东亚三角涡虫cDNA文库的构建及DjGon基因的进化、表达分析
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摘要
东亚三角涡虫(Dujesia japonica)是扁形动物门的代表动物。在动物演化的历史进程中,扁形动物是首次出现两侧对称、三胚层的类群。其所处的特殊演化地位,和强大的再生作用,为我们研究动物起源、进化、胚胎发育、染色体、行为以及细胞分化与脱分化分子机理提供了优良材料。
本文应用Clontech公司Creator~(TM) SMART~(TM) cDNA Library Construction Kit,构建了东亚三角涡虫cDNA文库。对文库克隆进行序列测定和生物信息学分析,共获得208个rRNA基因,148个编码蛋白基因,78个位于染色体上片段基因。编码蛋白大体分为:38个和酶类有关蛋白,27个参与基因调节与信号传导蛋白,11个位于线粒体细胞器中的蛋白,18个维持细胞结构,负责细胞内物质转运的蛋白,7个与肿瘤相关蛋白,5个泛素蛋白家族成员。这些基因在涡虫中多数均为首次报道,并且许多具有完整的开放性阅读框(ORF),甚至有些极有可能是涡虫中的特有基因,对这些基因的深入研究,可能为将来揭示涡虫再生作用过程的分子机制和发育进化中的特殊地位提供物质基础。
从东亚三角涡虫文库中分离的克隆K30,经生物信息学分析表明和人、老鼠、马、牛等哺乳动物同源性高于28%,该蛋白具有ADAMTS家族某些成员的GON保守结构域。利用该基因编码的296个氨基酸残基组成的蛋白进行进化分析,表明东亚三角涡虫DjGon基因可能代表了脊椎动物ADAMTS-9和-20基因分化前的一种前体基因型。
通过Southern blot和整体原位杂交等技术,研究了东亚三角涡虫DjGon基因的表达图式。Southern blot表明该基因在涡虫体内为两拷贝;整体原位杂交表明该基因在幼虫发育不同时期不同组织的表达存在差异:肠中信号较强,而皮肌囊较弱,且皮肌囊内的信号由头向尾梯度减弱。
Dujesia japonica is the typical in the phylum Platyhelminthes and shares with vertebrates keys traits such as bilateral,symmetry,three germ layers-ectoderm,mesoderm and endoderm, and dorsoventral and anteroposterior polarities.Due to these characters and its remarkable regeneration,it is the model animal to study the origin and evolution of the vertebrates, embryogenesis,chromosomes,stem cells and regeneration.
In order to separate and identify the developmental and regenerative gene from Dujesia japonica,the total RNA as template are constructed the cDNA library,according to SMART~(TM) cDNA Library Construction Kit and Advantage 2 PCR Kit.The unamplified cDNA library contained 1.22×10~5 independent clones in capability.The recombination rate was more than 98%.The average inserted cDNA fragments were about 900 bp,with the majority ranging from 500 to 1500 bp.From the library,we randomly selected some DNA fragments to sequence and blast in NCBI database on line.The results showed that it contained 208 rRNA genes,148 encoding genes and 78 genes in the chromosome.It is undoubted that our result established a solid molecular basis for the depth study of development and regeneration in planarian.
Planarian DjGon encoding 249 amino acids were identified from the cDNA library of the Dujesia japonica.Structural analysis suggested that it was a member of ADAMTS protein family,with the conserved domain GON.The deduced planarian protein has more than 30% identity with some mammals.And the molecular analysis shows that the planarian gene may be represent the precursor gene that existed prior to the origin of ADAMTS-9 and-20.
The paper used the molecular blot to study the expression pattern of this DjGon gene in Dujesia japonica.The southern blot revealed that this gene has two copies in Dujesia japonica. The whole mount in situ hybridization blot suggested that DjGon gene expression is restricted mainly to the gut and dermo-muscular lac in the planarian larvae.And the signal in gut is stronger than that in dermo-muscular lac.
本文应用Clontech公司Creator~(TM) SMART~(TM) cDNA Library Construction Kit,构建了东亚三角涡虫cDNA文库。对文库克隆进行序列测定和生物信息学分析,共获得208个rRNA基因,148个编码蛋白基因,78个位于染色体上片段基因。编码蛋白大体分为:38个和酶类有关蛋白,27个参与基因调节与信号传导蛋白,11个位于线粒体细胞器中的蛋白,18个维持细胞结构,负责细胞内物质转运的蛋白,7个与肿瘤相关蛋白,5个泛素蛋白家族成员。这些基因在涡虫中多数均为首次报道,并且许多具有完整的开放性阅读框(ORF),甚至有些极有可能是涡虫中的特有基因,对这些基因的深入研究,可能为将来揭示涡虫再生作用过程的分子机制和发育进化中的特殊地位提供物质基础。
从东亚三角涡虫文库中分离的克隆K30,经生物信息学分析表明和人、老鼠、马、牛等哺乳动物同源性高于28%,该蛋白具有ADAMTS家族某些成员的GON保守结构域。利用该基因编码的296个氨基酸残基组成的蛋白进行进化分析,表明东亚三角涡虫DjGon基因可能代表了脊椎动物ADAMTS-9和-20基因分化前的一种前体基因型。
通过Southern blot和整体原位杂交等技术,研究了东亚三角涡虫DjGon基因的表达图式。Southern blot表明该基因在涡虫体内为两拷贝;整体原位杂交表明该基因在幼虫发育不同时期不同组织的表达存在差异:肠中信号较强,而皮肌囊较弱,且皮肌囊内的信号由头向尾梯度减弱。
Dujesia japonica is the typical in the phylum Platyhelminthes and shares with vertebrates keys traits such as bilateral,symmetry,three germ layers-ectoderm,mesoderm and endoderm, and dorsoventral and anteroposterior polarities.Due to these characters and its remarkable regeneration,it is the model animal to study the origin and evolution of the vertebrates, embryogenesis,chromosomes,stem cells and regeneration.
In order to separate and identify the developmental and regenerative gene from Dujesia japonica,the total RNA as template are constructed the cDNA library,according to SMART~(TM) cDNA Library Construction Kit and Advantage 2 PCR Kit.The unamplified cDNA library contained 1.22×10~5 independent clones in capability.The recombination rate was more than 98%.The average inserted cDNA fragments were about 900 bp,with the majority ranging from 500 to 1500 bp.From the library,we randomly selected some DNA fragments to sequence and blast in NCBI database on line.The results showed that it contained 208 rRNA genes,148 encoding genes and 78 genes in the chromosome.It is undoubted that our result established a solid molecular basis for the depth study of development and regeneration in planarian.
Planarian DjGon encoding 249 amino acids were identified from the cDNA library of the Dujesia japonica.Structural analysis suggested that it was a member of ADAMTS protein family,with the conserved domain GON.The deduced planarian protein has more than 30% identity with some mammals.And the molecular analysis shows that the planarian gene may be represent the precursor gene that existed prior to the origin of ADAMTS-9 and-20.
The paper used the molecular blot to study the expression pattern of this DjGon gene in Dujesia japonica.The southern blot revealed that this gene has two copies in Dujesia japonica. The whole mount in situ hybridization blot suggested that DjGon gene expression is restricted mainly to the gut and dermo-muscular lac in the planarian larvae.And the signal in gut is stronger than that in dermo-muscular lac.
引文
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