【作者】 王旭波；

【导师】 张全启；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2008， 博士

【摘要】 本研究对半滑舌鳎(Cynoglossus semilaevis)雌鱼进行了分子细胞遗传学分析,主要包括:半滑舌鳎雌鱼基因组结构特征分析、fosmid文库克隆的染色体荧光原位杂交分析、半滑舌鳎W染色体的激光显微切割及扩增、W染色体文库的构建及分析、半滑舌鳎雌鱼特异片段的获得及分析等。同时,要实现半滑舌鳎的全雌育种,有两个技术问题需要解决:(1)、如何区分ZZ和ZW,即如何鉴别雌鱼和雄鱼,从而筛选出性逆转的伪雄鱼;(2)、如何区分ZW和WW,即如何筛选出超雌鱼。本研究建立了双引物PCR法并获得了雌鱼特异性FISH探针,分别解决了这两个难题。主要结果如下:1、半滑舌鳎雌鱼基因组结构特征分析本研究利用流式细胞术对半滑舌鳎、褐牙鲆(Paralichthys olivaceus)、石鲽(Kareius bicoloratus)和圆斑星鲽(Verasper variegates)的基因组大小进行了测定并进行了比较,检测结果为:半滑舌鳎雄鱼基因组大小为586.80 Mb,雌鱼基因组大小为606.36 Mb。同时我们发现无法利用流式细胞仪通过检测红血细胞的DNA含量的方法来鉴别半滑舌鳎的雄鱼和雌鱼。随机挑取1,152个半滑舌鳎雌鱼fosmid文库克隆进行双末端测序,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‰。对这些序列进行如下分析:首先利用Tandem Repeats Finder (TRF)软件进行串联重复序列的查找,结果共得到889条重复序列,其中微卫星序列303条,小卫星序列586条,分别占串联重复序列总数的34.09%和65.91%,没有找到卫星序列。从长度上看,所有串联重复序列总长105,307 bp,其中微卫星序列19,363 bp,小卫星序列85,944 bp,分别占串联重复序列总长的18.39%和81.61%,占测序总长的1.01%和4.47%。其次,通过在线服务器RepeatMasker查找,共获得38条散布重复序列,总长4,983 bp,占测序总长的0.26%。查找到的散布重复序列共有4种类型:DNA转座子、LTR反转座子、LINE反转座子和SINE反转座子。与散布重复的其它类型相比,DNA转座子在数目和长度上均占有绝对优势。2、半滑舌鳎雌鱼fosmid克隆的染色体定位分析根据对fosmid克隆两端序列的分析及BLAST比对结果,我们选择了8个克隆进行FISH定位。其中5个克隆含有单拷贝基因,有4个克隆获得了稳定又清晰的单一信号,另外1个克隆的信号出现在了几乎所有的染色体上。余下的3个克隆均含有rRNA基因序列,我们尝试对它们进行染色体定位,结果有2个克隆在W染色体和3对常染色体上出现阳性信号,而另1个克隆只在1对常染色体上出现杂交信号。以上FISH杂交结果将有利于半滑舌鳎细胞遗传学研究,比如基因的染色体定位,性染色体的进化,染色体重排等工作的开展。3、半滑舌鳎雌鱼W染色体的显微分离及W染色体文库的构建和分析利用Leica LMD激光显微切割系统对半滑舌鳎W染色体的显微切割进行了探索,成功地将W染色体显微分离,进行了DOP-PCR扩增,并将PCR扩增产物定位在了W染色体和Z染色体上。同时利用W染色体的扩增产物构建了半滑舌鳎的W染色体文库,共获得了596个克隆。从中选取288个克隆测序,得到了259条序列。对这些序列进行了串联重复、散布重复及BLAST比对分析,结果发现共有52条串联重复序列,2条散布重复序列。其中串联重复序列中有12条微卫星序列,40条小卫星序列;而2条散布重复序列全部都是DNA转座子类型。值得一提的是,我们发现有9个克隆均含有一段几乎完全相同的近540 bp的序列,从而推断半滑舌鳎的性染色体可能含有大量的多拷贝重复序列。4、半滑舌鳎性别相关片段的获得及分析对W染色体文库克隆的序列进行引物设计,利用5条雌鱼和5条雄鱼的DNA为模板进行PCR筛选,共获得2对半滑舌鳎雌鱼的特异性引物,加上1对阳性对照引物,我们建立了双引物PCR法,该方法可以非常准确迅速地区分半滑舌鳎的雌鱼和雄鱼。同时利用这2对特异性引物对半滑舌鳎雌鱼fosmid文库的超级池和二级池进行筛选,最终获得了2个雌鱼特有的fosmid克隆。通过FISH定位分析,这2个克隆均定位在了W染色体的中部且序列比对无相似性。利用这2个克隆制成的雌鱼特异性FISH探针进行荧光原位杂交,将有助于我们筛选出半滑舌鳎WW超雌鱼。该成果将有助于推动半滑舌鳎全雌育种的研究实施及推广进程,从而极大地提高半滑舌鳎的养殖效益。更多还原

【Abstract】 In this study, we did some researches about molecular cytogenetic analysis in female half-smooth tongue sole (Cynoglossus semilaevis), including the character analysis of genome structure in female C. semilaevis, fluorescent in situ hybridization (FISH) on chromosomes with the clones of female C. semilaevis fosmid library, laser microdissection and amplification of chromosome W from C. semilaevis, construction and analysis of chromosome W library, and analysis of female specific fosmid clones. Moreover, to actualize the all female production of C. semilaevis, two technical problems should be solved: one is how to identify ZZ and ZW, viz. how to distinguish the sex reversion ZW female ones; the other is how to identify ZW and WW, viz. how to screen out the WW female ones. In this study, we developed a double primer PCR method and female-specific FISH probes which could solve these two questions, respectively. The major results are as follows:1. Analysis of female half-smooth tongue sole genomeGenome sizes of C. semilaevis, Paralichthys olivaceus, Kareius bicoloratus and Verasper variegates were estimated via flow cytometry method and compared. The male and female C. semilaevis genome sizes were estimated to be 586.80 Mb and 606.36 Mb, respectively. Additionally, we found that we could not distinguish the male and female C. semilaevis fishes via flow cytometry method.1,152 individual clones were sequenced, resulting in 2,247 sequences. A total of 1,921,341 bp of genomic sequences was generated, representing approximately 3.17‰of the female C. semilaevis genome. Using Tandem Repeats Finder (TRF) software to analyze the sequences, a total of 889 tandem repeats were found, including 303 microsatellites, 586 minisatellites, accounting for 34.09% and 65.91% of all tandem repeats, respectively. No satellites were found. The total length of tandem repeats was 105,307 bp, in which the length of microsatellites and minisatellites was 19,363 bp and 85,944 bp, accounting for 18.39% and 81.61% of the total length of tandem repeats, and 1.01% and 4.47% of the total length, respectively.RepeatMasker analysis showed that a total of 38 interspersed repetitive sequences were found, which account for 0.26% of the total length. There are 4 types in these sequences: DNA transposons, LTR retrotransposons, LINE retrotransposons and SINE retrotransposons. Among them, the type of DNA transposons is the most frequent and length abundant.2. Chromosome assignment of fosmid clones in C. semilaevisBased on the analysis of end sequences, we selected 8 clones for FISH. Of the 5 clones which were likely to be low or single copy sequences, 4 clones represent single locus and the other one showed signals on almost all chromosomes. Hybridization with 3 clones contained rRNA gene sequences, 2 showed signals on chromosome W and other 3 couples of euchromosomes and 1 showed signals only on one couple of euchromosomes. These results will undoubtedly be useful for many aspects of cytogenetic research in C. semilaevis including chromosomal assignment of genes, sex chromosome evolutions, and chromosomal rearrangements.3. Laser microdissection, amplification and chromosome library construction of C. semilaevis chromosome WLaser microdissection was performed on the chromosome W of female C. semilaevis using Leica LMD system. The isolated chromosomes were successfully amplified and reverse painted on the sex chromosomes of C. semilaevis. Chromosome W library was constructed and 288 clones were sequenced and analyzed for tandem repeats, interspersed repetitive sequences and BLAST. As a result, we found 52 tandem repeats containing 12 microsatellites and 40 minisatellites, and only 2 interspersed repetitive sequences. Additionally, we found that 9 clones contained almost the same 540 bp fragments, which indicated that the sex chromosomes of C. semilaevis might be enriched for repetitive DNA elements and high or moderate in copy number.4. The obtainment and analysis of sex-specific fragment in C. semilaevisPrimers were designed from chromosome W library sequences and 2 female-specific primers were selected using 5 female and 5 male individuals as PCR templates. After appended another positive primer, we developed a double primer PCR method which could distinguish the male and female C. semilaevis fishes truly and rapidly. After library screening from fosmid library, we got 2 female-specific fosmid clones. These clones were both mapped on the middle region of chromosome W without any sequence comparability. The female-specific FISH probes prepared by these two clones will help us screen out the WW female ones. These results would be great promotion for the all-female production and the culture benefits of C. semilaevis.更多还原