小贯小绿叶蝉水状唾液蛋白的鉴定及其参与茶树“叶蝉烧”症状形成的初步研究

Identification of Watery Saliva Protein from Empoasca onukii and Preliminary Study on the Involvement in the Formation of “Hopperburn” Symptoms in Tea Plants

小贯小绿叶蝉(Empoasca onukii Matsuda)唾液蛋白在茶树“叶蝉烧”(Hopperburn)形成中的作用研究尚属空白。利用双层膜夹营养液法和自制唾液收集装置采集小贯小绿叶蝉成虫的水状唾液;采用SDT裂解法和FASP(Filter-aided sample preparation)酶解法提取唾液蛋白质;并通过LC-MS/MS质谱对小贯小绿叶蝉水状唾液蛋白质的种类和成分进行检测与分析。结果显示,小贯小绿叶蝉水状唾液中共鉴定到107个肽段、92个蛋白质,按不同功能可分为7类,包括酶类、转运蛋白、离子结合蛋白、调节蛋白、骨架蛋白、其他或非酶蛋白和未表征蛋白。此外,以收集纯化的成虫唾液蛋白质处理机械损伤茶树叶片,并与纯机械损伤叶片、纯唾液处理叶片、血清蛋白处理叶片和虫害叶片进行比较,结果发现,叶蝉唾液蛋白处理后的叶片与小贯小绿叶蝉危害叶片的变化趋势基本一致,处理48 h均会出现叶蝉烧症状;而其他处理叶片均未出现此类症状。本研究为进一步了解小贯小绿叶蝉的唾液组成及其与茶树之间的互作机理提供了一定的基础信息。

The role of saliva protein of Empoasca onukii in the formation of “Hopperburn” symptom in tea plants has not been studied to date. In this paper, the double-layer parafilm clamping nutrient solution method and a simple self-made saliva collection device were developed to collect the watery saliva of E. onukii adults. The proteins were then extracted from the watery saliva using SDT splitting method and FASP(Filter-Aided Sample Preparation)enzymatic hydrolysis method, and then watery saliva proteins were detected by LC-MS/MS. The results showed that a total of 107 peptides and 92 proteins were identified, which could be divided into 7 groups according to their functions, including enzymes, transporter proteins, ion-binding proteins, regulatory proteins, cytoskeletal proteins,non-enzymatic proteins and uncharacterized proteins. In addition, the collected and purified salivary proteins were used to treat the mechanically damaged tea leaves and make comparison with the pure mechanically damaged leaves,the pure saliva-treated leaves, the serum protein treated leaves and E. onukii adults infested leaves. The results show that the saliva protein-treated leaves had the same trends as the leaves damaged by leafhopper, with “Hopperburn”symptoms appearing at 48 h of treatment. While no such symptoms appeared in the other treatments. This study provided some basic information for further understanding of saliva composition of E. onukii and the interaction mechanism between leafhopper saliva protein and tea plants.