Transgenic kailan expression a modified Bacillus Thuringiensis; cryIA(b) gene

• Main Authors: Wei-Kai Yang, 楊惟凱
• Other Authors: Ching-Long Lan
• Format: Others
• Language: zh-TW
• Published: 1995
• Online Access: http://ndltd.ncl.edu.tw/handle/13185138245011752161

碩士 === 輔仁大學 === 生物學研究所 === 83 === 5-AzaC(5-Azacytidine)是常用去甲基化 (demathylation) 藥劑,前人研 究指出 5-AzaC 處理可提高轉殖率, 然而過量濃度處理會誘發突變.發芽 中的芥藍種子以 10 uM, 20 uM,40 uM, 60 uM, 80 uM 五種5-AzaC濃度處 理 24 hr, 經過 3 個月的觀察, 處理組和對照組植株在外形上並沒有顯 著差異, 以8 支含 10 個鹽基序列的逢機引子, 檢視處理組和對照組的 RAPD(random amplified polymorphic DNA) 剖面圖譜沒有發現顯著差異. 將攜帶部份修飾過的蘇力菌 cryIA(b) 基因的表現載體 pBI121NII以直接 轉形法導入農桿菌菌系 LBA4404/pAL4404 和 EHA101/pAL133 後分別感染 圓葉白花芥藍, 尖葉白花芥藍, 圓葉黃花芥藍三品系. 培殖體經感染後於 含有 50 ug/ml kanamycin 及 500 ug/ml carbencillin 之誘導芽體培養 基 (改良式 MS 基礎培養基 NAA 0.2 mg/l, BAP 0.1 mg/l) 誘導發芽, 經過篩選後, 移至發根培養基 (改良式 MS 基礎培養基 NAA 0.2 mg/l, BAP 1.0 mg/l) 誘導發根, 根系完全後, 移至溫室, 供日後生物檢定用. 以LBA4404/pAL4404/pBT121NII 感染前述三芥藍品系擬轉殖百分比分別為 圓葉白花 0.5%(4/800), 尖葉白花 2.37%(11/404), 圓葉黃花 7.36%(32/435), 以EHA101/pAL133/pBT121NII 感染擬轉殖百分比分別為 圓葉白花 0.65%(5/800), 尖葉白花 3.71%(14/377), 圓葉黃花 12.58%( 62/493). 圓葉白花芥藍種子經 20 uM,40 uM, 5-AzaC 處理, 以 LBA4404/ pAL4404/pBT121NII 感染, 擬轉殖百分比分別為 1.75%(7/400), 4.44%( 5/136); 以EHA101/pAL133/pBT121NII 感染擬轉 殖百分比分別為 2.5%( 10/400), 5.18%(7/135). 擬轉殖植株經 PCR 鑑 定後, 產生 0.49kb 大小之 cry 基因 DNA 片段, 初步確定植物轉殖成功 者有 144 株, 其中圓葉白花較早感染成功者有 37 株, 經馴化後剩下 21 株, 其他品系植株, 因為較小, 而尚未馴化.抽取擬轉殖植株蛋白質共 有 51 個樣品, 經 ELISA 偵測發現有 7 個樣品偵測到 cryIA(b) 蛋白存 在, 將擬轉殖植株 #1-#2 之葉片剪下, 餵食二齡初小菜蛾幼蟲. 每 24 hr 換葉片, 連續觀察 72 hr, 結果顯示,檢試的基因轉殖株均無名顯殺蟲 效果. Germinating Kailan seeds were given 24 hr exposure to 10, 20 40, 60, 80 uM 5-Azacytidine. No significant phenotypic difference was observed among the treated and the untreated plants during 3 month. Eight 10-bp primers were used to generate RAPD markers. The genomic fingerprints were also undistinguishable among the treated and the untreated plants. Plasmid carrying a modified cryIA(b) gene was introduced into Agrobacterium tumefaciens LBA4404/pAL4404 or EHA101/pAL133. Hypocotyl explants of cultivars RWK, SWK, and RYK were cocultured with the engineered bacteria and then regenerated on the shoot-induceing medium(MS medium added 0.2 mg/l NAA,and 0.1 mg/l BAP) containing 50 ug/ml kana- mycin and 500 ug/ml carbenicillin. Survived shoots were grown on the root-inducing medium (MS medium added 0.2 mg/l NAA, and 0.1 mg/l BAP). Plants with well-developed root system were finally hardened and moved to greenhouse. Explants infected with LBA4404/ pAL4404/pBI121 NII produced 0.5% (4/800),2.37% (11/404) and 7.36% (32/435) putative transformants, respectively. Explants infected with EHA101/pAL133/pAB121NII, on the other hand, produced 0.65%( 5/800), 3.71% (14/377) and 12.58%(62/493) putative transformants, respectively. RWK explants, previously exposed to 20 and 40 uM 5- AzaC,infecred with LBA4404/pAL4404/pBI121NII yielded 2.5%(10/400) and 5.18%(7/135) putative transformants, respectively. The inte- gration of cry sequence into genome of the putative transformants were further examined using PCR method. Totally, 144 trans- formants were confirmed of them, 21 RWK-transgenic plants were hardened and evaluated their tolerances to the diamondback month. Unfortunately, none of them produced significant amount of cry protein. Total protein extracted from 51 putative transformants were also screened for the presence of cryIA(b) protein using the ELISA method. Seven transformants were ELISA-Positive.