臺灣博碩士論文加值系統

外來植物的入侵及歸化，常造成棲地生物多樣性之失衡，也可能影響人畜的健康，或關聯於進出口農產品貿易及國際利益等因素。因此如何建立正確、快速及靈敏的檢測方法，為杜防有害外來植物入侵的重要植物防檢疫工作。南美獨行菜(Lepidium bonariense L.)為十字花科(Brassicaceae)獨行菜屬一年生草本植物，原產地位於南美洲，現已成為臺灣中部非耕地的新歸化雜草。由於南美獨行菜與獨行菜(L. virginicum L.)的植株及種子外觀極為相似，造成形態鑑定上的困擾。分子標誌己普遍運用於植物種類之鑑別，本研究針對南美獨行菜與獨行菜的5.8S rRNA-ITS核酸序列進行選殖及解序，並利用此2序列差異處，衍生為Polymerase Chain Reaction-Restriction Fragment Length Polymorphisms (PCR-RFLP)、allele-specific PCR(AS-PCR)及核酸晶片(DNA chip)等檢測方法；同時建立檢測南美獨行菜的Inter-Simple Sequence Repeat (ISSR)及Random Amplified Polymorphic DNA (RAPD)分子標誌。
研究結果顯示南美獨行菜與獨行菜5.8S rRNA-ITS序列長度分別為620及621 bp，一致性(identity)為98.9%，二者之間有6個鹼基之差異，其中南美獨行菜ITS2較獨行菜少1個鹼基。經由ITS序列比對，南美獨行菜與獨行菜分別有BseY I及Rsr II限制酶切之序列，5.8S rRNA-ITS核酸片段經此2種限制酶反應及電泳圖譜分析，可明顯區別此2植物，衍生為PCR-RFLP之鑑定方法；另於二者ITS序列差異處設計專一性引子，分別於南美獨行菜與獨行菜增幅365 bp及263 bp核酸片段，建立AS-PCR之鑑定方法；亦可經由設計專一性探針，於60℃的雜合條件可明顯區別此2植物，延續發展為呈色系統之核酸晶片檢測法。此外利用ISSR UBC#807、818、820、823、846、849、855、873及881等9組引子，可於南美獨行菜與獨行菜基因組核酸分別增幅300-3,000 bp之間的2至7條多型性核酸條帶；亦可利用RAPD＃4、16、17、23、25、30、31及50等8組引子，可分別增幅400-2,000 bp之間的2至8條多型性核酸條帶，皆具有明顯區別南美獨行菜及獨行菜之效果。
本研究針對外來入侵植物南美獨行菜以獨行菜(Lepidium virginicum Ｌ.)為對照，建立5.8S rRNA-ITS序列比較、PCR-RFLP、AS-PCR、DNA chip、ISSR及RADP等鑑定方法，其中AS-PCR及ISSR兩種分子標誌具有簡易、快速及經濟等特點，適合少量樣品檢測之用；而核酸晶片法適用於高通量樣品的快速檢測，皆可應用於入侵植物的監測及管理，進而維護臺灣原生物種之生態多樣性與平衡。
關鍵詞：南美獨行菜、分子標誌、核糖體核酸、內轉錄間隔區、聚合酶鏈鎖反應-限制片段長度多型性、特定基因片段序列、核酸晶片、簡單序列重複區間、隨機增幅片段多型性

Lepidium bonariense L., an annual Brassicaceae plant native to South America, has become an naturalized plant in Taiwan in recent decades. As the morphological characteristics is similar, L. bonariense L. has been easily misidentified with L. virginicum L. DNA-based molecular markers have been used to detect the genetic diversity of invaded alien species. Novel methods for the identification of the invasive plant L. bonariense and L. virginicum at the early stage of plant development have established in this study, based on direct sequencing of the internal transcribes spacer (ITS) region of 18S-26S ribosomal DNA (rDNA), PCR-restriction fragment length polymorphism (RFLP), allele-specific PCR, DNA chips, inter-simple sequence repeat (ISSR) and Random Amplified Polymorphic DNA (RAPD) markers. The results from sequential difference analysis showed that the 5.8S rRNA-ITS regions of L. bonariense and L. virginicum were 620 bp and 621 bp, respectively. The PCR product on the 5.8S rRNA-ITS of L. bonariense and L. virginicum were digested with the restriction endonuclease Rsr II and BseY I. Each fragment gave unique electrophoretic profiles with PCR-RFLP analysis. By using specific designed primers and probes that comparing the differences of ITS1 and ITS2 between L. bonariense and L. virginicum with AS-PCR and DNA chips. The result of ISSR and RAPD analysis, only ISSR UBC primers #807, 818, 820, 823, 846, 849, 855, 873, 881 and RAPD primers # 4, 16, 17, 23, 25, 30, 31, 50 could significantly distinguish L. bonariense and L. virginicum by 2-8 different polymorphic markers. This result indicated that AS-PCR, ISSR and DNA chips methods can more rapid, accurate identification of L. bonariense at the molecular level and may assist the effective management in this invasion plant and maintain the balance of biodiversity in agricultural ecosystems.
Key words, Lepidium bonariense L., molecular marker, ribosomal RNA, Internal transcribes spacer (ITS), Polymerase Chain Reaction-Restriction Fragment Length Polymorphisms (PCR-RFLP), Allele-specific PCR (AS-PCR), DNA chip, Inter-Simple Sequence Repeat (ISSR), Random Amplified Polymorphic DNA (RAPD)

目錄
中文摘要……………………………………………………I
英文摘要 …………………………………………………..IV
目錄 ………………………………………………...VIII
表目錄 …………………………………………………..VI
圖目錄 ………………………………………………….VII
壹、前言 …………………………………………………….1
貳、前人研究 ………………………………………………….....4
参、材料與方法 ……………………………………………….......22
肆、結果 ....………………………………………………...34
伍、討論 …………………………………………………...38
陸、結論 …………………………………………………...46
柒、參考文獻 …………………………………………………...47
縮寫表 …………………………………………………..68
附錄 ………………………………………………......69
表目錄
表1、本研究使用之引子序列、基因來源及預增幅之核酸長度..66
表2、南美獨行菜及獨行菜5.8S r RNA、ITS1及ITS2序列長度比67
圖目錄
圖1、利用PCR增幅南美獨行菜及獨行菜5.8S r RNA-ITS序列
之電泳圖譜.…………………………………………………57
圖2、南美獨行菜及獨行菜5.8S rRNA-ITS序列之比較…………. 58
圖3、南美獨行菜及獨行菜之PCR-RFLP標誌………………….... 59
圖4、南美獨行菜及獨行菜5.8S rRNA-ITS核酸之Allelic-
specific PCR標誌…………………………………………….60
圖5、利用生物晶片檢測南美獨行菜及獨行菜之圖譜……………. 61
圖6、南美獨行菜及獨行菜之ISSR標誌圖譜……………………... 62
圖7、南美獨行菜及獨行菜之RAPD標誌…………………………. 63
圖8、以AS-PCR、ISSR及RAPD分子標誌檢測南美獨行菜及
獨行菜種子……………………………………………………... 64

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