摘要
Myf5是生肌调节因子家族成员之一。为研究该基因在大口黑鲈(Micropterus salmoide)肌肉生长发育中的作用,运用RT-PCR和RACE技术获得大口黑鲈Myf5 cDNA序列1093bp,其中开放阅读框长723bp,编码240个氨基酸,经分析该蛋白无信号肽序列,属核蛋白,含有一典型的碱性螺旋-环-螺旋结构域。使用Genome walker技术获得启动子区序列2690bp,预测含有肌肉特异性转录调控相关元件:E-框、肌细胞特异增强子2(MEF2)、核转录因子1(SP1)、上游激活因子(USF)、血清应答因子(SRF)等结合位点。含有早期生长应答因子α(EGRα)、早期快反应生长应答因子1,2(EGR-1,2)等结合位点,它们可能是Myf5能够在体节形成早期表达的主要原因。获得Myf5 3个外显子与2个内含子序列,内含子1中存在一个在不同鱼类中高度保守的长度为123bp的序列,推测参与调节了Myf5基因的时空和组织特异性表达。本研究旨为进一步研究该基因对大口黑鲈肌肉生长发育的作用机理奠定基础。
As a member of the myogenic regulatory factors （MRFs）, Myf5 which is responsible for the specification and proliferation of myoblasts, plays an essential role in the growth and development of skeletal muscle. The human Myf5 was found firstly in 1989, capable of converting mouse 10TI/2C3H fibroblast into myosin expressing myotubes. The Myf5 gene-knockout mice delayed development of epaxial musculature and died from severe rib abnormalities, suggesting that Myf5 should primarily regulate the epaxial musculature formation. In fish, Myf5 cDNA of the carp, rainbow traint, Atlantic salmon, pufferfish and the genomic sequence of zebrafish, striped bass, sea perch, flounder and Fugu rubripes have been cloned and their molecular mechanism on muscle development and growth have been partially revealed. Unlike mammals, most of fish skeletal muscles grow dramatically during the post-larval life, and muscle growth determines the growth and size of the fish. Uncovering the molecular basis of muscle development and growth is an important step in aquaculture. To better understand the function of Myf5 in largemouth muscle development and growth, the cDNA and genomic sequence are characterized. Using RT-PCR and RACE techniques, a 1093-bp Myf5 cDNA from largemouth bass （Micropterus salmoides） was isolated. Its ORF was 723bp, encoding 240 amino acids, with a conserved basic Helix-Loop-Helix （bHLH） domain. It has no signal peptide and belonged to nucleic protein. The amino acid sequence of largemouth bass Myf5 bHLH domain was exactly identical to that of other fishes and exhibited 80% or greater identity with that of other vertebrates such as African clawed frog, chickens, bovine, mice, and humans. It indicates that largemouth bass Myf5 probably acted as a transcriptional factor to regulate muscle-specific genes, like its homologs in other species. Using Genomewalker technology, a 2 690-bp promoter sequence was isolated. By bioinformatics analysis, the basic transcriptional regulatory elements such as TATA Box, GC Box, CCAAT Box, music specific transcriptional factors such as E-box, MEF2, SP1, USF, SRF, MTBF binding sites and the early growth related transcriptional factors such as early growth response gene a （EGRa）, Egr-1 early growth response gene, Egr-2 early growth response gene product, growth factor independence 1, TCF binding sites which may be responsible for the Myf5 expression in developing somite were found. For many E-boxes in promoter, we predict that the other MRFs such as MyoD may be a negative regulator to the transcription of Myf5. MEF2 family was reported to promote the muscle-specific gene transcription together with the MRFs family. The MEF2 binding sites were found in largemouth bass Myf5 promoter, and we predict MEF2, as a transcription factor, raise the expression of Myf5 in largemouth bass. SP1, USF and SRF were all revealed to regulate the transcription of MyoD. So we predict that they may be the potential regulators to Myf5. Meanwhile, Myf5 of largemouth bass consisted of three exons and two introns, a highly conserved 123-bp region in intron-1 of the different fishes which may regulate the temporal and tissue-specific expression of Myf5. Conclusion： cDNA and genomic sequence of largemouth bass Myf5 are isolated and characterized, and the regulatory elements and potential transcriptional factors are predicted. It will help us to better understand the expression regulation of Myf5 in fish and further study the different signal paths in complex myogenesis net.
出处
《中国水产科学》
CAS
CSCD
北大核心
2008年第4期568-576,共9页
Journal of Fishery Sciences of China
基金
国家科技支撑项目(2005DKA21103)
国家科技基础条件平台工作项目(2005DKA21103)
关键词
肌肉
Myf5基因
调节
大口黑鲈
muscle
Myf5
gene
regulation
largemouth bass （Micropterus salmoides）
