利用抑制消减杂交技术构建了潮间带绿藻孔石莼(Ulva pertusa)在干出胁迫状态下上调表达基因的消减cDNA文库.获得的阳性克隆经测序得到150条上调表达的EST片段,其中21条为冗余序列(RS),137条非冗余序列(NRS).片段中最长833 bp,最短106 bp,平均长度364 bp.参与比对的EST片段按照其注释功能可分类为:细胞生长与发育、蛋白合成与分解、代谢过程、胁迫应答、光合作用与光诱导、转录调节、信号转导等相关基因.未知功能和无对比结果序列共60条,占非冗余序列的43.80%.比对相似度主要分布在20%～60%之间,主要与已经完成基因组测序的藻类或高等植物的氨基酸序列具有较高的相似性.孔石莼对密码子第三位碱基的使用偏好无显著差别(P>0.05),GC使用总频率为50.66%,而对终止密码子则明显偏好TGA.实时荧光定量结果表明,部分分离的基因在干出胁迫状态下表达量上调.这些不同功能基因的表达构成了复杂的调控网络,能够为揭示孔石莼抵御潮间带不良环境的分子机制提供多方面的切入点.%The subtractive cDNA library of up-regulated genes from Ulva pertusa under emersed stress was constructed by suppression subtractive hybridization (SSH) technology. 150 EST fragments of up-regulated genes were isolated after sequencing the positive clones, in which 21 fragments were redundant (RS) and other 137 fragments were non-redundant sequences (NRS). PCR analysis indicated that the length of the EST inserts were in the range of 100 to 900 bp. The longest was 833 bp, and the shortest was 106 bp and the average length was 364 bp. Based on the functional similarity with homologous genes in algae and higher plants, the EST fragments could be divided into several functions: cell growth and development, protein synthesis and degradation, metabolism, stress response, photosynthesis and photoinduction, transcription regulation and signal transduetion. 60 sequences with unknown functions or no comparative results accounted for 43.80％ of total non-redundant sequences. The comparison similarity values were distributed between 20％ and 60％ and the sequences had higher similarity with the amino acid sequences of algae and higher plants whose genome sequencing had been completed. No contigs but whole singletons represented in the predicted EST fragments with the same function. It was shown that the eodon usage in the third codon position showed no significant difference ( P ＞ 0.05 ) in U. pertusa and the GC frequency of eodon usage was 50.66％ of the total. The stop codon usage preferred TGA especially. Beal-time fluorescence quantitative PCB demonstrated that the relative expression abundance of partial genes was isolated from the library up-regulated at mRNA level. The expression of these different genes constituted a complex regulatory network, and would provide wide range entry points to demonstrate the mechanism of stress tolerance for U. pertusa emersed in intertidal zone.
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