牡蛎非整倍体的制备及其生物学特性研究

【作者】 巩宁；

【导师】 相建海； 张国范；

【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， 2003， 博士

【摘要】 非整倍体是指染色体组中有个别染色体缺失或增加的类型。与高等动物不同，贝类能够耐受个别染色体数目的增减而存活。本研究中，我们通过二倍体和三倍体杂交的手段，人工制备并筛选了长牡蛎非整倍体，利用荧光原位杂交（FISH）技术和同工酶方法对非整倍体进行了鉴定，探讨了各种类型非整倍体的发生频率、存活能力和生长状况，并从生物能量学角度对生长差异进行了分析。结果如下：1、在 1 龄和 4 龄时对二倍体、三倍体杂交组长牡蛎进行了染色体计数，发现两 杂交组产生非整倍体的频率不同： （1） DTA 组（2n♀×3n♂）产生了较高比例的非整倍体。1 龄时占 82.5%， 4 龄时占 83.7%。其中 1 龄时 2n 水平的非整倍体占 75%，3n 水平的占 7.5%；4 龄时 2n 水平的占 81.6%，3n 水平的占 2.1%。除了非整倍体 外，还发现了一定比例的二倍体，1 龄时为 17.5%，4 龄时为 16.4%。 未发现三倍体。 （2） TDA 组（3n♀×2n♂）产生了较多的整倍体（88.1%）和少量非整倍 体（11.9%），4 龄时该比例变化不大。在整倍体中，三倍体的比例有 所增加（从 1 龄的 30.9%到 4 龄的 41.1%），二倍体的比例略有下降（从 1 龄的 57.2%到 4 龄的 47.1%）。 从以上结果可以看出，二倍体、三倍体杂交是进行贝类非整倍体制备的 有效手段。在牡蛎中，三倍体父本和母本产生非整倍体子代的比例不同，雄 性三倍体产生了较多的非整倍体后代。2、本研究共发现 2n+1、2n+2、2n+3、3n-2 和 3n-1 五种类型的长牡蛎非整倍体， 其中 2n+2 和 2n+3 两种类型为首次报道。染色体数为 24-27 的个体可能不能 存活。在 1 龄时发现的 2n+3 和 3n-2 两种类型在 4 龄时未发现，并且 2n+2 类 型非整倍体的比例有所下降。可能是个体死亡或发生了染色体丢失。但作为 一个整体，非整倍体的存活能力与整倍体接近。 - 1 -<WP=6>巩宁 牡蛎非整倍体的制备及其生物学特性研究 中国科学院研究生院博士学位论文3、利用 rDNA 的 ITS1、ITS2 序列作引物，PCR 扩增的方法制备了长牡蛎 10 号 染色体的特异性探针，对长牡蛎的三体 10 进行了荧光原位杂交（Fluorescence in situ hybridization，FISH）鉴定。FISH 方法是进行非整倍体鉴定的有效手段。同 时采用淀粉凝胶和聚丙烯酰胺凝胶电泳，对长牡蛎非整倍体进行了 8 种同工 酶的分析。其中，苹果酸脱氢酶（MDH）的一个位点上，两个非整倍体个体 （三体和双三体）出现了预期的酶谱。该结果对非整倍体的鉴定及利用非整 倍体进行酶基因的染色体定位打下了基础。4、对人工制备的长牡蛎非整倍体进行了生长研究。在一龄和四龄时，染色体数 目对长牡蛎生长的影响均极其显著。一龄时，三倍体的全湿重在比二倍体大 64.7%；四龄时，三倍体的全湿重在比二倍体大 79%，软体部湿重大 98%。 作为一个整体，非整倍体的各个生长参数均小于二倍体和三倍体。2n+2 型非 整倍体小于其它类型。三体之间的生长差异较大，说明不同染色体的增加对 生长的影响程度不同。对长牡蛎的二倍体、三倍体和非整倍体进行了能量学 研究。三倍体用于生长的能量大于二倍体和非整倍体，二倍体的生长能大于 非整倍体。非整倍体与二倍体能量利用上的差异可能是二者生长差异的原因 之一。5、对养殖的长牡蛎、近江牡蛎进行了染色体计数。这两种牡蛎的二倍体染色体 数均为 20，但制片中都发现了数目不足 20 的非整倍体细胞。并且个体小的 牡蛎表现出了较高的细胞非整倍体率。经回归分析发现，全湿重与细胞非整 倍体率呈负相关，回归关系显著。更多还原

【Abstract】 Aneuploids are organisms where member chromosomes of the haploid set arerepresented in unequal copies. Mollusks like plants and unlike most vertebrate speciescan tolerate a variety of aneuploid conditions. In this study, aneuploids were preparedwith diploid, triploid crossing, and the research has been done on the isolation andidentification of the aneuploid Pacific oyster (Crassostrea gigas) by fluorescence insitu hybridization (FISH) and isozyme analysis. At the same time, we studied thetypes, frequency and survival of aneuploids as well as their growth, trying to explaindifference of growth between aneuploids and diploids with bioenergetics analysis.The results of this study are listed as followings:1. Chromosome number analysis was conducted on surviving progeny from diploid female × triploid male (DT) and reciprocal (TD) crosses of Pacific oyster at one and four years of age. And the frequency of aneuploids was different in the two crosses: (1) There are more aneuploids in DT crosses. At Year 1, oysters from DT crosses composed of 17.5% diploids (2n=20) and 82.5% aneuploids that consisted 75% 2n level and 7.5% 3n level. At Year 4, there was no change in evidence. 16.4% diploids and 83.7% aneuploids were found. Aneuploids included 81.6% 2n level and 2.1% 3n level. No triploid was found in both ages. (2) In contrast, oysters from TD crosses consisted of more euploids (88.1%) and some aneuploids (11.9%). There was little change over time in the overall frequency of aneuploids, but the ratio of triploids increased from 30.9% to 41.2%, while that of diploids decreased from 57.2% to 47.1%. - 120 -<WP=125>巩宁 牡蛎非整倍体的制备及其生物学特性研究 中国科学院研究生院博士学位论文 (3) Diploids and triploids crossing is an effective method to prepare aneuploids in mollusks. Triploid females produced more euploid gametes and viable progeny than triploid males. The study suggested that triploidy is not an evolutionary dead end and may play a role in chromosome number evolution in mulluscs.2. Viable aneuploid chromosome numbers included 2n+1,2n+2,2n+3,3n-2 and 3n-1. Oysters with 2n+2 and 2n+3 chromosome were found at the first time. The absence of chromosome numbers between 24 and 27 suggests that they are lethal in the Pacific oyster. Oysters with 2n+3 and 3n-2 chromosomes were observed at Year 1,but absent at Year 4 due to some individuals dead or chromosomes loss. The proportion of oysters with 2n+2 chromosomes was decreased at Year 4. But as a group, the survival of aneuploids was similar as that of euploids.3. The Pacific oyster trisomy 10 was successfully identified with the aid of ITS1, ITS2 rDNA probe that was prepared with PCR amplify. Trisomy 10 from DTA1, DTA3 and AA groups were screened out by fluorescence in situ hybridization (FISH). Isozyme analysis was carried out with starch gel (SGE) and polyacrylamide gel (PAGE) electrophoresis in aneuploid Pacific oysters. In the locus of MDH-1, two aneuploids showed the expected bands, which can be used for the chromosomal mapping of isozyme gene when the extra chromosome of aneuploids was identified by FISH technique.4. Chromosome number influenced the growth significantly in Pacific oysters at both Year 1 and 4. Triploidy progenies were significantly larger than diploids by 79% in whole body weight and 98% in meat weight at four years of age. On the other hand, aneuploids were significantly smaller than normal diploids with almost all the growth parameters as a group. Oysters with 2n+2 chromosomes were smaller than other aneuploids. There was considerable variation in body size within each chromosome number group, especially in trisomy. The bioenergetics analysis suggested that triploids spent more energy for growth than diploids and aneuploids did, while aneuploids cost less energy for更多还原