摘要
为了分离与鉴定野生大豆优良基因,以双高型优质野生大豆的近成熟种子为材料,采用裂解法提取了总RNA;以Oligo(dT)为引物,经SA-PMPS法分离出mRNA,反转录酶催化合成cDNA,并以cDNA第一链为模板在DNA聚合酶Ⅰ的作用下合成cDNA第二链,双链cDNA经加接头等步骤,成功构建了野生大豆cDNA文库。文库的重组率约为93.7%,PCR检测重组克隆的插入片段平均大于1000bp,测序片断大于500bp,表明构建的近成熟种子cDNA文库质量较高,为进一步进行EST测序和全长克隆打下了基础。
In order to separate and identification the novel gene of high-quality wild soybean, total RNA from the sprout of wild soybean（Glycine soja） seeds. RNA was purified by using the improved Promega extraction method and the mRNA was isolated from total RNA by using Biotin-labeled Oligo（dT） and SAPMPs. The cDNA was synthesized and catalysed by reverse transcriptase. The cDNA was then linked with EcoR Ⅰ Adaptor. Finally, by ligating the cDNA fragment with pBluescript vector and transforming the recombinants into E.coli, cDNA was reclaimed. This library reaches 9.594x10s in capacity; the percentage of recombination is as high as 93.7%. PCR results showed that the average size of inserts was larger than 1 000 bp, and sequencing length of fragments being cloned is over 500 bp indicating the good quality of the library. This research provides a base for further study on EST and the full length cloning of important genes.
出处
《生物技术通讯》
CAS
2005年第5期509-511,共3页
Letters in Biotechnology
基金
河南师范大学大学生科研基金重点资助项目(0410401)
河南省杰出人才创新基金项目(0321001400)
