抗黑胫病烟草品种K326 cDNA文库的构建*

 以优质高抗黑胫病的烟草品种K326为材料,在苗期接种黑胫病菌丝,3d后取叶片用Trizol试剂提取总RNA,经电泳检测28S,18S条带清晰,无DNA污染。以总RNA为模板,采用SMART技术合成双链cDNA,其产物主要集中在0.5～3kb之间,最大片段超过3kb。合成的双链cDNA经SfiⅠ（A/B）酶酶切后,用CHROMA SPIN-400层析柱分离分级cDNA,将大于0.5kb的cDNA片段混合,载入λTriplEx2载体,构建成云南第一个烟草cDNA文库。该文库的大小为1.2×10⁷pfu。扩增文库的滴度达到1.2×10⁹pfu/mL。文库的重组率大于94%。取阳性克隆转化为质粒,通过PCR反应检测,重组体确有DNA插入, 其片断大小在0.5～1kb之间。

Construction of cDNA Library from Excellent Tobacco Variety K326 with Resistance to Black Stalk

Total RNA was extracted from seedling leaves of tobacco variety K326, which was incubated by pathogen of black stalk. The RNA showed clear bands of the 28S and the18S after electrophoresis, which indicated that the RNA was not polluted by DNA polluted. The single stand of cDNA and the double stands of cDNA were synthesized with SMART kit. The synthesized ds-cDNA appeared mainly between 0.5kb and 3kb after separation by electrophoresis. The cDNAs were ligated to λTriplEx2 vector digested by SfiⅠ, which was the first one of tobacco cDNA library constructed in Yunnan Province. The cDNA library had 94% of recombinants calculated on blue/white phage plaques of bacterial strain XL1-Blue. The cDNA library contained about 1.5×10⁷ clones. The title of the amplified library was 1.2×10⁹pfu/mL. The insert DNA fragments were between 0.5～1kb, which was demonstrated by PCR.