花生簇葉病菌質體rpsT，serS與hflB基因之選殖與分析

Cloning and Analysis of rpsT, serS and hflB Genes of Peanut witches'-broom Phytoplasmao

呂沛穎(P. Y. Lu) ； 林長平(C. P. Lin)

《植物病理學會刊》 18卷2期 (2009/06) Pp. 57-66

https://doi.org/10.6649/PPB.200906_18(2).0001

摘要

本研究以建構與篩選不同花生簇葉病菌質體次基因體基因庫(subgenomic library)之策略，針對其serS基因進行選殖與分析。利用本研究室先前獲得之部分花生簇葉病菌質體serS基因序列，以PCR製備探針並應用於由花生簇葉病菌質體次基因體基因庫之篩選，獲得一選殖株SI419，進一步以其選殖片段序列，利用PCR製備另一探針並用於另一次基因體基因庫之篩選，並獲得選殖株HIII236。經序列整合分析，得知二選殖株之選殖片段中包含有兩個完整之open reading frames (ORFs)，與兩個不完整之可能ORFs，由5'端至3'端之順序分別將其命名爲ORF1至ORF4。其中ORF1爲不完整之ORF但其序列與rpsT基因具有相似性，爲獲得完整之rpsT基因，即由植物菌質體rpsT之保守性區域設計簡併性引子(degenerate primer)，藉由PCR進行rpsT基因之選殖，並獲得選殖株CW67。將各選殖株嵌入片段之重疊序列接續，遂獲得一3,902bp花生簇葉病菌質體基因體之片段序列，其組成爲rpsT-serS-hflB及一不完整之ORF4。rpsT基因之轉譯產物爲3OS subunit ribosomal protein S20: serS沼基因轉譯產物爲絲膠基化TRNA合成酵素，兩者都參與於轉譯中，爲蛋白質生合成之相關基因；hflB基因其轉譯產物爲ATP-dependent Zn metalloendoprotease，具有分解多種蛋白質之能力。在RT-PCR試驗中，利用引子序列位於基因內部且其增幅片段橫跨rpsT-serS與serS-hflb基因間區域(intergenic region)之各引子對組合進行反應時，皆可獲得增幅產物，顯示三基因均有RNA表現，且可能位於同一多基因操縱組(polycistronic operon)內。

關鍵字

花生簇葉病菌質體 ； 次基因體基因庫 ； rpsT基因 ； serS基因 ； hflb基因

並列摘要

To investigate serS gene in peanut witches'-broom (PnWB) phytoplasma, subgenomic libraries of PnWB phytoplasma were constructed in this study. Clone SI419 was obtained from the PnWB phytoplasma SpeI-restriction subgenomic library by colony hybridization using a probe containing partial serS gene sequence of PnWB phytoplasma. To obtain the 5' end sequence of serS, another probe WS8-6 was amplified according to the sequence of cloned fragment of SI419. Clone HIII236 was then selected from the PnWB phytoplasma HindIII-restriction subgenomic library. Sequences of the cloned fragments of 5I419 and HIIII236 were integrated and analyzed to reveal that the sequences may cotain four putative open reading frames (ORFs 1-4). The deduced amino acid sequences of ORF1, ORF2 and ORF3 showed homology with those of rpsT gene, serS gene and hflB gene, respectively. On the other hand, amino acid sequence of the ORF4 probably encodes a hypothetical protein with no significant homology to any known sequences. To clone complete rpsT gene, a degenerate PCR primer was designed according to the 5' conserved region of phytoplasma rpsT gene, a clone CW67 with full length of rpsT gene was then obtained using PCR-based cloning strategy. The 3, 902 bp genomic DNA sequence of PnWB phytoplasma was determined from the sequences of the cloned fragments of SI419, HIII236 and CW67 using SeqMan sequence analysis program. The fragment contains full length of rpsT gene, serS gene, hflB gene and an incomplete ORF4 in order. PCR fragments that spanned the rpsT-serS and serS-hflB intergenic region were amplified separately in RT-PCR using the total RNA prepared from PnWB-affected periwinkle as template, and the sequences of PCR products were identical to the corresponding sequences of the 3, 902 bp PnWB phytoplasma genomic fragment. RT-PCR experiments indicate that the three genes are expressed as a single transcript, demonstrating that they constitute an operon.

並列關鍵字

peanut witches-broom phytoplasma ； subgenomic library ； rpsT gene ； setS gene ； hflB gene