黄瓜抗白粉病品系cDNA文库构建及EST分析

Construction of Cucumber Leaf cDNA Library and Analysis of Expressed Sequence Tag

采用SMART技术构建了高抗白粉病的黄瓜品系JIN5-508的叶片cDNA文库,并对其进行了质量鉴定。结果表明,该文库的重组率为95.3%,库容量为1.02×106pfu·mL-1,平均插入片段为0.5kb左右,是一个高质量的cDNA文库。利用该文库随机挑选的8352个克隆从5′端进行单向测序,共获得8035个有效的ESTs(expressed sequence tags),序列的平均长度为838bp。进一步的生物信息学分析表明:EST序列拼接出3467个unigenes,其中包括953个contigs和2514条singletons,文库冗余度较低,为56.9%;从文库中筛选出了大量的低丰度表达基因,约占unigene总数的72.5%,说明文库的复杂性较高;1991个unigenes获得分子功能注释,其中与蛋白结合活性和催化活性相关的基因分别达到了41.34%和38.72%;在获得功能注释的unigene中,有部分抗病抗逆相关基因高丰度表达,其中3个unigenes与拟南芥白粉病抗性基因Mlo2、Mlo8和Mlo14高度同源,表明该文库可为进一步研究黄瓜抗白粉病相关的特异基因的克隆及功能验证奠定基础。

We constructed a cDNA library from the leaf of cucumber （Cucumis sativus L.） line J1N5-508 with high powdery mildew resistance based on the SMART technology, and checked the quality of this library. The recombinant rate of present library was 95.3%, the titer was 1.02 ~ 106 pfu - mL1, and the average size of inserted cDNA fragments were about 0.5 kb. It indicated that one high quality cDNA library of cucumber was successfully constructed. Subsequently, 8 352 clones were chosen randomly from this library, and 8 035 valid expressed sequence tags were generated from the 5＇ end sequencing. The average length of ESTs were 838 bp. Bioinformatics analysis of EST data indicated the low redundancy of this library with a score of 56.9%, in which 3 467 unigenes including 953 contigs and 2 514 singletons were assembled. Genes that expressed with low abundance occupied 72.5% of total unigenes, indicating the good complexity of gene expression in this library, One thousand nine hundred and ninty-one unigenes could be assigned functional description. Among the unigenes with molecular function classification, binding activity and catalytic acitivity related proteins took up to 41.34% and 38.72%, respectively. Three unigenes with high homology to Arabidopsis thaliana powdery mildew resistance locus were also identified, which suggested this library could be useful for further identification and expression analysis of powdery mildew resistance genes in cucumber.