沉默尿苷二磷酸葡萄糖醛酸转移酶基因OfUGT逆转亚洲玉米螟对Cry1Ie蛋白的抗性

doi:10.16409/j.cnki.2095-039x.2022.11.006

摘要/Abstract

摘要： 转Bacillus thuringiensis(Bt)基因作物在害虫防治中应用较多,但长期种植转Bt基因作物,靶标害虫不可避免会对杀虫蛋白产生抗性,如何进行抗性治理是当前面临的难题。本研究基于前期转录组分析结果,定量分析了尿苷二磷酸葡萄糖醛酸转移酶基因(OfUGT)在亚洲玉米螟Cry1Ie抗性品系(ACB-IeR)及敏感品系(ACB-BtS)的表达量,利用RNA干扰技术(RNAi)对ACB-IeR品系OfUGT基因进行沉默,并通过毒力测定试验来检验干扰效果。OfUGT基因在ACB-IeR三龄幼虫中相对表达量高于ACB-BtS,并且注射dsUGT后该基因表达量下降。Cry1Ie抗性品系的致死中浓度大于1000.00 µg/g,在蛋白浓度为500.00µg/g时,注射dsUGT和dsGFP后的幼虫存活率没有显著性差异,但较空白对照组存活率显著降低;在蛋白浓度为1000.00 µg/g时,注射dsGFP后的幼虫存活率较空白对照组降低,注射dsUGT后的幼虫存活率较空白对照和注射dsGFP的都显著降低。由结果可以看出RNAi沉默OfUGT基因后可以降低抗性品系亚洲玉米螟对Cry1Ie蛋白的抗性,但对抗性的逆转程度还需要后续更进一步的研究。

关键词: 亚洲玉米螟, 尿苷二磷酸葡萄糖醛酸转移酶, RNA干扰, Cry1Ie, 抗性逆转

Abstract: Bacillus thuringiensis (Bt) genetically modified crops are widely used in pest control, but with long-term planting of Bt genetically modified crops, the target pests will inevitably develop resistance to insecticidal proteins, and how to management the resistance is a difficult problem. Based on the results of previous transcriptome analysis, this study quantitatively analyzed the expression of UDP-glucuronyltransferase gene (OfUGT) in Cry1Ie resistant Asian corn borer strain (ACB-IeR) and susceptible strain (ACB-BtS). The OfUGT gene in ACB-IeR was silenced by RNA interference (RNAi) technology, and the effect of silencing OfUGT gene on ACB-IeR was evaluated by bioassay. The relative expression of the OfUGT gene in ACB-IeR third instar larvae was higher than that in ACB-BtS, and decreased after injection of dsUGT. The lethal concentration of the protein was greater than 1000.00 µg/g in the Cry1Ie-resistant strain. When the protein concentration was 500.00 µg/g, the survival rates of the dsUGT and dsGFP injected larvae did not have significant difference with control group. When the protein concentration was 1000.00 µg/g, the survival rate of the dsGFP injected larvae was lower than control group, and the survival rate of dsUGT injected larvae was significantly lower than the control and dsGFP injected group. The results show that RNAi of the OfUGT gene could reduce the resistance of the resistant line of Asian corn borer to the Cry1Ie protein, but the reversal degree of resistance needs to be further studied.

Key words: Ostrinia furnacalis, UDP-glucuronyltransferase, RNA interference, Cry1Ie, resistance reversal

中图分类号:

S435.132

林雅玲, 何康来, 王振营, 尚素琴, 张天涛. 沉默尿苷二磷酸葡萄糖醛酸转移酶基因OfUGT逆转亚洲玉米螟对Cry1Ie蛋白的抗性[J]. 中国生物防治学报, 2023, 39(4): 834-841.

LIN Yaling, HE Kanglai, WANG Zhenying, SHANG Suqin, ZHANG Tiantao. Silencing of the UDP-glucuronyltransferase Gene (OfUGT) Reverses the Resistance of Ostrinia furnacalis to Cry1Ie[J]. Chinese Journal of Biological Control, 2023, 39(4): 834-841.

参考文献

[1] 宋立秋, 魏利民, 王振营, 等. 亚洲玉米螟与串珠镰孢菌复合侵染对玉米产量损失的影响[J]. 植物保护学报, 2009, 36(6):487-490.
[2] Jurat-Fuentes J L, Heckel D G, Ferré J. Mechanisms of resistance to insecticidal proteins from Bacillus thuringiensis[J]. Annual Review of Entomology, 2021, 66:121-140.
[3] Xu L, Ferry N, Wang Z, et al. A proteomic approach to study the mechanism of tolerance to Bt toxins in Ostrinia furnacalis larvae selected for resistance to Cry1Ab[J]. Transgenic Research, 2013, 22(6):1155-1166.
[4] Comas C, Lumbierres B, Pons X, et al. No effects of Bacillus thuringiensis maize on nontarget organisms in the field in southern Europe:a meta-analysis of 26 arthropod taxa[J]. Transgenic Research, 2014, 23(1):135-143.
[5] Gould F. Sustainability of transgenic insecticidal cultivars:integrating pest genetics and ecology[J]. Annual Review of Entomology, 1998, 43:701-726.
[6] Morin S, Biggs R, Sisterson M, et al. Three cadherin alleles associated with resistance to Bacillus thuringiensis in pink bollworm[J]. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100:5004-5009.
[7] 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.
[8] Carrière Y, Crickmore N, Tabashnik B E. Optimizing pyramided transgenic Bt crops for sustainable pest management[J]. Nature Biotechnology, 2015, 33(2):161-168.
[9] Kaduskar B, Kushwah R B S, Auradkar A, et al. Reversing insecticide resistance with allelic-drive in Drosophila melanogaster[J]. Nature Communications, 2022, 13(1):1-8.
[10] Howe G A, Herde M. Interaction of plant defense compounds with the insect gut:new insights from genomic and molecular analyses[J]. Current Opinion in Insect Science, 2015, 9:62-68.
[11] 陈欣, 付锐锐, 张鸿, 等. 药用植物中UGT家族研究进展[J]. 生物资源, 2018, 40(5):443-449.
[12] De Bruyn F, Maertens J, Beauprez J, et al. Biotechnological advances in UDP-sugar based glycosylation of small molecules[J]. Biotechnology Advances, 2015, 33(2):288-302.
[13] Tephly T R, Burchell B. UDP-glucuronosyltransferases:a family of detoxifying enzymes[J]. Trends in Pharmacological Sciences, 1990, 11(7):276-279.
[14] Ross J, Li Y, Lim E K, et al. Higher plant glycosyltransferases[J]. Genome Bioogy, 2001, 2(2):1-6.
[15] Mackenzie P I, Bock K W, Burchell B, et al. Nomenclature update for the mammalian UDP glycosyltransferase (UGT) gene superfamily[J]. Pharmacogenetics and genomics, 2005, 15(10):677-685.
[16] Israni B, Wouters F C, Luck K, et al. The fall armyworm Spodoptera frugiperda utilizes specific UDP-glycosyltransferases to inactivate maize defensive benzoxazinoids[J]. Frontiers in Physiology, 2020, 11:604754.
[17] Andrade E D, Hunter W B. RNA interference-natural gene-based technology for highly specific pest control (HiSPeC)[M]. RNA Interference, 2016, 391-409.
[18] Cagliari D, Dias N P, Galdeano D M, et al. Management of pest insects and plant diseases by non-transformative RNAi[J]. Frontiers in Plant Science, 2019, 10:1319.
[19] Yang J, Sun X Q, Yan S Y, et al. Interaction of ferulic acid with glutathione S-transferase and carboxylesterase genes in the brown planthopper, Nilaparvata lugens[J]. Journal of Chemical Ecology, 2017, 43(7):693-702.
[20] 靳婷婷, 戈峰, 吴杰. 亚洲玉米螟幼虫膜结合海藻糖酶基因RNAi效应[J]. 中国生物防治学报, 2020, 36(3):452-457.
[21] Lin Y, Gao Q, Wang Y, et al. Comparative transcriptome analysis of Bt resistant and susceptible strains in Ostrinia furnacalis (Guenée)(Lepidoptera:Crambidae)[J]. Agriculture, 2022, 12(2):298.
[22] 宋彦英, 周大荣, 何康来. 亚洲玉米螟无琼脂半人工饲料的研究与应用[J]. 植物保护学报, 1999, 26(4):324-328.
[23] Liu Y, Shen D, Zhou F, et al. Identification of immunity-related genes in Ostrinia furnacalis against entomopathogenic fungi by RNA-seq analysis[J]. PLoS ONE, 2014, 9(1):e86436.
[24] Livak K, Schmittgen T. Analysis of relative gene expression data using real-time quantitative PCR and the 2^{− ΔΔCT} method[J]. Methods, 2001, 25(4):402-408.
[25] Ingham V A, Tennessen J A, Lucas E R, et al. Integration of whole genome sequencing and transcriptomics reveals a complex picture of the reestablishment of insecticide resistance in the major malaria vector Anopheles coluzzii[J]. PLoS Genetics, 2021, 17(12):e1009970.
[26] Heidel-Fischer H M, Vogel H. Molecular mechanisms of insect adaptation to plant secondary compounds[J]. Current Opinion in Insect Science, 2015, 8:8-14.
[27] 王文龙. 菜粉蝶三大解毒酶基因的鉴定及GST基因表达模式分析[D]. 合肥:安徽农业大学, 2018.
[28] Despres L, David J P, Gallet C. The evolutionary ecology of insect resistance to plant chemicals[J]. Trends in Ecology & Evolution, 2007, 22(6):298-307.
[29] Li X, Shi H, Gao X, et al. Characterization of UDP-glucuronosyltransferase genes and their possible roles in multi-insecticide resistance in Plutella xylostella (L.)[J]. Pest Management Science, 2018, 74(3):695-704.
[30] Kojima W, Fujii T, Suwa M, et al. Physiological adaptation of the Asian corn borer Ostrinia furnacalis to chemical defenses of its host plant, maize[J]. Journal of Insect Physiology, 2010, 56(9):1349-1355.
[31] Pan Y, Wen S, Chen X, et al. UDP-glycosyltransferases contribute to spirotetramat resistance in Aphis gossypii Glover[J]. Pesticide Biochemistry and Physiology, 2020, 166:104565.
[32] Li X, Zhu B, Gao X, et al. Over-expression of UDP-glycosyltransferase gene UGT2B17 is involved in chlorantraniliprole resistance in Plutella xylostella (L.)[J]. Pest Management Science, 2017, 73(7):1402-1409.
[33] Du T, Fu B, Wei X, et al. Knockdown of UGT352A5 decreases the thiamethoxam resistance in Bemisia tabaci (Hemiptera:Gennadius)[J]. International Journal of Biological Macromolecules, 2021, 186:100-108.