棉铃虫磷脂酰乙醇胺结合蛋白的克隆及表达分析  

Cloning and Expression of Phosphatidylethanolamine Binding Protein in Helicoverpa armigera

作　　者：赵洁[1] 任苏伟 刘宁[2] 艾新宇[1] 马纪[1] 刘小宁[1] ZHAO Jie;REN SuWei;LIU Ning;AI XinYu;MA Ji;LIU XiaoNing(College of Life Science and Technology, Xinjiang University, Urumqi 830046;Institute of Crop Variety Resources, Xinjiang Academy of Agricultural Sciences, Urumqi 830091)

机构地区：[1]新疆大学生命科学与技术学院,乌鲁木齐830046 [2]新疆农业科学院农作物品种资源研究所,乌鲁木齐830091

出　　处：《中国农业科学》2018年第8期1493-1503,共11页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31471781);新疆维吾尔自治区青年科技创新人才培养工程(qn2015yx001)

摘　　要：【目的】克隆获得棉铃虫(Helicoverpa armigera)磷脂酰乙醇胺结合蛋白(phosphatidylethanolamine binding protein,PEBP)的c DNA序列,分析Ha PEBP在棉铃虫体内的表达规律,检测2-十三烷酮处理条件下棉铃虫体内Ha PEBP的变化情况,为进一步明确棉铃虫PEBP的功能和选择该基因作为调控棉铃虫种群数量的分子靶标提供依据。【方法】利用RACE技术从棉铃虫6龄幼虫的中肠组织中扩增得到Ha PEBP的c DNA序列,并对其氨基酸序列和蛋白结构进行分析。将Ha PEBP的ORF序列连接至p ET32a载体并转化大肠杆菌BL21(DE3),IPTG诱导后检测目的蛋白的表达形式,并通过镍柱的亲和层析纯化融合蛋白。最后通过实时荧光定量PCR检测棉铃虫幼虫不同时期和6龄幼虫不同组织中Ha PEBP的表达规律,以及2-十三烷酮处理棉铃虫6龄幼虫后中肠内Ha PEBP的变化规律。【结果】获得的Ha PEBP c DNA序列长760 bp,其中ORF为588 bp,编码195个氨基酸,蛋白质分子量和等电点分别为21.76 k D和5.93。氨基酸序列分析表明Ha PEBP无信号肽、跨膜结构域和二硫键,是一个由4个α螺旋和9个β折叠片组成的胞质单体蛋白。将BL21(DE3)-p ET32a-Ha PEBP重组菌用1 mmol·L-1的IPTG在37℃条件下诱导4 h,约40 k D的融合蛋白His-Ha PEBP能以可溶的形式存在于重组菌中。按照同样的方法诱导1 L的重组菌,将超声处理后的上清液与Ni-NTA柱结合,进行咪唑缓冲液梯度洗涤,200 mmol·L-1的咪唑洗涤两次后得到约40 k D的单一蛋白,与融合蛋白的大小一致。将此流出液超滤浓缩,获得183.3 ng·μL-1的融合蛋白,能被抗His-Tag的单克隆抗体识别发生免疫反应。Ha PEBP在棉铃虫幼虫的1—6龄期和预蛹期均表达,且6龄幼虫的表达量最高。该基因在6龄幼虫的脂肪体、中肠、头部和体壁中也均有表达,且脂肪体内的表达量最高。不同浓度的2-十三烷酮处理后,棉铃虫6龄幼虫中肠内Ha PEBP的表达量均有所降低;低【Objective】The objective of this study is to clone and analyze phosphatidylethanolamine binding protein（PEBP） gene from Helicoverpa armigera, investigate the temporal and spatial expression profile in H. armigera, and test the expression ofHa PEBP after 2-tridecanone treatment in the 6 th instar larval midgut of H. armigera. The results will provide a theoretical basis for further studying the function of Ha PEBP and select the gene as a molecular target to regulate the population of H. armigera. 【Method】 The c DNA sequence of Ha PEBP was obtained from midgut of 6 th instar larvae by using RACE technique, and its amino acid sequence and protein structure were analyzed. The recombinant vector p ET32 a-Ha PEBP was constructed and transformed it into Escherichia coli BL21（DE3） strain. The fusion protein was induced by IPTG and identified by SDS-PAGE to confirm its distribution. The His-Ha PEBP was purified using Ni-NTA affinity chromatography. The Ha PEBP expression profile at different developmental stages, tissues and under 2-tridecanone treatments was determined by q RT-PCR. 【Result】 The Ha PEBP c DNA sequence is 760 bp, and its ORF is 588 bp, encoding 195 amino acids. The predicted molecular weight and isoelectric point of the protein are 21.76 k D and 5.93, respectively. The Ha PEBP is a cytoplasmic monomer protein without signal peptide, transmembrane region and disulfide bond, which consists of 4 α-helixes and 9 β-sheets. The soluble fusion protein, which was about 40 k D consistent with predicted 39.1 k D, was synthesized in BL21-p ET32 a-Ha PEBP strain by 1 mmol·L-1 IPTG induced 4 h at 37℃. And then the pure His-Ha PEBP（183.3 ng·μL-1） was obtained through Ni-NTA column and imidazole gradient buffers. Ha PEBP was expressed at all larval stages（1 st-6 th instar larvae and prepupa）, and the highest expression level was observed in the 6 th instar larvae. It was also expressed in the fat body, midgut, head and integument of the 6 th instar larvae and the highest expression was

关 键 词：棉铃虫 磷脂酰乙醇胺结合蛋白 原核表达 时空表达 2-十三烷酮 

分 类 号：S433[农业科学—农业昆虫与害虫防治]