转LdOA1基因果蝇品系GSTs基因表达及对溴氰菊酯胁迫响应

舞毒蛾是重要林业食叶害虫,揭示其G蛋白偶联受体介导G蛋白对GSTs的调控,这对于挖掘分子靶标开发新型杀虫剂具有重要意义。本文构建转LdOA1基因果蝇载体,获得表达LdOA1基因果蝇品系,分析了转基因果蝇GSTs基因表达量及对溴氰菊酯胁迫的响应,为明确昆虫OA1基因功能提供理论依据。通过传统的酶切-连接方法构建重组载体pUAST-attB-LdOA1,采用转基因技术构建表达LdOA1基因的纯合果蝇品系,利用PCR技术验证转基因果蝇品系,并运用实时荧光定量RT-PCR技术测定转基因果蝇GSTs Delta家族基因表达量及低浓度溴氰菊酯胁迫对GSTs基因表达量的影响。PCR扩增条带显示转LdOA1基因果蝇品系均能检测到453bp目的基因条带。与非转基因果蝇相比,转LdOA1基因果蝇品系中GSTs Delta家族基因(除GSTd4和GSTd7)均上调表达,为非转基因组1.01~3.27倍。低浓度溴氰菊酯胁迫6h,非转基因果蝇GSTd6和GSTd10基因被显著诱导激活,而其他GSTs基因被显著抑制,抑制率为6.48%~95.84%,胁迫12~72h,GSTs基因(除GSTd1和GSTd10外)均被显著抑制。低浓度溴氰菊酯胁迫转基因果蝇GSTs表达量变化趋势与非转基因果蝇基本一致,但转基因果蝇品系GSTs表达量显著高于非转基因果蝇品系(除12~48h GSTd1和GSTd10),且胁迫6h表达量最大。这些结果表明LdOA1基因可能介导G蛋白信号通路调控下游GSTs Delta亚家族基因表达响应溴氰菊酯胁迫。 

Glutathione S-transferase gene expression in transformant Drosophila expressing ocular albinism type 1 from gypsy moth and its response to deltamethrin stress

The Asian gypsy moth ( Lymantria dispar) is a key forest defoliator throughout the world. Study on regulation of G protein for GST genes mediated by G protein coupled receptor contributes to exploring molecular targets for developing novel pesticides to control gypsy moth. In the present study, transformant Drosophila vector was constructed and transformant Drosophila lines expressing LdOA1 were successfully obtained;GSTs gene expression levels in transformant Drosophila and its response to deltamethrin stress were analyzed. These results provided basic information for elucidating insect OA1 gene functions. The recombinant vector pUAST-attB-LdOA1 was constructed by traditional enzyme digestion and ligation technology . The transformant Drosophila lines were obtained by transgenic technology and validated using PCR technology. The expression levels of GSTs Delta family genes and the effects of deltamethrin on GSTs gene expressions in transformant Drosophila were analyzed by real-time RT-PCR technology. The RT-PCR results showed that the 453 bp band of LdOA1 gene was detected in transformant Drosophila lines
expressing LdOA1 gene. Compared with untransformant Drosophila, the expression levels of GSTs Delta family genes, except for GSTd4 and GSTd7 genes, in transformant Drosophila lines were up-regulated with 1. 01-to 3. 27-fold of untransformant Drosophila using qRT-PCR analysis. Under low concentration of deltamethrin stress for 6 h, the GSTd6 and GSTd10 were obviously induced while other GSTs genes were significantly inhibited in untransformant Drosophila, and the inhibition ratios were 6. 48%--95. 84%. Under deltamethrin stress for 12--72 h, the expressions of GST genes, except for GSTd1 and GSTd10, were significantly inhibited in untransformant Drosophila. Compared with untransformant Drosophila, the expressions of GSTs genes in transformant Drosophila varied in similarity under low concentration of deltamethrin stress. However, the GST gene expressions in transformant Drosophila were significantly higher than those in untransformant Drosophila except for GSTd1 and GSTd10 for 12--48 h;and the GST gene expressions in transformant Drosophila were highest under low concentration of deltamethrin stress for 6 h. These results indicated LdOA1 may mediate G protein signal transduction to regulate GSTs Delta subfamily genes in response to deltamethrin stress.