李小食心虫GfunOBP2的原核表达及气味配体结合特性  

Expression and Ligand Binding Characteristics of GfunOBP2 from Grapholita funebrana

作　　者：年和粉 张钰析 李伯辽 陈秀琳 罗坤 李广伟 NIAN HeFen;ZHANG YuXi;LI BoLiao;CHEN XiuLin;LUO Kun;LI GuangWei(Shaanxi Key Laboratory of Chinese Jujube(Yan’an University),Yan’an 716000,Shaanxi;College of Life Sciences,Yan’an University,Yan’an 716000,Shaanxi)

机构地区：[1]陕西省红枣重点实验室(延安大学),陕西延安716000 [2]延安大学生命科学学院,陕西延安716000

出　　处：《中国农业科学》2023年第12期2302-2316,共15页Scientia Agricultura Sinica

基　　金：国家自然科学基金(32160636);延安大学产学研合作培育项目(CXY202118);2021年延安大学校级大学生创新创业计划训练项目(D2021099)。

摘　　要：【目的】通过测定李小食心虫(Grapholita funebrana)Plus-C气味结合蛋白2(odorant binding protein,GfunOBP2)结合性信息素和苹果树挥发化合物的能力,分析GfunOBP2的嗅觉功能,为阐释李小食心虫定位寄主植物的嗅觉分子机理打下基础。【方法】利用RT-PCR扩增GfunOBP2的ORF序列,通过同源注释和比对氨基酸序列中半胱氨酸(Cys)的分布模式确定GfunOBP2属于Plus-C OBP亚家族;利用RT-qPCR检测GfunOBP2在李小食心虫3日龄成虫触角、头、胸、足、翅、腹部和性腺中的相对表达量;构建pET30a(+)/GfunOBP2原核表达载体,在大肠杆菌(Escherichia coli)BL21(DE3)细胞中表达GfunOBP2重组蛋白。利用荧光竞争结合试验测定重组GfunOBP2蛋白对5种性信息素和35种苹果树挥发化合物的结合能力;通过分子对接预测GfunOBP2与具有强结合能力的气味配体的相互作用力及关键氨基酸残基。【结果】克隆获得GfunOBP2(GenBank登录号:OQ054799.1)的全长序列,共编码183个氨基酸,氨基酸序列中有12个保守的Cys,其分布模式表明GfunOBP2属于Plus-C OBP。GfunOBP2主要在成虫触角中表达,在雄虫触角中的相对表达量显著高于雌虫触角(P<0.05)。重组GfunOBP2蛋白对反-2-己烯-1-醇、苯甲醇、1-庚醇、1-癸醇、己醛、庚醛、乙酸-顺-3-己烯酯、2-甲基丁酸叶醇酯、α-罗勒烯、β-石竹烯、α-蒎烯和柠檬烯具有强结合能力,抑制常数Ki均小于5.0μmol·L^(-1)。分子对接结果显示氢键、Donor-Donor相互作用和烷基相互作用是GfunOBP2结合反-2-己烯-1-醇、1-庚醇和1-癸醇的主要弱相互作用力,氢键和碳氢键是GfunOBP2结合乙酸-顺-3-己烯酯和2-甲基丁酸叶醇酯的主要弱相互作用力,烷基相互作用是GfunOBP2结合α-罗勒烯和β-石竹烯的唯一弱作用力。疏水性氨基酸Ile、Pro、Phe、Ala、Leu和Val在GfunOBP2结合气味配体中起着重要作用。【结论】GfunOBP2主要在李小食心虫成虫触角中表达,重组GfunOBP2蛋【Objective】The objective of this study is to determine the binding affinities of the Plus-C odorant binding protein 2 of Grapholita funebrana(GfunOBP2)to sex pheromones and volatile compounds from apple trees,and to provide a basis for explaining the olfactory molecular mechanism of locating the host plants of G.funebrana.【Method】The ORF of GfunOBP2 was cloned by RT-PCR,and GfunOBP2 was identified as a Plus-C OBP subfamily protein through homology annotation and alignment of cysteine distribution patterns in amino acid sequences.The relative expression level of GfunOBP2 in the antenna,head,thorax,leg,wing,abdomen,and sex gland of the 3-day-old adults of G.funebrana was detected by RT-qPCR.The prokaryotic expression vector pET30a(+)/GfunOBP2 was constructed,and the recombinant GfunOBP2 protein was expressed in Escherichia coli BL21(DE3)cells.The binding affinity of recombinant GfunOBP2 protein to five sex pheromones and 35 plant volatiles of apple trees was determined by using a fluorescence competitive binding assay.The interaction force and key amino acid residues of GfunOBP2 interacting with odorant ligands with strong binding affinities were predicted by molecular docking.【Result】The full-length ORF sequence of GfunOBP2(GenBank number:OQ054799.1)was cloned,encoding 183 amino acids.It was found that GfunOBP2 has 12 conserved cysteines,and the distribution motif of cysteine residues indicated that GfunOBP2 belongs to the Plus-C OBP subfamily.GfunOBP2 was mainly expressed in the antennae of adults,and the relative expression level in male antennae was significantly higher than that in female antennae(P<0.05).Recombinant GfunOBP2 protein exhibited strong binding affinities to(E)-2-hexen-1-ol,benzyl alcohol,1-heptanol,1-decanol,hexanal,heptanal,cis-3-hexenyl acetate,cis-3-hexenyl 2-methylbutanoate,α-ocimene,β-caryophyllene,α-pinene and limonene,and the inhibition constant(Ki)for each ligand above was less than 5.0μmol·L^(-1).The molecular docking results showed that hydrogen bonds,donor-donor inter

关 键 词：李小食心虫 化学感受 气味结合蛋白 寄主植物挥发物 分子对接 

分 类 号：S433.4[农业科学—农业昆虫与害虫防治]