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【目的】通过对棉铃虫Polycalin基因的克隆及其表达谱分析,进一步明确Polycalin基因在抗性中发挥的作用,为棉铃虫Helicoverpa armigera的抗性治理提供理论依据。【方法】通过RACE结合PCR技术克隆获得了棉铃虫Polycalin基因的全长序列,利用实时荧光定量PCR技术测定了在棉铃虫不同发育时期、幼虫肠道不同部位的Polycalin基因表达量,比较了棉铃虫取食含Cry1Ac蛋白的饲料后,Polycalin基因表达量的变化。【结果】该基因全长序列为2 955 bp,开放阅读框为2 781 bp,编码926个氨基酸(Gen Bank登录号为KP100652);预测蛋白的分子量为101.68 ku,等电点为4.57。推导的氨基酸序列中,N末端含有20个氨基酸组成的信号肽,含有8个O-糖基化位点,3个N-糖基化位点,C末端存在2个GPI结合位点。Polycalin在棉铃虫所有发育阶段都可以表达,幼虫期表达量较高,尤其在1~3龄幼虫体内表达量最高,在卵、成虫和蛹中的表达量较低。棉铃虫4龄幼虫取食含活化Cry1Ac蛋白的人工饲料后,Polycalin基因的表达受到抑制。【结论】研究结果可以为进一步揭示Polycalin基因的功能及其在Bt杀虫机制中的作用奠定基础。
【Objective】 The purpose of this study was to clone the Polycalin gene of Helicoverpa armigera and analyze the expression profile of Polycalin gene, further clarify the role of Polycalin gene in resistance and provide a theoretical basis for the resistance management of Helicoverpa armigera. 【Method】 The full-length sequence of Polycalin gene was cloned by RACE-PCR technique. The expression of Polycalin gene in different parts of larvae intestinal tract was detected by real-time fluorescence quantitative PCR. Changes in Polycalin Gene Expression After Feeding Cry1Ac-Containing Feeds. 【Result】 The full length of the gene was 2 955 bp with an open reading frame of 2 781 bp encoding 926 amino acids (Gen Bank Accession No. KP100652). The predicted molecular weight was 101.68 ku and the isoelectric point was 4.57. The deduced amino acid sequence contains 20 amino acid signal peptides at the N terminus, 8 O-glycosylation sites, 3 N-glycosylation sites and 2 GPI binding sites at the C-terminus. Polycalin was expressed at all developmental stages of Helicoverpa armigera, and was expressed at larval stage. Polycalin had the highest expression level in larvae of 1 ~ 3 instar larvae, but lower in egg, adult and pupae. The 4th instar larvae of H. armigera fed with artificial diet containing activated Cry1Ac protein inhibited the expression of Polycalin gene. 【Conclusion】 The results of this study may lay the foundation for further revealing the function of Polycalin gene and its role in insecticidal mechanism of Bt.