摘要
提取新鲜鲮(Cirrhinus molitorella)肌肉总RNA,采用RT-PCR技术分段克隆鲮Myf5基因核心序列,获得1104bp的目的片段,其中开放阅读框为723bp,编码240个氨基酸。分析表明,鲮Myf5蛋白具有MRF家族基因的典型性碱性bHLH螺旋-环-螺旋(Basic helix-loop-helix,bHLH)结构。设计特异性引物扩增获得2个内含子,大小分别为1311bp和90bp。用单链构向多态性SSCP的方法分析了Myf5基因在珠江水系3个不同江段鲮中的遗传变异,分布及群体杂合性等群体遗传信息。发现其编码区非常保守,仅在外显子412位点处发生突变C→T。对该位点进行基因型分析,结果表明:珠江北江段鲮中该位点符合Hardy-Weinberg平衡,而东江和西江段种群不符合Hardy-Weinberg平衡,3个江段种群总体上符合Hardy-Weinberg平衡,并存在较大基因流。以Myf5基因该位点的多态性分析表明,3个种群未出现明显分化;通过该位点的遗传结构分析表明:3个江段种群杂合度都较高,中度信息含量(PIC)及多态信息指数,即该位点有较好的变异性及遗传多态性。3个江段种群总体上该位点在不符合Hardy-Weinberg的情况下AA>BB,并由该位点群体杂合性及中性分析表明该位点突变群体具体良好的多态性且不遵循中性选择模式,说明BB型个体受到自然选择。本研究结果表明,鲮Myf5基因的C412T与个体生长有相关性,可作为候选基因标记,用于鲮生长相关分子标记辅助育种研究。
Several molecular markers were used in mud carp genetic diversity research. There is no choiceness varietal mud carp（ Cirrhinus molitorella） in present culture. Three part of Pearl-river drainage wild mud carp （Cirrhinus molitorella） were sampled as experimentation fish,and PCR-SSCP（single strand conformation polymorphism,SSCP）was used to screen Myf5 ORF（open reading frame） DNA sequence,expecting to find SNPs relative to growth and trait. Mud carp（ Cirrhinus molitorella） belongs to Cypriniformes,Cyprinidae,distributing in Pearl River,Ming River,Han River,Hainan. Taiwan and southeast of Asian. It is an important fishery production in Pearl-river drainage area. Myf5 gene（ myogenic factor 5） is a member of MRFs gene family,which include Myf5,MyoG,MyoD and MRF4. The primary function of the gene family is advance myoblast multiplication and differentiation. The function of MyoD and Myf5 genes is determined to initiate the differentiation process of that muscular satellite cells to be muscle derived stem cell. And that of MyoG and MRF4 genes was thought to be involved in regulating the differentiation process of muscle derived stem cell to be myotube and muscle fibers. All of those functions were implementing by the typical trait b-HLH structure of MRF family,which form dimeric by combining with E-protein. Then Key gene is activated and pressed by the dimeric recognize upstream E-box. Many species of Myf5 gene or cDNA have been cloned. Such as Mus musculus,Bos Taurus,Sus scrofa,Ovis ammon,Gallus gallus, Danio rerio,Cyprinus carpio,Micropterus salmoide and Oncorhynchus mykiss. Myf5 gene of mud carp（ Cirrhinus molitorella） has been cloned,sequenced and analyzed by PCR-SSCP. Mud carp Genome DNA and total RNA was extracted from flesh muscle of mud carp. Myf5 gene was cloned including three exons and two introns of which the length is 453 bp,76 bp,194 bp and 1 311 bp and 90 bp（GeneBank accession： GU289508）. The cDNA sequence of Myf5 gene was obtained by RT-PCR. The length of the cDNA is 1 104 bp, and 723 bp ORF（ open reading frame） was identified,which shared 93.6% homology with that of Cyprinus carpio, 85.9% homology with Danio rerio. It encodes a peptide of 240 amino acids whose calculated molecular weight is 26.288 31 kD and theoretical isoelectric is 6.08. AA sequence analysis showed that the Myf5 protein have the typical trait b-HLH structure of MRF family. The population genetic information about genetic distribution,variation and heterozygosity of Myf5 gene was analyzed by using PCR-SSCP in three part of the Pearl River. The only one mutation C/T at base position 412 of the whole CDS was found among individuals in whole group. Through further analysis of the position,it could be inferred that the genotypes distribution of Myf5 in three parts of the Pearl River were not in accordance with the Hardy-Weinberg equilibrium except the north of the Pearl River subgroup,and not in accordance with the Hardy-Weinberg equilibrium in whole group. All of the three groups have high heterozygosity and moderate PIC,which indicate that the mutation is high degree in the groups and fine genetic diversity. Furthermore,there is obvious differential between observed values and expected values that indicate the evolution is discord with theory of neutral selection. In the whole of the Pearl River,A and B alleles are that not in accordance with the Hardy-Weinberg equilibrium,high degree mutation,fine genetic diversity and discord with theory of neutral selection. The number of AA type is more than that of BB type evidently,and the conclusion can be made that BB type individual has been selected. Consequently,the SNP can be used in the breeding process,avoiding the BB type as parents From the molecular level,to speculate,phosphorylation switch site changed or disappeared because of the mutation,resulting in decline in individual life force. This is in the need of further experiments.
出处
《中国水产科学》
CAS
CSCD
北大核心
2010年第4期681-688,共8页
Journal of Fishery Sciences of China
基金
国家自然科学基金资助项目(30271022)
国家公益性行业(农业)科研专项(200903045)
国家重点基础研究发展计划项目(2004CB117401)
广东省科技计划(2005B20301007)
广东省科技厅农业攻关子项目(2005A20105001)
国家科技基础条件平台建设子项目(2006DKA30470-008)
