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《Chinese Journal of Zoology》 2007-01
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Construction of Testis cDNA Library and Sequence Analysis of Ubiquitin/L40e Extension Gene in Bufo bufo gargarizans

CHEN Wen ZHENG Ping-Ping NIE Liu-Wang* (College of Life Sciences,Anhui Normal University,Wuhu Anhui 241000,China)  
A full-length cDNA library from the testis of Chinese Large Toad (Bufo bufo gargarizans) was constructed with the SMART (switching mechanism at 5′ end of RNA transcript) technique.Total RNA was extracted from the testis and reversely transcripted into full-length cDNA using Powerscript TM reverse transcriptase.First-strand cDNA was amplified by long-distance PCR.After Sfi Ⅰ digestion and Chroma spin-400 fractionation,cDNAs (500 bp) were ligated to λ TriplEx2 vector and were packaged with Gigapack D○R Ⅲ Gold Packaging Extract.The optimal primary library contained 2.21×106 pfu/ml clones and the amplified library had a titer of 2.94×109 pfu/ml,in which over 90% clones were recombinant and the average size of inserted cDNAs was about 1.0 kb.These results show that the testis cDNA library can be used for screening genes.A full-length gene with 5′ and 3′ untranslated regions was isolated from the cDNA library.Sequence analysis showed that this 561 bp cDNA encoded a 128-amino acid ubiquitin/L40e extension protein.
【Fund】: 国家自然科学基金(No.30640048);; 生物环境与生态安全安徽省高校重点实验室专项基金;; 安徽省学术与技术带头人专项基金
【CateGory Index】: Q78;Q953
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