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 0.5kb and 3kb 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 1.5×10~7 clones.The title of the amplified library was 1.2×10~9pfu/mL.The insert DNA fragments were between 0.5～1kb,which was demonstrated by PCR.