大丽轮枝菌分泌蛋白提取方法比较

Comparison of Secretome Extraction Methods in Verticillium dahliae

【目的】采用超滤法(UF)、三氯乙酸沉淀法(TCA)、离子交换富集法(IEX)分别从大丽轮枝菌(Verticillium dahliae)高致病性菌株VdG1培养上清液中提取分泌蛋白,对提取样品进行质谱鉴定。寻找适合大丽轮枝菌分泌蛋白的提取方法,为大丽轮枝菌分泌蛋白组深入研究奠定基础。挖掘大丽轮枝菌分泌蛋白中潜在的致病相关蛋白,为其深入功能验证及致病机理研究提供平台。【方法】3种方法提取大丽轮枝菌VdG1分泌蛋白,shot-gun方法进行质谱鉴定。利用生物信息学软件WoLFPSORT、SignalP、TMHMM、PHOBIUS对其中经典分泌蛋白进行预测。通过CAZy和PHI数据库进行经典分泌蛋白中碳水化合物水解酶(CAZy)和病原菌寄主互作因子(PHI)注释,BLASTp软件进行RxLx、LysM、Cys Pattern等模体蛋白比对分析。提取的蛋白样品接种感病棉种(军棉1号)进行样品生物学活性及致病性检测。【结果】TCA法、IEX法和UF法制备大丽轮枝菌分泌蛋白,其提取效率分别为1.18、0.96和0.56 mg·L-1。SDS-PAGE电泳检测显示UF法提取的蛋白样品条带少而模糊。而TCA法和IEX法提取的蛋白样品条带较多,样品的纯度也较高;经生物信息学软件预测分析,提取的蛋白样品中含有经典分泌蛋白分别为124、112和75个;质谱鉴定的分泌蛋白中含有潜在致病因子分别为98、85和57个;另外,IEX法制备大丽轮枝菌分泌蛋白效果较好,具有小分子偏好性,且能够保持样品的生物学活性,该蛋白样品接种棉花叶片能够引起萎蔫、坏死的症状。【结论】3种方法提取大丽轮枝菌分泌蛋白,UF法提取效率最低,纯度最差,蛋白数量最少,蛋白样品中含有潜在的毒素蛋白数量也最少,而且对于大体积处理相当耗时。TCA法和IEX法都适合大丽轮枝菌分泌蛋白的制备,但是TCA法制备过程中不能保持样品的生物学活性,后续适合于2-D电泳检测等试验。IEX法首次应用于病原菌分泌蛋白的制备,操作简单、方便,全部过程均由仪器系统完成。制备的大丽轮枝菌分泌蛋白具有生物学活性,能够引起棉花叶片的萎蔫、坏死病症。另外,数据库及软件比对分析发现大丽轮枝菌分泌蛋白组中含有CAZy酶类61个、PHI相关蛋白38个、RxLx模体蛋白13个、LysM模体蛋白1个、Cys Pattern的模体蛋白15个、VdNEP蛋白家族2个。该结果将为致病相关蛋白的筛选和深入功能验证提供重要依据。

【Objective】The objective of this study is to identify the high efficient method for secreted protein extraction in Verticillium dahliae, provide a technique basis for further functional characterization of V. dahliae secretome, and to construct a platform for functional verification of virulence factors and the mechanism research of V. dahliae. 【Method】Secreted proteins of the high virulent V. dahliae strain VdG1 were extracted by three methods, including ultra-filtration（UF）, TCA precipitation and ion-exchange（IEX）, followed by LC-MS/MS analysis for protein identification. The bioinformatics softwares WoLFPSORT, SignalP, TMHMM and PHOBIUS were used to predict classical secreted proteins. The classical secreted proteins were annotated by using CAZy（carbohydrate-active enZYmes） and PHI（pathogen-host interaction） databases. In addition, motif searchs was conducted via BLASTp, such as RxLx, LysM and Cys Pattern. The extracted proteins were used to detect the biological activity and pathogenicity on susceptible cotton. 【Result】By the three methods of TCA precipitation, IEX and UF, the extracted efficiency of V. dahliae secretome was 1.18, 0.96 and 0.56 mg·L-1, respectively. The SDS-PAGE result showed that TCA and IEX extracted samples had more efficiency and purity than that of UF. The numbers of classical secreted proteins were 124, 112 and 75, respectively, of which 98, 85 and 57 were possible pathogenic factors. In addition, ion-exchange had a high extraction efficiency for small molecular weight proteins and the extracted protein could induce wilt and necrosis symptom to cotton. 【Conclusion】To extract secreted proteins of V. dahliae by three methods, the lowest efficiency and purity, the minimum amount of classical secreted proteins, the fewest number of potential pathogenic factors were the UF extracted samples, and such method was quite time-consuming for large volume processing. TCA and IEX methods were fit to extract secreted proteins of V. dahliae, but TCA method couldn＇t keep the sample＇s biological activities and the extracted sample was just for 2-D electrophoresis detection. IEX method was firstly applied in the preparation of pathogenic fungi, and the operation was simple, convenient, all processes were completed by the instrument system. The extracted proteins had biological activity, and could cause cotton leaf wilt and necrotic symptom. In addition, the database and software comparative analysis found that the identified secreted proteins of V. dahliae contain 61 CAZy enzymes, 38 PHI proteins, 13 RxLx motif proteins, 1 LysM motif protein, 15 Cys Pattern motif proteins, and 2 VdNEP family proteins. The results will be used for screening the pathogenic factors and providing an important basis for further functional verification.