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申本昌, 金治平, 赵德修, 乔传令. 2003: 小菜蛾全长cDNA文库的构建及质量分析. 动物学研究, 24(3): 215-219.
引用本文: 申本昌, 金治平, 赵德修, 乔传令. 2003: 小菜蛾全长cDNA文库的构建及质量分析. 动物学研究, 24(3): 215-219.
SHEN Ben-chang, JIN Zhi-ping, ZHAO De-xiu, QIAO Chuan-ling. 2003. Construction of a Full-length cDNA Library of the Diamondback Moth,Plutella xylostella. Zoological Research, 24(3): 215-219.
Citation: SHEN Ben-chang, JIN Zhi-ping, ZHAO De-xiu, QIAO Chuan-ling. 2003. Construction of a Full-length cDNA Library of the Diamondback Moth,Plutella xylostella. Zoological Research, 24(3): 215-219.

小菜蛾全长cDNA文库的构建及质量分析

Construction of a Full-length cDNA Library of the Diamondback Moth,Plutella xylostella

  • 摘要: 采用SMART(switching mechanism at 5 end of RNA transcript)技术,构建了小菜蛾(Plutella xylostella)的全长cDNA文库。按TRIzol试剂盒的操作程序提取4龄期小菜蛾总RNA,以此为模板,通过PowerScript逆转录酶逆转录合成第一链cDNA;再以第一链cDNA产物为模板,用LD-PCR合成第二链cDNA。该cDNA产物经分级分离和体外包装,即获得小菜蛾cDNA原始文库,其滴度为2.6×106 pfu/mL。扩增后的cDNA文库的滴度为2.4×1010 pfu/mL。取适量扩增文库稀释并铺平板,挑取60个独立噬菌斑,用PCR方法测得文库的重组率大于96%,插入cDNA的长度平均为1.6 kb。对60个独立噬菌体所插入的cDNA进行测序,克隆到了一个具有5 和3 非编码区的L27A核糖体蛋白基因。

     

    Abstract: In order to understand the molecular mechanism of the pesticide resistance of diamondback moth,Plutella xylostella,we used the SMART (switching mechanism at 5 end of RNA transcript) technology to construct full-length cDNA libraries.The PowerScript reverse transcriptase was used to synthesize and anchor the first-strand cDNA.Following reverse transcription,LD-PCR was performed using a modified oligo (dT) and an anchor primer to enrich the cDNA population for full-length sequences.The unamplified cDNA library contained 2.6×106 independent clones,in which the recombinant clones with an average insert size of 1.6 kb was > 96%.The titer of the amplified library was 2.4×1010 pfu/mL.PCR analysis of 60 randomly selected clones showed that 65% of cDNA clones contained inserts of above 1.0 kb.From the cDNA library,a full-length ribosomal protein L27A cDNA with 5 and 3 untranslated regions was isolated.

     

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