萍乡红鲫转铁蛋白研究
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摘要
萍乡红鲫(Pingxiang red-transparent crucian carp,Carassius auratus var.Pingxiangnesis)是江西省萍乡的一种野生鲫鱼突变种。经过七年的工作,已经完成了对其6代的选育。选育后的萍乡红鲫具有生长速度快、适应性强、营养价值高、易养易繁、抗病力强等优良经济性状,2008年被全国水产原、良种审定委员会审定为新品种。实验以萍乡红鲫的血清转铁蛋白(TF)为对象,通过电泳研究该鱼TF的表型、分子量等遗传性质;通过同源克隆得到该鱼TF-cDNA的序列,以期了解其遗传背景,为进一步的科学研究提供资料。
本文的工作主要为:
一、对萍乡红鲫的血清转铁蛋白(Transferrin,简称TF)进行电泳分析。聚丙烯酰胺凝胶电泳(PAGE)发现随机选取的12尾萍乡红鲫TF表型完全的相同,个体间没有差异;萍乡红鲫的TF表型为两个条带,推测可能由一个座位上的二个等位基因遗传决定。采用十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)对TF进行分析得出其分子量大约为71KDa。经人工选育的萍乡红鲫TF表型一致,种群内未见多样性,认为其遗传性状趋于稳定纯系;萍乡红鲫不同个体间完全_致的TF表型及分子量,可以利用TF雌核发育鉴定的遗传标记揭示萍乡红鲫具有雌核发育的生殖方式。
二、为进一步了解了萍乡红鲫TF的分子机制,利用RT-PCR和RACE-PCR克隆出萍乡红鲫TF互补基因,克隆得到的TF-cDNA包含完整的开放阅读框(open reading frame,ORF),肉红鲫TF-cDNA长2364碱基对(base pair,bp),包括8bp的5′非编码区(untranslated region,UTR),355bp的3'UTR,编码666个氨基酸,理论信号肽切割位点位于15-16个氨基酸之间,预测的分子量为71.9,没有潜在的糖基化位点,还有34个半胱氨酸残基。结果分析表明萍乡红鲫TF遗传性质与彩鲫A、银鲫E同源性较高。
最后对萍乡红鲫TF蛋白序列进行二级结构理论分析和三级结构的同源建模。
Pingxiang red-transpanent crucian carp,Carassius auratus var.Pingxiangnesis had been considered as a variant of wildness diploid spieces of crucian carp in Jiangxi Province.The sixth generation was successive produced by selective breeding for seven years.The fish had many good economic characters,such as fast growth,easily adaptable,rich nutrition,easily cultured,strong anti-resistance.It had been examined and authorized as a new aquaculture variety in the whole country.Transferrin of the fish was studied in this paper.The phenotypes of the TF was got by the method of Polyacrylamide gel electrophoresis(PAGE) and its molecular weights was analyzed through Sodium dodecyl sulfate(SDS) Polyacrylamide gel electrophoresis.In order to understand the genetic background of the fish and got more scientific data,the length TF-cDNA was produced by RT-PCR and RACE-PCR.
The main researches were as follows:
Firstly,12 specimens were collected for electrophoretic analysis.Polyacrylamide gel electrophoresis was carried out to separate TF.All electrophoretic phenotypes of TF were same;Two bands are observed,one of them denser,the other fainter and they were considered encoded by two co-dominant alleles.Sodium dodecyl sulfate Polyacrylamide gel electrophoresis was utilized to determine the molecular weights of TF and the result showed all the molecular weights were about 71KDa.The high homogeneity of TF might indicated the population were stable pure line and might prove the natural gynogenesis in this population.Results suggested that the TF may be a good genetic marker of gynogenesis for the fish.
Secondly,in order to understand the TF molecular mechanism,the length TF-cDNA was produced by RT-PCR and RACE-PCR.The TF-cDNA was 2364 bp long,with an complete open reading frame(ORF),8-bp 5'UTR and a 355-bp 3'UTR. The cDNA encoded a 666 amino acid with a potential signal peptide,for which the splicing site was located between the 15th and 16th amino acid residues.The deduced molecular weight(Mw) of the mature(after cleavage of the signal peptide) was 71.9 kDa,which was consistent with data from SDS-PAGE analysis.No possible N-linked glycosylation site was found.There were 34 cysteine(Cys) residues in mature TF-cDNA.Result showed there was similar genetic background among the colour crucian carp A,the silver crucian carp E and the Pingxiang red-transparent crucian carp.
Lastly,we made theoretic analysis of the secondary structure of TF-cDNA and proceeded to homology modeling to generate similar tertiary structures of TF-cDNA.
本文的工作主要为:
一、对萍乡红鲫的血清转铁蛋白(Transferrin,简称TF)进行电泳分析。聚丙烯酰胺凝胶电泳(PAGE)发现随机选取的12尾萍乡红鲫TF表型完全的相同,个体间没有差异;萍乡红鲫的TF表型为两个条带,推测可能由一个座位上的二个等位基因遗传决定。采用十二烷基磺酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)对TF进行分析得出其分子量大约为71KDa。经人工选育的萍乡红鲫TF表型一致,种群内未见多样性,认为其遗传性状趋于稳定纯系;萍乡红鲫不同个体间完全_致的TF表型及分子量,可以利用TF雌核发育鉴定的遗传标记揭示萍乡红鲫具有雌核发育的生殖方式。
二、为进一步了解了萍乡红鲫TF的分子机制,利用RT-PCR和RACE-PCR克隆出萍乡红鲫TF互补基因,克隆得到的TF-cDNA包含完整的开放阅读框(open reading frame,ORF),肉红鲫TF-cDNA长2364碱基对(base pair,bp),包括8bp的5′非编码区(untranslated region,UTR),355bp的3'UTR,编码666个氨基酸,理论信号肽切割位点位于15-16个氨基酸之间,预测的分子量为71.9,没有潜在的糖基化位点,还有34个半胱氨酸残基。结果分析表明萍乡红鲫TF遗传性质与彩鲫A、银鲫E同源性较高。
最后对萍乡红鲫TF蛋白序列进行二级结构理论分析和三级结构的同源建模。
Pingxiang red-transpanent crucian carp,Carassius auratus var.Pingxiangnesis had been considered as a variant of wildness diploid spieces of crucian carp in Jiangxi Province.The sixth generation was successive produced by selective breeding for seven years.The fish had many good economic characters,such as fast growth,easily adaptable,rich nutrition,easily cultured,strong anti-resistance.It had been examined and authorized as a new aquaculture variety in the whole country.Transferrin of the fish was studied in this paper.The phenotypes of the TF was got by the method of Polyacrylamide gel electrophoresis(PAGE) and its molecular weights was analyzed through Sodium dodecyl sulfate(SDS) Polyacrylamide gel electrophoresis.In order to understand the genetic background of the fish and got more scientific data,the length TF-cDNA was produced by RT-PCR and RACE-PCR.
The main researches were as follows:
Firstly,12 specimens were collected for electrophoretic analysis.Polyacrylamide gel electrophoresis was carried out to separate TF.All electrophoretic phenotypes of TF were same;Two bands are observed,one of them denser,the other fainter and they were considered encoded by two co-dominant alleles.Sodium dodecyl sulfate Polyacrylamide gel electrophoresis was utilized to determine the molecular weights of TF and the result showed all the molecular weights were about 71KDa.The high homogeneity of TF might indicated the population were stable pure line and might prove the natural gynogenesis in this population.Results suggested that the TF may be a good genetic marker of gynogenesis for the fish.
Secondly,in order to understand the TF molecular mechanism,the length TF-cDNA was produced by RT-PCR and RACE-PCR.The TF-cDNA was 2364 bp long,with an complete open reading frame(ORF),8-bp 5'UTR and a 355-bp 3'UTR. The cDNA encoded a 666 amino acid with a potential signal peptide,for which the splicing site was located between the 15th and 16th amino acid residues.The deduced molecular weight(Mw) of the mature(after cleavage of the signal peptide) was 71.9 kDa,which was consistent with data from SDS-PAGE analysis.No possible N-linked glycosylation site was found.There were 34 cysteine(Cys) residues in mature TF-cDNA.Result showed there was similar genetic background among the colour crucian carp A,the silver crucian carp E and the Pingxiang red-transparent crucian carp.
Lastly,we made theoretic analysis of the secondary structure of TF-cDNA and proceeded to homology modeling to generate similar tertiary structures of TF-cDNA.
引文
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