棉铃虫Polycalin基因的克隆及其表达分析
Cloning and expression analysis of the polycalin gene in the cotton bollworm Helicoverpa armigera
王亚楠1, 2** 钟 丰1 魏纪珍1 谢丙堂1 张万娜1 陈利珍2*** 梁革梅1*
点击：1630次 下载：39次
DOI：10.7679/j.issn.2095-1353.2015.063
作者单位：1. 华中农业大学植物科学技术学院, 武汉 430070；2. 中国农业科学院植物保护研究所，植物病虫害生物学国家重点实验室，北京 100193
中文关键词：棉铃虫，Polycalin基因，表达，Bt
英文关键词：Helicoverpa armigera, polycalin gene, expressin, Bt toxin
中文摘要： 【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（GenBank登录号为P100652）；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。
英文摘要： [Objetives] To further define the role of Polycalin in the Cry1Ac resistance mechanism of Helicoverpa armigera in and thereby develop a feasible resistance management strategy for this pest. [Methods] The full-lengthsequence of the polycalin coding gene was obtained using RT-PCR (reverse transcription-polymerase chain reaction) and RACE (rapid amplification of cDNA ends) technology. The expression levels of polycalin in different development stages and different tissues of the larval digestive tract were analyzed using real-time quantitative PCR. Changes in the expression of polycalin in the larval midgut in 4th instar larvae fed on an artificial diet containing the Bt insecticide protein Cry1Ac were also compared. [Results] The results indicate that the full-length of the polycalin from H. armigera coding gene was 2 955 bp (GenBank accession number KP100652), the open reading frame was 2 781 bp in length, encoding 926 amino acid residues. The predicted molecular weight was 101.68 ku and the isoelectric point was 4.95. The putative protein sequence contained a N-terminal signal peptide of 20 amino acids, three N-linked and eight O-linked glycosylation sites, and a GPI anchor signal peptide with 2 amino acids at the C-terminus. Polycalin was expressed in all growth stages of H. armigera, and expression in larvae was higher than that in other stages, especially in 1st-3rd instar larvae. The lowest expression levels occurred in eggs, adults and pupae. The expression of the polycalin gene was dramatically suppressed after larvae were fed on food containing the Cry1Ac toxin. [Conclusion] These results provide a basis for further clarifying the function of polycalin with respect to the Bt toxin.