乌珠穆沁羊细菌人工染色体（BAC）文库的构建、鉴定及IP-10基因的筛选、表达研究
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摘要
为了永久、有效地保存我国优良地方绵羊品种乌珠穆沁羊的遗传资源,本研究利用低熔点琼脂糖包埋法制备了高分子量基因组DNA,将高分子量DNA经HindⅢ部分酶切后进行两次脉冲电泳分离所需大片段DNA,电洗脱回收,与Copy Control pCC1BAC载体连接,转化Transfor Max EPI300 Electrocompetent E. coli感受态细胞,挑取和保存克隆获得了乌珠穆沁羊细菌人工染色体(BAC)文库。该文库包含了153,600个BAC克隆,共保存在400块384孔板里,平均插入片段大小为90-100 kb,其中平均空载率为3.2%,假阳性克隆比例为0.86%。绵羊的基因组按3×10~9计算,根据文库的平均插入片段和克隆数,计算出文库的基因组覆盖率约为5倍〔100×10~3 bp(153,600×0.86)/ 3×10~9 bp〕,预计从文库中筛选到单拷贝基因的机率为99.4%。经连续转接培养100代后的BAC克隆DNA指纹图谱显示,文库克隆稳定性好,无重排缺失现象。
为了有效利用所构建的乌珠穆沁羊细菌人工染色体文库,本研究采用Qpix多功能全自动机械手臂通过菌液的合并和混合克隆DNA的提取法建立了PCR筛选系统,PCR筛选系统分为菌液工作文库和DNA工作文库,分别包括40个超级池、400个板池、16个行池及10个列池的菌液和DNA。利用BM6465等26对覆盖绵羊染色体的微卫星标记物引物,对构建的乌珠穆沁羊细菌人工染色体(BAC)文库进行PCR筛选,筛选结果表明在文库中这些标记物均得到了阳性克隆,阳性克隆数在3-9个不等,平均克隆数为5.1,这与文库的5倍基因组覆盖率基本吻合。
为了永久、有效地保存我国优良地方绵羊品种乌珠穆沁羊的遗传资源,并为外源基因在成纤维细胞中的表达与调控提研究供优质的靶细胞,本研究以乌珠穆沁羊耳缘组织为实验材料,采用贴壁培养法和冷冻生物学技术构建了样本含量为33的群体成纤维细胞库,共保存优质细胞147份,每份细胞数量为4×10~6,冻存代数在6代以内,细胞生物学各项检测都达到了美国典型培养物保藏中心(ATCC)的细胞系鉴定标准。
为了验证所构建的乌珠穆沁羊细菌人工染色体文库的质量,本研究利用干扰素诱导蛋白10基因对文库进行超级池、行池、列池及板池的PCR筛选,结果得到了6个阳性超级池,共获得8个阳性克隆,编号分别为USB0501F18/041、USB0501L18/041、USB1209A8/119、USB1405P10/135、USB2201C3/211、USB2206C3/216、USB2503D17/243及USB2902G8/282,阳性克隆的平均插入片段均在90kb左右,菌体PCR检测后得到大小片段约为322bp的条带。重组体pGEM-T-IP-10的测序报告显示,所得到的序列为绵羊IP-10基因序列。真核表达重组质粒pEGFP-N3-IP-10用Xhol I和BamH I双酶切后可见309bp大小的目的片段与4.7kb大小的载体带。重组表达载体pEGFP-N3-IP-10和没有插入外源基因的对照组pEGFP-N3质粒均能在乌珠穆沁羊体外培养的细胞中得到良好的表达,转染后48h表达效率最高(P<0.05),绿色荧光在细胞中均匀分布,细胞核的荧光较亮,可以看到转染的靶细胞正在增殖分裂。
This work was aimed to seek an efficient approach for the conserving of important domestic animals in imminent danger or on the edge of extinction. A bacterial artificial chromosome (BAC) library of the nationally protected Chinese Ujumqin sheep was successfully established using the Hind III site of the vector pCC1BAC, comprising of 153,600 clones arrayed in 400 384-well microplates, with an average insert size of 90-100 kb and approximately fivefold coverage of the sheep genome, which yielded a theoretical probability of 99.4% of isolating a particular DNA sequence. The percentage of non-insert clones was 3.2%, false positive ratio was 0.86%. BAC clones of the library were stable in the bacterial host for at least 100 passages. These results suggested that the Chinese Ujumqin sheep BAC library provided a high-quality pool for conserving the nationally protected breed resource at genomic level for long term storage.
In order to effectively using the Chinese Ujumqin sheep bacterial artificial chromosome library, in this study, we using Qpix multi-functional automatic mechanical arm to combine and mix the BAC clones to establish the Ujumqin sheep bacterial artificial chromosome library PCR screening system. The PCR screening system is divided into bacteria library and DNA library, which including bacteria and DNA of 40 super-pools, 400 plate pools, 16 row pools and 10 column pools , respectively. 26 pairs of microsatellite marker primers including BM6465 covering sheep chromosome were screened the Ujumqin sheep bacterial artificial chromosome library, showed each of these markers were positive in the library, the positive clone number were 3 to 9, average number was 5.1, which was consistent with fivefold coverage of the sheep genome.
A Ujumqin sheep ear marginal tissue (USEM) fibroblast line, containing 147 cryovials with 4×10~6 cell each, was successfully established from 33 Ujumqin sheep ear marginal tissues using explant culture and cryopreservation techniques. The cell line met all the criteria from the American Type Culture Collection (ATCC). Not only has the germline of this important sheep breed been preserved at the cell level, but also valuable material had been provided for genome, postgenome and somacloning research. Moreover, the establishment of this technical platform may provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.
In order to verify the quality of Chinese Ujumqin sheep bacterial artificial chromosome library, in this study, the the super pool, pool row, column and plate pools pool were PCR screened by interferon inducible protein-10 gene was, the results indicated there were 6 positive superpools and eight positive clones were obtained, USB0501F18/041, USB0501L18/041, USB1209A8/119, USB1405P10/135, USB2201C3/211, USB2206C3/216, USB2503D17/243 and USB2902G8/282, respectively. The average insert size of positive clones was about 90kb. The fragments of PCR product was about 322bp bands. The sequencing report of recombinant pGEM-T-IP-10 indicated that the result was sheep IP-10 gene sequences. The eukaryotic expression plasmid pEGFP-N3-IP-10 was digested with Xhol I and BamH I indicated an visible fragment of 309 bp and 4.7 kb vector band. Recombinant expression vector pEGFP-N3-IP-10 was highly expressed in Ujumqin sheep fibroblast cells, the green fluorescence was uniformly distributed in the cells, and the target cells were proliferated and divided. pEGFP-N3-IP-10 and the control group of no insert pEGFP-N3 vector both expressed in target cells, the expression efficiencies were highest 48h after transfection (P <0.05).
为了有效利用所构建的乌珠穆沁羊细菌人工染色体文库,本研究采用Qpix多功能全自动机械手臂通过菌液的合并和混合克隆DNA的提取法建立了PCR筛选系统,PCR筛选系统分为菌液工作文库和DNA工作文库,分别包括40个超级池、400个板池、16个行池及10个列池的菌液和DNA。利用BM6465等26对覆盖绵羊染色体的微卫星标记物引物,对构建的乌珠穆沁羊细菌人工染色体(BAC)文库进行PCR筛选,筛选结果表明在文库中这些标记物均得到了阳性克隆,阳性克隆数在3-9个不等,平均克隆数为5.1,这与文库的5倍基因组覆盖率基本吻合。
为了永久、有效地保存我国优良地方绵羊品种乌珠穆沁羊的遗传资源,并为外源基因在成纤维细胞中的表达与调控提研究供优质的靶细胞,本研究以乌珠穆沁羊耳缘组织为实验材料,采用贴壁培养法和冷冻生物学技术构建了样本含量为33的群体成纤维细胞库,共保存优质细胞147份,每份细胞数量为4×10~6,冻存代数在6代以内,细胞生物学各项检测都达到了美国典型培养物保藏中心(ATCC)的细胞系鉴定标准。
为了验证所构建的乌珠穆沁羊细菌人工染色体文库的质量,本研究利用干扰素诱导蛋白10基因对文库进行超级池、行池、列池及板池的PCR筛选,结果得到了6个阳性超级池,共获得8个阳性克隆,编号分别为USB0501F18/041、USB0501L18/041、USB1209A8/119、USB1405P10/135、USB2201C3/211、USB2206C3/216、USB2503D17/243及USB2902G8/282,阳性克隆的平均插入片段均在90kb左右,菌体PCR检测后得到大小片段约为322bp的条带。重组体pGEM-T-IP-10的测序报告显示,所得到的序列为绵羊IP-10基因序列。真核表达重组质粒pEGFP-N3-IP-10用Xhol I和BamH I双酶切后可见309bp大小的目的片段与4.7kb大小的载体带。重组表达载体pEGFP-N3-IP-10和没有插入外源基因的对照组pEGFP-N3质粒均能在乌珠穆沁羊体外培养的细胞中得到良好的表达,转染后48h表达效率最高(P<0.05),绿色荧光在细胞中均匀分布,细胞核的荧光较亮,可以看到转染的靶细胞正在增殖分裂。
This work was aimed to seek an efficient approach for the conserving of important domestic animals in imminent danger or on the edge of extinction. A bacterial artificial chromosome (BAC) library of the nationally protected Chinese Ujumqin sheep was successfully established using the Hind III site of the vector pCC1BAC, comprising of 153,600 clones arrayed in 400 384-well microplates, with an average insert size of 90-100 kb and approximately fivefold coverage of the sheep genome, which yielded a theoretical probability of 99.4% of isolating a particular DNA sequence. The percentage of non-insert clones was 3.2%, false positive ratio was 0.86%. BAC clones of the library were stable in the bacterial host for at least 100 passages. These results suggested that the Chinese Ujumqin sheep BAC library provided a high-quality pool for conserving the nationally protected breed resource at genomic level for long term storage.
In order to effectively using the Chinese Ujumqin sheep bacterial artificial chromosome library, in this study, we using Qpix multi-functional automatic mechanical arm to combine and mix the BAC clones to establish the Ujumqin sheep bacterial artificial chromosome library PCR screening system. The PCR screening system is divided into bacteria library and DNA library, which including bacteria and DNA of 40 super-pools, 400 plate pools, 16 row pools and 10 column pools , respectively. 26 pairs of microsatellite marker primers including BM6465 covering sheep chromosome were screened the Ujumqin sheep bacterial artificial chromosome library, showed each of these markers were positive in the library, the positive clone number were 3 to 9, average number was 5.1, which was consistent with fivefold coverage of the sheep genome.
A Ujumqin sheep ear marginal tissue (USEM) fibroblast line, containing 147 cryovials with 4×10~6 cell each, was successfully established from 33 Ujumqin sheep ear marginal tissues using explant culture and cryopreservation techniques. The cell line met all the criteria from the American Type Culture Collection (ATCC). Not only has the germline of this important sheep breed been preserved at the cell level, but also valuable material had been provided for genome, postgenome and somacloning research. Moreover, the establishment of this technical platform may provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.
In order to verify the quality of Chinese Ujumqin sheep bacterial artificial chromosome library, in this study, the the super pool, pool row, column and plate pools pool were PCR screened by interferon inducible protein-10 gene was, the results indicated there were 6 positive superpools and eight positive clones were obtained, USB0501F18/041, USB0501L18/041, USB1209A8/119, USB1405P10/135, USB2201C3/211, USB2206C3/216, USB2503D17/243 and USB2902G8/282, respectively. The average insert size of positive clones was about 90kb. The fragments of PCR product was about 322bp bands. The sequencing report of recombinant pGEM-T-IP-10 indicated that the result was sheep IP-10 gene sequences. The eukaryotic expression plasmid pEGFP-N3-IP-10 was digested with Xhol I and BamH I indicated an visible fragment of 309 bp and 4.7 kb vector band. Recombinant expression vector pEGFP-N3-IP-10 was highly expressed in Ujumqin sheep fibroblast cells, the green fluorescence was uniformly distributed in the cells, and the target cells were proliferated and divided. pEGFP-N3-IP-10 and the control group of no insert pEGFP-N3 vector both expressed in target cells, the expression efficiencies were highest 48h after transfection (P <0.05).
引文
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