絲瓜簇葉病植物菌質體可能的ABC轉運系統基因之分離及特性分析

Abstract

本實驗參考Mycoplasma genitalium的ribonuclease III，設計一組PCR引子，以植物菌質體DNA為範本獲得之PCR產物，為DNA探針去篩選絲瓜簇葉病植物菌質體genomic library，結果非預期地篩選到推測含ATP-binding cassette（ABC）轉運系統（transport system）基因的選殖片段。 經序列的分析比對，此一選殖片段共3,903bp，G＋C比例為23.5mole％，符合已知植物菌植體之G＋C比例，並預測有三個完整的open reading frames（ORFs），此三個ORFs轉譯之蛋白質顯示並無如Mycoplasma及Spiroplasma利用UGA為色胺酸密碼之特性；其中ORF1及ORF2和ABC轉運系統中可結合ATP以提供主動運輸能量的ATP-binding protein有相當高的相似性；ORF3由蛋白質序列的比對並無具體之結果，但由二級結構、親水性質之分析及可能形成lipoprotein的胺基酸序列，推測ORF3可能為ABC轉運系統中對受質專一性結合的solute-binding lipoprotein；目前所知細菌的ABC轉運系統，除了獲取養份的功能外，還具有訊息傳遞、抗藥性及可能和寄生細菌之毒性有關；本文並就植物菌質體、革蘭氏陽性菌及革蘭氏陰性菌的系統，對ABC轉運系統這類蛋白質之生理功能及和寄主的交互作用進行探討。

Using the enriched phytoplasma DNA from diseased periwinkle total DNA as template and a pair of oligonucleotides (RN1, RN2) designed according to DNA sequence of ribonuclease III of Mycoplasma genitalium as primers, an 170-bp PCR product named RN-P was amplified. The loofah witches' broom phytoplasma λ ZAP genomic library was screened with the RN-P probe. After analyzing, and comparing the sequence of the cloned DNA fragment with the sequence in the databank, we found the DNA fragment contains the putative ATP-binding cassette (ABC) transport system genes instead of ribonuclease. That is because the cloned DNA contained the partial sequences of the primers. The cloned DNA fragment contains 3 open reading frames (ORFs). ORF1 and ORF2 have high homology with the ATP-binding proteins of the ABC transport system. There was no counterpart of ORF3 found in the protein databank. However, several properties including putative secondary structure, hydrophilicity and the amino acid sequence for acrylic site showed that ORF3 might encode a specific solute-binding lipoprotein of ABC transport system. In addition to obtain nutrients, the functions of known ABC transport system of bacteria include signal transduction, drug-resistance, and toxicity of parasitic bacteria. In this study, it possible implications to the unique traits of phytoplasmas were discussed.