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標題: | 甘藷Sporamin基因轉殖於菸草內之表現及其抗蟲特性之探討 Study on Gene Expression and Insect-Resistance of Sweet Potato Sporamin in Transgenic Tobacco |
作者: | 林美吟 |
出版年 : | 1996 |
學位: | 碩士 |
摘要: | Sporamin為甘藷塊根中含量豐富的儲藏性蛋白質,因具有trypsin inhibitor活性,推論可能具有抗蟲能力。本實驗利用轉殖植物來研究sporamin在栽培菸草(Nicotiana tabacum cv Wisconsin 38)的表現情形及抗蟲效應。 將sporamin cDNA(簡稱為TIA)之BamHI/XbaI片段接至雙向載體(binary vector)pBI121上,以直接轉型法(direct transformation)殖入農桿菌(Agrobacterium tumefaciens)LBA 4404菌株中,得到的轉殖菌株,以TIA cDNA為探針,進行南方雜合反應(Southern hybridization)確認無誤後,再利用葉圓片轉型法(leaf disc transformation)感染栽培菸草,誘導植株再生,經由100 μg/ml kanamycin篩選,得到六棵轉殖植株(Tb1?3,Tb 6?8),在這些轉殖植株中,TIA::pBI121插入菸草基因組(gemone)的數目及位置皆不同,除Tb8外,均能轉錄TIA mRNA(0.9 kb)及少量的TIA-GUS mRNA(2.7 kb)。菸草轉殖植株經自交產生的種子,以含200 μg/ml kanamycin培養基測定是對kanamycin的抗性,顯示TIA::pBI121能穩定遺傳給子代。由蛋白質活性分析得知,除Tb6外,轉殖植株均含有trypsin inhibitor活性。最後以斜紋夜盜蟲(Spodoptera litura)幼蟲進行轉殖植株抗蟲試驗,分析葉片被取食面積發現轉殖植株對斜紋夜盜蟲的抗性較未轉殖植株為佳,且斜紋夜盜蟲在取食轉殖菸草後,其存活率及體重均受影響,顯示sporamin具有抗蟲性質,可作為生物試劑及作物基因轉殖的材料。 Sporamin is an abundant storage protein in tuberous root of sweet potato. Because of its trypsin inhibitory activity, it is suggested to be a protein with insect-resistant character. In this report, the gene expression and protein production of sporamin in transgenic and untransgenic tobacco (Nicotiana tabacum cv. Wisconsin 38) were studied. The insecticidal activity of the transgenic tobacco was also evaluated. To initiate this study, a BamHI/XbaI fragment of the cDNA clone, TIA (trypsin inhibitor A), encoding sporamin was introduced to the binary vector pBI121. The TIA::pBI121 construct was then transferred into Agrobacterium tumefaciens LBA4404 by direct transformation. Clones containing this construct were identified by Southern blot using TIA insert as a probe and were further used to transform leaf discs of tobacco. Transformants were selected by kanamycin (100 μg/ml) resistance. After selection and regeneration, 6 transgenic plants were obtained. As shown by Southern blot analysis, TIA::pBI121 inserted into different position of the tobacco genome with different copy number in transgenic plants. All transgenic plants transcribed TIA mRNA (0.9kb) and a little TIA-GUS mRNA (2.7kb) except the Tb8 plant. The seeds produced by self-fertilization of individual transgenic plant (R0) were germinated on media containing 200 μg/ml kanamycin for genetic test. The segregation of T1 seedlings according to their kanamycin resistance indicates that the inserted genes are stably inherited. Protein activity stain showed that all transgenic plants except Tb6 contained trypsin inhibitory activities. The presence of TIA in transgenic tobacco showed significant effects on the growth and viability of Spodoptera litura larvae according to the feeding experiments. The leaf consumed by the larvae was much less in transgenic than in untransgenic plants. The results above indicate the insect-resistant character of sporamin. Sporamin is, therefore, a potential candidate for biopesticide, ie. insectcides, and for genetic engineering of insect-resistant crops. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76213 |
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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