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標題: | 作物物種間葉綠體DNA保留序列之搜尋及應用 The Search and Application of Conservative Chloroplast DNA Sequences among Crop Species |
作者: | Ying Chong 鍾瑩 |
指導教授: | 林順福 |
關鍵字: | 作物,葉綠體,基因體,保留序列, crop,chloroplast,genome,conserved sequence, |
出版年 : | 2009 |
學位: | 碩士 |
摘要: | 目前可利用於植物分類及演化研究之基因序列非常有限,而由於葉綠體基因體較核內基因體小,較容易獲得完整之序列,且一般具有母系遺傳之特性,故適合於基因體序列比對及應用於物種間之鑑別或演化研究。本研究目的在葉綠體基因體中的非保留區中搜尋保留序列,做為設計引子之依據,增幅非保留區序列以應用在物種鑑定,並且以13個物種及在NCBI網站公開之128個已知序列驗證應用可行性。
於NCBI資料庫中搜尋葉綠體基因體完整定序之6種物種,包含單、雙子葉模式植物(水稻及阿拉伯芥)及4種豆科植物(百脈根、疏花苜蓿、大豆及菜豆),進行比對分析後發現各物種葉綠體DNA總長度介於124,033 bp至154,478 bp,基因數則介於65至159個;基因體長度差異與葉綠體基因體中反轉重複序列(inverted repeat,IR)缺失有關,而基因數的差異則與資料庫中已註解之基因數有關。將搜尋之7物種條件劃分為保留區與非保留區,可劃分出92個非保留區域,其總長度佔整體基因體88.73% ∼ 91%,而92個非保留區域中各別長度差異極大,小則包含了僅1 bp的單一核苷酸變異,大則為65,183 bp之大區域變異。與保留區相較之下,非保留區域則有較多的片段位於基因間之區域(保留區域1.1%;非保留區域16.3%),亦即位於非編碼區之比例較高。 於非保留區域內部搜尋具連續性相似之保留序列供設計引子使用,因多數小片段之保留區緊鄰變異區,符合條件之搜尋結果僅佔1.69% ∼ 2.10%,而無論是整個非保留區域亦或是非保留區域內部的保留序列,G或C所佔比例皆介於30% ∼ 40%,較短保留序列及較低G或C所佔比例使得在非保留區之內設計引子的困難度提高。經過設定條件篩選後於適當之保留序列中設計6組引子對F89/R89、F134/R134、F167/R167、F223/R223、F245/R245及F289/R289,預期能增幅200 bp至1600 bp片段。依據這6組引子所涵蓋之DNA序列進行6物種之分群結果與APG分類結果相符。實際應用於13物種(17品種)發現, F134/R134及F167/R167引子對可能因G或C所佔比例偏低或是DNA序列包含過多的poly A或poly T,而無法順利定序進行分析。以F89/R89、F223/R223、F245/R245及F289/R289等4組引子對序列進行分群分析結果,與本研究室先前研究及現有之分類法均一致。另外將NCBI資料庫中所有之其他128個物種完整葉綠體基因體序列與可順利增幅條帶之4組引子對做序列比對,其中F289/R289可對應到較多的物種,且其增幅之DNA序列在物種間具有高比例(71.05%)之變異性,故有最佳物種鑑定應用潛力。 在相同植物物種內不同品種間之6組DNA序列中僅具有1∼3個變異,仍不足以做為物種內區分的依據,必須再設計更多引子對或修改部分引子序列,以提高物種內鑑定效果。 There is limited amount of gene sequence available for species identification and evolutionary. The chloroplast genomic sequence is suitable for alignment, and application in specific identification and evolutionary studies due to its characteristics of smaller size than the nuclear genome, easier to obtain the complete sequence, and maternal inheritance. The purposes of this study were to search the conserved sequences in non-conserved regions of the chloroplast genomes, to design primers, and to amplify non-conserved sequences for identifying different species. The feasibility of designed primers were verified by testing 13 species and 128 known sequences published in NCBI website. The complete chloroplast genome sequences of four legumes (Lotus japonicus, Medicago truncatula, Glycine max, and Phaseolus vulgaris), the model plant of dicotyledon (Arabidopsis thaliana), and the model plant of monotyledon (Oryza sativa ssp. japonica and Oryza sativa ssp. indica ) were searched in NCBI database. The total length of each chloroplast DNA was ranged from 124,033 bp to 154,478 bp, and the number of the genes was between 65 and 159. The difference in the length of chloroplast genome is related to the deficiency of one inverted repeat (IR) sequence; however, the difference of gene number is related to the number of annotated gene of a species. A total of 92 non-conserved regions, covering 88.73% ∼ 91% of the whole genomes, were identified. However, the length of each non-conserved region was quite different. The smallest piece contained only one single nucleotide polymorphism and the largest region contained 65,183 bp long. In comparison with the conserved sequences, more non-conserved regions were located in the intergenic region (1.1% in conserved region; 16.3 % in non-conserved region), namely, higher proportion of non-conserved sequences were located in non-coding regions. Only 1.69% ~ 2.10 % of conserved sequences were suitable owing to most short conserved sequences were closely connected with non-conserved regions. Moreover, about 30% ~ 40% of G or C contents were found whether in the conserved or non-conserved regions. The short conserved sequences and low G or C content made the difficulty of designing the primers between non-conserved regions. The pairs of designed primers F89/R89, F134/R134, F167/R167, F223/R223, F245/R245 and F289/R289 were expected to amplify 200 bp ~ 1600 bp fragments. According to the DNA sequences between the primer pairs, the clustering result of 7 plant species was same as APG classification. The primer pairs were verified by testing with thirteen crop species. Fragments amplified by F134/R134 and F167/R167 primer pairs were unable to be sequenced successfully due to the low G or C contents or containing too many poly A or poly T. Other sequences amplified by F89/R89, F223/R223, F245/R245 and F289/R289 primers revealed the same phylogeny result with previous studies in our laboratory and the existing plant classification. In addition, the sequences of the four primer pairs were probed in all 128 complete chloroplast sequences of plant species. More matched events (34.38%) on the primer sequences and high proportion (71.05%) of sequence variation among species were observed in the primer pairs F289/R289, inferring the superior application potential in specific identification. Only one to three sequence variations among varieties of a species were identified in the four amplified regions. It is necessary to design more primers or to modify primers sequences for variety identification. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44141 |
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