Abstract:Abstract The high-efficiency expression and stable inheritance of Bacillus thuringiensis gene (Bt) are crucial to transgenic rice breeding and commercial production. A transgenic line B2A68 (Oryza sativa) was obtained by introducing biplaphos resistance gene (Bar) and insecticidal crystal proteins gene (Cry2Aa) into the early season rice restorer line D68 via Agrobacterium mediated method. In this study, the temporal and spatial expression profile of Cry2Aa protein between homozygous and heterozygous genotype, different tissues and various developmental stages were studied by enzyme linked immunosorbent assay (ELISA), and the relationship between expression level, stage and tissue of insecticidal protein and insect resistance was revealed by referring bioassay results, in order to provide technical parameters for developing excellent lepidopteran resistant rice. The results showed that the expression level of Cry2Aa protein in homozygous plant leaf was significantly higher than that in heterozygous plant leaf (P<0.01). At milk stage, the contents of Cry2Aa protein in each organ were: leaf > glume and hulled grain > stem and sheath (P<0.05). At hard dough stage, the contents of Cry2Aa protein in each organ were: leaf > hulled grain > stem and glume (P<0.05), but there were no significant differences in the contents of Cry2Aa protein between hulled grain and sheath, sheath and stem, and stem and glume, respectively. From seedling to ripening phase, the Cry2Aa protein content in leaf increased first, then decreased, and later increased again, fluctuating in a same pattern in T3 and T4 generation. The mortality of striped rice borer larvae was highly correlated with the Cry2Aa protein content in different organs at milk stage (r=0.837), and the mortality of rice leaf roller larvae was highly correlated with the Cry2Aa protein content in leaves at different stages (r=0.988). For the heterozygous plant leaf with low Cry2Aa protein content, the lethal time of the rice leaf roller feeding with heterozygous plant leaf was slightly longer than that of feeding with homozygous plant leaf, but shorter than 5 days. This study will contribute to controlling Lepidopteran pests in field and developing transgenic rice in the future.
Alinia F, Ghareyazie B, Rubia L, et al. 2000. Effect of plant age, larval age, and fertilizer treatment on resistance of a Cry1Ab-transformed aromatic rice to lepidopterous stem borers and foliage feeders[J]. Journal of Economic Entomology, 93: 484-93.Bai Y, Jiang M, Cheng J. 2005. Temporal expression patterns of Cry1Ab insecticidal protein in Bt rice plants and its degradation in paddy soils[J]. Acta Ecologica Sinica, 25: 1583-90.Bakhsh A, Shahzad K, Husnain T. 2011. Variation in the spatio-temporal expression of insecticidal genes in cotton[J]. Czech Journal of Genetics and Plant Breeding, 47: 1-9.Bourdon V, Ladbrooke Z, Wickham A, et al. 2002. Homozygous transgenic wheat plants with increased luciferase activity do not maintain their high level of expression in the next generation[J]. Plant Science, 163: 297-305.Chandrasekaran S, Rajathi DS, Saravanan PA, et al. 2008. Pesticide residue, maximum residue limit (MRL) and safety to environment. Chen H, Tang W, Xu C, et al. 2005. Transgenic indica rice plants harboring a synthetic Cry2A* gene of Bacillus thuringiensis exhibit enhanced resistance against lepidopteran rice pests[J]. Theoretical and Applied Genetics, 111: 1330-1337.Duan X, Li X, Xue Q, et al. 1996. Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant[J]. Nature Biotechnology, 14: 494.Fujimoto H, Itoh K, Yamamoto M, et al. 1993. Insect resistant rice generated by introduction of a modified δ-endotoxin gene of Bacillus thuringiensis[J]. Bio/technolgy, 11: 1151-1155.German MA, Kandel-Kfir M, Swarzberg D, et al. 2003. A rapid method for the analysis of zygosity in transgenic plants[J]. Plant Science, 164: 183-187.Jin YM, Ma R, Yu ZJ, et al. 2015. Development of lepidopteran pest-resistant transgenic japonica rice harboring a synthetic Cry2A* gene[J]. Journal of Integrative Agriculture, 14: 423-429.Liu WQ, Meng QC, Weng LS, et al. 2016. A comparative study of two-line early season hybrid rice with lepidopteran resistance[J]. Filed Crops Research, 187:107-112.Manikandan R, Balakrishnan N, Sudhakar D, et al. 2016a. Development of leaffolder resistant transgenic rice expressing Cry2AX1 gene driven by green tissue-specific rbcS promoter[J]. World Journal of Microbiology and Biotechnology, 32: 37.Manikandan R, Balakrishnan N, Sudhakar D, et al. 2016b. Transgenic rice plants expressing synthetic cry2AX1 gene exhibits resistance to rice leaffolder (Cnaphalocrosis medinalis) [J]. Biotech, 6 (1): 10.Nayak P, Basu D, Das S, et al. 1997. Transgenic elite indica rice plants expressing CryIAc delta-endotoxin of Bacillus thuringiensis are resistant against yellow stem borer (Scirpophaga incertulas) [J]. Proceedings of the National Academy of Sciences of the United States of America, 94: 2111-2116.Ribeiro TP, Arraes FBM, Silva MS, et al. 2017. Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil[J]. Plant Biotechnol J, 15: 997-1009.Sridevi G, Parameswari C, Rajamuni P, et al. 2006. Identification of hemizygous and homozygous transgenic rice plants in T1 generation by DNA blot analysis[J]. Plant Biotechnology, 23: 531-34.Tang W, Chen H, Xu C, et al. 2006. Development of insect-resistant transgenic indica rice with a synthetic cry1C gene[J]. Molecular Breeding, 18: 1-10.Wang Z, Shu Q, Ye G, et al. 2002. Genetic analysis of resistance of Bt rice to stripe stem borer ( Chilo suppressalis ) [J]. Euphytica, 123: 379-386.Weng LS, Deng LH, Lai FX, et al. 2014. Optimization of the Cry2Aa gene and development of insect-resistant and herbicide-tolerant photoperiod-sensitive genic male sterile rice[J]. Czech Journal of Genetics & Plant Breeding, 50: 19-25.Wu G, Cui H, Shu Q, et al. 2001. Expression Patterns of cry1Ab Gene in Progenies of "Kemingdao" and the Resistance to Striped Stem Borer[J]. Scientia Agricultura Sinica, 34 (5): 496-501.Wu G, Cui H, Ye G, et al. 2002. Inheritance and expression of the Cry1Ab gene in Bt ( Bacillus thuringiensis) transgenic rice[J]. Theoretical and Applied Genetics, 104: 727-734.Ye GY, Shu QY, Cui HR, et al. 2000. A leaf-section bioassay for evaluating rice stem borer resistance in transgenic rice containing a synthetic Cry1Ab gene from Bacillus thuringiensis Berliner[J]. Bulletin of Entomological Research, 90: 179-182.Ye R, Huang H, Yang Z, et al. 2009. Development of insect-resistant transgenic rice with Cry1C*-free endosperm[J]. Pest Management Science, 65(9): 1015-1120.Zhang Y, Zhang J, Lan J, et al. 2016. Temporal and spatial changes in Bt toxin expression in Bt-transgenic poplar and insect resistance in field tests[J]. Journal of Forestry Research, 27: 1249-1256.Zhao QC, Liu MH, Zhang XW, et al. 2015. Generation of insect-resistant and glyphosate-tolerant rice by introduction of a T-DNA containing two Bt insecticidal genes and an EPSPS gene[J]. Journal of Zhejiang University-SCIENCE B, 16(10): 824-831.崔旭红, 张国安. 转Bt基因水稻对水稻螟虫和稻纵卷叶螟的抗性[J]. 中国农学通报, 2008, 24(1): 355-358.(Cui XH, Zhang GA. 2008. Resistance of transgenic Bt rice against Chilo suppressalis, Scirpophaga incertulas and Cnaphalocrocis medinalis[J]. Chinese Agricultural Science Bulletin, 24(1): 355-358.)蒋利平, 翁绿水, 肖国樱. 转基因水稻B2A68事件特异性检测方法的建立[J]. 杂交水稻, 2013, (5): 60-67.(Jang LP, Weng LS, Xiao GY. 2013. Establishment of an event-specific method to detect transgenic rice B2A68[J]. Hybrid rice, (5): 60-67.)李冬虎, 傅强, 王锋,等. 转sck/crylAc双基因抗虫水稻对二化螟和稻纵卷叶螟的抗虫效果[J]. 中国水稻科学, 2004, 18(1): 43-47.(Li DH,Fu Q, Wang F, Yao Q, Lai FX, Wu JC, Zhang ZT. 2004. Resistance of transgenic rice containing both sck and cry1Ac genes against Chilo suppressalis and Cnaphalocrocis medinalis[J]. Chinese J Rice Sci, 18(1): 43-47.)翁绿水, 蒋利平, 肖国樱. 抗虫抗除草剂转基因水稻恢复系B2A68的培育[J]. 杂交水稻, 2013, 28(1): 63-67.(Weng LS, Jiang LP, Xiao GY. 2013. Development of an insect-resistant and herbicide-resistant transgenic restorer line B2A68 in rice[J]. Hybrid Rice, 28(01): 63-67.)