利用改进的SMART法构建花生种皮全长cDNA文库

Establishment of Full-length Peanut Testa cDNA Library by Using Modified SMART

SMART法是目前全长cDNA文库构建的重要方法之一。在研究多种构建全长cDNA文库的方法基础上,吸收和改进了SMART法,对特异引物和PCR条件进行可行性的优化,成功构建了高质量的花生种皮全长cDNA文库,改进后的方法更经济、简便易行。经过涂平板测定和酶切反应快速鉴定表明,原始文库的滴度为1.23×106cfu.mL-1,重组率达93.3%,全长率为59%。插入片段大小多在1.0～2.0kb,所占比例为70%,平均大小在1.1kb左右,采用涂平板均匀扩增得到的扩增文库滴度达到了3.84×109 cfu.mL-1,可用于长期保存。

SMART is one of the most important methods for constructing a full-length cDNA library.In order to discover new genes from the peanut testa,the SMART method published previously was modified to improve its efficiency,reduce cost,simplify operation,and minimize test time.Using the optimized protocol,a full-length cDNA library for peanut testa was established.The new method produced an estimated titer of the amplified cDNA library as high as 3.84×109 cfu·mL-1,which was increased from 1.23×106 cfu·mL-1,with a recombinant rate of 93.3%.Furthermore,approximately 70% of the inserts,varying from 1.0 to 2.0 kb with an average size of about 1 100 bp,were obtained.The sequence analysis indicated that the full-length cDNA proportion was as high as 55%.It was concluded that the library was of desirable quality for full-length screening and cloning of the target genes.