鳜碱性肌球蛋白轻链基因cDNA的克隆及其发育表达分析
CLONING AND ONTOGENETIC EXPRESSION ANALYSIS OF THE ALKALI MYOSIN LIGHT CHAIN GENE IN SINIPERCA CHUASTI
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摘要: 肌球蛋白轻链是构成鱼类肌纤维主要组成部分,在鱼类肌肉生长和收缩过程中具有重要作用。鳜鱼具有生长快、肉质细嫩、味道鲜美、营养成分高等优良的性状。研究通过构建鳜肌肉组织cDNA文库分离到两个碱性肌球蛋白轻链基因,即MLC1和MLC3基因。序列分析显示MLC1和MLC3基因cDNA序列全长分别为1237bp和1070bp,分别编码192和150个氨基酸,除去MLC1N端多出的42个氨基酸残基,MLC1与MLC3氨基酸序列同源性为80.3%。通过PROSITEtools软件预测显示两种轻链都具有两个保守的EF-手相结构,其中第二个EF-hand结构除前三个氨基酸外同源性达100%。鱼类MLC3轻链N端没有高等脊椎动物MLC3特有标志序列。采用实时荧光定量PCR方法对鳜鱼MLC1和MLC3发育性表达分析表明,在原肠期开始有低量表达,与原肠期、尾芽期和肌肉效应期相比,心搏期和仔鱼期MLC1和MLC3表达量显著升高。研究结果首次提供了鳜肌肉组织肌球蛋白主要结构基因的分子生物学信息以及它们在鳜肌肉组织发生和功能的相关性。Abstract: Myosin light chains are major components of fish muscle fibers, and they play an important role in the process of muscle growth and contraction. The mandarin fish has been recently becoming one of the most important aquaculture fish species in China because of its good meat quality and protein composition. To a better understanding of the muscle development and its genetic controls, we constructed a cDNA library of the fast skeletal muscles the mandarin fish, S. chuatsi and successfully isolated two myosin light chains genes, MLC1 and MLC3. The length of the MLC1 and MLC3 cDNA was 1237 bp and 1070 bp, predicated encoding protein of 192 and150 amino acids, respectively. Excepting the 42 amino acid residues of the N terminal of the MLC1, the homology of the deduced amino acids sequences of the MLC1 and MLC3 was 80.3%. The two alkaline chains included two conservative EF-hand structures analyzed by PROSITE tools. The homology of the second EF-hand structure was 100% between the two genes, except the first three amino acids. However, MLC3 in fish did not discover the N-terminal sequence that was specific signs of MLC3 in higher vertebrates. Homologous comparison of the amino acid sequences of MLC1 and MLC3 in S. chuatsi with those the other fish species revealed certain variances in amino acid composition and species-specific characterization. To investigate the relationship the gene expression and muscle formation and development, we applied a real-time PCR technique to assay the ontogenetic expression of the two genes, and the results confirmed that the MLC1 and MLC3 mRNA were first detected in gastrula stage and its expression gradually increased from the muscular effect stage to larval stage, especially the two genes were highly expressed in the muscle effect and larval stages, that suggested their biological functions related to muscle differentiation and muscle motility. Our study provided detail information of the two alkaline MLC gene structure and its function on muscle development in S. chuasti.
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Keywords:
- Siniperca chuasti /
- Alkali myosin light chain gene /
- cDNA library /
- Cloning /
- Ontogenetic expression
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