银松素合酶基因地高辛标记RNA探针的合成  

The Synthesis of DIG-Labelled Pinosylvin Synthase Gene RNA Probe

作　　者：郭晋隆[1] 叶冰莹[2] 黄庆煌[1] 黄国强[1] 许玉芬[2] 林志楷[2] 陈由强[1] 陈如凯[1] 

机构地区：[1]农业部甘蔗生理生态与遗传改良重点开放实验室,福建福州350002 [2]福建师范大学生命科学学院

出　　处：《生物技术通讯》2006年第6期891-894,共4页Letters in Biotechnology

基　　金：国家自然科学基金项目(30371177);福建省科技厅项目(2005N037;K04030);国家部委项目(JA05225/wkj2)

摘　　要：目的:体外转录合成银松素合酶(PS)基因地高辛标记反义RNA探针。方法:通过双酶切把PScDNA全长序列接到pBlueskriptⅡKS(+)的多克隆位点上;同时,经由NCBI比对确定PScDNA的特异序列,设计含有SalⅠ和XbaⅠ这2个酶切位点的上、下游引物,用PCR法扩增含有该位点的目的片段,并连接到pBlueskriptⅡKS(+)的多克隆位点上;最后利用pBlueskriptⅡKS(+)上的启动子T7,用T7RNA聚合酶在体外转录合成地高辛标记PS全长及特异序列的反义RNA探针。结果:通过对构建探针的单、双酶切,PCR及电泳鉴定,表明成功合成了PS基因地高辛标记的全长及特异序列的反义RNA探针。结论:RNA探针的合成为后续马尾松PS基因表达的RNA原位杂交研究打下了基础。Objective： To synthesize DIG-labelled pinosylvin synthase（PS） gene RNA anti-sense probe by in vitro transcription. Methods： Link the PS cDNA full-length sequence to the multiple cloning site of pBlueskript Ⅱ KS（＋） by double restriction enzyme digest. Meanwhile, the PS cDNA specific fragment was determined by NCBI BLAST, primers with the two restriction enzymes of Sal Ⅰ and Xba Ⅰ were designed and PCR was used to amplify the target fragment, and was eventually linked to the multiple cloning site of pBlueskript Ⅱ KS （＋）. In the end, by means of the T7 promoter in the pBlueskript Ⅱ KS （＋） and T7 RNA polymerase, DIG-labelled PS gene full-length and specific fragment RNA anti-sense probe was synthesized by in vitro transcription. Results： Identify the construction and probe by single, double enzyme digest, PCR and electrophoresis, DIG-labelled PS gene full-length and specific fragment RNA anti-sense probe was synthesized successfully. Conclusion： The synthesis of RNA probe lays the foundation for the research of the expression of PS gene in Pinus massoniana Lamb by RNA in situ hybridization later on.

关 键 词：银松素合酶基因 探针 马尾松 芪合酶 

分 类 号：Q943[生物学—植物学]