报道了国内第一个茶树cDNA文库的构建及其EST测序成功率分析。以Trizol一步法从龙井43春茶新梢中提取总RNA,分离纯化富含Poly (A)的mRNA,LD-PCR反转录合成双链cDNA,以λTripEX2为载体,构建了龙井43新梢cDNA文库。以XL1-Blue为受体菌测定原始文库的滴度为6.8×105 pfu/ml,总克隆数为3.5×105个,重组率为98.05%,扩增后文库总滴度为7.2×109 pfu/ml。对随机挑取的噬菌斑进行PCR鉴定,表明插入片段大多分布在0.5-2.0 kb之间,绝大部分在1.0-1.5 kb左右。文库质量鉴定结果表明,构建的茶树新梢cDNA文库具有较好的库容量、较高的重组率以及较大的插入片段。对4320个克隆的序列测定表明,获得有用序列2963个,测序成功率为68.5%,剔除短序列后,首批共获得1687个茶树ESTs。
The construction of the first cDNA library of tea plant [Camellia sinensis cv. Longjing 43] in China and the analysis of expressed sequence tags sequencing successful ratio were reported. Total RNA was isolated from tender tea shoots using TRIZOL single-step method, mRNA separated and then the double-strand cDNA amplified by LD-PCR. After size fractionation, the ds-cDNA was ligated to λTripEX2 and recombinant bacteriophages were packaged. The cDNA library was tittered and amplified using XL1-Blue as receptor bacterium. The titer of the original library was 6.8×105pfu/ml with a recombinant rate of 98.05% and 3.5×105 clones in total, the amplified titer was 7.2×109pfu/ml. PCR amplification suggested the inserted cDNA fragments ranged from 0.5 kb to 2 kb, mostly from 1.0 kb to 1.5 kb. The data indicate that the tea cDNA library has high titer, high recombinant percentage and large inserted fragments. Totally, 4320 clones were sequenced, 2963 useful sequences were obtained, corresponding to 68.5%. The first batch of 1687 valid tea plant ESTs were generated.
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