亚洲玉米螟miR-1a-5p表达与Cry1Ab蛋白敏感性的关系

Relationship between miR-1a-5p expression and sensitivity of Cry1Ab protein of Ostrinia furnacalis

【目的】探索亚洲玉米螟miR-1a-5p表达与Cry1Ab蛋白敏感性的关系,为揭示miRNA在昆虫Bt抗性产生过程中的作用提供依据。【方法】通过荧光定量PCR,研究亚洲玉米螟Cry1Ab抗性品系(ACB-AbR)和Bt敏感品系(ACB-BtS)表皮及中肠组织中miR-1a-5p的表达差异。以Sf9细胞为研究对象,通过转染miR-1a-5p促进剂(Mimics)或抑制剂(Inhibitor)改变细胞中miR-1a-5p的表达量,测定不同处理细胞对Cry1Ab蛋白的敏感性,探讨miR-1a-5p表达量改变后Sf9细胞对Cry1Ab蛋白的敏感性变化。通过荧光素酶报告试验,研究miR-1a-5p对潜在靶标基因Unigene12370_All的调控作用。【结果】ACB-AbR表皮组织中miR-1a-5p的表达量显著高于ACB-BtS表皮组织,ACB-AbR中肠组织中miR-1a-5p的表达量显著低于ACB-BtS中肠组织;ACB-AbR和ACB-BtS幼虫表皮组织中miR-1a-5p的表达量均显著高于其中肠组织。Sf9细胞miR-1a-5p表达量升高导致Sf9细胞对Cry1Ab蛋白的敏感性增强,反之亦然。荧光素酶报告试验结果提示,miR-1a-5p与靶标基因Unigene12370_All可以结合。【结论】亚洲玉米螟miR-1a-5p表达量的变化与其对Cry1Ab蛋白敏感性的改变有关。

【Objective】This study explored the relationship between Ostrinia furnacalis miR-1a-5p expression and Cry1Ab sensitivity to provide basis for clarifying the mechanism of Bt resistance in insects.【Method】The expression levels of miR-1a-5p in cuticle and midgut of ACB-AbR and ACB-BtS were determined by real-time quantitative PCR.The sensitivity of Sf9 cells to Cry1Ab protein was analyzed by cell transfected with miR-1a-5p mimics or inhibitor and Cry1Ab protein sensitivity bioassay.The regulatory effect of miR-1a-5p on target transcript Unigene12370_All was verified by the dual luciferase reporting test.【Result】The expression level of miR-1a-5p in cuticle of ACB-AbR was significantly higher than that in cuticle of ACB-BtS.In midgut of ACB-AbR,the expression level of miR-1a-5p was significantly lower than that in midgut of ACB-BtS.The expression levels of miR-1a-5p in cuticle of ACB-AbR and ACB-BtS larvae were significantly higher than those in midgut.Transfection with miR-1a-5p mimics increased the expression of miR-1a-5p in Sf9 cells,resulting in increased sensitivity to Cry1Ab protein.Conversely,the transfection with miR-1a-5p inhibitor decreased the expression of miR-1a-5p in Sf9,and the sensitivity to Cry1Ab insecticidal protein decreased.Luciferase assay showed that miR-1a-5p could bind with the transcript Unigene12370_All.【Conclusion】The difference of miR-1a-5p in O.furnacalis were related to the changes of O.furnacalis to Cry1Ab.