苦豆子cDNA文库的构建  

Construction of cDNA library from Sophora alopecuroides L.

作　　者：罗淑萍[1] 张智俊[2] 许键[1] 喻梅辉[1] 

机构地区：[1]新疆农业大学农学院,乌鲁木齐830052 [2]南京大学生命科学学院,南京213092

出　　处：《新疆农业大学学报》2004年第2期8-11,共4页Journal of Xinjiang Agricultural University

基　　金：国家自然科学基金资助项目(39660022);国家863计划资助项目(Z17-01-01-6)

摘　　要：　以产于新疆豆科碟形花亚科槐属植物苦豆子(Sophoraalopecuroides.L)幼苗为材料,用改进的一步抽提法提取了总RNA,再以生物素标记的Oligo(dT)为引物,经过磁性球珠法,分离出mRNA,反转录酶催化合成cD NA。与EcoRIAdaptor连接,再经SeptacrylS 400分级,磷酸化后将双链cDNA克隆于λgt11载体中,经体外包装和感染Y1090菌株,成功构建了效价为0.9×106pfu·ml-1苦豆子幼苗cDNA文库。随机挑选5个重组噬斑,PCR法检查出重组克隆的插入片段平均大于1000bp,说明cDNA文库质量较高。Total RNA from the sprout of Sophora alopecuroides L. was purified by using the improved one-step extraction method. The mRNA was isolated from it by using Biotin-labeled Oligo(dT) and SA-PMPs. The cDNA was synthesized and catalysed by reverse transcriptase. The cDNA was then linked with EcoRI adaptor.After Sephacryl S-400 was fractionized and small fragment of cDNA and excess adaptors were phosphorated, the double strand cDNA was colonized into lamb λgt11 vector which was later packaged with bacteirophage proteins to transform the host bacteria strain Y1090 forming cDNA library with the titer of 0.9×10~6 pfu·ml^(-1). PCR of DNA from 5 randomly picked recombinant placques from the library. It showed that the average size of inserts was larger than 1000bp indicating the good quality of the library.

关 键 词：苦豆子 CDNA文库 PCR 

分 类 号：S551.01[农业科学—作物学]