【作者】 陈禹保；

【导师】 刘少军；

【作者基本信息】 湖南师范大学 ， 发育生物学， 2011， 博士

【摘要】 异源四倍体鲫鲤(Allotetraploid crucian-carp,简称AT)是通过红鱼即(Carassius auratus red var.,(?))与鲤鱼(Cyprinus carpio L.,(?))远缘杂交首次在脊椎动物中人工培育的两性可育、遗传性状稳定的四倍体鱼。目前该四倍体鲫鲤品系目前已繁殖到第18代,形成了一个庞大的新型四倍体鲫鲤鱼群体,该群体的形成在鱼类演化和鱼类遗传育种具有重要意义。研究表明,四倍体鲫鲤与其二倍体父母本在基因型和表现型均发生了改变。因此,有必要从基因组和转录组水平对其生物学特征进行较为系统的研究,并深入分析功能基因的筛选、遗传标志的发现定位及经济形状与基因功能关联连锁确定。并可为鱼类多倍化形成机制提供新的信息,最终为鱼类分子设计育种提供一个技术服务指导性平台。本研究对F18异源四倍体鲫鲤新品系进行了BAC文库构建及鉴定,对F18四倍体鲫鲤、红鲫鱼和鲤鱼的肝脏转录组进行高通量测序研究、并对转录组序列进行生物信息学分析,获得的主要研究结果如下：1、首次构建新品系异源四倍体鲫鲤的BAC文库,该文库包含43200个克隆。构建BAC文库采用全血细胞包埋琼脂糖法制备HMWDNA,经HindⅢ限制性酶切,脉冲场电泳(PFGE)分离选择获得150-300kb的DNA片段,通过电洗脱和透析纯化HMW DNA,连接到线性磷酸化的克隆载体p CC1BAC上,转化到大肠杆菌中。对构建的BAC文库进行质量鉴定评估,插入DNA片段大小在110-310kb之间,平均插入片段大小为190kb左右,具有约2.6倍左右的基因组覆盖率。获得的BAC文库克隆,保存在450块96孔板中,存放在-80℃冰柜备用。所建立的BAC文库基本能满足功能基因筛选、基因定位、进化机制、数量形状连锁分析及转基因等研究的需要。2、异源四倍体鲫鲤、红鲫和鲤鱼的肝脏转录组测序共得到异源四倍体鲫鲤原始数据1.2GB,红鲫原始数据934MB,鲤鱼原始数据为1.1GB,用CLC BIO软件对F18异源四倍体鲫鲤转录组数据经基本处理后产生Unigene数为9554条。3、使用EDEGR对F18与红鲫拼接好的contigs进行基因差异表达分析(阈值设定为logFC>2),结果显示：F18与红鲫相比,在肝脏转录组中6192个contigs表达上调(logFC>2,P<05),有217个contigs表达下调(logFC>2,P<0.05)。F18与鲤鱼相比,在肝脏转录组中4867个contigs表达上调(logFC>2, P<0.05),有578个contigs表达下调(logFC>2, P<0.05).4、F18与红鲫和鲤鱼肝脏相比有差异contigs通过KEGG代谢途径分析其功能差异。发现上调的基因主要在碳水化合物代谢、氨基酸代谢、脂代谢和能量代谢作用途径中。GO分析表明,在分子生物学功能层次中binding和catalytic activity的差异基因占主要部分。在细胞组成水平层次中cytoplasm和organelle占主要部分。在生物过程层次中,metabolic process和regulation of biological process的差异表达基因最多。5、与红鲫的转录组及鲤鱼转录组进行SV (structure variation)分析,结果表明：在异源四倍体鲫鲤的Unigenes中有185条Unigenes与红鲫转录组的contig有基因融合,有192条Unigenes与鲤鱼转录组的contig有基因融合。有24条Unigenes包含有红鲫、鲤鱼的基因片段。6、对F18与红鲫和鲤鱼肝脏转录组进行SNP分析,首次发现：对比F18和红鲫,共发现有SNP位点46449个；而与鲤鱼相比其SNP位点共发现有59436个。相比较于红鲫,F18与父本鲤鱼的遗传变异位点更多,证明了F18偏母性遗传的特征。其中A型转铁蛋白transferrin variantA和MHC class I antigen的变异位点较多,提示这两个基因在F18中发生的变化较大,这可能对其抗逆能力的增加有作用。7、Transferrin variant A和MHC class I antigen的演化分析；从transferrin variant A构建的进化树可以看出,该基因F18更偏向红鲫,与红鲫聚为一支,而与鲤鱼相差较大；从MHC class I antigen构建的进化树可以看出,F18的MHC class I antigen从序列上看更偏向鲤鱼,而与红鲫差异较大。从分子水平说明多倍体化过程中,基因偏向性选择,每个基因面对被演化选择的压力。通过对异源四倍体鲫鲤BAC文库构建,为今后的分子育种研究及功能基因的筛选提供新的技术平台。另外利用高通量测序技术,对异源四倍体鲫鲤及其父母本肝脏转录组进行测序并运用生物信息学研究分析其结构及表达差异,并对其所测到的序列进行功能注释比较,为多倍体鱼演化研究和分子育种提供新的信息。更多还原

【Abstract】 A bisexual fertile and stable inheritabl the allotetraploid (AT) crucian-carp population was obtained originally by crossing red crucian carp(Carassius auratus red var., ,2n=100) with common carp (Cyprinus carpio L., S,2n=100). Up to date,18th generation of the allotetraploid crucian-carp was produced and became a new large allotetraploid folk. The formation of these folk has a great significance in understanding of fish evolution and genetic breeding. Studies indicated that there are lot of changes in genotype and phenotype between Allotetraploid crucian-carp and their diploid parents. Thus, it is essential to make deep systematic studies on its characterization in genome and transcriptome. For futher study of the Allotetraploid crucian-carp genomics, it is important to identify functional genes and biomarker relative to genetic traits and genetic mapping. It is benefical for us to establish a platform for molecular breeding.Here we constructed BAC (Bacterial artificial chromosome) library of Fig Allotetraploid crucian-carp, sequenced transcriptome of the allotetraploid crucian-carp liver, red crucian carp liver and common carp liver using 454 GS FLX,analyzed bioinformaticaly the data produced by 454 GS FLX. The major results were presented as following:1、For the first time we constructed a highly redundant genomic BAC library using high molecular weight(HMW) DNA from the allotetraploid crucian-carp produced in our fish development bioloy Lab by crossing red crucian carp(Carassius auratus red var.,9,2n=100) with common carp(Cyprinus carpio L.,,2n=100). the BAC library we constructed consists of 43200 clones in total. High quality genomic HMW DNA extracted from blood cells embedded in low-melting-point agarose plug was partially digested with Hind III,150-300kb fragments were isolated by pulsed field gel electrophoresis (PFGE), then 150-300kb fragment were purified with dialysis.ligated to linearized and phosphorylased vector pCClBAC, transformed into E.coli.the constructed BAC library with the insert fragments size about 110-310kb,the average size around 190kb as estimated by PFGE. The representation of the constructed BAC library is about 2.6 equivalents to the Fig Allotetraploid crucian-carp genome. All these BAC clones arepreserved in 450 pieces of 96-well plates in-80℃refrigerator. The se reusts show that the BAC library will be usefull for identifying functional genes, genetic mapping, sequencing of the Allotetraploid crucian-carp genome, evolutional molecular machnism and trangenetic study.2、The transcriptomic raw data of the Allotetraploid crucian-carp liver, red crucian carp liver and common carp liver produced by 454 GS FLX are respectively about 1.2Gb,934Mb and 1.1Gb. After basical analysis,9554 Unigene were obtained.3、By using EDEGR, differential expression was analysized between Fi8 and red crucian carp. The result showed that 6192 contigs were upregulated and 217 contigs were downgulated (cutoff:logFC> 2, P< 0.05). Compared with common carp,4867 contigs were upregulated and 578 contigs were downregulated (cutoff:logFC> 2, P<0.05)4、Based on KEGG pathway analysis, the differential expression genes were identified. The result showed that the upregulated genes in F18 were mainly in Carbohydrate Metabolism, Amino Acid Metabolism, Lipid Metabolism and Energy Metabolism. Go analysis indicated that in molecular function level differential genes are mainly in binding and catalytic activity. Cytoplasm and organelle are the most of celluar component, while metabolic process and regulation of biological process contribut the most part of biological process.5、F18 contigs were mapped with red crucian carp and common carp reads. The results showed that 185 Unigenes of F18 contains fragments of red crucian carp, while 192 Unigenes of F18 contains fragments of common carp.24 Unigenes contains fragments from both red crucian carp and common carp.6、Between F18 and red crucian carp,46449 SNP sites were found, while 59436 SNP sites were found between F18 and common carp. Compared with red crucian carp, more SNP sites observed in common carp. This result proved that partial maternal inheritance of F18 in genetic characteristics. Transferrin variant A and MHC class I antigen had the most SNP sites, which potentially due to its increased resilience.7、Evolution analyis of transferrin variant A and MHC class I antigen:Phylogenetic tree of transferrin variant A showed, the sequence of F18 was more similar to red crucian carp compared with common carp While, MHC class I antigen of F18 showed high similarity with, common carp. This result suggestted that in F18 was in inherit both genetic information from parents and different genes show different deflection. From molecular level to decipher ploidy evolution process, all the genes face selection pressure of evolutionFor further study of molecular breeding and identifying functional genes, F18 BAC library was constructed。In addition, using 454 high throughout sequence technology, F18 and its parents liver transcripton were sequenced and then analysized using bioimformatics tools. The structure and differential expression of genes were studied. With the genes functional annotation, we offered more information for understanding ploidy evolution of fishes and molecular breeding更多还原