4H2在褐飞虱的原位杂交分析.doc

4H2在褐飞虱的原位杂交分析 摘要 本研究采用原位杂交技术，对褐飞虱取食抗虫和感虫水稻后，其唾液腺基因表达发生明显变化的4H2基因进行表达分析，分析该基因产生在褐飞虱体内的具体位置。采用将带有靶序列和SP6、T7启动子的PGEM-T-4H2质粒转入大肠杆菌DH-5α，通过氨苄青霉素抗性筛选得到重组质粒，并以ScaI限制性内切酶酶切并且纯化后得到的线性化质粒为模板，DIG RNA Labeling Mix为底物，经T7、SP6聚合酶体外转录，分别制得DIG标记的正义链和反义链RNA探针，最后进行靶基因在褐飞虱的原位杂交定位。结果显示，4H2基因在褐飞虱的脂肪体和唾液腺表达。与其作为卵黄蛋白在脂肪体合成卵黄，并进一步转运到卵中的作用相一致，本研究结果对于进一步分析4H2基因产物在水稻与褐飞虱互作中是否作为激发子奠定了基础。 关键词 褐飞虱；原位杂交；RNA探针；石蜡切片 Location of 4H2 gene in the brown planthopper tissues by RNA in-situ hybridization Huanghanlin School of Life Sciences，Guangzhou University 4H2 was studied in brown planthopper(BPH) tissues by RNA in-situ hybridization because of its expression remarkable changed, when feeding the BPH-resistant rice and BPH- susceptible rice type. PGEM-T-4H2 with target sequences and SP6, T7 promoters was transfered into the Escherichia coli DH-5α, and screened recombinant plasmids by ampicillin resistance; using the product of ScaI restriction and purification as the template, the DIG RNA Labeling Mix as the substrate, through in-vitro transcription by the T7, SP6 polymerase, producing the positive-sense strand marked by DIG and the antisense strand RNA probe respectively, and then finally, it applies the in-situ hybridization technique to locate the target genes in the rice brown planthopper. The result showed that 4H2 gene expressed in the fat body and the salivary glands of the brown planthopper. It has the same role as a yolk protein becoming yolk in the fat body, which is further transported to the egg, the results of this study lay the foundation for further analysis of whether gene 4H2 is acted as elicitors in rice brown planthopper interactions. KEY WORDS the brown planthopper; in-situ hybridization; paraffin section 目录 前言 ……………………………………………………………………………1 材料与方法 ……………………………………………………………………2 2.1 材料 ………………………………………………………………………2 2.1.1 材料 ……………………………………………………………………2 2.1.2 试剂 ……………………………………………………………………2 2.1.3 仪器 ……………………………………………………………………2 2.1.4 试剂配制 ………………………………………………………………2