地高辛标记的黄鳝F64基因cRNA探针的制备及其应用  

Preparation and Application of cRNA Probe Labeled with Digoxingenin for Gene F64 of Rice Field Eel

作　　者：蒋骄云[1] 田文斐[2] 冯龙 曲宪成[4] 

机构地区：[1]广西师范大学生命科学学院,桂林541004 [2]桂林理工大学博文管理学院,桂林541006 [3]泗阳县水产技术指导站,泗阳223700 [4]上海海洋大学水产与生命科学学院,上海201306

出　　处：《生物技术通报》2016年第3期137-141,共5页Biotechnology Bulletin

基　　金：广西师范大学实验室管理创新与技术革新项目(2014GJ02);上海高校知识服务平台上海海洋大学水产动物遗传育种中心基金资助项目(ZF1206)

摘　　要：以黄鳝F64基因序列为模板设计引物,扩增用于c RNA探针合成的模板,构建F64/p GM-T重组质粒并线性化,利用RNA聚合酶体外转录合成正、反义c RNA探针,并对其进行地高辛标记,利用原位杂交方法检测F64基因在黄鳝性腺发育过程中的表达变化情况。结果显示,正义探针未检测到阳性信号,反义探针检测到该基因在黄鳝性腺发育早期不表达,于V期性腺开始表达。研究结果表明,体外转录法可以有效合成c RNA探针,制备的c RNA探针可以准确检测F64基因的时空表达。The specific gene primers were designed based on the sequences of gene F64 of rice field eel,then the templates for synthesizing the cRNA probe were amplified,then recombinant F64/p GM-T plasmids were constructed and linearized by restriction enzymes,further the sense and anti-sense c RNA probes were synthesized by in vitro transcription with RNA polymerase,and finally labeled with Digoxigenin. The expression changes of gene F64 during the gonadal development of rice field eel were detected by in situ hybridization. According to our results,the expression was only detected by the anti-sense F64 cRNA probe,and the earliest signal was observed in stage V of ovarian,but no more in early stage of ovarian formation. Our study indicated that the cRNA probes can be synthesized using in vitrotranscription,and the cRNA probes prepared in this study can be used for accurately detecting the spatial and temporal expression of gene F64.

关 键 词：黄鳝F64基因 性腺发育 c RNA探针 原位杂交 

分 类 号：Q78[生物学—分子生物学]