转cry1Ab+cry1C双价抗虫水稻对二化螟的抗性评价

doi:10.16409/j.cnki.2095-039x.2018.01.008

摘要/Abstract

摘要： 为明确转cry1Ab+cry1C双价抗虫水稻对二化螟的抗性及其杀虫蛋白的时空表达，本研究采用离体水稻组织生测法系统评价了cry1Ab、cry1C、正交cry1Ab+cry1C和反交cry1C+cry1Ab 4种抗虫水稻品系对二化螟的杀虫效果，并用ELISA方法测定了Cry1Ab、Cry1C蛋白在各个抗虫水稻品系中的表达情况。结果显示，二化螟在不同生育期的4种转基因抗虫水稻上取食的叶面积显著低于非转基因对照亲本明恢63。4个转基因抗虫水稻品系在生长前期（苗期、分蘖和拔节期）对二化螟表现极高的杀虫效果，生长后期（孕穗期和成熟期）杀虫效果有所下降。两双价抗虫水稻杀虫效果最好，其次为转cry1Ab和cry1C水稻。Bt蛋白表达水平随着抗虫水稻生育期的变化而变化，且差异显著；Cry1Ab的蛋白表达量在水稻整个生长期均显著高于Cry1C。Cry1Ab蛋白在单价抗虫水稻叶片和茎杆中均表现出随生育期先升高后下降的趋势，但在双价抗虫水稻中表现出随生育期逐渐下降的趋势。Cry1C蛋白在单、双价抗虫水稻的叶片组织中均表现出逐渐升高的趋势。与单价抗虫水稻相比，双价抗虫水稻中的Cry1Ab、Cry1C的蛋白表达水平并没有表现出显著的降低。因此，双价抗虫水稻不仅能高效防治害虫，还能延缓害虫抗性，在生产上表现出良好的应用前景。该研究结果可望为转Bt基因抗虫作物的环境安全评价提供科学数据和理论依据。

关键词: 二化螟, 转基因抗虫水稻, 抗虫效率, 杀虫蛋白表达

Abstract: Control efficacies of four transgenic rice lines, cry1Ab, cry1C, cry1Ab+cry1C and cry1C+cry1Ab, against the main target pest, Chilo suppressalis, were evaluated through detached rice tissues bioassay, and Cry1Ab and Cry1C protein contents in these transgenic lines were measured by enzyme-linked immunosorbent assays. The results showed cumulative feeding areas of C. suppressalis feeding on transgenic rice lines for 48 h and 96 h were significantly lower than those on non-transgenic rice line, MH63. Four insect-resistant transgenic rice lines presented very high resistant level against C. suppressalis at early developmental stage (seedling, tillering and elongation stages), while control efficacy of transgenic lines obviously decreased at later growth stages (booting and maturing stages). Double insect-resistant genes pyramided rice lines exhibited the highest resistant level among the four transgenic lines, second followed by cry1Ab and cry1C lines. Both Cry1Ab and Cry1C protein levels showed significantly different in respective transgenic rice lines at five different developmental stages. Additively, Cry1Ab protein expressing levels in transgenic lines were significantly higher than Cry1C protein. Cry1Ab levels were shown firstly increased and then decreased with the increase of growth stages in single-gene line, however, they presented the gradually decreasing trend with the increasing developmental stages both in leaf and stem tissues in double-gene lines. Cry1C levels showed the gradually rising trend with the increasing developmental stages in leaf tissues both in single-and double-gene lines. Moreover, Cry1Ab or Cry1C levels did not exhibit the significant decrease in double-gene lines, when compared with that in single-gene lines. Therefore, two-gene pyramided plants have robust prospects for commercial use, because they not only provide the efficient control against the pests, but also delay the insect resistance evolution. These results will provide the scientific data and theoretical basis for environmental safety assessment of transgenic Bt crops.

Key words: Chilo suppressalis, insect-resistant transgenic rice, control efficacy, insecticidal protein expression

中图分类号:

S435.112

陈耕, 何珊, 韩兰芝, 彭于发. 转cry1Ab+cry1C双价抗虫水稻对二化螟的抗性评价[J]. 中国生物防治学报, 2018, 34(1): 71-78.

CHEN Geng, HE Shan, HAN Lanzhi, PENG Yufa. Evaluation of Transgenic cry1Ab+cry1C Rice Lines for Its Resistance to Chilo suppressalis[J]. journal1, 2018, 34(1): 71-78.

参考文献

[1] 全国农业技术推广服务中心病虫测报处. 2015年全国农作物重大病虫害发生趋势预[J]. 中国植保导刊, 2015, 35(2):43-48.
[2] He Y, Zhang J, Gao C, et al. Regression analysis of dynamics of insecticide resistance in field populations of Chilo suppressalis (Lepidoptera:Crambidae) during 2002-2011 in China[J]. Journal of Economic Entomology, 2013, 106(4):1832-1837.
[3] Chen M, Shelton A, Ye G Y. Insect-resistant genetically modified rice in China:from research to commercialization[J]. Annual Review of Entomology, 2011, 56(1):81-101.
[4] Han L Z, Jiang X F, Peng Y F. Potential resistance management for the sustainable use of insect-resistant genetically modified corn and rice in China[J]. Current Opinion Insect Science, 2016, 15:139-143.
[5] Li Y H, Hallerman E M, Liu Q S, et al. The development and status of Bt rice in China[J]. Plant Biotechnology Journal, 2016, 14(3):839-848.
[6] Liu Q S, Hallerman E, Peng Y F, et al. Development of Bt rice and Bt maize in China and their efficacy in target pest control[J]. International Journal of Molecular Sciences, 2016, 17(1561):1561-1576.
[7] Tabashnik B E, Gassmann A J, Crowder D W, et al. Insect resistance to Bt crops:evidence versus theory[J]. Nature Biotechnology, 2008, 26(2):199-202.
[8] Zhao J Z, Cao J, Li Y X, et al. Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution[J]. Nature Biotechnology, 2003, 21(12):1493-1497.
[9] Bravo A, Soberón M. How to cope with insect resistance to Bt toxins[J]. Trends in Biotechnology, 2008, 26(10):573-579.
[10] Bates S L, Zhao J Z, Roush R T, et al. Insect resistance management in GM crops:past, present and future[J]. Nature Biotechnology, 2005, 23(1):57-62.
[11] Tabashnik B E, Brévault T, Carrière Y. Insect resistance to Bt crops:lessons from the first billion acres[J]. Nature Biotechnology, 2013, 31(6):510-521.
[12] Storer N P, Thompson G D, Head G P. Application of pyramided traits against Lepidoptera in insect resistance management for Bt crops[J]. GM Crops and Food:Biotechnology in Agriculture and the Food Chain, 2012, 3(3):154-162.
[13] Yang Z, Chen H, Tang W, et al. Development and characterisation of transgenic rice expressing two Bacillus thuringiensis genes[J]. Pest Management Science, 2011, 67(4):414-422.
[14] Abbott W S. A method of computing the effectiveness of an insecticide[J]. Journal of Economic Entomology, 1925, 18(2):265-267.
[15] SAS Institute. JMP® Statistics and Graphics Guide, Version 4[M]. Cary, NC, USA, 2000.
[16] 彭于发, 张永军, 刘信, 等. 中华人民共和国国家标准农业部953号公告:转基因植物及其产品环境安全检测抗虫水稻-第1部分:抗虫性[R]. 2007-12-18.
[17] Alcantara E P, Aguda R M, Curtiss A, et al. Bacillus thuringiensis δ-endotoxin binding to brush border membrane vesicles of rice stem borers[J]. Archives of Insect Biochemistry and Physiology, 2004, 55(4):169-177.
[18] Fiuza L M, Nielsen-Leroux C, Gozé E, et al. Binding of Bacillus thuringiensis Cry1 toxin to the midgut brush border membrane vesicles of Chilo suppressalis (Lepidoptera:Pyralidae):evidence of shared binding sites[J]. Applied and Environmental Microbiology, 1996, 62(5):1544-1549.
[19] Wang Y N, Zhang L, Li Y H, et al. Expression of Cry1Ab protein in a marker-free transgenic Bt rice line and its efficacy in controlling a target pest, Chilo suppressalis (Lepidoptera:Crambidae)[J]. Environmental Entomology, 2014, 43(2):528-536.
[20] Wang Y N, Ke K Q, Li Y H, et al. Comparison of three transgenic Bt rice lines for insecticidal protein expression and resistance against a target pest, Chilo suppressalis[J]. Insect Science, 2016, 23(1):78-87.
[21] Christensen A H, Sharrock R A, Quail P H. Maize polyubiquitin genes:structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation[J]. Plant Molecular Biology, 1992, 18(4):675-689.
[22] Cornejo M J, Luth D, Blankenship K M, et al. Activity of maize ubiquitin promoter in transgenic rice[J]. Plant Molecular Biology, 1993, 23(3):567-581.
[23] Matzke A J, Neuhuber F, Park Y D, et al. Homology-dependent gene silencing in transgenic plants:epistatic silencing loci contain multiple copies of methylated transgenes[J]. Molecular Genetics and Genomics, 1994, 244(3):219-229.
[24] 何珊, 徐杨洋, 韩兰芝, 等. 二化螟Cry1Ac汰选品系对不同Bt蛋白的敏感性[J]. 植物保护学报, 2017, 44(3):371-376.
[25] Han L Z, Han C, Liu Z W, et al. Binding site concentration explains the differential susceptibility of Chilo suppressalis and Sesamia inferens to Cry1A-producing rice[J]. Applied and Environmental Microbiology, 2014, 80(16):5134-5140.