利用5.8S rRNA-ITS序列發展3種鑑定外來入侵植物南美獨行菜(Lepidium bonariense L.)與獨行菜(L. virginicum L.)之分子標誌

Three Molecular Markers Developed from 5.8S rRNA-ITS Sequences Used in Rapid Detection of Invasive Plants Lepidium bonariense L. and L. virginicum L

袁秋英(Chiou-Ing Yuan) ； 蘇承鋐(Cheng-Hung-Su) ； 林李昌(Li-Chang Lin) ； 蔣慕琰(Mou-Yen Chiang)

《作物、環境與生物資訊》 8卷2期 (2011/06) Pp. 119-128

https://doi.org/10.30061/CEB.201106.0004

摘要

外來植物的入侵及歸化，常造成棲地生物多樣性失衡，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，易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，本研究利用5.8SrRNA-ITS序列之差異，衍生為AS-PCR、PCR-RFLP及核酸晶片技術，建立南美獨行菜與獨行菜之檢測方法。試驗結果顯示南美獨行菜及獨行菜5.8S rRNA-ITS序列長度分別為620及621bp，獨行菜於ITS2多1個鹼基，二者於5.8S rRNA-ITS序列的一致性(identity)為98.9%，僅有6個鹼基之差異，其中2個鹼基位於ITS1，4個鹼基位於ITS2。經由比對南美獨行菜及獨行菜5.8S rRNA-ITS核酸之異同，設計AS-PCR的特異性引子，PCR反應可於南美獨行菜與獨行菜分別增幅365及263bp核酸片段；另可利用核酸差異處之BseY I及Rsr II限制酶反應，將南美獨行菜與獨行菜18S rRNA-26SrRNA之PCR核酸片段，切割為555及152 bp或是551及157bp之多型性條帶；亦可經由設計專一性探針，延續發展為呈色系統之核酸晶片檢測法。本研究所建立之AS-PCR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，後續可應用於進口作物種子中夾帶雜草種子之檢驗，或入侵來源比對之用，協助外來入侵植物之風險評估、監測及管理，進而維護臺灣原生物種之生態多樣性與平衡。

關鍵字

南美獨行菜 ； 獨行菜 ； 分子鑑定 ； 核糖體核酸 ； 間隔區 ； 聚合酶鏈鎖反應－限制酶片段長度多型性 ； 等位基因特異性之聚合酶鏈鎖反應 ； 核酸晶片

並列摘要

Invasive alien species impose one of the greatest biological threats on biodiversity, in addition to affecting ecosystems, native species and human health. Thus, developing accurate, fast and sensitive methods to detect harmful exotic plants is important for plant inspection and quarantine works. Lepidium bonariense L., an annual Brassicaceae plant native to South America, has naturalized in Taiwan in recent years. Its morphological characteristics are similar to L. virginicum L. and can be easily mixed at the early stage of plant growth. DNA-based molecular markers have been widely used to detect the genetic diversity of invaded alien species. In this study, we developed 18S-26S ribosomal DNA (rDNA), based on direct sequencing of the internal transcribes spacer (ITS) region, to differentiate these two weed species by the methods of allelic-specific polymerase chain reaction (AS-PCR), PCR-restriction fragment length polymorphism (RFLP) and DNA chips. The 5.8S rRNA-ITS regions of L. bonariense and L. virginicum were 620 and 621 bp, with the similarity of 98.9%. The specific primers of AS-PCR were designed from the 5.8S rRNA-ITS nucleotide polymorphism via multiplex PCR, to produce unique 365 bp and 263 bp single bands, respectively. Through PCR-RFLP method, the PCR products of 18S-26S rRNA in L. bonariense and L. virginicum were digested with the restriction of endonucleases BseY I and Rsr II. Each fragment gave unique electrophoretic profiles. DNA chips assay of amplified DNA fragments from AS-PCR products used specific oligo nucleotide probes. Visible spots could be detected on the nylon slides. Results suggest that AS-PCR markers and DNA chips are effective for management of invasive plant L. bonariense and maintaining the balance of biodiversity in agricultural ecosystem.

並列關鍵字

Lepidium bonariense L. ； L. virginicum L. ； Molecular marker ； Ribosomal DNA ； Internal transcribed spacer ITS ； Polymerase chain reaction-Restriction Fragment Length Polymorphism PCR-RFLP ； Allele-specific polymerase chain reaction AS-PCR ； DNA chip