几种对虾染色体及多倍体诱导研究

IOCAS-IR > 海洋环流与波动重点实验室

	几种对虾染色体及多倍体诱导研究
	张晓军
学位类型	博士
	2001
学位授予单位	中国科学院海洋研究所
学位授予地点	中国科学院海洋研究所
学位专业	海洋生物学
关键词	对虾染色体荧光原位杂交基因定位多倍体性腺发育
摘要	本文利用染色体制片、流式细胞术、显微荧光、组织学切片、透射电镜、及荧光原位杂交等技术，从细胞遗传学角度，研究了对虾的染色体和多倍体诱导等问题。以胚胎、无节幼体、卵巢、精巢等为材料，采用空气干燥法制片，获得了刀额新对虾、凡纳对虾和细角对虾的体细胞和减数分裂染色体中期分裂相。结果表明，刀额新对虾的染色体数目为2n = 78, n = 39，核型为2n = 78 = 40M + 10SM + 14ST + 14T。凡纳对虾和细角对虾染色体数目相同，为2n = 88, n = 44。以鸡血细胞为对照，用流式细胞仪（Partec CCAII)对刀额新对虾的细胞核DNA含量进行了测定，其值为鸡血细胞的1.75倍，绝对含量为4.025pg/2c。对细角对虾、日本对虾、刀额新对虾和中国对虾的基因组大小进行了比较，发现四种对虾基因组由大到小的顺序为细角对虾、日本对虾、刀额新对虾、中国对虾，由此推导出对虾科在进化过程中DNA发生丢失的假设。应用荧光原位杂交技术研究了中国对虾18SrDNA序列和（AG）n微卫星序列在染色体上的定位。初步研究的结果发现18SrDNA序列和（AG）n序列分布在中国对虾一对染色体的端粒区域；（AG）n微卫生序列分布在5对染色体上，着丝粒和端粒区域都有分布。本文对分布在热带和亚热带的日本对虾，斑节对虾及刀额新对虾的多倍体诱导进行了研究，成功地诱导出了三种对虾的三倍体幼体，诱导率最高分别达到68.52%、59.99%、49.70%。并进一步探讨了热带对虾多倍体诱导的特点，认为对产卵温度较市制热带和亚热带对虾，冷休克的诱导效果较好。采用热休克、冷休克、CB、6-DMAP、秋水仙素及咖啡因等方法，进行了中国对虾四倍体诱导研究。通过抑制第一极体排放或抑制第一次卵裂等途径，上述方法都获得了四倍体胚胎，最高诱导率达100%。实验中优化了诱导条件，筛选了诱导方法，认为热休克方法最佳。利用显微荧光技术研究精子入卵，原核形成、移动、结合以及再分离的过程，微管、微丝的活动及纺锤体等的动态变化，有助于深入了解多倍体的诱导机理。本文在中国对虾三倍体、四倍体诱导过程中，观察了诱导处理后染色体行为的变化，进一步分析了多倍体的形成过程。实验中发现四倍体诱导中存在细胞形态的特殊变化，认为是由于诱导处理导致的中心体复制异常所致，这也是诱导率降低的主要原因。文中讨论了四倍体诱导的最佳时机的选择即选择染色体已经分开，中心体尚未复制的时刻，并由此讨论了中心体的复制事件。利用组织学切片和透射电镜观察了三倍体成虾不同时期的性腺。结果表明，中国对虾三倍体的卵巢外观上退化明显，在组织学上可分为增殖期、阻滞期和退化期，卵巢细胞的发育大多停滞在卵原细胞阶段，尚未观察到有形态成熟的卵子形成，除少数生殖细胞进入卵黄形成前期，大多数为不定性细胞。三倍体精巢形态发育和组织学结构与二倍体相近，但产生的精子数明显少于二倍体。且输精管、贮精囊也呈退化趋势。精巢中所发现的染色体数在60-80、100-120和230-250区间的非整倍体细胞所占比例很大，其原因可能是减数分裂过程中，染色体发生了丢失。在超微结构上，观察到三倍体卵巢内的卵原细胞线粒体退休。核糖体较少，内质网和高尔期体比较发达，在晚期少数卵母细胞有卵黄颗粒存在。三倍体精巢中精原细胞和精母细胞的细胞器不发达，线粒体少，高尔基体较多；滤泡腔和输精管内精子很少，大小不一。
其他摘要	Penaeid shrimps are very important commercial species in the marine aquaculture industry and their production has been increasing in the world. But the supply of shrimps is still not enough to satisfy consumer's need because of the over fishing of wild stock and the serious viral diseases in shrimp-farming. The sustainable aquaculture of shrimp becomes more and more important. But because of a lack of knowledge on fundamental aspects of their biology, progress in penaeid genetics and biotechnology research is slow. In cytogenics, there is little research undertaken on the chromosome number, structure, composition and chromosome manipulation in shrimp. A careful study of shrimp chromosomes is not only useful to taxonomy and evolution studies of crustacean, but also essential to its genetic and breeding researches for the shrimp aquaculture. In this paper, we studied the chromosome, ploidy manipulation and gene location using chromosome preparation, flow ctyometery, fluorescence microscopy, histological methods, electronic microscopy and fluorescence in situ hybridization. High qualitative somatic and meiotic chromosomes from embryo, larvae and adult of three shrimps: Metapenaeus ensis, Litopenaeus vannamei and L. stilyrostris were obtained. The somatic chromosomes were prepared from embryo, nauplius larvae and adult tissues including ovaries and testes. The testis lobes contained both meiotic and mitotic cells, so diploid and haploid chromosomes can be obtained. Diploid chromosomes can be observed in ovaries of M. ensis, but the majority of the chromosomes in testes and ovaries were the "dot" shapes. The chromosome number of M. ensis is 2n = 78, n = 39, preliminary results of karyotype is 40 M + 10SM + 14ST + 14T. L. vannamei and L. stilyrostris have the same diploid chromosome number 2n = 88. Using of DNA of chicken erythrocyte (2.30pg/2c) as a standard, diploid nucleus DNA content of M. ensis was measured from the blood, ovaries, muscle and gills, by flow cytometer (Patarc-PCAII). DNA content of M.ensis is 4.025 pg/2c and the ratio to that of chickens is 1.75. The genome size was compared in four species shrimps L. stilyrostris, Marsupenaeus japonicus, M. ensis and. Fenneropenaeus chinensis by flow cytometery. The L. stilyrostris have the largest genome size, it is 1.48 times to M. japonicus, 1.61 to M. ensis, and 1.65 to F. chinensis. We presumed DNA lose may occur in the Penaeoidae evolution. Fluorescence in situ hybridization (FISH) is a promising technique used in cytogenetic researches at the molecular level. It regards to such fields as gene localization, sex identification, chromosome variation, and interspecies hybridization. In this paper, FISH was attempted in shrimp. 18SrRNA gene was applied into localization in two chromosomes of Chinese shrimp F. chinensis, which not only enriched the cytogenetic studies of shrimps but also provided implication for genome maping. Microsatelate sequence (AG)_(10) was also been localized in 10chromosomes of F. chinensis. The triploid of three species tropical shrimps: M. japonicus, P. monodon, and M. ensis were induced by heat shock, cold shock and 6-DMAP. Optimal condition of temperature shock was determined in the M japonicus and P. monodon. The eggs were spawn at about 28 ℃, cold shock treatment at 5-9 ℃ for 10-20 min duration initiated at different time 6-18min after fertilization. 68.52% and 84.70% triploid nauplii were produced in M. japonicus and P. monodon.. 6-DMAP was used to induced triploid in M.japonicus. P. monodon, and M. ensis with 400-600 μmol/L 6-DMAP for 15-20min initiated at 6-18 min after spawning, the induction rate was 52.42%, 59.99%, 49.70% triploid juveniles respectively. Ploidy levels of nauplii were determined by flow cytometer with DAPI staining. These experiment show that cold shock treatment have a higher percentage of triploids than that of 6-DMAP in tropical shrimp. Tetraploidy was induced by inhibiting the first cleavage or PB1 release with heat shock, cold shock, cytochalasin B (CB), 6-DMAP, colchicine or coffeine respectively. the tetraploids were obtained in embryo stage in all these treatment. The effects of treatment duration and timing of treatment after fertilization on embryonic hatching rate and the percentage of tetraploid were examined. The heat-shock is the better than other treatment methods. Optimal treatment duration time in heat-shock was 3-5min; optimal starting time of treatment was 3-5min before cleavage. To establish the efficient procedures for polyploid induction in F. chinensis, the effect of heat shock treatment on meiosis and cleavage of eggs was investigated with fluorescence microscopy. Three pronuclei were observed in the treated eggs and that will develop into triploid embryos. Cleavage could be inhibited with heat-stock tetraploid treatment, two nuclei stop separating and gradually moved together then intermixed to become one nucleus. We observed some abnormal chromosome behavior of heat shocked eggs. Centrosome duplication is a key step for bipolar spindle formation and correct segregation of chromosomes during cleavage after tetraploid induction. Centrosomes can nucleate microtubules and duplicate once and only once per cell cycle. This duplication and subsequent segregation in mitosis results in maintenance of the one centrosome/cell ratio. Centrosome duplication occurs during the G1/S transition in somatic cells and must be coupled to the events of the nuclear cell cycle; failure to coordinate duplication and mitosis results in abnormal numbers of centrosomes and aberrant mitosis. Many abnormal cleavages with multipolar were observed in this experiment, which debase the tetraploid rate. But how centrosome duplication is regulated and coordinated with other cell-cycle functions remains poorly understood. The gonad of triploid F. chinensis at different stage was histoloically studied. Compared with diploid, the development of ovary in triploids was degenerate and it was divided into three stages: proliferating stage, stopping stage, and degenerating stage. But male triploids can produce less gametes than those diploid. Otherwise, its gamete mature was absent, which resulted in sterile gonad. Future work is aimed at identifying the chromosomes of penaeid shrimp, cloning chromosome-specific libraries. Efforts are underway to implement a genomic mapping project aimed at developing a high efficient aquaculture for shrimp.
页数	151
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/459
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	张晓军. 几种对虾染色体及多倍体诱导研究[D]. 中国科学院海洋研究所. 中国科学院海洋研究所,2001.

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