长牡蛎可存活四倍体的诱发机制和异源三倍体的诱发

IOCAS-IR > 海洋环流与波动重点实验室

	长牡蛎可存活四倍体的诱发机制和异源三倍体的诱发
	阙华勇
学位类型	博士
	1998
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	长牡蛎近江牡蛎染色体的分离行为杂交异源三倍体
摘要	采用乙酸地衣红染色技术（Acetic orcein staining technique）较系统地研究了长牡蛎 Crassostrea gigas (Thunberg)三倍体产生的卵子在受精且第一极体释放受抑制后的减数分裂过及染色体的分离行为以阐明可存活四倍体的产生体的机制。用浓度为0.5 mg/L的细胞松驰素B （CB）处理受卵以抑制其第一极体的释放。在观察到个别受精卵出现第一极体时开始CB处理，持续至对照组中50％的受精卵出现的第一极体。对处理组和对照组的受精卵从受精后隔5分钟取样一次，用卡诺氏液（Carnoy's fixative, 冰醋酸和甲醇按1：3的体积比充分混合）固家样品。采用0.5%的乙酸－地衣红染料进行受精卵的染色，而后压片观察受精卵染色体行为。长牡蛎三倍体产生的卵子，中期I同源染色体构型呈现单价体（Univalents）,二价体（Bivalents）、三价体（Trivalents）以及大于三价体的多价体（Multivalents）混合出更的特征。在第一极体释放受抑制的受精卵的第二次减数分裂过程中，可确认四种染色体分离类型：三极分离（Tripolar segregation） (54.5%)、联合二极分离（United bipolar segregation） (12%)、独立二级分离（Incomplete united bipolar segregation）(4%)。其余卵子的染色体分离行为（23％）不规律，呈现不同程度的紊乱，但总体看来介于上述四种分离类型之间。此外，某些特定的独立二级也可能是四位体形成的最主要的细胞遗传学体制。此外，某些特定的独立二极分离也可能产生四倍体。轻细胞体驰素B 处理的受精卵的减数分裂过程具有显著的不同步性，表现在三个方面：第一，在两个重复组之间，即两个雌体之间，存在第二次减数分裂的时间进程的不同步性；第二，同一个雌体产生的卵子之间的发育速度不同步性，表现为不同的卵子进入第一次减数分裂的时间不同；第三，同一卵子内的染色体之间，其行为有时存在的不同步性。另外，探讨了中心类在支配第二减数分裂时各种染色体分离行为的可能机制。以长牡蛎二倍体与近江牡蛎二倍体的染交作为对照，探讨了能长牡蛎四倍体与近江牡蛎二倍体杂并诱导异源三倍体的可行性。长牡蛎Crassostrea gigas (Thunberg)四倍体和二倍体与近江牡蛎Crassostrea rivularis (Gould)二倍体的杂交以及相应的对照组共进行了三批重复实验，杂交实验采用高密度的精子。研究结果表明，自交组平均受精率依次为94％（GG）、77％（RR）,88% （G／GG）和85％（GG／G）。双方差分析（ANOVA）表明，各自交组之间受精率没有显著差异（F＝3.118, P=0.132）。在杂交组，直接授精后180分钟，尚未观察到受精迹象，因而无法估计受精率。授精后48小时的孵化率各组之间差异很大，并经双方差分析（ANOVA）表明存在显著性差异，（F＝3.188, P=0.018）。其中GGR和RGG组的孵化率相近似，产生的幼虫数量明显少于对照组。在四种类型的杂交实验中，二倍体C. gigas (雌体) ＊二倍体 C. rivularis (雌体)（ＧＲ）早最成功的。虽基ＧＲ组幼虫的生长率低于对照组，但其存活率接近于对照组。长牡蛎四倍体与近江牡蛎二倍体杂交组（GR），在授精后两天的孵化率较低，但幼虫的生长状况与对照组接近。另外两个杂交组，即近江牡蛎二倍体与长牡蛎四倍体（RGG），二倍体近江牡蛎江与二倍体长牡蛎（RG），授精后两天的孵化率很低，幼虫生长得缓慢。三个重复组的GR杂交组和一个重复组的GGR杂交组获得稚贝。聚合酶链式反应／限制性酶切片段长度的多态性（PCR／PFLP）检支分析结果证实这些稚贝均是杂交种；流式细胞术分析结果证明GGR获得的稚贝是三倍体，从而证明获得了长牡蛎与近江牡蛎的异源三倍体。有迹象表明三倍体与二倍体杂交种之间（GGR对GR）存在生长上的差异。首先，GGR的眼点幼虫大约比GR组早出现5－7天即仅次于对照组GG，G／GG，和GG／G；第二，尽管仅获得少量GGR幼贝，这些幼贝在授精后90天的大小显著大于GR组的个体。在RGG和RG组中，幼虫没能存活到眼点幼点阶段。细胞学检查结果表明，杂交组的绝大多数卵子发育停滞在第一次减数分裂中期（Metaphase I）,这一过程至少持续到授精后180分钟。仅有2％的GGR 组的卵子在授精后180分钟进入第一次减数分裂后期）（Anaphase I）. 而在此时期，GR，RGG和RG组的卵子中，仍只观察到10第二价体（Bivalents）.
其他摘要	Chromosome segregation in fertilized eggs from triploid Pacific oyster Crassostrea gigas following inhibition of first polar body (PB1) was studied with acetic-orecein staining techniques. To block the release of PB1, frertilized eggs were treated with 0.5 mg/l of cytochalasin B (CB). To study chromosome segregation, samples of developing zygotes were taken every 5 minutes until 60 min PF and fixed with Carnoy's solution (1:3 glacial acetic acid and absolute methanol). Fixatives were changed twice by centrifugation. Chromosomes were observed by acetic orcein stain (Guo et al., 1992). Briefly, drops of fixed sample were spread on a slide, mixed with 2-3 drops of orcein stain (0.5% orcein in 60% acetic acid) and covered with a cover glass. After staining for 5 minutes, the cover glass was pressed gently and sealed with cytoseal mouting medium. Synapsis in eggs from triploid Pacific oyster was characterized by the presence of a mixture of t rivalents, bivalents, and univalents. Four types of segregation were observed, namely, "tripolar segregation" (54.5%), "united dipolar segregation" (12%), "separated bipolar segregation" (2.5%) and "incomplete united bipolar segregation" (4%) during the second meiosis following inhibition of chromosome disorganization. It seemed that the predominant chromosome segregation pattern that gave rise to tetraploids was united bipolar segration, although certain separated bipolar segregation might also lead to the formation of tetraploids. The sequential events of meioses observed in CB-treated eggs are described. Our observations also suggest that the meiotic events in CB-treated triploid eggs are remarkably asynchronous. Asynchrony was observed at three levels. First, there was variation in the timing of meiosis II between the two replications, that is, between the two females. Second, among eggs from a single female, asynchrony was observed as different eggs entered meiosis I at different times. Third, asynchrony sometimes occurred among chromosomes of the same eggs. We speculate that the behavior of centrioles might be responsibe for vrious patterns of chromosome segregation observed in this study. As to the tripolar segregation, the centrioles normally taken into the first polar body would participate in the organization of one pole of the tripolar spindle. The other centrioles normally remained in the zygotes divided and was responsible for the organization of two poles of the tripolar spindle. The united bipolar segregation could be the consequence of either the dysfunction of the peripheral centrioles or the non-division of inner centrioles; separated bipolar segregation could be arisen by the division of peripheral centrioles. Three replicates of hybrid crosses of tetraploid and diploid C. gigas (Thunberg) with diploid C. rivularis (Could) were produced with controls. Larval survival and growth were documented. Cytological evens were also revealed in oocytes from hybrid crosses following insemination.Mean fertilization rate in the pure crosses was 94% (GG,), 77% (RG), 88% (G/GG), and 85% (GG/G). There was no statistically significant difference among pure crosses (F = 3.118, P =0.132) by ANOVA. In hybrids, signs of fertilization did not appear till 180 min after insemination, precluding estimates of fertilization rate. Among four types of hybrid crosses, diploid C. gigas (female) * diploid C. rivularis (male) (GR) was the most successful. Survival of GR was about the same as controls, although its growth rate was lower. Crosses of tetraploid C. gigas (female) and diploid C. rivularis (male) (GGR) had poor yield at day 2 postinsemination but grew nearly as well as controls subsequently. The other two types of hybrids, i.e., diploid C. rivularis (female) and tetraploid C. gigas (male) (RGG), diploid C. rivularis (female) and diploid C. gigas (male) (RG) suffered very low yield at day 2 and grew very slowly. For RGG and RG crosses, mortality was severe and steady for 13 days, with no survival to eyed stage. Spat were obtained from all replicates of GR crosses and 1 of 3 replicates of GGR and proved to be hybrids by PCR/RFLP diagnosis. GGR hybrids were confirmed to be triploid by flow cytometry. It is also suggested that there is a difference in grwoth rate between triploid (GGR) and diploid (GR) hybrids. First, GGR eyed larvae appeared 5-7 days earlier than GR and right after those of controls GG, G/GG, and GG/G. Second, the size of spat from GGR was significantly greater than the size of GR at 90 days postinsemination, although the number of GGR spat was small. No larvae survived to eyed stage in RGG or RG crosses. Cytological examination revealed that the vast majority of oocytes from hybrid crosses had a prolonged meiotic prophase I or metaphase I at least through 180 min post-insemination. Only 2% of eggs from GGR had entered anaphase I by 180 min post-insemination. In GR, RGG and RG, 10 bivalents were still observed at this time. Chromosome aggregation was much more complicated in eggs from GGR crosses. In general, eggs contained an average of 10 quadrivalents although other types of synaptic chromosomes were also present, i.e., univalents, univalents, bivalents and trivalents. PCR/RFLP diagnosis was an effective means to verify putative hybrid progeny. ITS-1amplification/Hinf I digestion successfully distinguished among C. gigas, C. rivularis, and hybrids which show bands present from both parental species.
页数	86
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1481
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	阙华勇. 长牡蛎可存活四倍体的诱发机制和异源三倍体的诱发[D]. 中国科学院海洋研究所. 中国科学院海洋研究所,1998.

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