【作者】 郇聘；

【导师】 相建海；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2009， 博士

【摘要】 中国明对虾和栉孔扇贝是我国重要的海水养殖动物,在我国水产养殖业中占有重要的地位;同时二者还是我国的地方特有种,在生物资源保护方面具有重要的意义。目前,中国明对虾和栉孔扇贝养殖业面临种质退化和病害频发的问题。对中国明对虾和栉孔扇贝开展充分的基础研究具有重要的应用意义。由于染色体制备及分析难度较大,中国明对虾和栉孔扇贝的染色体研究进展较慢,尚停留在以普通光学显微镜观察为基础的染色体计数以及核型分析的水平。本研究将荧光原位杂交(Fluorescence in situ hybridization, FISH)技术引入中国明对虾和栉孔扇贝的染色体研究中来,建立了相关技术平台,在染色体上定位了部分功能基因,并对得到的实验结果在染色体鉴别、进化分析、功能基因组等方面的潜在应用价值进行了讨论,详述如下:1.在中国明对虾和栉孔扇贝中建立了FISH平台,通过对FISH各个参数的全面考察,建立了一种质量监控技术。利用该技术可以对FISH实验的主要实验环节进行质量控制,在实验出现阴性结果时迅速锁定可能的问题环节,对FISH技术在海洋生物中有更广泛的应用提供了技术支持。2.在中国明对虾染色体上成功定位了5S rDNA序列,发现5S rDNA定位于中国明对虾的一对同源染色体上,可以作为染色体特异性探针来使用。为了验证这种想法,在三倍体中国明对虾上进行了FISH实验,成功鉴定了包含有5S rDNA的三条同源染色体,证明5S rDNA探针可以用来鉴定中国明对虾染色体。此外,通过对5S rDNA在精巢染色体(减数分裂染色体)上信号分布特征的观察,认为中国明对虾的同源染色体在第一次减数分裂中期可能发生了末端联会(Terminal Synapsis)。3.在栉孔扇贝染色体上定位了18S rDNA和组蛋白序列,发展了双色FISH技术平台,首次对栉孔扇贝18S rDNA和组蛋白序列进行了同时定位,发现二者各定位于一对同源染色体的短臂上。栉孔扇贝18S rDNA和组蛋白的染色体定位可以为双壳贝类的进化研究提供基础资料,并均可作为染色体特异性探针使用。4.在对多拷贝序列(包括rDNA和组蛋白序列)成功定位后,以本实验室建立的栉孔扇贝BAC基因组文库为基础,发展了BAC-FISH技术。在栉孔扇贝染色体上定位了包含热休克蛋白70(HSP70)、丝氨酸蛋白酶(Serine Protease)和脂多糖葡聚糖结合蛋白(LGBP)等免疫相关基因的BAC克隆,发现它们均定位于一对同源染色体的长臂上。进一步建立了双色BAC-FISH技术,对包含LGBP基因的6个BAC克隆进行了同时定位,发现6个包含栉孔扇贝LGBP基因的BAC克隆在间期细胞核上共定位,形成融合信号,提示这6个BAC克隆可能是片段重叠的。双色BAC-FISH平台的建立和免疫相关基因的染色体定位,为栉孔扇贝功能基因的染色体定位提供了经验和技术支撑,推动了栉孔扇贝免疫系统的深入研究;同时这些BAC克隆可以作为染色体特异性探针,在栉孔扇贝染色体鉴别中发挥作用。5.对M-FISH技术进行了尝试。对栉孔扇贝18S rDNA、组蛋白基因h3以及包含免疫相关基因的3个BAC克隆共5个探针进行了同时定位。发现探针数目的增加导致阳性信号率下降,难以找到全部信号都出现的分裂相。通过对一个分裂相的分析,发现其上包含10个阳性信号中的9个,所有信号可根据颜色以及在染色体上的位置相互区分开来,其分布模式与实验设计相同,在部分程度上证明了M-FISH技术的可行性,为M-FISH技术平台在栉孔扇贝中的应用以及栉孔扇贝细胞遗传图谱的构建提供了基础支持。6.在栉孔扇贝免疫相关基因中发展了SNP标记,对栉孔扇贝图谱整合工作进行了探索。在对包含栉孔扇贝热休克蛋白70(HSP70)、丝氨酸蛋白酶(Serine Protease)和脂多糖葡聚糖结合蛋白(LGBP)等免疫相关基因的BAC克隆进行了染色体定位后,利用PCR产物直接测序、多序列比对等方法分别在这三个基因内部发掘了数个潜在的SNP位点。其中,由于不同DNA材料间良好的对应关系,利用PCR产物直接测序法在丝氨酸蛋白酶和脂多糖葡聚糖结合蛋白基因内部发掘的13个碱基变异位点有很高的可信度,应该是真实存在的SNP位点;而利用多序列比对法在热休克蛋白70内部发掘的6个碱基变异位点则可信度稍差,需要进一步的验证其是否是SNP位点。在有合适家系材料的情况下,这些SNP位点可以被定位到遗传连锁图谱上,从而实现遗传连锁图谱与细胞遗传图谱(染色体定位)的初步整合。此外,本研究首次报道了栉孔扇贝SNP标记的筛选工作,展示了栉孔扇贝SNP位点的丰富性,认为栉孔扇贝SNP标记的研究将大大促进栉孔扇贝图谱构建、遗传育种方面的工作。更多还原

【Abstract】 Chinese shrimp (Fenneropenaeus chinensis) and Zhikong scallop (Chlamys farreri), distributing mainly around China, are economically important marine aquaculture species. Diseases and degeneration of cultured breeding lines are the main obstacles hampering the culture of the two species in the past severl years. Many efforts were devoted to resolve these problems.The chromosome studies in F. chinensis and C. farreri have been progressed very slowly because the chromosomes are difficult to obtain and to analyze. In this study, Fluorescence in situ hybridization (FISH) was employed to promote the studies on chromosomes of F. chinensis and C. farreri. Some genes were mapped to the chromosomes, and SNP markers were developed among these genes. The potential applications of these results were discussed.1. FISH technology platform was constructed in F. chinensis and C. farreri successfully. One test method for FISH was developed, through which researchers can catch the factors which had interfered with FISH experiments. The test method would facilitate the applications of FISH to other marine invertebrates.2. 5S rDNA was localized on one pair of homologous chromosomes of F. chinensis, which made it an ideal chromosome-specific probe. To further verify that, FISH attempts were carried out on chromosomes obtained from triploid F. chinensis. The results showed that three 5S rDNA-bearing chromosomes were identified from 132 chromosomes, which certified the hypothesis that 5S rDNA can be used to identify chromosomes of F. chinensis. Besides, terminal synapsis was observed to occur in the meiosis of spermatocytes based on the analysis of signal distribution of 5S rDNA on chromosomes obtained from testis.3. 18S rDNA and histone gene h3 were mapped to the chromosomes of C. farreri simultaneously through double-color FISH. The two probes were revealed to localize on short arms of each pair of homologous chromosomes. Single locus of 18S rDNA and h3 in the genome of C. farreri indicates their potential applications in chromosome identification. On the other hand, the chromosomal localization of 18S rDNA and h3 can provide useful information for evolutional analysis of bivalve.4. BAC-FISH were developed in C. farreri with the aid of BAC genomic DNA libraries, which ensure the chromosomal localization of low-copy functional genes. Three BAC clones containing immune-related genes, including heat shock protein 70 gene (hsp70), serine protease gene (sp) and lipopolysaccharide and beta-1,3-glucan binding protein gene (lgbp), were mapped to the chromosomes of C. farreri. The results revealed that the three clones located on long arms of each pair of homologous chromosomes respectively.Two-color BAC-FISH were developed for mapping more than one BACs to explore the comparative localizations of these BACs. The results revealed that six lgbp-containing BACs co-localized with each other because merged signals were detected for every probe combination. The localizations of immune-related genes would promote researches on immune system of C. farreri. And these BACs can also be used as chromosome-specific probes for chromosome identification in C. farreri. Optimizations were carried out to employ M-FISH technology in C. farreri. Five probes mapped previously, namely 18S rDNA, histone gene h3 and three immune gene-containing BACs (CFB123C08 for hsp70, CFB040H03 for sp and CFB094J04 for lgbp) were mapped to the chromosomes simutaneously. Although the results revealed a dramatically decrease for positive signals, nine of ten signals were detected in an incomplete metaphase, which can be distinguished from each other basing on the colors of the signals and their locations on the chromosomes. The distribution characters of the signals were consistent with which had been imaged according to the experiment design, indicating that M-FISH was feasible in C. farreri with further optimizations. On the other hand, a cytogenetic map would be constructed accordingly when M-FISH technology was developed successfully.5. Several SNP markers were developed in immune genes of C. farreri to promote the construction of integration maps. Both sequencing of PCR products and multi-alignment were used to develop SNPs in hsp70, sp, and lgbp. Six base mutations in hsp70, six in sp and seven in lgbp were detected. The 13 base mutations detected in sp and lgbp were believed to be SNPs because high consistencies on the mutation sites were revealed in three DNA samples either from single or mixed individuals. And the six base mutations detected in hsp70 should be further verified before they were taken as SNPs because false positives can be caused by mismatching in PCR reaction and sequencing, which can not be avoided in multi-alignment. The SNPs can be localized to the genetic linkage map when a proper family of C. farreri is available, leading to the integration of genetic linkage map and cytogenetic map (chromosomal mapping). Besides, the abundance of SNP revealed in this work would make it possible to develop SNPs in any given region of the genome of C. farreri.更多还原