【作者】 王永平；

【导师】 郭希明； Rutgers University；

【作者基本信息】 中国科学院海洋研究所 ， 海洋生物学， 2001， 博士

【摘要】 本研究应用显带技术和荧光原位杂交（Fluorescence in situhybridization,FISH）技术，鉴定了牡蛎的染色体；应用FISH方法定位了一系列的重复序列和大分子的P1克隆DNA；制备了染色体特异性探针。应用FISN特异性探针成功地鉴定了长牡蛎的三体10。结果如下： 1．分析了G带和C带在美洲牡蛎染色体上的分布。G带在每一条染色体上的带型不同，某些染色体间（如第1对和第4对染色体，第7对和第9对染色体）的带型差别不是很明显。G带型容易受染色体收缩程度的影响。C带型重复性较好，染色体带型较清楚，分布在染色体的端粒区域和着丝粒区域。G带和C带带型能够用来鉴定牡蛎的染色体，但是重复性低和带型差异不显著，并不适合常规的染色体鉴定。 2．早期胚胎和担轮幼虫制备的染色体适合于FISH分析。染色体制备方法重复性好，可适用于其它贝类的染色体制备。 3．研究了重复序列基因—rDNA的定位： 1）18S-5.8S rDNA在研究的五种巨蛎属Crassostrea牡蛎均只有一个位点。太平洋种（C.gigas，C.ariakensis和C.plicatula）中，杂交信号位于最短的染色体—第10对染色体长臂的端粒区域，在大西洋种（C.virginica和C.rhizophorae）中，同一序列定位在第2对染色体短臂的端粒区域。 2）18S-28S rDNA在两种蛤中有两个位点。rDNA探针定位在侏儒蛤（MulinisLateralis）的第15对和第19对染色体的端粒区域，同一序列定位在硬壳蛤（Mercenaria mercenaria）的第10对染色体的长臂和第12对染色体短臂的端粒区域。信号强度在两对染色体之间有差异。 3）5S rDNA位于美洲牡蛎的第5对染色体的短臂上靠近着丝粒区域和第6对染色体的短臂的中间区域。信号强度在两对染色体之间没有显著差异。5SrDNA探针可以作为鉴定和识别第5对和第6对染色体的特异性探针。 4．研究了一些重复序列的定位 1）两个短的重复序列1G8，1P2均产生很强的荧光信号分布在美洲牡蛎所有的染色体上。在低严谨条件下，这些序列均产生很强的信号散布在所有的染色 王永平 中国科学院海洋研究所博士论文 体上。在高严谨条件下，信号强度大大减弱，但是信号仍散布在所有的染色体 上。这些重复序列散布在美洲牡顿的整个基因组中。 2）高度重复序列 Cgl 70产生的信号分布在长牡顿的 7对染色体的着丝粒区 域，没有发现间区信号。在第1对，第二对，第4对和第7对染色体上的荧光 信号强且稳定。在第5对，第8对和第10对染色体上的信号相对弱且不稳定。 在剩余的染色体上（第3对，第6对和第9对染色体）没有检测到荧光信号。 结果表明此卫星序列是一个着丝粒卫星序列。在美洲牡顿的染色体上没有检测 到荧光信号，表明了这个着丝粒卫星序列在这两种牡顿中的分布存在着显著的 差异。 3）脊椎动物端粒序列（ITAGGG切的FISH信号局限在四种双壳贝类（美洲 牡领，the mangrove oyst口，硬壳蛤，株儒蛤）所有染色体的端粒区域，没有发 现间区信号的存在。研究结果与己报道的研究结果表明脊椎动物端粒序列或许 存在于所有双壳贝类的染色体末端。双壳贝类是目前研究过的唯一含有脊椎动 物端粒序列DNA的无脊椎动物。 4）研究了RAPD探针在美洲牡领染色体上的定位。大多数RAPD探针产生了 多个信号散布在间期细胞核和所有的染色体上。引物 OPX个3，OPX刁4，OPX－ 06，OPG－02，OPM－04，OPM－11，OPS－02制备的探针在适宜的条件下产生特异性荧光 信号，分布在牡物的特定的染色体上。PCR特异性带产生的探针 OPX刁6上 和 OPG－02－300产生了特异性的荧光信号：OPX－06－310产生的信号位于第5对染色 体的短臂的近端粒区域，OPG－02－300探针定位到第3对染色体的短臂上。这两 个探针是鉴定美洲牡烟单条染色体的特异性探针。 5．研究了大分子n克隆DNA（插人片断为80—100 kb）在美洲牡绳染色体 上的定位。PI克隆DNA通过切口平移方法标记digoxigenin－lldUTP用作FISH 的探针。COt—IDNA作为竟争剂有效地抑制了N克隆序列中的重复序列产生 的信号。杂交信号用fluorescein标记的anti－digoxigenin抗体来检测，用两 层抗体 rabbit一anti一sheep抗体和 FITC anti－rabbit抗体来扩增信号。9个 PI探针成功地定位在特定的染色体上。46－l探针杂交到第1对染色体的长臂靠 近着丝粒区域；47司 探针定位到第2对染色体的长臂近端粒区域；CVpl和 48d 两探针定位到第3对染色体上：Cvpl位于短劳的端粒区域，48l3探针 位于长臂的近着丝粒区域；48?更多还原

【Abstract】 In this study the traditional banding technique and fluorescence in situ hybridization (FISH) technique were used to characterize and identif the chromosomes of oysters. We developed chromosome-specific FISH probes by mapping a variety of repetitive DNA and P1 clone DNAs by FISH. The Pacific oyster trisomy 10 was successfully identified using chromosome-specific probes. The results of this study are listed as followings: I .The (3 and C banding distribution were analyzed in the eastern oyster chromosomes. Each chromosome has its unique banding characteristics. Differences in banding characteristics among some chromosomes (such as Chromosome 1 and 4, Chromosome 7 and 9) were not outstanding. G banding is susceptible to interference by variations such as the state of chromosome contraction. C banding pattern is highly reproducible and clear, C banding mainly distributed at the centromere and telomere region. Although C and 0 banding identified all chromosomes of the eastern oyster after intensive screening and careful analysis, they were not suitable for routine identification of oyster chromosomes. 2. Metaphase chromosomes prepared from early embryos and trocorphore stage were adequate for use in FISH analysis. The methods of chromosome preparing are highly producible and easily applied to other marine mollusc species. 3. Chromosomal localization of multiple gene DNA were performed by FISH. 1) 1 8S-5.8S rDNA was assigned to one locus in 5 Crassostrea oyster species studied, in the Pacific species (C.gigas, C. ariakensis and C. plicatula) , rDNA was localized to the telomeric region of the long arm of Chromosome 10; And in the Atlantic species (C. virginica and C. rhizophorae), rDNA was localized to the telomerie region of the short arm of Chromosome 2. .2) 1 8S-28S rDNA was assigned to two loci in 2 clam species studied. rDNA was assigned to the telomezic region of Chromosome 15 and Chromosome 19 in dwarf-surf clani (Mulinis Lateralis Say), and the same sequence was assigned on the proximal region of long arm of Chromosome 10 and on the telomere of short arm of Chromosome 12 in the hard clam (Me rcenaria mercenaria Linnaeus). The signal intensity was variable between two pairs of chromosomes. 3) 55 rDNA was assigned to the interstitial region inirnediately next to the centromere on the short arm of Chromosome 5 and the interstitial region of the short arm of Chromosome 6 in the eastern oyster. The signal intensity was not significantly variable between two pairs of chromosomes. 5S rDNA can serve as a specific marker for the identification of two pairs of homologous chromosomes in the eastern oyster. 4 Chromosome localization of some repetitive sequences were studied. 1) Two short repetitive sequences 1 P2 and I G8 produced strong hybridization signals dispersed on every chromosome in the eastern oyster. The signals were dispersed through all the chromosomes under low stringency, although the signals intensity was much lower under high stringency, the signals were still on every chromosome, suggesting this two sequences are dispersed in the oyster genome. 2) A satellite repetitive sequence Cgl 70 was mapped to centromeric regions of seven pairs of the Pacific oyster chromosomes. No interstitial site was found. FISH signals were strong and consistent on Chromosomes I, 2, 4 and 10, but weak or variable on Chromosomes 5, 8 and 10. No signals were observed on Chromosomes 3, 6 and更多还原