摘要
【目的】克隆Q型烟粉虱(Bemisia tabaci)化学感受蛋白1(chemosensory protein 1,CSP1)基因,诱导表达Q型烟粉虱CSP1重组蛋白(以下简称Bt CSP1),研究其与主要寄主植物挥发性气味分子的结合特性。【方法】利用全长引物通过RT-PCR扩增并克隆Q型烟粉虱CSP1基因ORF全长,连接并构建p ET-30a(+)原核表达载体,转化入BL21(DE3)大肠杆菌感受态细胞,并用IPTG诱导表达Bt CSP1重组蛋白。收集菌液后超声破碎细胞,离心取上清,经Ni2+-琼脂糖柱结合梯度浓度咪唑洗脱纯化后,经PBS反复透析获得重组蛋白,并用Bradford法测定重组蛋白浓度。采用常见的N-苯基-1-萘胺(N-phenyl-1-naphthylamine,1-NPN)荧光探针作为报告子,利用荧光竞争结合法研究重组Bt CSP1蛋白与植物挥发物的结合功能。首先用1 mmol·L-1 1-NPN滴定Bt CSP1蛋白溶液,直至蛋白最大发射波长处的荧光值完全猝灭为止,然后再以各供试配基滴定Bt CSP1-1-NPN体系,通过配基竞争猝灭1-NPN最大发射波长,并用Scatchard等方程计算表征Bt CSP1与配基亲和力大小的解离常数KD。【结果】克隆了Q型烟粉虱CSP1基因ORF全长,经双酶切和连接构建了p ET-30a(+)/CSP1重组质粒,在IPTG终浓度为1 mmol·L-1的条件下诱导获得了Bt CSP1重组蛋白,Ni2+-琼脂糖柱纯化透析后测定重组蛋白浓度,稀释至1.5μmol·L-1作为工作浓度。在荧光光谱试验中,Scatchard方程线性化后(相关系数达到0.9967),显示Bt CSP1与1-NPN的解离常数K1-NPN为2.78μmol·L-1,结合位点数n为0.82,表明两者结合较好,且基本是1:1结合,适合作为本试验中竞争性荧光结合试验的报告子。在荧光竞争结合试验中,有多种供试植物挥发物分子能使1-NPN的相对荧光强度降低到50%以下,其中包括能引起烟粉虱趋避行为的化合物,如3-蒈烯、p-伞花烃、顺-3-己烯-1-醇和α-蒎烯(KD值分别为26.47、39.43、54.01和83.46μmol·L-1),且3-蒈烯具有较强的竞争结合能力,能在200μmol·L-1时将1-NPN报告子相对荧光值竞争至约40%。【结论】Q型烟粉虱CSP1蛋白能与测试的多种寄主植物挥发物产生较为广谱的结合能力,尤其与对烟粉虱有趋避性的挥发物的结合更强,表明CSP1很有可能参与Q型烟粉虱对非寄主植物的趋避行为,这对揭示其入侵寄主选择行为生理机制具有重要意义。
[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＆amp;nbsp;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.
出处
《中国农业科学》
CAS
CSCD
北大核心
2015年第10期1955-1961,共7页
Scientia Agricultura Sinica
基金
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(2012BAD19B00)
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
Bemisia tabaci
chemosensory proteins （CSPs）
prokaryotic expression
plant volatiles
binding characterization
