【作者】 杨东；

【导师】 余来宁；

【作者基本信息】 华中农业大学 ， 水产养殖， 2006， 博士

【摘要】 尼罗罗非鱼（Oreochromis niloticus）已成为世界性的主要养殖对象之一，研究表明其遗传性别决定类型为XX／XY型。对不同遗传型尼罗罗非鱼的有丝分裂中期相染色体进行荧光原位杂交表明，在尼罗罗非鱼中，X与Y染色体之问存在差异。但是到现在为止，没有发现性别特异分子标记。遗传性别的快速鉴定问题是尼罗罗非鱼性别控制研究中的一个难点。本文从DNA水平对雌雄尼罗罗非鱼进行比较，研究其性别控制机理，筛选能区分雌雄的分子标记。主要研究结果如下：1．本文参照不同物种DM结构域设计了一对兼并引物，以尼罗罗非鱼精巢RNA为模板，通过RT-PCR方法，扩增出一条带，长约140bp。对扩增产物进行克隆，阳性克隆采用SSCP分析筛选不同基因片段，进一步对筛选的不同基因进行了DNA测序。结果获得2个不同的Dmrt基因，与GenBank联机，采用BLAST方法与人类相应DMRT基因进行序列比对，发现与人类相应DMRT基因的氨基酸序列一致性分别为：89.1％、100％，显示出Dmrt基因在系统进化上具有高度的保守性。根据尼罗罗非鱼（Oreochromis niloticus）的拉丁文名，按习惯将其命名为onDmrt1，onDmrt2。为了探讨Dmrt1和Dmrt2两个基因的功能，根据获得的序列，分别设计一对特异引物，采用RT-PCR技术，对尼罗罗非鱼不同组织Dmrt1和Dmrt2基因的表达进行了研究。结果发现Dmrt2基因在精巢，卵巢，脑，眼，鳃，心脏中都有表达，暗示Dmrt2基因的功能可能主要是参与器官发育，而不是性别决定过程。而Dmrt1只在精巢中特异表达，在其它组织中无表达，显示Dmrt1基因主要在性别决定及性状维持中有重要功能。根据扩增出的Dmrt1 DM-domain的序列，设计上游引物P5，P6，用3‘RACE（rapid amplification of cDNA ends）法从尼罗罗非鱼精巢中分离和测定了Dmrt1 cDNA的序列，3’-RACE扩增得到1108bp的片段，共编码262个氨基酸，奥利亚罗非鱼、虹鳟、新月鱼、青鲻等动物的Dmrt 1的氨基酸序列进行比较，同源性分别为：99％，62％，73％，41％。以Dmrt 1基因组序列为基础，采用Garnier-Robson方法、Chou-Fasman方法和Karplus-Schulz方法预测蛋白质的二级结构；按Kyte-Doolittle方案、Emini方案和Jameson-Wolf方案预测DMRT1蛋白的B细胞表位，为精确地研究DMRT1蛋白的结构和功能，从而揭示他们的性别调控机能提供参考。2．参照人SRY基因HMG-box保守区序列，设计一对兼并引物，采用PCR技术扩增了尼罗罗非鱼Sox基因，在雌雄尼罗罗非鱼个体中均扩增出五条带，大小分别约为200 bp，400 bp，600 bp，800 bp，1200bp，回收200bp左右扩增产物进行克隆，阳性克隆采用SSCP分析，筛选不同基因片段。进一步对筛选的不同基因进行DNA测序，结果获得了五个不同的228bp Sox基因，与GenBank联机，采用BLAST方法与人类相应SOX基因进行序列比对，发现其与人类相应SOX蛋白的氨基酸序列一致性分别为97.2％、97.2％、94.4％、96％、88.2％，显示出该基因在分子进化上具有高度的保守性。根据尼罗罗非鱼（Oreochromis niloticus）的拉丁文名，按习惯将其命名为onSox1a、onSox1b、onSox3、onSox4 and onSox12。采用RT—PCR技术，研究了尼罗罗非鱼精巢、卵巢、脑、眼、鳃和心脏等组织中Sox3基因的表达。结果显示，Sox3基因在精巢、卵巢、脑和眼中有不同程度表达，而在鳃和心脏中无表达，，证实Sox3基因在其中枢神经系统的发育，性腺发生和功能维持上有着重要功能。本研究为探索尼罗罗非鱼的性别决定机制和Sox基因表达模式提供了资料。3．采用RAPD技术对尼罗罗非鱼雌、雄各11尾个体共22个样本进行性别和遗传多样性检测。从80个寡聚核苷酸随机引物中筛选出17个扩增重复性好、条带清晰、特异性强的引物，对每个个体基因组DNA进行了扩增。得到RAPD产物的分子量在200-2000bp之间，产物总计127个位点，其中，多态位点45个（占35.43％）。计算个体间遗传相似系数平均为0.8648，个体间遗传距离平均为0.1352。用Shannon多样性指数量化的遗传多态度（H₀），雄性群体（0.1910）高于雌性群体（0.1797），平均遗传多态度（Hpop）为0.1854。尼罗罗非鱼遗传多态度所占的比例在群体内为13.63％，而雌、雄群体间为86.37％。在尼罗罗非鱼雌、雄个体RAPD产物中，电泳图谱上不能读出明显的雄性特征带。4．利用AFLP技术，应用49个引物组合，检测了尼罗罗非鱼雌雄基因组DNA的多态性，筛选与尼罗罗非鱼性别相关的分子标记。实验中共扩增出2694条带，筛选出候选差异DNA片段748条。挑选出20对引物进行雌雄两代个体进一步的分析。其中引物E4／M5组合产生1个大小约为150bp的片段，在所有两代雄性尼罗罗非鱼均出现这一片段，在F1代的15尾雌尼罗罗非鱼中有仅有1尾出现该片段，对F2代15尾雌尼罗罗非鱼也只有2出现，由此推断此条带可能与性别相连锁。对该特异扩增带经回收和测序，为了将AFLP标记转化为重复性和特异性更好的SCAR（Sequence Characterized Amplified Regions）标记，根据序列分析结果，分别合成了一对20碱基特异性引物，用该特异性引物进行PCR扩增，结果雄性有一条SCAR标记，而雌性没有。尼罗罗非鱼的SCAR分子标记能用于筛选全雄品系。更多还原

【Abstract】 The Nile tilapia（Oreochromis niloticus） is one of most important species in worldwide fish aquaculture. Research on the sex determination system in O.niloticus has provided evidence for genetic sex determination（GSD） with an XX female and XY male sex determination system. Analysis of the hybridization of probes drived from X and Y chromosomes to different genotype has demonstrated that the sequence differents exist between the sex chromosomes of O.niloticus. The inability to rapidly determine the genotypic sex of an individual is a problem in aquculture, where precocious maruration and subsequent reproduction can be a significant problem under certain conditions. The article tested male and female O. niloticus to search for molecular markers associated with sex chromosome. The major research results of this study are as follows:1. the Dmrt genes of O. niloticus were amplified by using a pair of degenerated primers which were designed based on the conservative DM domain of different species. The amplification band of Dmrt gene familay was-observed in testis of O. niloticus whose length was 140bp. Then, PCR products were cloned. To detect the different clones, SSCP technique was used. Two different Dmrt genes were obtained and sequenced respectively. After sequence analysis, those two Dmrt genes are named as onDmrt1, onDmrt2 according to their high homology to corresponding Human DMRT genes because their identities to those human DMRT genes in the amino acid sequence are 89.1％, 100％respectively; This may be concluded that Dmrt genes are highly conserved in phylogenetic. To study the function of Dmrt1 and Dmrt2 in development, we designed specific primers according the sequence of onDmrt1 and onDmrt2 for amplification by RT-PCR of different tissues in O. niloticus Dmrt2 expression is detectable in the brain, eye, gill, heart, testis and ovary. Dmrt1 expression is only detectable in the testis. No expression was detected for other tissues. This indicated that Dmrt2 genes may only play roles in the development of organs and Dmrt1 mainly participates in sex determining and differentiation. Based on DM-domain of in Dmrt1 of O. niloticus, primer P5 and P6 were designed. A rapid amplification of cDNA ends（RACE） was used for isolation of the length cDNA of Dmrt1 from testis of Oreochromis niloticus. We amplified a fragment about 1108bp by 3’RACE-PCR, encoding 262 amino acid. Sequence analysis revealed that the homology of Dmrt1 in O. niloticus and O. aurea is 99％. The secondary structure of Dmrt1 protein was predicted by the methods of Gamier-Robson, Chou-Fasman and Karplus-Schulz based on the amino acid sequence of Dmrt1. And Hydrophilicity plot, Surface probability plot and Antigenic index for Dmrt1 protein were obtained by the methods of Kyte-Doolittle, Emini and Jameson-Wolf, respectively. Theses results are helpful for studies on sex control mechanism of Dmrt1 in O. niloticus2. In the part of studies, Using a pair of degenerate primers based on the conservative region, HMG-box, of Human SRY gene, the Sox genes of O. niloticus were amplified. Five DNA bands with the length 200bp, 400bp, 600bp, 800bp, 1200bp were observed in the PCR products of both male and female O. niloticus. To detect the different clones, SSCP technic was used. Five different Sox genes were obtained and sequenced respectively. After sequence analysis, those Five Sox genes are named afer O. niloticus as onSox1a, onSox1b, onSox3, onSox4 and onSox12, according to their high homology to corresponding Human SOX genes because their identities to those human SOX genes in the amino acid sequence are 97.2％、97.2％、94.4％、96％、88.2％respectively, This may be concluded that Sox genes are highly conserved in phylogeny. The Sox3 gene expression analysis of different tissues from O. niloticus was studied by using RT-PCR. An expression was observed in testis in male, ovary in female, brain, eye in both male and female, no expression in gill, heart in both male and female. Based on these results, we suggest that Sox3 should play a key role in central nervous system development and gonad of O. niloticus. This research provided molecular data for the sex-determining mechanism of O. niloticus and expression pattern of Sox gene.3. RAPD analysis was applied to study sex and genetic diversity in O. niloticus. A total of 20 samples, which are 10 males and 10 females, were used in the test. Of 80 random oligonucleotide primers for the amplification of O. niloticus genomic DNA, 17 could produce reproducible, distinctive and characteristic bands from 200-2000 bp. 127 sites were detected, 45 of which（35.43％） were polymorphic. The average genetic similarity index and the average genetic distance were 0.8688 and 0.1312 respectively. Genetic diversity quantified by Shannon index（H₀） was 0.1910（male） and 0.1797 （female） respectively, with an average of 0.1854 Partition of genetic variation indicated that 13.63％was distributed within groups and 86.37％among male and female groups.. Difference bands can not be detected between male and female from the electrophoresis profiles. 4. In this paper genomic DNA polymorphism of female and male O. niloticus were detected using amplified fragment length polymorphism（AFLP） technique with 49 primer combinations. It produced a total of 2694 and an average of 55 bands. 20 primer combina tions were selected for further analysis. One AFLP band about 150bp related to the male was obtained with primer E4/M5, which was present in all males and absent in 14 of the 15 females of F₁ and 13 of the 15 F₂, it could have relations with sex.. The male associateed fragment about 150bp was cloned and sequenced. In order to convert the AFLP marker into SCAR（Sequence Characterized Amplified Regions） marker, one pair of 20-mer specific primer was constructed and used for PCR amplifing. The male-linked dominant SCAR marker was obtained.更多还原