茶苗嫩根cDNA文库的构建和EST分析

Construction of cDNA library for tender roots of tea plant(Camellia sinensis(L.) O.Kuntze) and analysis of its expressed sequence tags

以茶苗嫩根为材料,采用SMART技术构建了cDNA文库并对其质量进行了鉴定。结果显示,该文库的重组率为92%,库容量为5×105,平均插入片段长度超过1 kb,是一个较高质量的文库。从文库中随机挑取克隆并从5′端单向测定2 651个克隆,获得2 600个有效的EST(expressed sequence tag),序列的平均长度为703 bp,用Phrap软件进行拼接,结果获得404个contigs、610个singlets。通过与NCBI的非冗余核酸数据库进行Blastx比对,初步确定已知功能基因734个,推定功能基因102个,未知功能基因178个。该结果既为进一步分离克隆茶树根部功能基因奠定了基础,又为以后的差异杂交获取茶树根部特定代谢途径的酶基因及相关的调控基因提供了平台。

Based on SMART technology, we constructed a cDNA library from the tissue of tender roots of tea plant （ Camellia sinensis）. The results showed： without amplication of the library, the cloning efficency was 5 ×10^5, the recombinant rate was 92% , and the average size of inserted cDNA fragment was over 1 kb. It suggested that one high quality tender roots cDNA library of tea plant was successfully constructed. Then a set of clones were randomly selected and single-pass sequenced from their 5＇ end. As a result, 2 651 clones were sequenced and 2 600 valid sequences were generated with average size of 703 bp after vector trimming and discarding the sequences less than 100 bp, in which 404 contigs and 610 singlets were obtained after initial assembly with Phrap program. Blastx was performed against a nonredundant protein sequence database in NCBI and filtered at e-value of 1 e-10, 734 known functional genes, 102 putative functional genes and 178 unknown mRNA were achieved. This study makes a foundation for isolation of functional genes especially those related with metabolism and control in the root of tea plant.