半滑舌鳎（Cynoglossus semilaevis）雌鱼fosmid基因组文库的构建及初步分析
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摘要
半滑舌鳎(Cynoglossus semilaevis)是我国优质的大型经济鱼类,其养殖业发展迅速,但目前基因组研究较少,限制了许多研究工作的深入开展。本研究构建了第一个半滑舌鳎雌鱼fosmid基因组文库,对文库的部分克隆进行了双末端测序和序列分析,并从中筛选了部分微卫星多态性位点和一个性别相关序列。主要结果如下:
1、半滑舌鳎雌鱼fosmid基因组文库的构建和鉴定
本研究用流式细胞术测得半滑舌鳎雌性基因组大小约为606.36 Mb;从半滑舌鳎雌鱼肌肉提取基因组DNA,选取大小合适的DNA片段,经末端修复并回收后,再以fosmid质粒作为载体,构建了半滑舌鳎雌鱼基因组文库。该文库含有49,920个克隆,重组率为96.88%;插入片段长度分布在33～45kb之间,平均为39.2kb,覆盖率为雌性半滑舌鳎基因组的3.23倍,从文库中筛选得到单拷贝DNA序列的概率为95.6%;对培养第一天和第六天的文库克隆进行比较检测结果表明,文库稳定性高,培养过程中没有发生变化。半滑舌鳎fosmid基因组文库的构建,为该物种基因组的研究工作奠定了基础,为开展测序、分子标记发掘、功能基因筛选与定位、物理图谱构建等研究工作提供了有效的工具。
2、半滑舌鳎雌鱼fosmid末端序列分析
本研究从半滑舌鳎雌鱼fosmid文库中随机选取1,152个克隆进行双末端测序,去除了大肠杆菌和载体的污染后获得2,247条序列。序列总长1,921,341 bp,大约占半滑舌鳎雌鱼基因组的3.17‰,A+T含量为57.9%,G+C含量为42.1%。对这些序列进行BLAST比对,结果表明在2,247条序列中,有848条序列与数据库现有的序列有明显的相似性(E< e-5),占测序总数的37.74%。BLASTN比对有明显相似性的464条序列中,与nr数据库序列相似的有232条,其中54条与斑马鱼Danio rerio序列相似,36条与小鼠Mus musculus序列相似,17条与三刺鱼Gasterosteus aculeatus序列相似;与EST数据库序列相似的有232条,其中的32条序列与三刺鱼的EST相似,31条与青鳉Oryzias latipes EST相似。BLASTX比对有明显相似性的序列有384条,占序列总数的17.09%,其有144条序列与青黑斑河豚Tetraodon nigroviridis相似,106条与斑马鱼Danio rerio相似。
3、从半滑舌鳎雌鱼fosmid文库中筛选微卫星标记
本研究使用软件TANDEM REPEATS FINDER (version 4.00)对随机挑取的384个fosmid克隆的双向测序结果进行微卫星扫描,共发现了168个微卫星序列。所得微卫星序列使用PRIMER PREMIER version 5.0软件共设计得到了101对扩增引物,引物优化后进行PCR反应,并以12%非变性聚丙烯酰胺凝胶电泳检测扩增结果。在所有成功扩增的位点中,有64个位点检测到了多态性,其中有50个符合哈迪-温伯格平衡。不同的引物获得的等位基因数为2～9个不等,平均每对引物获得4.1个等位基因。期望杂合度(HE)为0.05～0.94,观测杂合度(HO)为0.06～1.00。
4、从半滑舌鳎雌鱼fosmid文库中筛选性别相关序列
本研究从fosmid文库的测序结果中随机挑取384个克隆的双向测序结果,除去测序失败的克隆后,使用PRIMER PREMIER version 5.0软件设计PCR扩增引物。设计结束后,从每个克隆的两对引物中选取预期PCR扩增结果较好的一对进行合成,共得到了374对PCR扩增引物。利用雌鱼DNA池和雄鱼DNA池为模板对设计得到的引物进行初步PCR扩增筛选,退火温度统一设定为50℃。对扩增出差异的引物和没有扩增出明显条带的引物,利用温度梯度PCR优化每对引物的最佳退火温度,并在最佳退火温度下再次用雌鱼DNA池和雄鱼DNA池进行PCR扩增筛选和电泳检测。对仍然扩增出雌雄差异的引物,使用12个个体的DNA在其最佳退火温度下进行PCR扩增,以排除个体差异的可能。最终经个体验证后获得了1对雌雄差异引物。该引物所对应的克隆号为B169-47,所扩增出的差异片段长度为291bp,Blast比对结果没有发现高度相似序列。性别相关序列的获得可为该物种分子性别的判定和全雌育种的研究提供有效工具,并且可以对性别相关基因和性别决定机制的研究提供帮助。
Half-smooth tongue sole (Cynoglossus semilaevis) is an important marine flatfish in north China and its aquaculture industry has been booming quickly in recent years. However, the research on its genome is underdeveloped compared with the study of its culture and breeding technology. Genomic studies require the availability of large-insert genomic libraries, such as cosmid, fosmid, bacterial artificial chromosome (BAC), and yeast artificial chromosome (YAC) libraries. The fosmid system developed in 1992 has some significant advantages. First, fosmid clones are easier to handle than BAC and YAC for their smaller insert size. Second, the insert fragments were prepared by random shearing without specific restriction endonuclease digestion and thus there is no potential bias of insert DNA. Third, the fosmid vector allows maintenance and storage as single copy for better stability of cloned inserts while the copy number also can be induced to 10～50 copies per cell to improve DNA yields for applications. In this thesis, the first female Half-smooth tongue sole fosmid library construction and initial Analysis has been presented.The major results are as follows:
1. Construction and characterization of female Half-smooth tongue sole fosmid library
A female half-smooth tongue sole fosmid library has been constructed in this study. The DNA content of female half-smooth tongue sole blood cell has been determined to be 0.620±0.044pg via the flow cytometry method and the chicken (Gallus domesticus) erythrocyte (1C = 1.25 pg) was selected as the internal reference standard. The genome size of female half-smooth tongue sole was then calculated to be about 606.36 Mb. The genomic DNA was prepared from different tissues using different methods and the ones prepared from muscle via the phenol-chloroform procedure showed the best fragment size and DNA purity. The prepared DNA was end-repaired and then the size selected fragments which lager than 36kb were recovered from low melting point gel using different agarose enzyme. The ones using GelaseTM appeared to have better result. The collected fragments were ligated into the fosmid vectors and then packaged to construct the library. The final fosmid library consists of 49,920 clones with an average insert size of 39.2kb kb, amounting to 3.23 genome equivalents. Insert fragments assay showed 3 of 96 clones selected randomly have no insert DNA and the frequency of recombinant clones was calculated to be 96.88%.The reason for lower recombinant frequency might be cell contamination. The insert DNA may have restriction enzymes sites which resulted in 8 of the random 96 clones showed two DNA fragments after NotI digestion. The probability of recovering any specific sequence of interest was 95.6% and the fosmid stability assays indicated that inserted DNA was stable during propagation. The fosmid library will serve as a useful resource for end sequences, physical mapping, and positional cloning of half-smooth tongue sole. It provides a base for further genetic analysis in this species and will be important for a better understanding of the genome.
2. Analysis of fosmid end sequences
In the present study, 1,152 individual clones have been sequenced, resulting in 2,247sequences with the total of 1,921,341 bp. These 2,247sequences represent approximately 3.17‰of the female Half-smooth tongue sole fosmid library and 1,383 sequences in them has been found to significantly hit to sequences in GenBank database using BLASTN and BLASTX (E < e-5). BLASTN searches on the nr databases, EST databases and BLASTX searches on the nr database resulted 232, 232 and 384 significant hits, respectively.
3. Isolation and characterization of microsatellite markers from female half-smooth tongue sole fosmid library
Three hundred and eighty-four fosmid clones were randomly selected to sequence (double strand reading), and 168 sequences in one hundred and forty-three clones were found to contain microsatellites. Among 101 primer pairs which were designed, 64 gave polymorphic PCR products. Based on characterization with 36 individuals, the number of alleles ranged from two to nine. The values of observed and expected heterozygosities varied from 0.06 to 1.00 and from 0.05 to 0.94, respectively. These markers have the potential for population structure evaluation, ecological analyses and linkage map construction. 4. Isolation of sex-related sequence from female half-smooth tongue sole fosmid library
The sequence results of 384 fosmid clones were radomly selected to design primer pairs with PRIMER PREMIER version 5.0 and 374 pairs were finally produced. Among theses 374 pairs, 1 pairs amplifyed sex-related sequence after a series of PCR screens by using female DNA pool and male DNA pool as templates. This primer pair came from fosmid clone B169-47.The lenth of sex specific sequence is 291bp.BLASTN searches on the nr databases, EST databases and BLASTX searches on the nr database had no significant hit of this sequence.
1、半滑舌鳎雌鱼fosmid基因组文库的构建和鉴定
本研究用流式细胞术测得半滑舌鳎雌性基因组大小约为606.36 Mb;从半滑舌鳎雌鱼肌肉提取基因组DNA,选取大小合适的DNA片段,经末端修复并回收后,再以fosmid质粒作为载体,构建了半滑舌鳎雌鱼基因组文库。该文库含有49,920个克隆,重组率为96.88%;插入片段长度分布在33～45kb之间,平均为39.2kb,覆盖率为雌性半滑舌鳎基因组的3.23倍,从文库中筛选得到单拷贝DNA序列的概率为95.6%;对培养第一天和第六天的文库克隆进行比较检测结果表明,文库稳定性高,培养过程中没有发生变化。半滑舌鳎fosmid基因组文库的构建,为该物种基因组的研究工作奠定了基础,为开展测序、分子标记发掘、功能基因筛选与定位、物理图谱构建等研究工作提供了有效的工具。
2、半滑舌鳎雌鱼fosmid末端序列分析
本研究从半滑舌鳎雌鱼fosmid文库中随机选取1,152个克隆进行双末端测序,去除了大肠杆菌和载体的污染后获得2,247条序列。序列总长1,921,341 bp,大约占半滑舌鳎雌鱼基因组的3.17‰,A+T含量为57.9%,G+C含量为42.1%。对这些序列进行BLAST比对,结果表明在2,247条序列中,有848条序列与数据库现有的序列有明显的相似性(E< e-5),占测序总数的37.74%。BLASTN比对有明显相似性的464条序列中,与nr数据库序列相似的有232条,其中54条与斑马鱼Danio rerio序列相似,36条与小鼠Mus musculus序列相似,17条与三刺鱼Gasterosteus aculeatus序列相似;与EST数据库序列相似的有232条,其中的32条序列与三刺鱼的EST相似,31条与青鳉Oryzias latipes EST相似。BLASTX比对有明显相似性的序列有384条,占序列总数的17.09%,其有144条序列与青黑斑河豚Tetraodon nigroviridis相似,106条与斑马鱼Danio rerio相似。
3、从半滑舌鳎雌鱼fosmid文库中筛选微卫星标记
本研究使用软件TANDEM REPEATS FINDER (version 4.00)对随机挑取的384个fosmid克隆的双向测序结果进行微卫星扫描,共发现了168个微卫星序列。所得微卫星序列使用PRIMER PREMIER version 5.0软件共设计得到了101对扩增引物,引物优化后进行PCR反应,并以12%非变性聚丙烯酰胺凝胶电泳检测扩增结果。在所有成功扩增的位点中,有64个位点检测到了多态性,其中有50个符合哈迪-温伯格平衡。不同的引物获得的等位基因数为2～9个不等,平均每对引物获得4.1个等位基因。期望杂合度(HE)为0.05～0.94,观测杂合度(HO)为0.06～1.00。
4、从半滑舌鳎雌鱼fosmid文库中筛选性别相关序列
本研究从fosmid文库的测序结果中随机挑取384个克隆的双向测序结果,除去测序失败的克隆后,使用PRIMER PREMIER version 5.0软件设计PCR扩增引物。设计结束后,从每个克隆的两对引物中选取预期PCR扩增结果较好的一对进行合成,共得到了374对PCR扩增引物。利用雌鱼DNA池和雄鱼DNA池为模板对设计得到的引物进行初步PCR扩增筛选,退火温度统一设定为50℃。对扩增出差异的引物和没有扩增出明显条带的引物,利用温度梯度PCR优化每对引物的最佳退火温度,并在最佳退火温度下再次用雌鱼DNA池和雄鱼DNA池进行PCR扩增筛选和电泳检测。对仍然扩增出雌雄差异的引物,使用12个个体的DNA在其最佳退火温度下进行PCR扩增,以排除个体差异的可能。最终经个体验证后获得了1对雌雄差异引物。该引物所对应的克隆号为B169-47,所扩增出的差异片段长度为291bp,Blast比对结果没有发现高度相似序列。性别相关序列的获得可为该物种分子性别的判定和全雌育种的研究提供有效工具,并且可以对性别相关基因和性别决定机制的研究提供帮助。
Half-smooth tongue sole (Cynoglossus semilaevis) is an important marine flatfish in north China and its aquaculture industry has been booming quickly in recent years. However, the research on its genome is underdeveloped compared with the study of its culture and breeding technology. Genomic studies require the availability of large-insert genomic libraries, such as cosmid, fosmid, bacterial artificial chromosome (BAC), and yeast artificial chromosome (YAC) libraries. The fosmid system developed in 1992 has some significant advantages. First, fosmid clones are easier to handle than BAC and YAC for their smaller insert size. Second, the insert fragments were prepared by random shearing without specific restriction endonuclease digestion and thus there is no potential bias of insert DNA. Third, the fosmid vector allows maintenance and storage as single copy for better stability of cloned inserts while the copy number also can be induced to 10～50 copies per cell to improve DNA yields for applications. In this thesis, the first female Half-smooth tongue sole fosmid library construction and initial Analysis has been presented.The major results are as follows:
1. Construction and characterization of female Half-smooth tongue sole fosmid library
A female half-smooth tongue sole fosmid library has been constructed in this study. The DNA content of female half-smooth tongue sole blood cell has been determined to be 0.620±0.044pg via the flow cytometry method and the chicken (Gallus domesticus) erythrocyte (1C = 1.25 pg) was selected as the internal reference standard. The genome size of female half-smooth tongue sole was then calculated to be about 606.36 Mb. The genomic DNA was prepared from different tissues using different methods and the ones prepared from muscle via the phenol-chloroform procedure showed the best fragment size and DNA purity. The prepared DNA was end-repaired and then the size selected fragments which lager than 36kb were recovered from low melting point gel using different agarose enzyme. The ones using GelaseTM appeared to have better result. The collected fragments were ligated into the fosmid vectors and then packaged to construct the library. The final fosmid library consists of 49,920 clones with an average insert size of 39.2kb kb, amounting to 3.23 genome equivalents. Insert fragments assay showed 3 of 96 clones selected randomly have no insert DNA and the frequency of recombinant clones was calculated to be 96.88%.The reason for lower recombinant frequency might be cell contamination. The insert DNA may have restriction enzymes sites which resulted in 8 of the random 96 clones showed two DNA fragments after NotI digestion. The probability of recovering any specific sequence of interest was 95.6% and the fosmid stability assays indicated that inserted DNA was stable during propagation. The fosmid library will serve as a useful resource for end sequences, physical mapping, and positional cloning of half-smooth tongue sole. It provides a base for further genetic analysis in this species and will be important for a better understanding of the genome.
2. Analysis of fosmid end sequences
In the present study, 1,152 individual clones have been sequenced, resulting in 2,247sequences with the total of 1,921,341 bp. These 2,247sequences represent approximately 3.17‰of the female Half-smooth tongue sole fosmid library and 1,383 sequences in them has been found to significantly hit to sequences in GenBank database using BLASTN and BLASTX (E < e-5). BLASTN searches on the nr databases, EST databases and BLASTX searches on the nr database resulted 232, 232 and 384 significant hits, respectively.
3. Isolation and characterization of microsatellite markers from female half-smooth tongue sole fosmid library
Three hundred and eighty-four fosmid clones were randomly selected to sequence (double strand reading), and 168 sequences in one hundred and forty-three clones were found to contain microsatellites. Among 101 primer pairs which were designed, 64 gave polymorphic PCR products. Based on characterization with 36 individuals, the number of alleles ranged from two to nine. The values of observed and expected heterozygosities varied from 0.06 to 1.00 and from 0.05 to 0.94, respectively. These markers have the potential for population structure evaluation, ecological analyses and linkage map construction. 4. Isolation of sex-related sequence from female half-smooth tongue sole fosmid library
The sequence results of 384 fosmid clones were radomly selected to design primer pairs with PRIMER PREMIER version 5.0 and 374 pairs were finally produced. Among theses 374 pairs, 1 pairs amplifyed sex-related sequence after a series of PCR screens by using female DNA pool and male DNA pool as templates. This primer pair came from fosmid clone B169-47.The lenth of sex specific sequence is 291bp.BLASTN searches on the nr databases, EST databases and BLASTX searches on the nr database had no significant hit of this sequence.
引文
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