甜菜单体附加系M14花蕾cDNA文库的构建

Construction of cDNA Library of M14 Bud,Themonosomic Addition Lines of Beta vulgaris

为了克隆甜菜单体附加系M14无融合生殖相关基因 ,采用cDNA文库快速构建法制备了M14花蕾cDNA文库。根据甜菜单体附加系M14无融合生殖的细胞胚胎学研究结果 ,提取甜菜M14花蕾三个关键时期的RNA ,分离纯化mRNA ,以Oligo(dT)为引物 ,在逆转录酶的作用下 ,合成第一链cDNA ,进而合成第二链cDNA。含有EcoRⅠ和NotⅠ粘性末端的双链cDNA在T4DNA连接酶的作用下与载体λZAP臂相连 ,并对连接产物进行体外包装 ,得到噬菌体颗粒 ,即甜菜单体附加系M14花蕾cDNA文库。经大肠杆菌寄主菌株XL1-blueMRF’平板检测 ,三个文库滴度分别为 2 .8× 10 5pfu·mL-1、1.6× 10 5pfu·mL-1和 3.5× 10 5pfu·mL-1,克隆重组率为 83%、78%和 81%。

In order to clone apomixis related genes of M14 bud of the monosomic addition lines of Beta vulgaris,the cDNA library of M14 bud was constructed with a quick method.According to the embryological studies of the monosomic addition lines of Beta vulgaris-M14,extracted total RNA of three key periods of M14 bud and purificated mRNA.The first strand of cDNA was synthesized by superscript RNase H reverse transcriptase with the oligo(dT) primer,followed directly by second strand replacement synthesis.The double-stranded cDNA with EcoRⅠ和NotⅠcohensive ends was ligated to λZAP arms.The ligated DNA was added to a packing extract and the packaged phage referred to as original cDNA library was obtained.Titration of packed phage on LB plate containing E.coli XL1-blue MRF' indicated that the titre of the cDNA library was 2.8×10~5pfu·mL^(-1),1.6×10~5pfu·mL^(-1) and 3.5×10~5pfu·mL^(-1),and recombinant phage accounted for 83%,78% and 81%.The resultant cDNA library will be used for the screeing of target genes.