内蒙古绒山羊BMP-2基因DNA序列的克隆与荧光原位杂交定位
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摘要
本研究从内蒙古绒山羊血中提取基因组DNA,PCR扩增绒山羊的BMP-2基因片段,连接到pGM-T载体、转化,筛选鉴定阳性克隆提取质粒进行测序,和GenBank数据库进行比对,对它们的核苷酸和氨基酸序列进行生物信息学分析,并将克隆所得质粒标记探针,利用FISH技术确定BMP-2在染色体上的位置。试验结果充实了BMP-2基因图谱,为进一步了解BMP-2基因信息对绒山羊的选育提供了理论支持。结果如下:
1.克隆了内蒙古绒山羊BMP-2基因的外显子,外显子长1751bp。对绒山羊、绵羊、牛、猪、人和猴子的相应序列做同源性比较分析和进化树分析,所得结果与物种间的进化关系相符。
2.绒山羊、绵羊和牛的BMP-2基因第三外显子的这部分序列,序列间的变异完全是碱基的替换。其中绒山羊和绵羊序列之间,发生了3个碱基替换,相似率高达99.6%,而牛与绒山羊碱基替换数目为13个,相似率为98.1%,比绒山羊与绵羊的相似率要低,比较绒山羊、绵羊和牛第三外显子的氨基酸序列,有5处不同,其中与绵羊只有1变异,牛有4处,同源性非常高,可见,绒山羊、绵羊和牛之间的碱基突变绝大多数为同义突变,说明BMP-2基因保守性很强。
3.将克隆得到的绒山羊BMP-2基因片段质粒,根据试剂盒说明标记为探针,利用荧光原位杂交方法定位于染色体13q15。
In this study,the genome DNA were extracted from blood in Cashmere goat.The sequence were obtained by PCR and cloned into pGM18-T vector .The positive clones were sequenced.Then we compared the sequences of BMP-2 gene fragments among goats,sheep and other animals that have been submitted to GeneBank. The method of bioinformatics was used to aligment of both their nucleotide and amino acid sequence of Cashmere goat. Furthermore we made the probe with DIG using the cloning product of Cashmere goat, for studying the map of BMP-2 in Cashmere of Cashmere goat by fluorescence In situ hybrition. The results would enrich gene map and then offered the theoretical support to understand the effect of gene message of BMP-2 on breeding of Cashmere goat.The results showed as following:
1. Obtained the sequence of the exon of bmp2 which spaned 1751bp. The phylogenetic trees among goat, sheep, cow, pig, human and macaca conformted to taxonomy of tradition. It is suggested that BMP-2 gene can represent the character of Cashmere goat.
2. The replacement of Exon 3 of BMP-2 among goat, sheep and cow was only nucleotide. It was found 4 mutant among goat and sheep,99.6% identity between cashmere goat and sheep. It was found 13 mutant among goat and cow,98.1% identity between cashmere goat and cow. The sequence of the BMP-2 gene's Exon 3 was translated to amino acid sequence. The results of aligment of the amino acid showed that it was found 5 mutant among Cashmere goat, sheep and cow, but only 1 mutant between goat and sheep, the identity of goat and sheep was very high. It is thus clear that, the replacement of BMP-2 gene between goat and sheep is samesense mutation, furthermore explanation of BMP-2 gene’conservatism is very better.
3. The recombinant plasmid were marked, accroding to the guider of kit, then made a probe. BMP-2 gene was maped into chromosome 13q15 of Cashmere goat by fluorescence In situ hybridization.
1.克隆了内蒙古绒山羊BMP-2基因的外显子,外显子长1751bp。对绒山羊、绵羊、牛、猪、人和猴子的相应序列做同源性比较分析和进化树分析,所得结果与物种间的进化关系相符。
2.绒山羊、绵羊和牛的BMP-2基因第三外显子的这部分序列,序列间的变异完全是碱基的替换。其中绒山羊和绵羊序列之间,发生了3个碱基替换,相似率高达99.6%,而牛与绒山羊碱基替换数目为13个,相似率为98.1%,比绒山羊与绵羊的相似率要低,比较绒山羊、绵羊和牛第三外显子的氨基酸序列,有5处不同,其中与绵羊只有1变异,牛有4处,同源性非常高,可见,绒山羊、绵羊和牛之间的碱基突变绝大多数为同义突变,说明BMP-2基因保守性很强。
3.将克隆得到的绒山羊BMP-2基因片段质粒,根据试剂盒说明标记为探针,利用荧光原位杂交方法定位于染色体13q15。
In this study,the genome DNA were extracted from blood in Cashmere goat.The sequence were obtained by PCR and cloned into pGM18-T vector .The positive clones were sequenced.Then we compared the sequences of BMP-2 gene fragments among goats,sheep and other animals that have been submitted to GeneBank. The method of bioinformatics was used to aligment of both their nucleotide and amino acid sequence of Cashmere goat. Furthermore we made the probe with DIG using the cloning product of Cashmere goat, for studying the map of BMP-2 in Cashmere of Cashmere goat by fluorescence In situ hybrition. The results would enrich gene map and then offered the theoretical support to understand the effect of gene message of BMP-2 on breeding of Cashmere goat.The results showed as following:
1. Obtained the sequence of the exon of bmp2 which spaned 1751bp. The phylogenetic trees among goat, sheep, cow, pig, human and macaca conformted to taxonomy of tradition. It is suggested that BMP-2 gene can represent the character of Cashmere goat.
2. The replacement of Exon 3 of BMP-2 among goat, sheep and cow was only nucleotide. It was found 4 mutant among goat and sheep,99.6% identity between cashmere goat and sheep. It was found 13 mutant among goat and cow,98.1% identity between cashmere goat and cow. The sequence of the BMP-2 gene's Exon 3 was translated to amino acid sequence. The results of aligment of the amino acid showed that it was found 5 mutant among Cashmere goat, sheep and cow, but only 1 mutant between goat and sheep, the identity of goat and sheep was very high. It is thus clear that, the replacement of BMP-2 gene between goat and sheep is samesense mutation, furthermore explanation of BMP-2 gene’conservatism is very better.
3. The recombinant plasmid were marked, accroding to the guider of kit, then made a probe. BMP-2 gene was maped into chromosome 13q15 of Cashmere goat by fluorescence In situ hybridization.
引文
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