【作者】 陈建军；

【导师】 常重杰；

【作者基本信息】 河南师范大学 ， 动物学， 2011， 博士

【摘要】 本研究以黄河鲤为实验材料,选择220条10bp的随机引物、10对SSR引物及100条ISSR引物,利用RAPD-PCR、SSR-PCR及ISSR-PCR三种分子标记技术对黄河鲤基因组进行扫描,试图发现一些与黄河鲤性别相关的连锁标记。SSR结果显示,虽然在对其DNA池进行扫描的过程中,在1对引物的扩增谱带上发现了1条可能与性别连锁的疑似条带,但当利用这对引物在更多的个体中验证时,没有发现其性别特异性;而ISSR结果表明,在所有筛选出来的10条引物的扩增谱带上,未发现与性别连锁的疑似条带。利用RAPD技术对黄河鲤雌、雄基因池进行扫描的结果表明,其中的5条随机引物的扩增谱带中有疑似性别特异条带的存在,利用这5条随机引物在个体中进行反复验证,只有随机引物S2107的扩增产物中存在一条稳定的雄性特异条带。经回收、克隆、测序后获得了一条大小为909bp的候选雄性特异条带,命名为Ccmf1。BLAST检索发现该片段与位于斑马鱼连锁群1的一段重复序列(DKEY-24 H 22)有一定的相似性。此外,本研究还利用抑制性消减杂交技术,成功构建了黄河鲤鱼基因组DNA的消减文库,随机挑选150个克隆经巢式引物PCR验证后,获得88个阳性克隆,以正、反向二次PCR产物为探针,与88个阳性克隆质粒DNA进行Southern dot blotting杂交,从中挑选了22个候选性别特异阳性克隆进行序列测定,经特异引物的PCR验证后,获得了两个长度分别为387bp和183bp雄性特异的DNA片段,BLASTN和BLASTX检索未发现与其同源的核苷酸和氨基酸序列,将其命名为Ccmf2和Ccmf3。根据3个雄性特异片段的序列分别设计特异引物,以管家基因GAPDH为内部对照,雌雄个体基因组为模板进行特异PCR扩增。结果表明,在所有的雄性个体中均扩增出了稳定的目的特异性条带,而雌性个体中没有该条带的出现。由于分离到的三个片段都与雄性性别连锁,所以推测黄河鲤的染色体类型应该为XX/XY型。为了进一步了解三个片段的性别特异性,利用特异PCR技术对100个黄河鲤的雌、雄基因组进行了验证,结果发现,Ccmf1和Ccmf2的性别特异性分别为99%和98%;Ccmf3的性别特异性达到了100%,故3个雄性特异片段在两染色体(性染色体和常染色体)间的重组值都很低,可以用于黄河鲤的遗传性别的鉴别。分别以Ccmf1、Ccmf2和Ccmf3为探针,与鲤鱼的其他三个品种:野生黄河鲤、建鲤以及丰鲤的基因组进行斑点印迹杂交(雌、雄各5尾),以期判断这3个性别特异片段的分子性别有效性(即雌雄鉴别的效率)。结果发现,在雌性个体中,Ccmf1、Ccmf2和Ccmf3的分子性别有效性分别为67%,80%和93%;而在雄性个体中,其分子性别有效性分别为67%,100%,和73%。充分说明在鲤鱼不同种间性染色体的分子类型的多样性。3个雄性连锁标记的发现与鉴定,能够作为鉴别黄河鲤遗传性别以及识别该鱼种性染色体的分子切入点,从而为探讨该物种性别决定及分化机制提供依据。为了更加全面的了解黄河鲤性腺发育机制,本研究还从mRNA水平上,分别以精巢与卵巢互为验证方,成功构建了黄河鲤成熟期雌、雄性腺的双向差减文库,经菌液PCR对文库进行初步筛选,分别获得了280个(来自雄性文库)和240个(来自雌性文库)含有插入片段的阳性克隆;然后利用Southern dot bltting对随机挑选的180个克隆(雌、雄各90个)在正、反杂交膜上进行了鉴定,从中找到了64个(其中来自雄性文库21个,雌性文库43个)候选差异表达克隆,经序列测定后,通过BLASTN和BLASTX对这些候选差异基因片段进行功能分类,得到了50个ESTS序列。其中雄性文库的18个差异ESTS中,包括14个已知功能的和4个未知功能的基因片段。按照其功能,14个ESTS分属于结构蛋白基因、蛋白质翻译机制相关基因、离子通道和蛋白转运基因、肿瘤相关和肿瘤抑制因子及其他基因等;而来自雌性文库的32个差异ESTS代表了27个已知功能基因和5个未知功能的基因,在已知功能基因中,出现频率较高的是来自呼吸链上的相关基因片段,以及与蛋白合成有关的基因片段;除一些未知功能蛋白外,其他的则出现了1次。参照斑马鱼及其他脊椎动物基因功能分析,雌性文库中已知功能EST分属调节因子、结构蛋白、酶类、转录因子、信号调控等。根据部分同源基因在黄河鲤精、卵巢中的半定量表达情况,在雄性差异文库中找到10个差异表达基因片段:5个为精巢特异表达基因;5个为精巢高效表达基因;其中包括2个首次发现的未知功能基因。在雌性差异文库中找到8个差异表达基因片段:2个为卵巢特异表达基因;6个为卵巢高效表达基因,其中包括1个未知功能基因。根据各基因片段的性质及编码功能,选择了可能参与性腺发育的3个基因片段,利用RACE技术对其全长进行了扩增,分别获得了大小为2173bp、1763bp以及884bp的全长基因,他们分别是Hmwt1a基因、HmSetd6基因以及HmPsmb2基因。最后,通过在成体不同组织的半定量和荧光定量表达模式分析,发现这3个基因均为性腺差异表达基因,其中Hmwt1a基因主要在精巢中表达,HmSetd6基因则主要在卵巢中表达,且表达量远远高于精巢,而HmPsmb2基因则是卵巢特异表达基因;通过与脊椎动物性腺发育相关基因功能的对比,推测Hmwt1a基因可能是黄河鲤雄性性别发育的重要调控基因;Hmsetd6基因则作为表观调控基因参与了黄河鲤雌性性状的发育;而HmPsmb2基因是在生理上或行为上维持黄河鲤雌性性别的性别偏向基因;这些基因的发现及相关性腺差异表达EST文库的构建,将对了解黄河鲤性腺发育及性别分化的分子机制奠定基础,同时为黄河鲤性别控制机制的研究提供丰富的基因鉴定资源。更多还原

【Abstract】 In this study, we identified a sex-specific DNA marker using randomly amplified polymorphic DNA (RAPD) fingerprinting, simple sequence repeat(SSR) and inter-simple sequence repeat (ISSR) in Yellow River carps (C. carpio from the Yellow River). Two hundred twenty random primers for RAPD fingerprinting, ten primes for SSR and one hundred primes for ISSR were used in pooled DNA samples and individual DNA samples from male and female fish. The results of SSR-PCR showed one candidate band which were different between male and female pools, however, no sex-specific band was found when was tested repeatedly in male and female individuals by dot boltting hybridization. The results of ISSR-PCR showed as SSR, no sex-linked suspected bands were found among all the amplification bands with 10 primers selected.In RAPD-PCR, Five candidate bands, which were different between male and female, were tested five times repeatedly in male and female individuals to verify their stability. Ultimately we found that one of the primers, S2107 (CACCATCGTG), produced a stable sex-specific band in the DNA fingerprints of all males tested, but not females. DNA sequencing revealed that this 909 bp long DNA fragment has a low similarity to a repetitive sequence in zebrafish, which was named as Ccmf1. The BLAST search revealed that the sequence of the sex-specific fragment in Yellow River C.carpio had a low similarity to a repetitive sequence (DKEY-24 H 22) located in linkage group 1 of zebrafish.Furthermore, a sex subtractive genomic DNA library was successfully constructed using suppression subtractive hybridization (SSH) between male and female Cyprinus carpio. To eliminate the false positives from self-ligations, we screened 150 clones from the sex subtractive genomic DNA library by PCR with the nested primers and identified 88 out of 150 clones carrying the distinguishable insertions. These clones selected were dotted onto two duplicate nylon membranes and hybridized separately with DIG-labeled forward or reverse subtracted DNA. 22 clones exhibited distinct differences. These positive clones were the candidate sex-specific fragments. Specific primers were designed for the 22 clones. PCR was used to further confirm the SSH results by using specific primers to amplify the fragments from the individual male and female genomic DNA. Only the DNA fragments in clones f9 and f10 could be amplified in genomic DNA samples from all males but not in any of the females. which were named as Ccmf2 (387 bp) and Ccmf3 (183 bp) respectively. BLASTN and BLASTX search results showed there are no homologous sequences at the DNA level and no significant similarity (E-value>10-4) at the protein level in other species for the two sequences. As the three fragments isolated were linked to male sex, we speculate that the chromosome type of the Yellow River carp is XX / XY.To confirm male-specificity of Ccmf1, Ccmf2 and Ccmf3 sequences, we tested an additional 50 male and 50 female Yellow River C. carpio. The Ccmf1&2 reached 99% and 98% overall efficiency respective; the Ccmf3 was 100% accuracy. So they have the low number of presumed recombinants among the individuals tested as Ccmf1 and Ccmf2 markers in the Yellow River strain of C. carpio, and can be used to rapidly and accurately identify the gender of Yellow River C. carpio.Three sex-specific markers were used as probes in an attempt to search for homologous sequences in the genome of other common carp strains and C. idellus by a dot blotting hybridization experiment. in the three tested common carp strains the molecular sexing efficiency for Ccmf1, Ccmf2, and Ccmf3 was 67%, 80%, and 93% in the females and 67%, 100% and 73% in the males, respectively. The results revealed that the molecular diversity exists on the Y chromosome of common carps. We may provide a very efficient selective tool for practically breeding monosex female populations in aqua-cultural production, and the sex-specific chromosome region may be characterized and used to study mechanisms of chromosome evolution in this fish species.To comprehensive understand the mechanisms of gonadal development in Yellow River carp, the two-way cDNA subtractive libraries from mature male and female gonads was constructed. 280 clones from male libraries and 240 from female’s carrying the distinguishable insertions were identified by PCR with the nested primers. 180 clones (90 of each library for male and female) were screened with dot bltting, 64 positive clones were obtaind, of which 21 from male libraries and 43 from famales were sequenced. Blast anaysis was performed to find some differentially expressed genes related to gonad development in Yellow River carp. The results showed that of 14 ESTs of 21 sequenced clones shared significant similarities in Genbank datdbase. In accordance with their function, 14 ESTs belong to structural protein genes, the protein translation mechanism related genes, ionic channels proteins and transporters genes, tumor-related and tumor suppressor genes, etc. 4 ESTS of 21 had no significant similarities in Genbank datdbase, whose function were unknown. 32 differences ESTs from the female libraries represented 27 known genes and 5 unknown function genes. In the known genes, the occurence frequencies of respiratory chain-related and protein synthesis-related genes were higher than others. Reference to the gene function of zebra fish and other vertebrates, the known function genes from female EST library belong to regulatory factors, structural proteins, enzymes, transcription factors, signal control, etc.According to semi-quantitative expression in sperm and ovarian of the Yellow River carp, we found 10 differentially expressed gene fragments in the male libraries: 5 of which are testis-specific genes (Including 1 unknown gene discovered firstly); the others are highly expressed genes in the testis (Including 1 unknown gene discovered firstly). In the female libraries, we found 8 differentially expressed gene fragments: 2 of which are ovarian-specific gene; the others are highly expressed genes in the ovarian (Including 1 unknown gene discovered firstly).3 genes (Hmwt1a, HmSetd6, HmPsmb2) which may be involved in gonadal development were chosen to amplify their full-length sequences by RACE-PCR. The results shown Hmwt1a is 2173 bp long, HmSetd6 is 1763bp long, and HmPsmb2 is 884 bp long; Semi-quantisative RT-PCR and fluorescent quantitation RT-PCR were employed to analyse the expression of three genes in the different organizations of Yellow River carps at sexual maturity stages, and the result indicated that Hmwt1a was expressed at highest levels in the testis than ovary, so we speculated that the Hmwt1a gene may be important regulatory genes in male sexual development in Yellow River carp. On the contrary, the Hmsetd6 gene whose expression was higher in the ovary than testis, The HmPsmb2 gene differentially expressed genes in ovarian. Base on the comparison of gene function associated with gonadal development in vertebrates,we speculated that the Hmsetd6 gene involved in the female character development as epigenetic regulation of genes in the Yellow River carp and the HmPsmb2 gene maintained mainly the female characters in the physical or behavioral as sex-bias genes in the Yellow River carp. The discovery of these genes and the construction of gonadal differential EST library, which will help us to learn about of the molecular mechanism gonad development and sex differentiation in the Yellow River carp, as well as provide rich resources of genetic identification for understanding the mechanism of sex control in the Yellow River carp.更多还原