铜绿丽金龟气味结合蛋白AcorOBP11的表达纯化及功能分析

Expression,Purification and Functional Analysis of Odorant Binding Protein 11(OBP11)in Anomala corpulenta

【目的】通过研究铜绿丽金龟(Anomala corpulenta)气味结合蛋白11(odorant binding protein 11,OBP11)与寄主植物挥发物的结合特性,探讨AcorOBP11的功能,为阐明铜绿丽金龟识别寄主植物的嗅觉分子机制打下基础。【方法】设计特异性引物,通过RT-PCR克隆AcorOBP11,分析其序列特征并与相似序列进行对比;将目的基因OBP11连入原核表达载体pET28a后,重组质粒转入大肠杆菌感受态细胞BL21(DE3)。利用IPTG诱导AcorOBP11重组蛋白的表达,超声破碎菌体,取上清液过镍柱,利用重组肠激酶切除His标签后再次过镍柱纯化获得目的蛋白;纯化后的蛋白以1-NPN为荧光探针开展荧光竞争结合试验,测定AcorOBP11蛋白与37种寄主植物挥发物的结合特性;利用Modeller对AcorOBP11进行同源建模,以冈比亚按蚊气味结合蛋白AgamOBP48(PDB ID:4ij7)作为模板,获得其三维结构;利用Autodock半柔性对接将蛋白与化合物对接,模拟AcorOBP11与6种候选寄主植物挥发物的结合情况。【结果】克隆获得了AcorOBP11全长基因,开放阅读框共639 bp,N末端有17个氨基酸组成的信号肽,具有8个保守的半胱氨酸位点,属于Plus-C OBP亚家族,进化树结果表明其与HparOBP9进化关系最近。AcorOBP11成功连入pET28a表达载体,在0.25 mmol·L^(-1) IPTG、37℃条件下诱导表达,并通过两次镍柱纯化得到目的蛋白。竞争结合试验结果表明,AcorOBP11结合谱较广,对α-紫罗兰酮、异戊醛、丙烯酸-2-乙基己酯、乙酸龙脑酯、柠檬烯、反-2-己烯醛、2-乙基己醛、异戊酸等13种寄主植物挥发物有较好的结合能力。其中,与寄主植物挥发物α-紫罗兰酮结合能力最好,竞争解离常数为22.78μmol·L^(-1)。构建了AcorOBP11三维结构图并进行蛋白构象评估后,与6种化合物进行分子对接,结果表明AcorOBP11与α-紫罗兰酮结合自由能最低,为-24.74 kJ·mol^(-1),表明其与α-紫罗兰酮的结合能力最强,与荧光竞争结合结果一致;从对接图还可以看出,α-紫罗兰酮不仅与AcorOBP11在Cys163处形成氢键,而且其疏水端进入了蛋白的疏水腔,两个甲基与蛋白表面高度契合,因此,α-紫罗兰酮与蛋白间结合能力最强。【结论】AcorOBP11能够识别多种寄主植物挥发物,推测其在铜绿丽金龟成虫定位寄主植物中发挥重要作用,此研究结果为生态防控铜绿丽金龟提供了新思路。

【Objective】The objective of this research is to explore the function of Anomala corpulenta odorant binding protein 11(AcorOBP11)by studying the binding characteristics of AcorOBP11 with the host plant volatiles,and to lay the foundation for clarifying the olfactory molecular mechanism of AcorOBP11.【Method】Reverse transcription PCR(RT-PCR)was used to clone the full-length ORF of AcorOBP11 by specific primers,and sequences with high similarity were downloaded for sequence comparison and analysis by BLAST.Prokaryotic expression of AcorOBP11 was conducted by Escherichia coli protein expression system,the OBP11 was inserted into the expression vector pET28a,and the recombinant plasmid was transferred into E.coli competent cells BL21(DE3).The target protein was purified by using recombinant enterokinase to remove the His-tag and purified again by using a nickel column.The purified protein was diluted with 50 mmol·L^(-1) Tris-HCl(pH 7.4)to a dilution of 2μmol·L^(-1),and the odorant was diluted with chromatographic grade methanol to a final concentration of 1 mmol·L^(-1),and 1-NPN was used as a fluorescent probe to determine the binding characteristics of AcorOBP11 to 37 host plant volatiles by fluorescence competitive binding assay.Modeller was used to obtain the three-dimensional structure of AcorOBP11 by using AgamOBP48(PDB ID:4ij7)as a template.Autodock semi-flexible docking was used to simulate the binding of AcorOBP11 to host plant volatiles.【Result】The full-length ORF of AcorOBP11 was amplified,which is 639 bp in total,including 17 amino acids of signal peptide at the N-terminal.With eight conserved cysteine sites,AcorOBP11 belongs to the Plus-C OBP subfamily.The evolutionary tree results indicated that it had the closest evolutionarily relationship with HparOBP9.AcorOBP11 was successfully inserted into pET-28a and expressed at 0.25 mmol·L^(-1) IPTG and induced expression at 37.℃The target protein was obtained by nickel column purification twice.The results of competitive binding experiments showed that AcorOBP11 has a good binding ability to 13 kinds of host plant volatiles,such asα-ionone,3-methylbutanal,2-ethylhexyl acrylate,bornyl acetate,cinene,trans-2-hexenal,2-ethylhexanal,isovaleric acid.Among them,α-ionone has the best binding ability,and its competitive dissociation constant is 22.78μmol·L^(-1).The molecular docking results showed that AcorOBP11 has the lowest binding free energy withα-ionone,which is-24.74 kJ·mol^(-1),and forms a hydrogen bond at Cys163,indicating that it has the strongest binding ability withα-ionone,which is consistent with the fluorescence competition binding result.【Conclusion】AcorOBP11 can recognize a variety of host plant volatiles,and it is speculated that it plays an important role in locating host plants for A.corpulenta.The results will provide new insights for A.corpulenta ecological control.