, Hernández-Martínez P, van Rie J, et al., 2013. Shared midgut binding sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa proteins from Bacillus thuringiensis in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda. PLoS ONE, 8(7):e68164. https://doi.org/10.1371/journal.pone.0068164
Huang FN, Andow DA, Buschman LL, 2011. Success of the high-dose/refuge resistance management strategy after 15 years of Bt crop use in North America. Entomol Exp Appl, 140(1):1–16. https://doi.org/10.1111/j.1570-7458.2011.01138.x
Huang JK, Hu RF, Rozelle S, et al., 2005. Insect-resistant GM rice in farmers’ fields: assessing productivity and health effects in China. Science, 308(5722):688–690. https://doi.org/10.1126/science.1108972
Hunt TE, Buschman LL, Sloderbeck PE, 2007. Insecticide use in Bt and non-Bt field corn in the western corn belt: as reported by crop consultants in a mail survey. Am Entomol, 53(2):86–93. https://doi.org/10.1093/ae/53.2.86
Hutchison WD, Burkness EC, Mitchell PD, et al., 2010. Areawide suppression of European corn borer with Bt maize reaps savings to non-Bt maize growers. Science, 330(6001):222–225. https://doi.org/10.1126/science.1190242
James C, 2015. Global Status of Commercialized Biotech/GM Crops: 2015. ISAAA Brief No. 51, International Service for the Acquisition of Agri-biotech Applications (ISAAA), Ithaca, NY, USA.
Kota M, Daniell H, Varma S, et al., 1999. Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects. Proc Natl Acad Sci USA, 96(5):1840–1845. https://doi.org/10.1073/pnas.96.5.1840
Liu YG, Chen YL, 2007. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. BioTechniques, 43(5):649–656. https://doi.org/10.2144/000112601
Sambrook J, Russell DW, 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, New York.
Shen P, Zhang QY, Lin YH, et al., 2016. Thinking to promote the industrialization of genetically modified corn of our country. China Biotechnol, 36(4):24–29 (in Chinese). https://doi.org/10.13523/j.cb.20160404
Sohail MN, Karimi SM, Asad S, et al., 2012. Development of broad-spectrum insect-resistant tobacco by expression of synthetic cry1Ac and cry2Ab genes. Biotechnol Lett, 34(8):1553–1560. https://doi.org/10.1007/s10529-012-0923-6
Tabashnik BE, 1994. Evolution of resistance to Bacillus thuringiensis. Annu Rev Entomol, 39:47–79. https://doi.org/10.1146/annurev.en.39.010194.000403
Tabashnik BE, Gassmann AJ, Crowder DW, et al., 2008. Insect resistance to Bt crops: evidence versus theory. Nat Biotechnol, 26(2):199–202. https://doi.org/10.1038/nbt1382
Xu L, Wang Z, Zhang J, et al., 2010. Cross-resistance of Cry1Ab-selected Asian corn borer to other Cry toxins. J Appl Entomol, 134(5):429–438. https://doi.org/10.1111/j.1439-0418.2010.01517.x
Yang Z, Chen H, Tang W, et al., 2011. Development and characterisation of transgenic rice expressing two Bacillus thuringiensis genes. Pest Manag Sci, 67(4):414–422. https://doi.org/10.1002/ps.2079
Zhang Q, Yu H, Zhang FZ, et al., 2013. Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 14(10):867–874. https://doi.org/10.1631/jzus.B1300090
Zhao JZ, Cao J, Li YX, et al., 2003. Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nat Biotechnol, 21(12):1493–1497. https://doi.org/10.1038/nbt907
Zhao QC, Liu MH, Tan MM, et al., 2014. Expression of Cry1Ab and Cry2Ab by a polycistronic transgene with a self-cleavage peptide in rice. PLoS ONE, 9(10):e110006. https://doi.org/10.1371/journal.pone.0110006
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Project supported by the Fundamental Research Funds for the Central Universities (No. 2017FZA6011) and the National Key Transgenic Research Projects (No. 2016ZX08010003) of China
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Liu, Mm., Zhang, Xj., Gao, Y. et al. Molecular characterization and efficacy evaluation of a transgenic corn event for insect resistance and glyphosate tolerance. J. Zhejiang Univ. Sci. B 19, 610–619 (2018). https://doi.org/10.1631/jzus.B1700345
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DOI: https://doi.org/10.1631/jzus.B1700345