高GC含量的鳜鱼rRNA基因家族的克隆  

Cloning of the rRNA Genes Family with High GC Content of Siniperca chuasti

作　　者：唐冬生[1] 严霞[1] 张勇[1] 张细权[2] 李芳[1] 蒋泓[3] 李月琴[3] 周天鸿[3] 

机构地区：[1]佛山大学生命科学学院,佛山528000 [2]华南农业大学动物科学学院,广州510642 [3]暨南大学生命科学与技术学院,广州510632

出　　处：《中国生物工程杂志》2005年第8期60-64,共5页China Biotechnology

基　　金：国家自然科学基金资助项目(30170737;30470978);广东省自然科学基金资助项目(04011645)

摘　　要：动物rRNA基因是一种GC含量较高、结构复杂的重复序列。该研究结合亲缘生物法生物信息学技术,经反复摸索后选用LA PCR法即LA PCR Taq酶结合GC缓冲液来扩增鳜鱼复杂的rRNA基因重复序列,经测序鉴定最终克隆了鳜鱼的3个rRNA基因及其2个间隔序列。分析了鳜鱼与相关动物的rRNA基因序列的同源性和进化关系。探索了克隆复杂DNA序列时引物设计的特别规则、反应体系的改进、DNA聚合酶的选用、循环参数的调整等措施。The rRNA genes in animals are a kind of repetitive sequences with high GC content and complicated structure. Combining the bioinformatics technique of relative organism method, the DNA sequences of rRNA repetitive gene family of Tetraoden nigrovirdis were gained. Three pairs of primers （ P1/P2,P3/P4 and P5/P6） were designed based on the sequences. The LA PCR method with LA PCR Taq DNA polymerase and GC buffer was used to amplify the complicated repetitive sequences of rRNA genes in Siniperca chuasti after repeat pre-experiments. Furthermore, the PCR products were cloned into pUCm-T plasmid. Sequencing analysis identify the 3 rRNA genes and 2 internal transcribed spacers （ ITS1 and ITS2） have been cloned. The sequencing results showed that the length of three cloned fragments of rRNA gene were 3 321bp, 2 873bp and 1 502bp, respectively. The integrated contig with three sequences was exactly identified to be 7 139bp, which separately comprised Siniperca chuatsi rRNA genes of 18S, 1TS1, 5.8S, ITS2 and 28S with the length of 1 837bp, 745bp, 158bp, 434bp and 3 073bp. These above sequences were submitted to GenBank, and the corresponding accession numbers were granted to AY452489, AY452490, AY452491, AY452492, AY452493, AY452494 and AY452495, respectively. Based on the analysis of nucleotide sequence homology in phylogenetic rRNA repetitive gene family sequences from Siniperca chuatsi , Tetraoden nigroviridis , Oreochromis esculentus , Cyprinus carpio and Oncorhynchus mykiss （rainbow trout）, human, Norway Rat, the pairwise nucleotide sequence homology of 18S, 5.8S, 28S, ITS1 and ITS2 among 7 organisms were showed to be 93.8% - 98.4%, 95.6% - 98.1%, 90.0% -92.8%, 9.0% -62.9% and 11.9% -64.0%, respectively. In terms of that, the phylogeny tree was constructed. The evolution of rRNA gene sequences between Siniperca chuasti and the related organism have been analyzed. The measures to clone the complicated DNA sequences, such as the special rules of primer design, the improvement of reaction system, the selecti

关 键 词：RRNA基因 基因克隆 高GC含量 重复序列 鳜鱼 RRNA基因序列 GC含量 克隆 基因家族 DNA聚合酶 

分 类 号：Q959.4[生物学—动物学] Q987