中国美利奴细毛羊20号染色体MHC区段ClassⅡb延伸区域contig搭建
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摘要
旦的：绵羊是我国和世界上最有经济价值的家养动物之一。中国美利奴细毛羊更是我国优良的种质资源和经济物种。绵羊基因组主要组织相容性复合体(MHC),又叫绵羊白细胞抗原(Ovine Leukocyte Antigen, OLA),包含众多紧密连锁的免疫应答和调控基因,这些基因与动物抗病性以及疾病的易感性等密切相关。因此,对MHC区段的特征与功能研究至关重要。对该区段研究而中国美利奴细毛羊基因组BAC文库已经建好,并且MHC区段最小重叠度(Minimal Tiling Path, MTP)物理图谱已经搭建好,并完成测序以及对该区段进行了基因、重复序列以及microRNA等的预测,但是在MHCClassⅡa与MHC ClassⅡb之间因为染色体倒位存在一个大的gap。而这个gap所在区段的遗传连锁研究表明,是由于常染色体片段倒位产生,它将MHC ClassⅡ区域分割成两个不连续的区段ClassⅡa和ClassⅡb。2009年4月牛的全基因组序列公布,为绵羊基因组(特别是与牛相似的倒位区域)的研究提供了宝贵而丰富的资源。因此本研究的目的就是要根据牛的基因组序列和比较基因组学原理,利用PCR方法筛选中国美利奴细毛羊基因组BAC文库,最终利用有效阳性BAC克隆构建绵羊基因组MHC ClassⅡb延伸区域的MTP物理图谱,为绵羊基因组MHC区段倒位区域MTP物理图谱的一部分,进一步推动该倒位区域的研究,并推进整个绵羊基因组MHC区段完整的MTP物理图谱的构建,为今后的对绵羊的基础研究奠定基础。
方法：首先,通过比较基因组学方法,根据已公布的牛(Bos Taurus)的基因组序列,选择位于BoLA ClassⅡb延伸区域的一些有效Markers/Symbols,并据此设计引物。然后,利用PCR方法从已有的中国美利奴细毛羊基因组BAC文库中筛选出候选的阳性BAC克隆并进行限制性酶切指纹图谱分析(Fingerprinting Analysis),搭建出初步的克隆重叠群(contig);然后,对阳性BAC克隆进行末端测序,并据此设计BAC末端引物,对克隆间的重叠关系进行PCR确证,最终绘制出中国美利奴细毛羊OLA ClassⅡb延伸区的MTP物理图谱。
结果：(1)根据牛的基因组图谱,设计了26对用于文库筛选的候选PCR引物,最终条件优化好的可用于PCR筛选的引物为23对；(2)制备了已有基因组BAC文库的一(初)级384孔板混合池和二(次)级行混合池；(3)通过PCR方法筛选出58个阳性BAC克隆,并进行了限制性酶切指纹图谱分析和克隆间重叠关系的PCR确证,最终选择25个有效BAC克隆绘制出了OLA ClassⅡb延伸区的MTP物理图谱。
结论：(1)利用牛的BoLA ClassⅡb延伸区的一些Markers/Symbols,通过PCR方法能筛选出阳性BAC克隆,不仅说明绵羊与牛在该区域的基因组序列同源性很高,而且证明运用PCR方法进行基因组BAC文库筛选是快速而行之有效的。(2)在本研究中有3对根据牛的基因组序列设计的引物没有从绵羊基因组中扩增出PCR产物,此外,在多处区域发现了两个或三个不同的Markers筛选出同一个BAC克隆的现象。此结果暗示,绵羊与牛之间在以后的平行进化过程中,该区域的局部基因组序列也许发生了丢失。(3)绵羊基因组该区域MTP物理图谱的构建,推进了绵羊MHC区段到位区域MTP物理图谱的构建,不仅充实了绵羊基因组信息,而且可以大大地促进整个绵羊基因组测序和反刍动物基因组比较研究。
Object:The sheep is one of the best economic valued domestic animals in the world and China. Especially the Chinese fine-wool Merino sheep is an excellent genetic germplasma resource and economic breed. Same to other all jawed vertebrates, the major histocompatibility complex (MHC) of ovine genome, also designated as Ovine Leukocyte Antigen (OLA), harbors clusters of tightly linked immune response/regula-tion genes involved in overall resistance/susceptibility of an animal to various pathogenic viruses, bacteria and parasites. So it is very important to research the characters and functions of MHC. A BAC Library of Chinese Fine-Wool Merino Sheep was constructed, and the MTP pysical map of The Ovine MHC Region had been done, but there is a big gap between MHC ClassⅡa and ClassⅡb. Genetic linked studies shows that OLA is similar to BoLA in structure in that the classⅡis split into two discontinuous subregions,'Ⅱa' and'Ⅱb',separated by a large piece of non-MHC autosomal inversion. The aim of this study is to screen the genomic BAC library previously constructed of Chinese fine-wool Merino sheep using PCR strategy based on bovine genomic sequence and comparatively genomics theory, ultimately assemble BAC clone-based MTP physical map of OLA ClassⅡb extended region utilizing effective positive BAC clones selected. The MTP physical map OLA ClassⅡb extended region is a part of OLA inversion region MTP physical map, improving the research on inversion region and improving the construction of the whole OLA MTP physical map.
Methods:Firstly, we selected some effective Markers/Symbols in BoLA ClassⅡb extended region based on bovine genomic sequence published and designed corresponding primer pairs for PCR. Then we screened the genomic BAC library previously constructed of Chinese fine-wool Merino sheep using PCR strategy and selected some candidated positive BAC clones. Primary contig was constructed by analyzing enzymic fingerprints of candidated positive BAC clones. Subsequently, we sequenced the ends of effective BAC clones among primary contig and designed corresponding primers-end for PCR in order to determine the overlapping relationships and orders of these clones. Finally, we constructed the MTP physical map of OLA ClassⅡb extended region.
Results:(1) A total of 26 pairs of PCR primers weie designed and optimized 23 pairs of primers were used for PCR screening. (2) BAC DNA mixture pools (primary 384-well plate pool and secondary row pool) of genomic BAC library previously constructed were prepared for screening. (3)A total of 58 positive BAC clones were selected utilizing PCR strategy. The overlapping relationships and orders of the positive BAC clones were determined by BAC DNA fingerprinting analysis, BAC-end sequencing and sequence-specific PCR. A total of 25 effective BAC clones were selected to cover the OLA ClassⅡb extended region. Therefore, a complete BAC-based MTP physical map of the OLA ClassⅡb extended region was constructed.
Conclusions:(1) we designed corresponding primer pairs for PCR screening through Markers/Symbols of BoLA ClassⅡb extended region. Then we screened the BAC DNA mixture pools of genomic BAC library previously constructed and selected positive BAC clones. The results show that this region of OLA has somewhat greater sequence identity to that of BoLA and it is rapid and effective to screen genomic BAC library utilizing PCR strategy. (2) For three primer pairs designed based on bovine genomic sequence published, the PCR product was not detected when PCR amplification was performed with ovine genome DNA as template. Moreover, an identical BAC clone was selected with two or three distinct markers as probes in several subregions. The results give a hint that local sequence in OLA ClassⅡb extended region may arise from parallel divergence subsequently. (3) The MTP physical map constructed in this study is not only enriched the information of ovine genome, will also facilitate strongly the sequencing of ovine entire genome and comparative studies of genome in ruminants.
方法：首先,通过比较基因组学方法,根据已公布的牛(Bos Taurus)的基因组序列,选择位于BoLA ClassⅡb延伸区域的一些有效Markers/Symbols,并据此设计引物。然后,利用PCR方法从已有的中国美利奴细毛羊基因组BAC文库中筛选出候选的阳性BAC克隆并进行限制性酶切指纹图谱分析(Fingerprinting Analysis),搭建出初步的克隆重叠群(contig);然后,对阳性BAC克隆进行末端测序,并据此设计BAC末端引物,对克隆间的重叠关系进行PCR确证,最终绘制出中国美利奴细毛羊OLA ClassⅡb延伸区的MTP物理图谱。
结果：(1)根据牛的基因组图谱,设计了26对用于文库筛选的候选PCR引物,最终条件优化好的可用于PCR筛选的引物为23对；(2)制备了已有基因组BAC文库的一(初)级384孔板混合池和二(次)级行混合池；(3)通过PCR方法筛选出58个阳性BAC克隆,并进行了限制性酶切指纹图谱分析和克隆间重叠关系的PCR确证,最终选择25个有效BAC克隆绘制出了OLA ClassⅡb延伸区的MTP物理图谱。
结论：(1)利用牛的BoLA ClassⅡb延伸区的一些Markers/Symbols,通过PCR方法能筛选出阳性BAC克隆,不仅说明绵羊与牛在该区域的基因组序列同源性很高,而且证明运用PCR方法进行基因组BAC文库筛选是快速而行之有效的。(2)在本研究中有3对根据牛的基因组序列设计的引物没有从绵羊基因组中扩增出PCR产物,此外,在多处区域发现了两个或三个不同的Markers筛选出同一个BAC克隆的现象。此结果暗示,绵羊与牛之间在以后的平行进化过程中,该区域的局部基因组序列也许发生了丢失。(3)绵羊基因组该区域MTP物理图谱的构建,推进了绵羊MHC区段到位区域MTP物理图谱的构建,不仅充实了绵羊基因组信息,而且可以大大地促进整个绵羊基因组测序和反刍动物基因组比较研究。
Object:The sheep is one of the best economic valued domestic animals in the world and China. Especially the Chinese fine-wool Merino sheep is an excellent genetic germplasma resource and economic breed. Same to other all jawed vertebrates, the major histocompatibility complex (MHC) of ovine genome, also designated as Ovine Leukocyte Antigen (OLA), harbors clusters of tightly linked immune response/regula-tion genes involved in overall resistance/susceptibility of an animal to various pathogenic viruses, bacteria and parasites. So it is very important to research the characters and functions of MHC. A BAC Library of Chinese Fine-Wool Merino Sheep was constructed, and the MTP pysical map of The Ovine MHC Region had been done, but there is a big gap between MHC ClassⅡa and ClassⅡb. Genetic linked studies shows that OLA is similar to BoLA in structure in that the classⅡis split into two discontinuous subregions,'Ⅱa' and'Ⅱb',separated by a large piece of non-MHC autosomal inversion. The aim of this study is to screen the genomic BAC library previously constructed of Chinese fine-wool Merino sheep using PCR strategy based on bovine genomic sequence and comparatively genomics theory, ultimately assemble BAC clone-based MTP physical map of OLA ClassⅡb extended region utilizing effective positive BAC clones selected. The MTP physical map OLA ClassⅡb extended region is a part of OLA inversion region MTP physical map, improving the research on inversion region and improving the construction of the whole OLA MTP physical map.
Methods:Firstly, we selected some effective Markers/Symbols in BoLA ClassⅡb extended region based on bovine genomic sequence published and designed corresponding primer pairs for PCR. Then we screened the genomic BAC library previously constructed of Chinese fine-wool Merino sheep using PCR strategy and selected some candidated positive BAC clones. Primary contig was constructed by analyzing enzymic fingerprints of candidated positive BAC clones. Subsequently, we sequenced the ends of effective BAC clones among primary contig and designed corresponding primers-end for PCR in order to determine the overlapping relationships and orders of these clones. Finally, we constructed the MTP physical map of OLA ClassⅡb extended region.
Results:(1) A total of 26 pairs of PCR primers weie designed and optimized 23 pairs of primers were used for PCR screening. (2) BAC DNA mixture pools (primary 384-well plate pool and secondary row pool) of genomic BAC library previously constructed were prepared for screening. (3)A total of 58 positive BAC clones were selected utilizing PCR strategy. The overlapping relationships and orders of the positive BAC clones were determined by BAC DNA fingerprinting analysis, BAC-end sequencing and sequence-specific PCR. A total of 25 effective BAC clones were selected to cover the OLA ClassⅡb extended region. Therefore, a complete BAC-based MTP physical map of the OLA ClassⅡb extended region was constructed.
Conclusions:(1) we designed corresponding primer pairs for PCR screening through Markers/Symbols of BoLA ClassⅡb extended region. Then we screened the BAC DNA mixture pools of genomic BAC library previously constructed and selected positive BAC clones. The results show that this region of OLA has somewhat greater sequence identity to that of BoLA and it is rapid and effective to screen genomic BAC library utilizing PCR strategy. (2) For three primer pairs designed based on bovine genomic sequence published, the PCR product was not detected when PCR amplification was performed with ovine genome DNA as template. Moreover, an identical BAC clone was selected with two or three distinct markers as probes in several subregions. The results give a hint that local sequence in OLA ClassⅡb extended region may arise from parallel divergence subsequently. (3) The MTP physical map constructed in this study is not only enriched the information of ovine genome, will also facilitate strongly the sequencing of ovine entire genome and comparative studies of genome in ruminants.
引文
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[2]郭健,杨博辉,赵有璋,等.我国肉用绵羊新品种的培育及分子辅助育种技术应用[J].家畜生态学报,2007,28(6)：116-121.
[3]梁小军,李颖康,郑丽霞.我国引入的国外肉用种羊及其应用效果(上)[J].中国草食动物,2003,23(6)：47-49.
[4]梁小军,李颖康,郑丽霞.我国引入的国外肉用种羊及其应用效果(续完)[J].中国草食动物,2004,24(2)：58-59.
[5]梁小军.我国引入的国外肉用种羊及其应用效果(中)[J].中国草食动物,2004,24(1)：57-58.
[6]何其宏,魏荣安,马春萍,等.中国美利奴多胎肉用细毛羊羊毛品质分析[J].中国草食动物,2004,24(5)：52-53.
[7]王金富,邓湘泉,等.中国美利奴军垦型细毛羊繁殖周期中NAG活力与发情排卵的关系[J].中国草食动物,2001,3(3)：6-8.
[8]储明星.Booroola羊Fec(?)B基因图谱研究进展[J].中国草食动物,2001,3(2)：43-46.
[9]Borland R J. The chromosomes of domestic sheep[J]. Hereditas,1964(55):61-64.
[10]Ansari H A, Maher D W, Pearce P D, et al. Resolving ambiguities in the karyotype of domestic sheep (Ovis aries). Ⅱ. G-, Q-, and R-banded idiograms, and chromosome-specific molecular markers [J]. Chromosoma.1996,105(1):62-67.
[11]Crawford A M, Dodds K G, Ede A J, et al. An autosomal genetic linkage map of the sheep genome[J]. Genetics.1995,140(2):703-724.
[12]de Gortari M J, Freking B A, Cuthbertson R P, et al. A second-generation linkage map of the sheep genome[J]. Mamm Genome.1998,9(3):204-209.
[13]Maddox J F, Davies K P, Crawford A M, et al. An enhanced linkage map of the sheep genome comprising more than 1000 loci[J]. Genome Res.2001,11(7):1275-1289.
[14]潘爱銮.生物技术与绵山羊育种[J].四川草原,1999(3)：46-49.
[15]Cockett N E. Current status of the ovine genome map[J]. Cytogenet Genome Res.2003, 102(1-4):76-78.
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