摘要：将以下五种已构建成功的转基因载体pUAST-attB-CYP6CS2v1、pUAST-attB-CYP6BD10v2、pUAST-attB-CYP439A1v3、pUAST-attB-CYP6FJ1v2和pUAST-attB-CYP4C72利用显微注射的方法将其分别导入果蝇胚胎，通过筛选和纯化获得纯合的转基因果蝇品系UAS-CYP6CS2v1、UAS-CYP6BD10v2、UAS-CYP439A1v3、UAS-CYP6FJ1v2和UAS-CYP4C72。通过GAL4/UAS系统，将带有β-tubulin强启动子的tub-gal4果蝇品系分别与UAS-CYP6CS2v1、UAS-CYP6BD10v2、UAS-CYP439A1v3、UAS-CYP6FJ1v2和UAS-CYP4C72杂交，获得全身超量表达灰飞虱CYP6CS2v1、CYP6BD10v2、CYP439A1v3、CYP6FJ1v2和CYP4C72基因的果蝇品系tub>CYP6CS2v1、tub>CYP439A1v3、tub>CYP6FJ1v2、tub>CYP4C72和tub>CYP6BD10v2。利用实时荧光定量PCR的方法，检测了超量表达目标基因的实验组果蝇和未启动目标基因过量表达的对照组果蝇中灰飞虱CYP6CS2v1、CYP6BD10v2、CYP439A1v3、CYP6FJ1v2和CYP4C72基因的表达量。结果显示：实验组目标基因CYP6CS2v1、CYP6BD10v2、CYP439A1v3、CYP6FJ1v2和CYP4C72的表达量分别是对照组表达量的9.95、30.39、19.10、33.30和38.42倍，与目标基因序列相似的果蝇内源基因在实验组和对照组中的表达量显著低于目标基因的表达量，同时无显著性差异（P＞0.05）。因此，灰飞虱CYP6CS2v1、CYP6BD10v2、CYP439A1v3、CYP6FJ1v2和CYP4C72基因在果蝇中得到了超量表达。36747
关键词：灰飞虱；细胞色素P450基因；转基因果蝇；定量PCR
Construction of 5 transgenic Drosophila strains with 5 CYP genes from Laodelphax striatellus and Q-PCR verification
Abstract：Constructed transgenic vectors of pUAST-attB-CYP6CS2v1, pUAST-attB-CYP6BD10v2, pUAST-attB-CYP439A1v3, pUAST-attB-CYP6FJ1v2 and pUAST-attB-CYP4C72, were introduced into Drosophila embryos respectively, by microinjection. Homozygous transgenic Drosophila strains of UAS-CYP6CS2v1, UAS-CYP6BD10v2, UAS-CYP439A1v3, UAS-CYP6FJ1v2 and UAS-CYP4C72 were obtained by screening and purification. The tub-gal4 Drosophila melanogaster strain with β-tubulin promoter was genetic crossed with UAS-CYP6CS2v1, UAS-CYP6BD10v2, UAS-CYP439A1v3, UAS-CYP6FJ1v2 and UAS-CYP4C72 respectively to obtain the systemic overexpression strains of tub>CYP6CS2v1, tub>CYP439A1v3, tub>CYP6FJ1v2, tub>CYP4C72 and tub>CYP6BD10v2. By using the real-time quantitative PCR method, the expression levels of target genes in the experimental groups were detected. The results showed that the expression levels of CYP6CS2v1, CYP6BD10v2, CYP439A1v3，CYP6FJ1v2 and CYP4C72  in the experimental groups were 9.95, 30.39, 19.10, 33.30 and 38.42 times than that in control groups. The expression levels of endogenous related CYP genes in Drosophila were significantly lower than the expression level of target genes. At the same time, there was no significant difference (P > 0.05). Therefore, the CYP6CS2v1, CYP6BD10v2, CYP439A1v3, CYP6FJ1v2 and CYP4C72 genes of small brown planthopper were overexpressed in the Drosophila.
Key words: Laodelphax striatellus; cytochrome P450 ; transgenic Drosophila；Q-PCR
目  录
摘要    1
关键词    1
Abstract    1
Key words    1
引言    1
1  材料与方法    2
1．1 供试果蝇品系及饲养方法    2
1．1．1  供试果蝇品系    2
1．1．2  果蝇的饲养    2
1．2 主要试剂与仪器    3
1．2．1 实验主要试剂    3
1．2．1 实验仪器设备    3
1.3显微注射    3
1.3.1制作产卵碟    3
1.3.2 新鲜胚胎的收集    3
1.3.3显微注射    4
1.4转基因果蝇品系的纯化    4
1.4.1与W1118果蝇杂交    4
1.4.2与平衡子杂交    4 5个转灰飞虱CYP基因果蝇品系的建立和定量PCR验证:http://www.youerw.com/shengwu/lunwen_35318.html