北京油鸡细菌人工染色体文库的构建与肉质风味相关基因的研究
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摘要
为了有效保存我国优质肉鸡品种资源——北京油鸡,本研究构建了北京油鸡细菌人工染色体(bacterial artificial chromosome)文库,并将ATIC(aminoimidazole carboxamide ribonucleotide transformylase and IMP cyclohydrolase)基因和L-FABP(liver fatty acid-binding protein)基因作为影响鸡肉质和风味的相关基因,对基因的结构、在体外成纤维细胞中的表达及亚细胞定位进行了研究,同时获得了稳定表达ATIC基因的阳性克隆细胞株。
本研究抽取雌性北京油鸡静脉血,用于制备大分子量基因组DNA,制备好的基因组DNA经HindⅢ部分酶切后,二次脉冲电泳分离所需DNA片段。电洗脱回收100～400kb范围的片段,与pBeoBAC11载体连接,转化DH10B感受态细胞,挑取和保存白色单克隆。本研究所构建北京油鸡基因组BAC文库,含有115,200个BAC克隆(保存在30个超级池,每个超级池由10个384孔板组成)。随机挑选了200个BAC克隆的插入片段进行估计,平均大小为158kb,其中空克隆的比例为0.9%,表明文库的覆盖率为13.7倍,预计从文库中筛选到单拷贝基因的机率为99.99%。文库中70%的克隆插入大于100kb,而且有部分克隆甚至大于300kb,这样大小的插入可以满足所有试验对于片段大小的要求。
本研究取北京油鸡鸡胚和阿勒泰羊的耳缘组织,通过组织块贴壁培养法,成功构建了北京油鸡和阿勒泰羊的成纤维细胞系,并对所建细胞系进行了各项生物学特性检测,同时对绿色荧光蛋白基因转染成纤维细胞的各项影响因素进行了研究。结果表明,细胞系的各项指标均达到美国典型培养物中心(American type Culture Collection, ATCC)细胞系鉴定标准,为研究外源基因在成纤维细胞中的表达与调控提供了优质的靶细胞。
本研究提取北京油鸡心、肝、脾、肺、肾、脑、腿肌与胸肌等不同组织的总RNA,利用RT-PCR方法扩增得到L-FABP基因,并检测ATIC基因mRNA在不同组织中的差异表达水平。将ATIC基因完整开放阅读框重组至真核表达载体pDsRed1-N1, pEYFP-N1及pEGFP-N3的多克隆位点EcoR I和BamH I之间,将L-FABP基因完整开放阅读框重组至pEGFP-N3的多克隆位点XhoI和EcoRI之间,成功构建带有报告基因的真核重组表达载体pDsRed1-N1-ATIC, pEYFP-N1-ATIC, pEGFP-N3-ATIC和pEGFP-N3-L-FABP。采用脂质体介导法,将pDsRed1-N1-ATIC、pEYFP-N1-ATIC和pEGFP-N3-ATIC导入北京油鸡体外培养成纤维细胞中,将pEGFP-N3-L-FABP导入阿勒泰羊体外培养成纤维细胞中。对ATIC基因和L-FABP基因在细胞中的表达、亚细胞定位、阳性细胞生长与增殖状况进行了研究,并利用G418药物筛选和进行单克隆化培养。结果发现,在转染后24h、48h和72h,各个重组融合蛋白的转染率在10.3%～53.2%之间。重组融合蛋白在北京油鸡和阿勒泰羊成纤维细胞的细胞核与细胞质均分布,但主要集中在细胞核周围。经RT-PCR扩增确认ATIC融合蛋白基因整合到北京油鸡成纤维细胞的基因组中,L-FABP融合蛋白也整合到阿勒泰羊成纤维细胞的基因组中,Western blot检测验证发现ATIC融合蛋白在北京油鸡成纤维细胞的基因组中获得正常表达。经药物筛选和单克隆化培养,获得了表达pDsRed-N1-ATIC,pEYFP-N1-ATIC与pEGFP-N3-ATIC融合蛋白的阳性克隆细胞株。
In order to preserve precious genetic material of Beijing fatty chicken, we have constructed a bacterial artificial chromosome (BAC) library of Beijing fatty chicken and studied these two genes of chicken, aminoimidazole carboxamide ribonucleotide transformylase and IMP cyclohydrolase (ATIC) and liver fatty acid-binding protein (L-FABP), which involved in meat quality and flavor. The genomic structure of ATIC and L-FABP were determined, and their expression in fibroblasts and Sub-cellular location were studied. Three cell lines that stably expressing pDsRed1-N1-TAIC, pEYFP-N1-ATIC and pEGFP-N3-ATIC fusion protein were cloned successfully.
High-molecular-weight (HMW) DNA was extracted from blood of female Beijing fatty chicken, with concentration of 3.0×108 cells/ml cell suspension, and then mixed with equal volume of 1% low-melting-point liquefied agarose, partially digested with HindШand separated using double size selection system. Digested DNA fragments within the range of 100～400kb was recovered by elctro-elution and ligated into pBeoBAC11 vector, and then was used to transform into DH10B competent cells. Individual white colone was picked and stored in -70℃refrigerator after incubation.
The Beijing fatty chicken BAC library consists of 115,200 BAC clones totally (conserved in 30 superpools, each in 10384-well plate). 200 clones were selected randomly and analyzed, and the results indicated that the average insert size of the library was estimated to be 158 kb, and the large inserts (>100kb) accounted for more than 70% and the non-recombinant clones was only 0.9%. It was calculated that the genome coverage of the library is 13.7 and the possibility to find a single-copy gene in the library is 99.99%. This Beijing fatty chicken BAC library would serve as a valuable resource in comparative and functional genomic research.
The present study established the target cell lines of Beijing fatty chicken and Aletai sheep successfully by direct adherent culture method. In order to study the exogenous gene expression, pEGFP-N3 fluorescent gene was transfected into the fibroblast cells. The results showed that the quality of the cell line met the quality requirements of the ATCC (American Type Culture Collection), and indicated that the cell line was outstanding target cells for gene expression.
L-FABP gene was amplified and the specific expression of ATIC gene in 8 different tissues (heart, liver, spleen, lung, kidney, brain, leg muscle and chest muscle) of Beijing fatty chicken was investigated by RT-PCR method in present study. The full length of ATIC cDNA was inserted into fusion expression vector pDsRed1-N1, pEYFP-N1 and pEGFP-N3 between multiple cloning sites of EcoR I and BamH I, while the full length of L-FABP cDNA was inserted into fusion expression vector pEGFP-N3 between multiple cloning sites of EcoR I and Xho I, and recombinant eukaryotic expression vector pDsRed1-N1-ATIC, pEYFP-N1-ATIC, pEGFP-N3-ATIC and pEGFP-N3-L-FABP were constructed fused with GFP as reporter gene. Lipofectin method was used to transfect these recombinant vectors of pDsRed1-N1-ATIC, pEYFP-N1-ATIC, pEGFP-N3-ATIC into Beijing fatty chicken and pEGFP-N3-L-FABP into Aletai sheep fibroblast cells. The resistant colonies were picked and sub-cultured until use after G418 screening. The results showed: at 24h, 48h and 72h after transfection, the expression efficiency of these 4 kinds of recombinant fusion protein were between 10.3% and 53.2%, and the fluorescence well distributed in cytoplasm and nucleus except cryptomere vesicle; three cell lines that stably expressing pDsRed1-N1-ATIC, pEYFP-N1-ATIC and pEGFP-N3-ATIC fusion protein were obtained by the G418 drug screening and monoclonal training. RT-PCR confirmed the pDsRed1-N1-ATIC, pEYFP-N1-ATIC and pEGFP-N3-ATIC had been integrated into the Beijing fatty chicken, fibroblast cell genome, and pEGFP-N3-L-FABP had been integrated into the Aletai sheep fibroblast cell genome, the western blotting results showed that pDsRed1-N1-ATIC, pEYFP-N1-ATIC and pEGFP-N3-ATIC has normal fusion protein expression. On the other hand, there was no significant effect(P>0.05)on cell shape, cell growth and reduplication state when compared with control groups.
Above all, The present work not only preserved the germplasm resources of the important Beijing fatty chicken at the gene level but also provided valuable material for genome, post-genome and somatic cell cloning research.
本研究抽取雌性北京油鸡静脉血,用于制备大分子量基因组DNA,制备好的基因组DNA经HindⅢ部分酶切后,二次脉冲电泳分离所需DNA片段。电洗脱回收100～400kb范围的片段,与pBeoBAC11载体连接,转化DH10B感受态细胞,挑取和保存白色单克隆。本研究所构建北京油鸡基因组BAC文库,含有115,200个BAC克隆(保存在30个超级池,每个超级池由10个384孔板组成)。随机挑选了200个BAC克隆的插入片段进行估计,平均大小为158kb,其中空克隆的比例为0.9%,表明文库的覆盖率为13.7倍,预计从文库中筛选到单拷贝基因的机率为99.99%。文库中70%的克隆插入大于100kb,而且有部分克隆甚至大于300kb,这样大小的插入可以满足所有试验对于片段大小的要求。
本研究取北京油鸡鸡胚和阿勒泰羊的耳缘组织,通过组织块贴壁培养法,成功构建了北京油鸡和阿勒泰羊的成纤维细胞系,并对所建细胞系进行了各项生物学特性检测,同时对绿色荧光蛋白基因转染成纤维细胞的各项影响因素进行了研究。结果表明,细胞系的各项指标均达到美国典型培养物中心(American type Culture Collection, ATCC)细胞系鉴定标准,为研究外源基因在成纤维细胞中的表达与调控提供了优质的靶细胞。
本研究提取北京油鸡心、肝、脾、肺、肾、脑、腿肌与胸肌等不同组织的总RNA,利用RT-PCR方法扩增得到L-FABP基因,并检测ATIC基因mRNA在不同组织中的差异表达水平。将ATIC基因完整开放阅读框重组至真核表达载体pDsRed1-N1, pEYFP-N1及pEGFP-N3的多克隆位点EcoR I和BamH I之间,将L-FABP基因完整开放阅读框重组至pEGFP-N3的多克隆位点XhoI和EcoRI之间,成功构建带有报告基因的真核重组表达载体pDsRed1-N1-ATIC, pEYFP-N1-ATIC, pEGFP-N3-ATIC和pEGFP-N3-L-FABP。采用脂质体介导法,将pDsRed1-N1-ATIC、pEYFP-N1-ATIC和pEGFP-N3-ATIC导入北京油鸡体外培养成纤维细胞中,将pEGFP-N3-L-FABP导入阿勒泰羊体外培养成纤维细胞中。对ATIC基因和L-FABP基因在细胞中的表达、亚细胞定位、阳性细胞生长与增殖状况进行了研究,并利用G418药物筛选和进行单克隆化培养。结果发现,在转染后24h、48h和72h,各个重组融合蛋白的转染率在10.3%～53.2%之间。重组融合蛋白在北京油鸡和阿勒泰羊成纤维细胞的细胞核与细胞质均分布,但主要集中在细胞核周围。经RT-PCR扩增确认ATIC融合蛋白基因整合到北京油鸡成纤维细胞的基因组中,L-FABP融合蛋白也整合到阿勒泰羊成纤维细胞的基因组中,Western blot检测验证发现ATIC融合蛋白在北京油鸡成纤维细胞的基因组中获得正常表达。经药物筛选和单克隆化培养,获得了表达pDsRed-N1-ATIC,pEYFP-N1-ATIC与pEGFP-N3-ATIC融合蛋白的阳性克隆细胞株。
In order to preserve precious genetic material of Beijing fatty chicken, we have constructed a bacterial artificial chromosome (BAC) library of Beijing fatty chicken and studied these two genes of chicken, aminoimidazole carboxamide ribonucleotide transformylase and IMP cyclohydrolase (ATIC) and liver fatty acid-binding protein (L-FABP), which involved in meat quality and flavor. The genomic structure of ATIC and L-FABP were determined, and their expression in fibroblasts and Sub-cellular location were studied. Three cell lines that stably expressing pDsRed1-N1-TAIC, pEYFP-N1-ATIC and pEGFP-N3-ATIC fusion protein were cloned successfully.
High-molecular-weight (HMW) DNA was extracted from blood of female Beijing fatty chicken, with concentration of 3.0×108 cells/ml cell suspension, and then mixed with equal volume of 1% low-melting-point liquefied agarose, partially digested with HindШand separated using double size selection system. Digested DNA fragments within the range of 100～400kb was recovered by elctro-elution and ligated into pBeoBAC11 vector, and then was used to transform into DH10B competent cells. Individual white colone was picked and stored in -70℃refrigerator after incubation.
The Beijing fatty chicken BAC library consists of 115,200 BAC clones totally (conserved in 30 superpools, each in 10384-well plate). 200 clones were selected randomly and analyzed, and the results indicated that the average insert size of the library was estimated to be 158 kb, and the large inserts (>100kb) accounted for more than 70% and the non-recombinant clones was only 0.9%. It was calculated that the genome coverage of the library is 13.7 and the possibility to find a single-copy gene in the library is 99.99%. This Beijing fatty chicken BAC library would serve as a valuable resource in comparative and functional genomic research.
The present study established the target cell lines of Beijing fatty chicken and Aletai sheep successfully by direct adherent culture method. In order to study the exogenous gene expression, pEGFP-N3 fluorescent gene was transfected into the fibroblast cells. The results showed that the quality of the cell line met the quality requirements of the ATCC (American Type Culture Collection), and indicated that the cell line was outstanding target cells for gene expression.
L-FABP gene was amplified and the specific expression of ATIC gene in 8 different tissues (heart, liver, spleen, lung, kidney, brain, leg muscle and chest muscle) of Beijing fatty chicken was investigated by RT-PCR method in present study. The full length of ATIC cDNA was inserted into fusion expression vector pDsRed1-N1, pEYFP-N1 and pEGFP-N3 between multiple cloning sites of EcoR I and BamH I, while the full length of L-FABP cDNA was inserted into fusion expression vector pEGFP-N3 between multiple cloning sites of EcoR I and Xho I, and recombinant eukaryotic expression vector pDsRed1-N1-ATIC, pEYFP-N1-ATIC, pEGFP-N3-ATIC and pEGFP-N3-L-FABP were constructed fused with GFP as reporter gene. Lipofectin method was used to transfect these recombinant vectors of pDsRed1-N1-ATIC, pEYFP-N1-ATIC, pEGFP-N3-ATIC into Beijing fatty chicken and pEGFP-N3-L-FABP into Aletai sheep fibroblast cells. The resistant colonies were picked and sub-cultured until use after G418 screening. The results showed: at 24h, 48h and 72h after transfection, the expression efficiency of these 4 kinds of recombinant fusion protein were between 10.3% and 53.2%, and the fluorescence well distributed in cytoplasm and nucleus except cryptomere vesicle; three cell lines that stably expressing pDsRed1-N1-ATIC, pEYFP-N1-ATIC and pEGFP-N3-ATIC fusion protein were obtained by the G418 drug screening and monoclonal training. RT-PCR confirmed the pDsRed1-N1-ATIC, pEYFP-N1-ATIC and pEGFP-N3-ATIC had been integrated into the Beijing fatty chicken, fibroblast cell genome, and pEGFP-N3-L-FABP had been integrated into the Aletai sheep fibroblast cell genome, the western blotting results showed that pDsRed1-N1-ATIC, pEYFP-N1-ATIC and pEGFP-N3-ATIC has normal fusion protein expression. On the other hand, there was no significant effect(P>0.05)on cell shape, cell growth and reduplication state when compared with control groups.
Above all, The present work not only preserved the germplasm resources of the important Beijing fatty chicken at the gene level but also provided valuable material for genome, post-genome and somatic cell cloning research.
引文
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