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標題: Molecular Cloning of a Glutathione S-Transferase GST-4 cDNA of Diamondback Moth, Plutella xylostella (L.)
小菜蛾麩胱甘轉基 GST-4 cDNA 之選殖
作者: 鍾威垣
Chung, Wei-Yuan
關鍵字: glutathione S-transferase;麩胱甘轉基;molecular cloning;insect resistance;分子選殖;昆蟲抗藥性
出版社: 昆蟲學系
Four glutathione S-transferase ( GST, EC 2. 5. 1. 18 ) isozymes have been characterized from larvae of the diamondback moth, Plutella xylostella (L.). Among them, GST-4 has biochemical, toxicological and immunological properties similar to GST-3. Both GSTs are involved in this insect resistance to some insecticide. The present work used GST-3 cDNA as a probe to screen midgut cDNA library of CME strain, and eight clones were obtained. While 4 cDNA clones were found identical to that of GST-3, the other 4 clones were the same and different from GST-3 cDNA only at 610th nucleotide, with a change of amino acid at Gln204 to Lys. The calculated pI of GST-4 was 8.78, and this increase of pI ( 8.5 for GST-3 ) coincided with the observed values in isoelectric focusing ( Fig. 1 ). The open reading frame of GST-4 was constructed into the expression vector, pET-21b, and then transferred to E. coli BL21 ( DE3 ). The activity of both GST-3 and GST-4 toward DCNB was significantly inhibited by teflubenzuron, implying a possible binding of the latter to the enzyme.

在十字花科害蟲小菜蛾Plutella xylostella ( L. ) 幼蟲體內,已知有4種麩胱甘轉基 ( GST, EC 2. 5. 1. 18 ) 的同功異構。其中GST-3和GST-4 2個生化毒理性狀相近且有免疫相關性,與小菜蛾對一些殺蟲劑的抗藥性有關。本工作以GST-3 cDNA為探針,篩選CME品系小菜蛾中腸cDNA library,共篩到8個clones。其中C2、C4、C12及C18這4個clones,在DNA序列第610個nucleotide與GST-3不同,序列由C變成A,並使胺基酸Gln204變為Lys。這個胺基酸的改變使計算GST的pI值自8.5增至8.78,與所觀測之pI值關係相似。目前已將GST-4的開放讀架構築在表現載體pET-21b,並送入表現寄主E. coli BL21 ( DE3 ) 中。而GST對受質活性的測定結果,顯示GST-3和GST-4代謝DCNB的活性明顯受到teflubenzuron抑制,顯示後者可能與這2個GST isozyme有binding作用。
Appears in Collections:昆蟲學系

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