二温式PCR在特异短核酸探针合成中的应用  

Synthesis of Specific and Short RNA Probes with Two-temperature PCR

作　　者：董倩[1] 黄国强[2] 叶冰莹[3] 陈由强[4] 陈如凯[2] 

机构地区：[1]福建师范大学福清分校生物与化学工程系,福建福清350300 [2]福建农林大学甘蔗综合研究所,福建福州350002 [3]福建师范大学生命科学学院 [4]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

出　　处：《花生学报》2008年第3期5-10,共6页Journal of Peanut Science

基　　金：农业部“948”重大项目资助(2006-G37);国家公益性行业(农业)科研专项项目(nyhyzx07-019);国家“863”计划项目(2007AA100701)

摘　　要：经基因库序列比对确定花生白藜芦醇合酶基因(RS)中的特异序列(长度约108 bp)。以此为模板设计分别含有SalI和SacI酶切位点的一对上、下游引物,并应用二温式PCR扩增得到该特异短片段。将该特异片段正向连接到pBlueskriptIIKS(+)的多克隆位点上,利用T7 RNA聚合酶在体外转录合成地高辛标记的反义特异短核酸探针。该特异短核酸探针的合成为后续RS基因在花生组织器官中表达的RNA原位杂交研究打下了基础。The Arachis hypogaea resveratrol synthase （RS） mRNA specific fragment was determined by NCBI BLAST. Primers with the two restriction enzymes of Sal Ⅰ or Sac Ⅰ were designed and two-temperature PCR was used to amplify the specific and short fragment. The fragment was eventually linked to the multiple cloning site of vector pBlueskript Ⅱ KS （＋）. By means of the T7 promoter in the pBlueskript Ⅱ KS （＋） and using the specific fragment as a templet to synthesize Digoxigeninlabeled antisense specific and short RNA probe in vitro transcription with T7 RNA polymerases. The synthesis of probes laid a foundation for the research of the expression of RS gene in peanut by RNA in situ hybridization.

关 键 词：二温式PCR 花生 白藜芦醇合酶 核酸探针 

分 类 号：S565.2[农业科学—作物学] Q784[生物学—分子生物学]