花生簇葉病菌質體group II intron之選殖與分析

Abstract

本研究利用逢機定序策略與基因選殖技術，由花生簇葉病菌質體基因庫中篩選雜配訊號較強之轉形株，共獲得七個含有matR基因之不同group II intron選殖株，分別為限制酵素EcoRI構築之EI42, EI47及EI49, EcoRV構築之EV9, EV10, EV33及HindIII構築之HIII57選殖株。經由定序與分析，此七個group II intron間之核酸與胺基酸序列皆具有保守性。除了EI42缺乏group II intron 5’端之序列外，其他六個選殖株皆具有group II intron保守之5’及3’ boundary sequences。又除了EV33選殖株，其他五個選殖株皆具有專一性辨識之IBS2 (intron binding site 2), IBS1, EBS2 (exon binding site 2) 及EBS1序列，推估花生簇葉病菌質體之group II intron主要係以retrohoming之機制插入基因體內。而其內之matR基因轉譯出IEP (intron-encoded protein) 之胺基酸序列亦具有matR基因之保守性RVT (reverse transcriptase), X (maturase), D (DNA binding) 及En (endonuclease) 等區塊 (domain)，但此等選殖株之matR基因在進行轉譯 (translation) 時，相對於洋蔥黃萎病菌質體之matR基因序列均可發現有提早出現之終止密碼 (stop codon)，而且除了EV33選殖株外，其餘選殖株在其序列皆會出現translational frameshift之特性，再者EV9及EV10選殖株之matR基因具有大片段之缺失，顯示吾人所獲得之各matR基因均具讀取架 (reading frame) 不連續之現象，又因各選殖株相互間之核酸序列具明顯差異性，故此七個花生簇葉病菌質體之group II intron應屬於基因體上之不同套組。而選殖株EV9及HIII57之matR基因胺基酸序列與其他套組具明顯差異，經序列比對可將此七個group II intron區分為兩群，顯示花生簇葉病菌質體group II intron具序列異質性 (sequence heterogeneity)。當以matR基因核酸探針R1-1進行北方氏雜配時，可獲得四條大小不一之雜配訊號，其中一條位於約2.4-2.5 kb之片段其訊號較強，此片段大小與選殖株group II intron之預期長度相近，顯示此片段可能為與group II intron上游基因一同轉錄後並經由RNA splicing切下之group II intron RNA。而5 kb之雜配訊號，則可能是group II intron與上游基因一同轉錄產生之一整條pre-RNA；此外，位於約1.5 kb大小之二條雜配訊號，則可能為具有matR基因大片段缺失之group II intron RNA。當針對罹病植株之全RNA進行反轉錄聚合酵素連鎖反應 (reverse transcription PCR, RT-PCR)，可成功增幅出EV33選殖株group II intron RNA約2.4 kb之序列，顯示EV33選殖株之group II intron具有RNA表現，但其matR基因序列於D domain開始處即出現轉譯終止密碼，故推論EV33轉譯出之IEP蛋白在缺乏En domain功能之情況下輔助group II intron RNA於基因體中進行移動複製。

A random ('shotgun') sequencing strategy was conducted to investigate the genome libraries of peanut witches' broom (PnWB) phytoplasma in this study. After screening the PnWB-phytoplasma libraries by using R1-1 probe amplified from the conserved region of partial matR gene in PnWB phytoplasma, a total of seven clones of matR gene containing group II intron sequences of peanut witches' broom (PnWB) phytoplasma were obtained and analyzed. Except clone EI42 that lacked 5’ sequence of group II intron, the group II intron sequences of other six clones contained both 5’ and 3’ boundary sequences. Except clone EV33, the conserved sequences of IBS2 (intron binding site 2), IBS1, EBS2 (exon binding site 2) and EBS1 were identified in these six clones. This may be inferred that these six group II introns may transpose in the genome of PnWB phytoplasma mainly through retrohoming mechanism. After sequence alignment, it was revealed the matR genes in these seven group II introns may comprise the conserved RVT (reverse transcriptase), X (maturase) and En (endonuclease) domains. Premature translation stop codon, translational frameshift, and sequence deletion of matR genes were identified in various clones. Based on the sequence analyses of these seven clones, they were implied to be the various versions of group II intron located in different parts of PnWB genome. Besides, the amino acid sequences of matR gene in EV9 and HIII57 were distinguishable to those of the others, and these group II introns were therefore classified to two classes in PnWB phytoplasm based on the sequence heterogeneity. In Northern hybridization analyses by using R1-1 probe, several RNA transcripts of matR gene were revealed. The hybridization signal of 2.4-2.5 kb in size was the expected length of group II intron and was inferred to be the group II intron RNA excised from the pre-RNA. The longer 5.0 kb Northern hybridization signal fragment was inferred to the unspliced pre-RNA and the two shorter fragments about 1.5 kb in size were inferred to be the deficient group II intron RNAs. A 2400 bp-PCR fragment containing almost the complete sequence of group II intron was amplified in reverse transcription-PCR (RT-PCR) with the total RNA prepared from PnWB-affected periwinkle as template and the sequence was revealed to be identical to that of EV33 clone. This implied that the group II intron in clone EV33 may carry out gene expression. Though there is one advance stop codon found in the D domain in matR gene of EV33, it was revealed that the IEP may still be a functional protein lacking En domain to assist group II intron RNA movement. The results indicate that group II introns have high rates of intron gain and loss and thus create the diversity of group II introns in PnWB-phytoplasma genome.