作物葉綠體DNA序列變異之辨識及應用

Abstract

本研究由NCBI的GenBank資料庫中搜尋四種豆科植物(百脈根、疏花苜蓿、毛豆及菜豆)、雙子葉模式植物(阿拉伯芥)及單子葉模式植物(水稻)之葉綠體整個基因體DNA序列，並且進行序列比對分析，結果發現葉綠體DNA序列之內含子(intron)與外顯子(exon)交界不符合GT/AG序列法則；辨識出93個保留區長度佔基因體總長8.59%~10.70%，大都分布於基因區，且編碼區保留性較高，位於rRNA與tRNA基因之保留區在物種間相似性較高。經由上述比對分析後設計出5引子對，位於葉綠體基因體的位置分別為psbA~trnK、psbB~psbH、rpl23~trnI、trnR-ACG~trnN-GUU及trnY-GUA~ trnD-GUC，預期實際應用在其他物種上所能增幅之變異序列長度分別約為500~600 bp、800~900 bp、650~700 bp (豆科以外物種為350 bp)、700~800 bp (雙子葉植物為400 bp)及500~600 bp。上述5組引子對實際應用在包括豌豆、落花生、綠豆、毛豆(兩個品種)、豇豆(兩個亞種)、紅豆、蠶豆、甜瓜、茶樹、馬鈴薯、水稻(三個品種)、玉米、大芻草及薏苡等14個物種之PCR長度變異，具有良好之效果。選擇其中三組引子對(基因體位置為psbA~trnK、psbB~psbH及trnR-ACG~trnN-GUU)增幅片段之序列進行親緣分析，以最大簡約法及鄰接法之分群結果皆與目前植物分類相符，故三組引子對在物種間親緣關係分析及物種鑑定上都有不錯的效果，位於psbA~trnK及psbB~psbH之引子對效果尤佳，但是三組引子對在物種內變異的辨識效果不甚理想，在兩個毛豆品種間(高雄5號與高雄8號)、兩個豇豆亞種間(短豇豆與長豇豆)及三個水稻品種間(台農67號、台稉9號與台中秈10號)的增幅片段沒有明顯差異存在。本研究設計之引子對可有效辨識物種間差異性，但是欲辨識物種內差異性則需再搜尋其它變異區域重新設計引子並加以驗證。

The complete chloroplast genome sequences of four legumes (Lotus japonicus, Medicago truncatula, Glycine max, Phaseolus vulgaris), the model plant of dicotyledon (Arabidopsis thaliana), and the model plant of monotyledon (Oryza sativa ssp. japonica) were searched from the GenBank database of NCBI for identifying chloroplast DNA sequence variation among plant species. Result from sequence alignment and comparison indicated that the junctions between intron and exon of the chloroplast genes didn’t follow the GC/AG rule. A total of 93 conserved regions accounting for 8.59%~10.70% length of the whole genome were identified. Most of conserved sequences were located in the coding regions. The conserved regions of rRNA and tRNA genes exhibited higher sequence similarity among plant species. Five primer pairs were designed from the conserved regions distributing in psbA~trnK, psbB~psbH, rpl23~trnI, trnR-ACG~trnN-GUU and trnY-GUA~trnD-GUC, respectively to amplify sequences about 500~600 bp, 800~900 bp, 650~700 bp (350 bp, not legume species), 700~800 bp (400 bp in dicotyledon), and 500~600 bp. The application of above primer pairs were verified from the length variation in PCR products of 14 tested species, including pea (Pisum sativum), peanut (Arachis hypogaea), mungbean (Vigna radiata), vegetable soybean (Glycine max), cowpea (Vigna unguiculata), azuki bean (Vigna angularis), broad bean (Vicia faba), melon (Cucumis melo), tea (Camellia sinensis), potato (Solanum tuberosum), rice (Oryza sativa), maize (Zea mays), teosinte (Euchlaena mexicana), and Jobs tears (Coix lacryma-jobi). Three primer pairs located in psbA~trnK, psbB~psbH and trnR-ACG~trnN-GUU regions were applied in phylogenetic analysis according to Maximum parsimony and Neighbor-joining methods. The consistent results between the phylogenetic analysis and taxonomy indicated the application potential of the three primer pairs in phylogenetic analysis and species identification. However, due to no sequence variation observed in amplified regions between two vegetable soybean varieties (KS5 and KS8), two cowpea subspecies (Vigna unguiculata ssp. cylindrica and Vigna unguiculata ssp. sesquipedalis), and among three rice varieties (TNG67, TK9, and TCS10), the three primer pairs are not effective in identifying variation within species. The designed primers in this study were effective in identifying the differences inter species. It is necessary to screen chloroplast DNA sequence variation and design new primers if we want to identify difference within species.