棉铃虫叉头框蛋白A类似蛋白基因HarmFoxAl的克隆及表达谱分析

doi:10.16380/j.kcxb.2019.06.003

摘要/Abstract

摘要：

【目的】本研究旨在克隆并分析一种棉铃虫Helicoverpa armigera叉头框蛋白A (forkhead box protein A, FoxA) 类似蛋白基因HarmFoxAl，探讨2-十三烷酮胁迫下棉铃虫中肠中HarmFoxAl的表达情况，为进一步明确棉铃虫FoxA的功能和参与棉铃虫生长发育的调控通路提供依据。【方法】从棉铃虫幼虫中肠中扩增得到HarmFoxAl的cDNA序列，并对其氨基酸序列和蛋白结构进行分析。将HarmFoxAl的ORF序列连接至pET32a载体并转化大肠杆菌 Escherichia coli Transetta菌株，IPTG诱导后检测目的蛋白的表达形式，并利用镍柱亲和层析法纯化融合蛋白。通过qPCR检测棉铃虫不同发育阶段(1-6龄幼虫和预蛹)，6龄幼虫不同组织(脂肪体、中肠、体壁和头部)以及10 mg/g 2-十三烷酮处理6龄幼虫不同时间后中肠中HarmFoxAl的表达谱。【结果】HarmFoxAl (GenBank登录号: XM_021331806)的开放阅读框为669 bp，编码222个氨基酸，蛋白的相对分子质量和等电点分别为25.03 kD和6.34。氨基酸序列分析表明，HarmFoxAl单体蛋白无信号肽、跨膜区和二硫键，核心区域是由4个α螺旋和3个β折叠组成的球状结构。将重组的Transetta (pET32a-HarmFoxAl)菌株用0.5 mmol/L IPTG在25℃条件下诱导5 h，约45 kD的融合蛋白His-HarmFoxAl能以可溶的形式存在于重组菌中，这与预测的分子量(42.8 kD)相一致。发育阶段特异性表达谱表明，HarmFoxAl在棉铃虫1-3龄幼虫期、6龄幼虫期和预蛹期均有表达，且预蛹期的表达量最高。组织表达谱结果表明，该基因在6龄幼虫的脂肪体、中肠和体壁中表达，且脂肪体内的表达量最高，而在头部中不表达。10 mg/g 2-十三烷酮处理棉铃虫6龄幼虫后中肠中HarmFoxAl的表达量显著降低，但随着时间延长其表达量逐渐升高，处理48 h后表达量显著高于对照。【结论】棉铃虫HarmFoxAl在预蛹期和幼虫脂肪体中表达量最高，2-十三烷酮处理幼虫后HarmFoxAl的表达量急速降低后逐渐升高，推测其在棉铃虫变态发育和解毒代谢过程中发挥重要作用。

关键词: 棉铃虫, 叉头框蛋白, 原核表达, 时空表达, 2-十三烷酮

Abstract: 【Aim】 This study aims to clone and analyze the forkhead box protein A (FoxA)-like protein gene HarmFoxAl from Helicoverpa armigera, and to investigate its expression profiles after the larval midgut was subjected to 2-tridecanone treatment, so as to provide a theoretical basis for further studying the function and regulatory pathway of FoxA involved in the growth and developmental process of H. armigera. 【Methods】 The cDNA sequence of HarmFoxAl was cloned from the larval midgut of H. armigera, and its amino acid sequence and protein structure were analyzed. The recombinant vector pET32a-HarmFoxAl was constructed and transformed into Escherichia coli Transetta strain. The fusion protein His-HarmFoxAl was induced by IPTG in Transetta (pET32a-HarmFoxAl) strain, and was purified using the Ni-NTA affinity chromatography. The expression profiles of HarmFoxAl in different developmental stages (1st-6th instar larva and prepupa), tissues (fat body, midgut, integument and head) of the 6th instar larva, and midguts of the 10 mg/g 2-tridecanone treated 6th instar larvae of H. armigera at different time post treatment were detected by qPCR. 【Results】 The open reading frame of HarmFoxAl (GenBank accession no.: XM_021331806) is 669 bp, encoding 222 amino acids. The predicted molecular weight and isoelectric point of HarmFoxAl protein are 25.03 kD and 6.34, respectively. HarmFoxAl is a monomer protein without signal peptide, transmembrane region and disulfide bond, and its core sequence consists of four α-helixes and three β-sheets. After the Transetta (pET32a-HarmFoxAl) strain was induced by 0.5 mmol/L IPTG for 5 h at 25℃, a ~45 kD soluble fusion protein His-HarmFoxAl was expressed in the strain, which is consistent with the predicted molecular weight (42.8 kD). Developmental stage-specific expression profiles revealed that HarmFoxAl was expressed in the 1st, 2nd, 3rd and 6th instar larva and prepupa, with the highest expression level in the prepupa. Tissue expression profiles revealed that HarmFoxAl was expressed in the fat body, midgut and integument of the 6th instar larva, with the highest expression level in fat body, while not expressed in the head. When the 6th instar larvae were exposed to 10 mg/g 2-tridecanone, the expression level of HarmFoxAl in the midgut was significantly decreased first, and then gradually increased with the exposure time, being significantly higher than that of the control group at 48 h after treatment. 【Conclusion】 HarmFoxAl is highly expressed in the prepupal stage and larval fat body of H. armigera. After the H. armigera larvae are exposed to 2-tridecanone, the expression level of HarmFoxAl significantly decreases first and then gradually rises in larval midgut. Therefore, we presume that HarmFoxAl may play an important role in metamorphosis and metabolic detoxification of H. armigera.

Key words: Helicoverpa armigera, forkhead box protein, prokaryotic expression, temporal and spatial expression, 2-tridecanone

赵洁, 魏倩, 任苏伟, 刘小宁. 棉铃虫叉头框蛋白A类似蛋白基因HarmFoxAl的克隆及表达谱分析[J]. 昆虫学报, 2019, 62(6): 672-684 .

ZHAO Jie, WEI Qian, REN Su-Wei, LIU Xiao-Ning. Cloning and expression profiling of the forkhead box protein A-like protein gene HarmFoxAl in Helicoverpa armigera (Lepidoptera: Noctuidae)[J]. Acta Entomologica Sinica, 2019, 62(6): 672-684 .

[1]	陈昊, 罗巧玉, 马秋雨, 初旭, 袁超, 刘颖, 余伟, 吴松青, 王荣, 梁光红, 张飞萍, 胡霞. 松墨天牛漆酶基因MaLac1的克隆、原核表达及表达谱分析[J]. 昆虫学报, 2020, 63(4): 381-389.
[2]	巩雪芳, 杨平, 王延来, 郭莉, 陈迪, 谢寿安, 吕淑杰. 花椒窄吉丁气味结合蛋白基因AzanOBP3的克隆、原核表达及组织表达谱分析[J]. 昆虫学报, 2020, 63(4): 390-400.
[3]	马百伟, 刘晓岚, 常亚军, 谢桂英, 陈文波, 刘杨, 赵新成, 王桂荣. 利用单感器记录与神经元示踪结合对棉铃虫主要性信息素感器内神经元投射的鉴定[J]. 昆虫学报, 2020, 63(4): 413-420.
[4]	王婷婷, 索倩, 孙晓燕, 马跃敏, 刘凯于, 彭建新, 彭蓉 . 棉铃虫激活增强子结合蛋白AP-4基因的原核表达、多克隆抗体制备、表达谱分析及功能鉴定[J]. 昆虫学报, 2019, 62(9): 1028-1037.
[5]	鲁艳辉, 白琪, 郑许松, 田俊策, 吕仲贤. 四种鳞翅目害虫精氨酸激酶基因的表达谱及基于RNAi的功能分析[J]. 昆虫学报, 2019, 62(8): 901-911.
[6]	王芹芹, 崔丽, 王奇渊, 王立, 谢娜, 袁会珠, 芮昌辉. 茚虫威亚致死浓度对茚虫威敏感性降低的棉铃虫生物学参数及解毒酶活性的影响[J]. 昆虫学报, 2019, 62(8): 960-969.
[7]	朴君, 许春玲, 朴敬爱, 周益军, 李硕. 灰飞虱内生病毒HiPV外壳蛋白VP1多克隆抗体的制备及在病毒检测中的应用[J]. 昆虫学报, 2019, 62(7): 823-829.
[8]	张亚坤, 邓中原, 谷少华, 李显春. 棉铃虫剂量补偿相关基因Hamsl1的鉴定与功能分析[J]. 昆虫学报, 2019, 62(7): 799-813.
[9]	杨晓霞, 徐鑫, 刘忠渊毛新芳. 光滑鳖甲肽聚糖识别蛋白ApPGRP的表达与功能分析[J]. 昆虫学报, 2019, 62(6): 653-662.
[10]	刘小飞, 孟建玉, 赵晓超, 张长禹. 棉铃虫c-Jun氨基末端激酶基因的克隆、表达谱及对UV-A胁迫的响应[J]. 昆虫学报, 2019, 62(4): 407-417.
[11]	姚雪, 李琳红, 魏纪珍, 席玉强, 杜孟芳, 崔江宽, 安世恒, 刘晓光. 棉铃虫酪氨酸羟化酶基因的分子特性及功能分析[J]. 昆虫学报, 2019, 62(3): 294-303.
[12]	麦麦提艾力·阿卜杜纳斯尔, 马纪, 刘宁, 王威, 李梦鸽, 古新蓉, 王新, 刘小宁. 棉铃虫Ⅳ型几丁质酶的基因克隆、酶学性质及表达谱[J]. 昆虫学报, 2019, 62(2): 170-180.
[13]	赵旭东, 孙宇航, 陈昌宇, 田朔, 陶蓉, 郝德君. 美国白蛾丝氨酸蛋白酶基因HcSP1的克隆、时空表达及对取食不同寄主植物的表达响应 [J]. 昆虫学报, 2019, 62(2): 160-169.
[14]	梁振普, 王亮, 李鹏娟, 王朝兴, 肖宇博, 张小霞. 棉铃虫围食膜的蛋白质组鉴定[J]. 昆虫学报, 2019, 62(2): 181-192.
[15]	苏旭, 张永军, 耿亭, 李静, 谷少华. 小地老虎化学感受蛋白AipsCSP8的表达及配体结合特性分析[J]. 昆虫学报, 2019, 62(12): 1369-1378.

棉铃虫叉头框蛋白A类似蛋白基因HarmFoxAl的克隆及表达谱分析

Cloning and expression profiling of the forkhead box protein A-like protein gene HarmFoxAl in Helicoverpa armigera (Lepidoptera: Noctuidae)

PDF (PC)

赞

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

Metrics

本文评价

推荐阅读 10