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題 名 | 蘇力菌臺灣株B11-30a之營養期殺蟲基因vip3Aa的選殖與表現=Cloning and Expression of the Vegetative Insecticidal Crystal Protein Gene, vip3Aa, from Bacillus Thuringiensis Taiwan Isolate B11-30a |
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作 者 | 石傑宇; 郭雪; 高穗生; 曾經洲; | 書刊名 | 植物保護學會會刊 |
卷 期 | 49:4 2007.12[民96.12] |
頁 次 | 頁299-309 |
分類號 | 433.85 |
關鍵詞 | 蘇力菌; 營養期殺蟲基因; 基因選殖及表現; Bacillus thuringiensis; Vip3Aa; Vegetative insecticidal protein; VIP; Cloning; Expression; |
語 文 | 中文(Chinese) |
中文摘要 | 分離自台灣各地穀倉之111個蘇力菌菌株,利用vip3A基因特異性引子,進行1.1 kb片段的PCR篩選,獲得66%的篩獲率。由菌株B11-30a選殖vip3A相關基因,並進行基因的定序及比對,所得之2,370 bp的vip3A相關基因,與已發表之vip3Aa15具有99.4%同源性,推測是vip3Aa類型基因。轉譯後之胺基酸序列,與Vip3Aa15有7個胺基酸的差異,顯示本基因應該具有地區獨特性,且非因境外移入所產生。選殖此vip3Aa基因至pQE82蛋白質表現載體,進行IPTG的誘導及表現培養,經蛋白質電泳及西方墨點法分析,顯示vip3Aa基因,在大腸桿菌宿主中,可受IPTG誘導及表現,產物約為88 kDa。可能由於表現量不高、形成包涵體(inclusion bodies)導致蛋白質立體結構錯誤的摺疊,或胺基酸序列的些微差異,造成本研究之Vip3Aa蛋白質產物,對於小菜蛾(Plutella xylostella)、擬尺蠖(Trichoplusia ni)、斜紋夜盜(Spodoptera litura)及甜菜夜蛾(Spodoptera exigua)幼蟲的生物活性試驗,均不具明顯的殺蟲效果。 |
英文摘要 | Using vip3A-specific primers to detect vip3A gene from 111 isolates of the Bacillus thuringiensis obtained in Taiwan by PCR, 73 isolates were positive for the vip3A product. A full-length gene encoding vegetative insecticidal protein (Vip) was amplified from B11-30a isolate and cloned into pSC-A vector. Nucleotide sequence analysis revealed that 99.4% of the vip gene was identical to the published vip3Aa15 gene. There were seven amino acid residues in the Vip protein sequence which was different from the Vip3Aa15 protein. Results showed that vip3A gene has the local distinctive characterization and eliminated the possibility that it was imported from abroad. The vip3Aa gene we identified was subcloned into pQE82 expression vector for protein expression in Escherichia coli. Western blot analysis showed that Vip3Aa protein was expressed as expected with a size of 88 kDa. The bioassay data showed that the expressed Vip3Aa protein did not have significant activity against the larvae of Plutella xylostella, Trichoplusia ni, Spodoptera exigua, and S. litura, probably because of the protein misfolding in the inclusion body. |
本系統中英文摘要資訊取自各篇刊載內容。