棉铃虫磷脂酰乙醇胺结合蛋白的克隆及表达分析

doi:10.3864/j.issn.0578-1752.2018.08.007

摘要/Abstract

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cDNA序列，分析HaPEBP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HaPEBP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HaPEBP的cDNA序列，并对其氨基酸序列和蛋白结构进行分析。将HaPEBP的ORF序列连接至pET32a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HaPEBP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HaPEBP的变化规律。【结果】获得的HaPEBP cDNA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 kD和5.93。氨基酸序列分析表明HaPEBP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)-pET32a-HaPEBP重组菌用1 mmol·L^-1的IPTG在37℃条件下诱导4 h，约40 kD的融合蛋白His-HaPEBP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 kD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。HaPEBP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HaPEBP的表达量均有所降低；低浓度(2.5和5 mg·g^-1)在6 h就能降低HaPEBP的表达量，其mRNA含量随着时间的延长逐渐增加；而高浓度(10和15 mg·g^-1)在12 h才会降低HaPEBP的表达量，其mRNA含量随着时间的延长先下降后上升。【结论】克隆分析了HaPEBP，利用大肠杆菌系统表达出可溶的融合蛋白His-HaPEBP，并通过镍柱-咪唑纯化法得到了高纯度的融合蛋白，时空表达分析显示HaPEBP在棉铃虫6龄幼虫和脂肪体中表达量最高，2-十三烷酮处理6龄幼虫后中肠内HaPEBP的表达量显著降低。

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

Abstract: 【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 of HaPEBP after 2-tridecanone treatment in the 6th instar larval midgut of H. armigera. The results will provide a theoretical basis for further studying the function of HaPEBP and select the gene as a molecular target to regulate the population of H. armigera. 【Method】 The cDNA sequence of HaPEBP was obtained from midgut of 6th instar larvae by using RACE technique, and its amino acid sequence and protein structure were analyzed. The recombinant vector pET32a-HaPEBP 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-HaPEBP was purified using Ni-NTA affinity chromatography. The HaPEBP expression profile at different developmental stages, tissues and under 2-tridecanone treatments was determined by qRT-PCR. 【Result】 The HaPEBP cDNA 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 kD and 5.93, respectively. The HaPEBP 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 kD consistent with predicted 39.1 kD, was synthesized in BL21-pET32a-HaPEBP strain by 1 mmol·L^-1IPTG induced 4 h at 37℃. And then the pure His-HaPEBP (183.3 ng·μl^-1) was obtained through Ni-NTA column and imidazole gradient buffers. HaPEBP was expressed at all larval stages (1st-6th instar larvae and prepupa), and the highest expression level was observed in the 6th instar larvae. It was also expressed in the fat body, midgut, head and integument of the 6th instar larvae and the highest expression was found in the fat body. The expression of HaPEBP in the midgut of 6th instar larvae decreased after treatment with different concentrations of 2-tridecanone. In the low concentration groups (2.5 and 5 mg·g^-1), the expression of HaPEBP significantly decreased at 6 h and gradually increased over time. However, the expression of HaPEBP decreased at 12 h in the high concentrations (10 and 15 mg·g^-1), the mRNA content decreased firstly and then increased over time. 【Conclusion】The HaPEBP cDNA was cloned and analyzed. The fusion protein His-HaPEBP was synthesized and purified using the prokaryotic expression system. The results of temporal and spatial expression showed that the HaPEBP was highly expressed in 6th instar larvae and fat body. The expression of HaPEBP significantly decreased after treatment with 2-tridecanone in the midgut of 6th instar larvae.

Key words: Helicoverpa armigera, phosphatidylethanolamine binding protein, prokaryotic expression, temporal and spatial expression, 2-tridecanone

赵洁，任苏伟，刘宁，艾新宇，马纪，刘小宁. 棉铃虫磷脂酰乙醇胺结合蛋白的克隆及表达分析[J]. 中国农业科学, 2018, 51(8): 1493-1503.

ZHAO Jie, REN SuWei, LIU Ning, AI XinYu, MA Ji, LIU XiaoNing. Cloning and Expression of Phosphatidylethanolamine Binding Protein in Helicoverpa armigera[J]. Scientia Agricultura Sinica, 2018, 51(8): 1493-1503.

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