牛Meg8基因的克隆、组织表达及印记状态分析
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摘要
基因组印记是一种表观遗传现象,来自亲本的等位基因或染色体在发育过程中产生专一性的加工修饰,导致后代细胞中两个亲本来源的等位基因有不同的表达活性,具有这种亲本差异性表达现象的基因称为印记基因。Dlk1-Dio3区域是在哺乳动物中相对保守的一个印记基因簇,该区域内的印记基因参与调控胚胎、胎盘发育和肌肉形成。Dlk1-Dio3印记区域在人和鼠中研究的较多,由于基因序列及多态信息的限制,在牛中相关报道较少。
本研究选取Dlk1-Dio3印记域中在鼠和羊中被鉴定为母源表达印记的Meg8基因为研究对象。首先采用RT-PCR和RACE方法得到该基因的cDNA序列(Genebank登录号:HQ407410-HQ407422)。序列分析表明牛Meg8基因至少存在12种可变剪接形式,DNA序列全长约43 kb,cDNA序列全长1062 bp,包括7个外显子,外显子表现出与羊高度的相似性(82.8%~97.7%)。牛Meg8基因5′侧翼区未发现与Kozak规则相匹配的翻译起始序列,推测该基因是一种非编码RNA,可能作为一种RNA调解分子调控靶基因的转录。以上研究结果将为进一步分析该基因的生物学功能以及揭示其分子调控机制奠定基础。
通过RT-PCR方法分析了Meg8基因在成年牛各个组织中的表达情况,结果表明,Meg8基因在成年牛的心、肝、脾、肺、肾、脑、肌肉和脂肪都表达RNA,未表现出在鼠和羊中所报道的组织特异性。
印记基因的表达是基于基因的单等位基因表达,为了研究基因是单等位基因表达还是双等位基因表达,必须找出一个表达的多态,并且要求被研究的动物是杂合子,由此能辨别是哪一个亲本基因被转录。哺乳动物基因组中最常见的多态是SNP,包括DNA点突变,即碱基对的插入或缺失,可以在基因组水平上检测出这种突变。SSCP技术是一种简单、快速、可重复性好的方法,广泛用于寻找基因突变和DNA分型。本研究首先应用PCR-SSCP方法寻找Meg8基因中表达的多态,鉴定杂合子,通过RT-PCR及直接测序法分析杂合子牛的心、肝、脾、肺、肾、大脑、肌肉和脂肪组织中该多态位点的表达情况。研究结果表明Meg8基因在成年牛被检测的八个组织中均表现为单等位基因表达,表明Meg8基因在牛中为印记基因。
Genomic imprinting is an epigenetic phenomenon that results in an allele-specific expression from a single in a parent-of-origin-dependent manner. Imprinted genes are genes that preferentially expressed from either the maternally allele or the paternally allele. Dlk1-Dio3 imprinted domain is conserved among mammals, and involved in embryonic, placental and muscle growth. Dlk1-Dio3 imprinted domain has been widely studied in mouse and human. But due to the lack of sequence and polymorphism information in coding regions, few imprinted genes have been reported in cattle.
In this study, we cloned the cDNA sequence and analyzed the expression and imprinted status of Meg8 gene, which is identified as a maternally expressed gene in both mouse and sheep. The cDNA sequences were obtained by RT-PCR and RACE (GenBank accession number: HQ407410-HQ407422). Sequence analyse showed that 12 transcript variants occured in cattle Meg8 gene. The DNA and cDNA sequences were about 43 kb and 1062 bp, respectively. There are 7 exons in cattle Meg8 gene,the exons had high identity ranged from 82.8 to 97.7% when aligned with the sheep orthologue. Bioinformatics analysis showed that none of the ATGs is consistent with Kozak consensus sequence. So the cattle Meg8 gene may be a noncoding RNA and act as a regulated factor. These results are essential to analyze the biological function and the molecular regulation mechanism of cattle Meg8 gene.
We used RT-PCR to analyse the tissue-specific distribution of the cattle Meg8 gene in different tissues.The results indicated that Meg8 was expressed in all investigated tissues, including heart, liver, spleen, lung, kidney, brain, skeletal muscle and subcutaneous fat of the adult cattle. Our data showed that no specific expression pattern existed in cattle Meg8 gene which is not consistent with mouse and sheep.
Expression patterns of imprinted genes are studied based on the fact that the genes are monoallelically expressed. To study if gene expressed as monoallelic or biallelic, an expressed polymorphism (polymorphism in the mRNA or proteins) must be found to distinguish which parental allele is transcribed. In addition, the animals being studied must be heterozygous for the gene/polymorphism of interest. In the mammalian genome single nucleotide polymorphisms (SNP) are the most common polymorphisms, which are DNA point mutations (base-pair change or insertions/deletions) and can be distributed in the genome level. Single strand conformation polymorphism(SSCP) is a quite simple, rapid and reproducible method and widely used in the detection of known mutations and the analysis of DNA variation.
We found an SNP in the coding sequence of cattle Meg8 gene by PCR-SSCP and then analysed the cDNA of the heterozygous individuals using RT-PCR and direct sequencing. Results revealed that the Meg8 gene was monoallelically expressed in the heart, liver, spleen, lung, kidney, brain, skeletal muscle and subcutaneous fat in adult cattle. This is the first time to determine that the Meg8 gene is imprinted in cattle.
本研究选取Dlk1-Dio3印记域中在鼠和羊中被鉴定为母源表达印记的Meg8基因为研究对象。首先采用RT-PCR和RACE方法得到该基因的cDNA序列(Genebank登录号:HQ407410-HQ407422)。序列分析表明牛Meg8基因至少存在12种可变剪接形式,DNA序列全长约43 kb,cDNA序列全长1062 bp,包括7个外显子,外显子表现出与羊高度的相似性(82.8%~97.7%)。牛Meg8基因5′侧翼区未发现与Kozak规则相匹配的翻译起始序列,推测该基因是一种非编码RNA,可能作为一种RNA调解分子调控靶基因的转录。以上研究结果将为进一步分析该基因的生物学功能以及揭示其分子调控机制奠定基础。
通过RT-PCR方法分析了Meg8基因在成年牛各个组织中的表达情况,结果表明,Meg8基因在成年牛的心、肝、脾、肺、肾、脑、肌肉和脂肪都表达RNA,未表现出在鼠和羊中所报道的组织特异性。
印记基因的表达是基于基因的单等位基因表达,为了研究基因是单等位基因表达还是双等位基因表达,必须找出一个表达的多态,并且要求被研究的动物是杂合子,由此能辨别是哪一个亲本基因被转录。哺乳动物基因组中最常见的多态是SNP,包括DNA点突变,即碱基对的插入或缺失,可以在基因组水平上检测出这种突变。SSCP技术是一种简单、快速、可重复性好的方法,广泛用于寻找基因突变和DNA分型。本研究首先应用PCR-SSCP方法寻找Meg8基因中表达的多态,鉴定杂合子,通过RT-PCR及直接测序法分析杂合子牛的心、肝、脾、肺、肾、大脑、肌肉和脂肪组织中该多态位点的表达情况。研究结果表明Meg8基因在成年牛被检测的八个组织中均表现为单等位基因表达,表明Meg8基因在牛中为印记基因。
Genomic imprinting is an epigenetic phenomenon that results in an allele-specific expression from a single in a parent-of-origin-dependent manner. Imprinted genes are genes that preferentially expressed from either the maternally allele or the paternally allele. Dlk1-Dio3 imprinted domain is conserved among mammals, and involved in embryonic, placental and muscle growth. Dlk1-Dio3 imprinted domain has been widely studied in mouse and human. But due to the lack of sequence and polymorphism information in coding regions, few imprinted genes have been reported in cattle.
In this study, we cloned the cDNA sequence and analyzed the expression and imprinted status of Meg8 gene, which is identified as a maternally expressed gene in both mouse and sheep. The cDNA sequences were obtained by RT-PCR and RACE (GenBank accession number: HQ407410-HQ407422). Sequence analyse showed that 12 transcript variants occured in cattle Meg8 gene. The DNA and cDNA sequences were about 43 kb and 1062 bp, respectively. There are 7 exons in cattle Meg8 gene,the exons had high identity ranged from 82.8 to 97.7% when aligned with the sheep orthologue. Bioinformatics analysis showed that none of the ATGs is consistent with Kozak consensus sequence. So the cattle Meg8 gene may be a noncoding RNA and act as a regulated factor. These results are essential to analyze the biological function and the molecular regulation mechanism of cattle Meg8 gene.
We used RT-PCR to analyse the tissue-specific distribution of the cattle Meg8 gene in different tissues.The results indicated that Meg8 was expressed in all investigated tissues, including heart, liver, spleen, lung, kidney, brain, skeletal muscle and subcutaneous fat of the adult cattle. Our data showed that no specific expression pattern existed in cattle Meg8 gene which is not consistent with mouse and sheep.
Expression patterns of imprinted genes are studied based on the fact that the genes are monoallelically expressed. To study if gene expressed as monoallelic or biallelic, an expressed polymorphism (polymorphism in the mRNA or proteins) must be found to distinguish which parental allele is transcribed. In addition, the animals being studied must be heterozygous for the gene/polymorphism of interest. In the mammalian genome single nucleotide polymorphisms (SNP) are the most common polymorphisms, which are DNA point mutations (base-pair change or insertions/deletions) and can be distributed in the genome level. Single strand conformation polymorphism(SSCP) is a quite simple, rapid and reproducible method and widely used in the detection of known mutations and the analysis of DNA variation.
We found an SNP in the coding sequence of cattle Meg8 gene by PCR-SSCP and then analysed the cDNA of the heterozygous individuals using RT-PCR and direct sequencing. Results revealed that the Meg8 gene was monoallelically expressed in the heart, liver, spleen, lung, kidney, brain, skeletal muscle and subcutaneous fat in adult cattle. This is the first time to determine that the Meg8 gene is imprinted in cattle.
引文
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