Development of protein markers for the identification of Bemisia tabaci biotypes B and Q
담배가루이의 B와 Q 바이오타입 진단을 위한 단백질 마커의 개발
Graduate Program for Nanomedical Science/석사
The whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), is one of the most destructive pests damaging more than 500 crop species worldwide. They caused direct damages by feeding in plant phloem, removing plant sap, reducing plant vigor and excreting honeydew which promote sooty mold. Whiteflies also caused indirect damages by transmitting a great number of plant viruses. Whiteflies were reported about 25 biotypes according to various ecological, physiological and genetic factors such as their different traits with respect to host range, host-plant adaptability, feeding ability, and plant virus-transmission capabilities among its populations. Especially biotypes B and Q are most widely spread in Korea but they are not distinguishable based on morphological characters. Recently reliable discrimination of B. tabaci biotypes rely on molecular diagnosis such as specific PCR. These methods, however, must be performed in laboratory and required relatively long time commitment. Comparative protein chip-based diagnosis is not only simple and rapid but also can is able to use in the field. In this study, in order to search for protein biomarkers that can be employed for rapid and accurate diagnosis of B. tabaci biotypes, we compared total proteome catalogues of two biotypes of B. tabaci by the combined methods of 2DE, quantitative image analysis, and nano-LC tandem mass analysis. Eleven biotype-specific spots were repeatedly identified during three times 2DE and were analyzed by nano LC-MS/MS. One of the B type-specific protein spots was identified as carboxylesterase 2 (COE2). The transcription level of coe2 was determined to be 6 times higher in B biotype than in Q biotype by quantitative real-time PCR. The recombinant protein, COE2 (rCOE2), was expressed by using an E. coli expression system and was purified by using DEAE-Sepharose chromatography and Ni2+-NTA agarose chromatography. In order to generate polyclonal antibodies specific to B biotype, the purified rCOE2 was injected to mice as an immunogen. We also performed Western blot analysis with the whole extracts of B and Q biotype collected from 4 different regions in Korea to evaluate for specificity of anti-rCOE2 serum. It showed that the anti-rCOE2 serum specifically discriminated B biotype from Q biotype B. tabaci. It would be a good protein biomarker for the diagnostic kits to discriminate biotypes of B. tabaci at the local farms.