| 摘要: |
| 采用RT-PCR 及RACE 法, 分离、克隆鳜鱼β-肌动蛋白基因cDNA 全序列, 再用基因组步行法(Genome Walker)克隆鳜鱼β-肌动蛋白基因5′调控区。序列分析结果表明, 鳜鱼β-肌动蛋白基因全长1897bp, 其中5′-UTR 长94bp, 3′-UTR 长675bp, 编码区长1128bp, 编码375 个氨基酸。将所得序列与其它动物类群的β-肌动蛋白基因序列进行比较分析显示, 鱼类、两栖类、鸟类、哺乳类等不同类群脊椎动物β-肌动蛋白氨基酸序列同源性均在96%以上, 说明该基因在生物进化过程中高度保守。通过鳜鱼与其它脊椎动物β-肌动蛋白基因的核苷酸序列构建的进化树显示, 脊椎动物β-肌动蛋白聚类成3 个分支, 鱼类β-肌动蛋白基因形成一个独立的分化群, 说明鱼类β-肌动蛋白基因起源于一个共同祖先。克隆得到的鳜鱼β-肌动蛋白基因5'侧翼序列长1399bp, 对其进行序列分析, 在其起始密码字ATG 上游200bp 范围内发现含有CAAT box、CC(A/T)6GG(CArG box)、TATA box 对转录调控起重要作用的顺式元件, 同时在侧翼区也发现含有GC box、MYOD、YY1、SP1、GATA 等多个潜在调控元件。鳜鱼β-肌动蛋白基因5′侧翼序列的克隆成功, 为今后转基因鳜鱼的研究工作奠定了基础。
|
| 关键词: β-肌动蛋白基因, cDNA 序列, 5′调控区, 克隆, 鳜鱼 |
| DOI：10.11693/hyhz200901017017 |
| 分类号: |
| 基金项目:国家自然科学基金项目, 30670367 号; 广东省自然科学基金项目, 031886 号; 国家高技术研究发展计划项目(863), 2007AA09Z437 号; 广东省科技计划项目, 2005B20301005 号、2007B020701002 号; 广州市科技计划项目, 2006Z3-E0551 号。 |
附件 |
|
| CLONING AND ANALYSIS OF THE FULL-LENGTH cDNA SEQUENCE AND 5′-FLANKING REGION OF CHINESE PERCH SINIPERCA CHUATSI β-ACTIN GENE |
|
LIU Xiu-Xia, LIANG Xu-Fang, WANG Lin, DUAN Jin-Xia, LI Guang-Zhao, LIAO Wan-Qin
|
|
College of Life Science and Technology, Jinan University
|
| Abstract: |
| Referring to the deduced amino acid sequences of β-actin from other vertebrates, two degenerated primers were designed and synthesized for cloning the cDNA of Chinese perch (Siniperca chuatsi) β-actin gene by RT-PCR. The full-length cDNA sequence was further obtained with the method of 3′- and 5′- rapid amplification of cDNA ends (RACE). Genome walker method was applied to get 5′-flanking region of Chinese perch β-actin gene. The complete Chinese perch β-actin cDNA was 1897bp in length, containing an open reading frame of 1128bp (encoding 375 amino acids), flanked by 94bp 5′UTR and 675bp 3′UTR. The sequence analysis reveals that, the identities of β-actin amino acid sequence among Chinese perch, African clawed frog (Xenopus laevis), chicken (Gallus gallus), Norway rat (Rattus norvegicus), house mouse (Mus musculus), and human (Homo sapiens) are more than 96%. This suggests that β-actin is highly conserved. The phylogenetic tree constructed on the basis of β-actin nucleotide sequence describes accurately the relationship among fish, amphibian, bird and mammals. The result supports that β-actin may be investigated further as a good phylogenetic marker. A 1399bp 5′-flanking region of Chinese perch β-actin gene was obtained using genome walker method, a proximal promoter element, containing highly conserved CAAT, CArG, and TATA boxes, has been found within the first 200bp upstream of the start codon ATG. Other potential regulatory elements including GC box, MYOD, YY1, SP1, and GATA also have been found in this 5′-flanking region. The successful cloning of 5′-flanking region of Chinese perch β-actin gene, lays a foundation for future study on transgenic Chinese perch.
|
| Key words: β-actin gene, cDNA sequence, 5′-flanking region, Cloning, Chinese perch |
