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標題: | 組合型胰蛋白?抑制因數轉殖於阿拉伯芥的抗蟲能力分析 Testing Combined Trypsin Inhibitor Genes for Insect Resistance using Transgenic Arabidopsis |
作者: | 張善喻 |
出版年 : | 2001 |
學位: | 碩士 |
摘要: | 面對自然界環境逆境〔stress〕,昆蟲亦能不停地進化出抗性族群。為了能夠減緩昆蟲產生具抗性族群,若利用兩個抗蟲基因轉殖進入作物體內,應該可以有效的輔助植物來抵抗昆蟲,並使昆蟲不易產生抗性族群。 在本實驗中,我們建構三個不同的基因,分別是含SPoramin基因的pCAMBIA1301/sporamin、含Arrowhead Proteinase inhibitor〔API〕基因的pcAMBIA1301/API和結合兩者的 pcAMBIA1301/AL,利用農桿菌〔Agrobacterium tumefaciens〕將基因轉殖進入阿拉伯芥〔Agrobidopsis〕中。經由抗生素hygromycin篩選後,再利用南方點墨法〔Southern blot〕和北方點墨法〔Northern blot〕確認基因有插入及轉錄作用〔transcription〕成mRNA。經由GUS活性分析亦發現具有GUS活性,其胰蛋白抑制脢活性分析〔trypsin inhibitor assay〕亦有活性表現,但卻無法由西方點墨法〔Westhern blot〕和膠體胰蛋白脢活性染〔in situ gel trypsin activity assay〕偵測到蛋白質的存在。 利用阿拉伯芥轉殖株的葉片餵食-齡小菜蛾〔Plutella xylostela〕分析轉殖株的抗蟲能力,發現經過四天的餵食,sporamin轉殖株的存活率53.5%,優於API 轉殖株的59%、AL轉殖株的64.5%,蟲重的分析亦顯示sporamin轉殖株比API和AL轉殖株有效。因此具有雙基因的阿拉伯芥轉殖株並不具有更好的抗蟲效果。 阿拉伯芥轉殖株其蛋白質表現量不佳,可能是載體中的ADH intron所造成。另外一方面,具有sporamin和API的雙基因轉殖株可能有未知的因素造成基因表現的互相幹擾,致使雙基因表現較單一基因差。 Facing on the environmental stress, insects evolve the resistant population rapidly. This causes the chemical pesticide and resistant genes invalid shortly. One possible strategy to delay the selection of resistant population is to engineer multiple resistant genes into a single crop cultivar. It is also the way to effectively confer insect resistance on plants. In this study, we use three constructs to perform plant transformation. They are either containing sporamin cDNA gene, i.e. pCAMBIA1301/sporamin, or containing Arrowhead proteinase inhibitor 〔API〕, i.e. pCAMBIA1301/API, and a combinatory construct containing both sporamin and API together, i.e. pCAMBIA13O1/AL. These three constructs were delivered into Arabidopsis respectively by the Agrobacterium tumefaciens-mediated transformation. Many independent transformants of sporamin, API and AL gene were obtained from hygromycin-selection medium. Further identification by Southern blot and Northern blot showed that most transformants were integrated with transgene, and expressed mRNA transcripts effectively. Confirmation by β-glucuronidase 〔GUS〕 activity assays also showed positive results. On the contrary, the presence of TI was not detected from Western blot and in situ gel activity assay. In the bioassay of insect resistance, first-instar insects of Plutella xylostela was fed on leaves of transgenic Arabidopsis. After 4-day feeding, the insect survival rate fed on sporamin is 53.5%, whereas 59% for feeding on API transformants, and 64.5% for feeding on combined API and sporamin transformants. The insect weight also showed that sporamin transformants are more effective than API transformants. Moreover, the insect resistance of transgenic Arabidopsis with combined transgenes was lower than that of plants expressing sporamin or API alone. The low level of transgene expression in Arabidopsis is possibly caused by the presence of ADH intron. However, there might be some unknown factors that interact the gene expression of co-existence of API and sporamin. Therefore, it caused combined gene expressing less than that of the single gene alone. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75178 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
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