木榄幼苗叶片cDNA文库的构建及其表达序列标签分析

应用SMART技术构建了25‰盐度下生长4个月的木榄叶片的cDNA文库,文库滴度为10~6 cfu mL~(-1),重组率为94.4%,插入片断长度为1～2 kb.从cDNA文库中随机挑选了96个重组克隆进行序列分析,共获得94个表达序列标签(ESTs),经质量控制和聚类拼接后得到81个unigenes,包括5个片断聚合群和76个单一序列.Blastx分析结果表明这些unigencs与GenBank的Nr数据库中已报道的基因具有较高的同源性(E<10~(-5)),它们参与呼吸代谢、光合作用、糖代谢、氨基酸代谢、脂肪酸代谢以及不饱和脂肪酸生物合成等重要的生理过程,并与机体的损伤修复、胞吞作用以及PPAR信号途径等相关.

Construction of cDNA Library from Leaves of Bruguiera gymnorrhiza Seedlings and Its Expressed Sequence Tags Analysis

The seedlings of Bruguiera gymnorrhka were cultured under 25‰ salinity for 4 months, then the total RNA were extracted from leaves. Following the protocol of SMART~(TM) cDNA Library Construction kit, the higher quality cDNA library was constructed. The primary titer of the cDNA library was about 1 ×10~6 cfu mL~(-1), its recombinant efficiency was calculated as 94.4%, and the size of the insert DNA fragments was ranged from 1 kb to 2 kb. Ninety-six randomry selected recombinant cbnes from the cDNA library were sequenced to produce 94 expressed sequence tags (ESTs), among which 81 unigenes (including 5 contigs and 76 singletons) were generated after quality control and assembly of sequences, they were all homologous to the sequences in NCBI Nr database by blastx analysis (E< 10~(-5)). These unigenes involved in the processes of respiration, photosynthesis, glucose metabolism, amino acid metabolism, fatty acid metabolism, biosynthesis of unsaturated fatty acids, etc, and had relation to the damage repair, endocytosis, and PPAR signaling pathway.