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:XM021331806)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
【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 pET32 a-HarmFoxAl was constructed and transformed into Escherichia coli Transetta strain. The fusion protein His-HarmFoxAl was induced by IPTG in Transetta(pET32 a-HarmFoxAl) strain, and was purified using the Ni-NTA affinity chromatography. The expression profiles of HarmFoxAl in different developmental stages(1 st-6 th instar larva and prepupa), tissues(fat body, midgut, integument and head) of the 6 th instar larva, and midguts of the 10 mg/g 2-tridecanone treated 6 th instar larvae of H. armigera at different time post treatment were detected by qPCR.【Results】 The open reading frame of HarmFoxAl(GenBank accession no.: XM021331806) 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(pET32 a-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 1 st, 2 nd, 3 rd and 6 th instar larva and prepupa, with the highest expression level in the prepupa. Tissue expression profiles revealed that HarmFoxAl was expres
作者
赵洁
魏倩
任苏伟
刘小宁
ZHAO Jie;WEI Qian;REN Su-Wei;LIU Xiao-Ning(College of Life Science and Technology, Xinjiang University, Urumqi 830046, China;College of Agriculture, Shihezi University, Shihezi, Xinjiang 832003, China)
出处
《昆虫学报》
CAS
CSCD
北大核心
2019年第6期672-684,共13页
Acta Entomologica Sinica
基金
国家自然科学基金项目(31471781)
新疆维吾尔自治区青年科技创新人才培养工程(qn2015yx001)
关键词
棉铃虫
叉头框蛋白
原核表达
时空表达
2-十三烷酮
Helicoverpa armigera
forkhead box protein
prokaryotic expression
temporal and spatial expression
2-tridecanone
