摘要
本实验以新红星苹果为研究材料，采用CTAB法成功的从苹果果实里提取了高质量的RNA。利用MMLV反转录酶把总RNA中的mRNA反转录成cDNA，用特异引物进行LD—PCR扩增合成双链cDNA。然后用CHROMA SPIN—400Column对cDNA进行分级分离，去除小于500bp的cDNA片段。用带有粘性末端双链cDNA与λ Trip Ⅰ Ex2载体连接，利用λ噬菌体包装蛋白对合成的双链cDNA进行体外包装。包装产物侵染E.coli XL-Blue后构建cDNA文库，最后进行文库的扩增。该cDNA文库的滴度为1.191×10~6，文库的克隆数为1.112×10~6，扩增后滴度为1.532×10~10，插入片段平均大小在1 kb左右，蓝白斑筛选证实其重组率大于95％。同时，由于反转录过程中采用附加序列的方法使全长mRNA得到反转录，所以得到的cDNA是完整的，完全符合cDNA文库的要求。本实验成功构建了新红星苹果果实cDNA文库。
A high quality cDNA library of M. Red stark ing fruit has been constructed. The total RNA of apple fruits has been extracted by using CTAB method. The single strand cDNA was synthesized from mRNA with MMLV reverse transcriptase, then double strand cDNA was synthesized by using PCR. Double strand cDNA size fractionation was completed by CHROMA SPIN-400 column, so the low molecular weight cDNA fragments shorter than 500 bp could be removed out. cDNA was packaged using the A. phage packaging system after being ligated to the blunted A. Trip I Ex2. The cDNA library has been constructed and amplified through transducting E.coli XL1-Blue. The constructed cDNA library has the liter of 1. 191+106 and the clone number is 1. 112 +106. While the average insert sizes of the library are 1 kb with the recombination efficiency more than 95%, and the amplified titer of the library is 1.532+1010. Therefore, a cDNA library of apple fruit has been successfully constructed.
引文
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