中国农业科学Issue(10)：1955-1961,7.
Q型烟粉虱化学感受蛋白CSP1与植物挥发物的结合特征
Binding Characterization of Chemosensory Protein CSP1 in the Bemisia tabaci Biotype Q with Plant Volatiles
摘要
Abstract
[Objective]The objective of this study is to clone the ORF (open reading frame) of chemosensory protein 1 (CSP1) from Bemisia tabaci biotype Q, and characterize the binding profiles of CSP1 with some candidate plants volatiles.[Method] By means of full-length ORF primer, reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the full-length ORF BtCSP1, which was then cloned into the pET-30a (+)/BL21 (DE3) prokaryotic expression vector after double enzyme digestion. The recombinant BtCSP1 protein was expressed and purified by the method of Ni2+-agroase affinity chromatograph. The protein concentration was measured with Bradford method. The competitive fluorescence assay was used to analyze the binding properties of BtCSP1 with general plant volatiles with different chemical structures. As a suitable fluorescence reporter in studies of insect GOBPs’ function in vivo, N-phenyl-1-naphthylamine (1-NPN) was used to titrate the BtCSP1 solution until the fluorescence emission peak at maximum wavelength of BtCSP1 completely quenched. Then all plant volatiles were added into BtCSP1-1-NPN complex, respectively. The dissociation constants K D data represented the affinity of BtCSP1 with ligands were calculated by the Scatchard equation. [Result] BtCSP1 protein was successfully expressed after induction of 1 mmol·L-1 of IPTG, then purified by Ni2+affinity column with gradient imidazole as washing solutions, finally dialyzed sufficiently using PBS buffer. The working concentration of BtCSP1 was diluted to 1.5 µmol∙L-1. In the competitive fluorescence assay, the dissociation constants K1-NPN and the number of binding sites n of BtCSP1 and 1-NPN were 2.78μmol·L-1and 0.82, respectively. It indicated that the binding of BtCSP1 with 1-NPN is strong and the binding ratio is almost 1:1. In all candidate chemicals, some plant volatiles preferred to bind with BtCSP1 with high affinity, such as 3-carene, 4-cymene, HL-cis-3-hexen-1-ol andα-pinene, which are reported to be related to the repellent characteristics of B. tabaci. Their binding affinity with BtCSP1 was strong, with the dissociation constant KD of 26.47, 39.43, 54.01 and 83.46μmol·L-1, respectively. Especially 3-carene reduced the relative fluorescence intensity of 1-NPN by about 40%at the concentration of 200μmol·L-1. [Conclusion] BtCSP1 exhibited a strong binding affinity with some repellent plant volatiles, indicating that BtCSP1 may be involved in the recognition of the repellent plants of host selection in the process of expanding into the invasion fields.关键词
烟粉虱/化学感受蛋白/原核表达/植物挥发物/结合特性Key words
Bemisia tabaci/chemosensory proteins (CSPs)/prokaryotic expression/plant volatiles/binding characterization引用本文复制引用
吴帆,张晓曼,赵磊,崔旭红,李红亮,罗晨..Q型烟粉虱化学感受蛋白CSP1与植物挥发物的结合特征[J].中国农业科学,2015,(10):1955-1961,7.基金项目
国家自然科学基金（31471773）、北京市叶类蔬菜创新团队建设（BLVT-13）、国家科技支撑计划 （）
