烟粉虱MED隐种气味结合蛋白OBP8的克隆、原核表达及与植物挥发物的结合特性

Cloning and prokaryotic expression of odorant binding protein OBP8 in MED cryptic species Bemisia tabaci and the binding characteristics with plant volatiles

烟粉虱为重要的入侵性经济作物害虫,对入侵地寄主植物存在选择性和偏好性。为明确烟粉虱气味结合蛋白(odorant binding protein,OBPs)的相关特性,通过克隆其OBP8基因Btab OBP8,进行同源基因进化关系分析,构建原核表达载体诱导表达重组蛋白,并利用基于荧光探针N-苯基-1-萘胺(N-phenyl-1-naphthylamine,1-NPN)的荧光竞争结合试验测试了Btab OBP8与不同寄主植物挥发物的结合功能。结果表明,Btab OBP8基因的开放阅读框全长480 bp,编码159个氨基酸,与半翅目其它害虫的OBP8基因进化关系较远。Btab OBP8蛋白重组后,4种化合物即β-紫罗兰酮、月桂烯、柠檬烯和P-伞花烃将1-NPN的相对荧光强度降低到50%以下,其中β-紫罗兰酮和月桂烯与Btab OBP8的结合能力最强,解离常数分别为13.32μmol/L和21.53μmol/L。表明β-紫罗兰酮和月桂烯与Btab OBP8具有良好的亲和力,推测Btab OBP8对β-紫罗兰酮和月桂烯的识别可能参与了烟粉虱对绿色开花植物的吸引和选择作用。

Bemisia tabaci（ Gennadius） is one of the most important pests on a series of economic crops and exhibits obvious host plant selection and preference. To characterize odorant binding proteins（ OBPs）,in the present study,one novel OBP gene of B. tabaci,named Btab OBP8,was cloned and analyzed. After phylogenetic analysis, a prokaryotic expression vector was constructed, and the expression of recombinant protein Btab OBP8 was induced. The competitive fluorescence assay using Nphenyl-1-naphthylamine（ 1-NPN） as a fluorescence probe was also conducted to test the function of Btab OBP8 binding with different volatiles. The results showed that the full-length open reading frame（ ORF） of Btab OBP8 was 480 bp,encoding 159 amino acids. Btab OBP8 was distinctly different from other known OBP8 in hemipteran pests. Based on the recombinant protein,there were four plant volatiles preferred to bind with Btab OBP8 with half affinity,including β-ionone,myrcene,（ ＋ /-）-limonene and4-cymene,and β-ionone and myrcene had the strongest binding with Btab OBP8,and the dissociation constants were 13. 32 μmol / L and 21. 53 μmol / L,respectively. It was assumed that the recognition of Btab OBP8 to β-ionone and myrcene might be involved in host-preference mechanisms which were associated with the olfactory recognization in B. tabaci.