【作者】 王昌留；

【导师】 张士璀；

【作者基本信息】 中国海洋大学 ， 海洋生物， 2003， 博士

【摘要】 文昌鱼被认为是和脊椎动物亲缘关系最近的动物。自Stricht 1895年报道文昌鱼的染色体以来，迄今只报道了Branchiostoma lanceolatum、B．floridae、B．belcheri三种文昌鱼染色体数目。有关文昌鱼染色体核型和带型的研究尚未见任何报道，文昌鱼染色体核型和带型的研究将为比较基因组学和细胞遗传学研究提供珍贵的背景资料。本文首次报道了青岛文昌鱼染色体的核型和带型。 1．染色体数目(2n)和臂数(FN) 用青岛文昌鱼的胚胎细胞作材料，对其染色体数目和核型进行研究。计数150个染色体分散好的中期相，发现染色体数目的变化范围是18至38条，长度为1.4至3.7μm，染色体的众数是36，占150个中期相的66％，说明青岛文昌鱼染色体的数目是36。在36条染色体中，除第一对染色体属亚端部着丝粒染色体(st)外，其余均为端部着丝粒染色体(t)，未见中部着丝粒染色体(m)和亚中部着丝粒染色体(sm)及随体。因此，青岛文昌鱼染色体的核型是2n=36，2st+34t，FN=36，这是首次头索动物核型的报道。从文昌鱼染色体的核型看，似乎形态较小的端部着丝粒染色体是脊椎动物染色体的远祖。 2．G带型 用稍作修改的标准的胰酶显带技术，进行GTG带纹的显示，它们能较好地显示头索动物青岛文昌鱼的晚期囊胚和早期原肠胚的中期染色体的G带，并且重复性好。G显带时胰酶的浓度及处理时间是获得良好显带效果的关键因素，而染色体的片龄似乎并不太重要。经G显带技术处理后，65.5％的染色体区域呈阳性染色，共显示了149条带，其中阳性带为77，阴性带为65，可变带为7。 3．R带型青岛文吕鱼染色体的核型及带型研究 参照nutrillaux和Lejeune(1971)的R带显示方法，稍作修改。文昌鱼染色体共显示100条R带，其R带数目大约是脊椎动物的1/4至1/5。在100条R带中阳性带有57个，阴性带有33个，可变带有10个，约有722%的染色体区域显示R带阳性结果。R带的数目与基因的多少相关，许多哺乳动物的基因已精确地进行了染色体定位，大量报道证明它们一般分布在R带上，而不是G带上，文昌鱼R带的数目恰恰与脊椎动物起源时发生了两次基因倍增的假设相吻合。4.G、R带型的互补性 一般认为，染色体的带纹与其碱基组成密切相关，R阳性带对应于GC含量丰富的区域，G阳性带则对应于AT含量丰富的区域。因此，G阳性带通常对应于R阴性带，反之亦然。精确计算和比较每条染色体的G、R阳性和阴性带纹特征，发现有些染色体呈一定的互补性，但这种互补性在文昌鱼染色体上较低，大约只有38%的部位存在互补现象。为何存在这种现象，其原因现在尚不清楚。可能是文昌鱼的GC和Ar“丰富区”和脊椎动物不一致，其DNA的碱基组成是一种“中间类型”，即GC和AT碱基组成没有明显的区段划分或者是碱基组成丰富程度相同，使许多G、R阳性带在染色体上重叠。5.C带型 用Sumne:(1 972)介绍的C显带方法，稍作修改后显示文昌鱼的染色体，在所有染色体上都存在C带，大约54.3%的染色体区域显示C阳性结果，其总带数是64个。其存在位置上除经典的着丝粒区、近着丝粒区、中间区、端部有其分布外，还发现某些染色体全部为C带着色及某些区域的C带是可变的。同脊椎动物相比，文昌鱼染色体的C带所显示区域的比率要高。众所周知，C带显示的是结构异染色质，虽然有人提出它具有许多功能，但一般认为异染色质是由大量的“静止”基因或“无功能”的DNA片段构成，含很少的活动基因。因此，文昌鱼染色体含有较少的活动基因也许是其C带比率高的原因。6. NOR带型 核仁组织者是染色体上的核糖体RNA(rRNA)的基因，可用银染的方青岛文昌鱼染色体的核型及带型研究法证实其存在。按照稍作修改的Howen和Black(1 980)的方法进行NOR带显色，发现NOR带位于第十二对染色体的着丝粒区，NOR带的数目是2个。按照Amemiya(1 990)和Martins(1995)的观点，从文昌鱼NOR带的特征看，头索动物是较鱼类、两栖类、爬行类、哺乳类和人等低等的类群。7.性染色体 文昌鱼第二对染色体分为2种类型A和B，在30个中期相中，有14个中期相的第二对染色体属于B类型，该类型的染色体无论形状还是相对长度都相同，定名为ZB。30个中期相的另16个的第二对染色体属A类型，虽然它们的形状相同，但其相对长度有极显著的差异(P<0.01)。将较长的第二对染色体的一个命名为2A’，另一个命名为ZA。进一步分析ZA染色体时，发现它在相对长度上与2A’染色体有很大的不同，但其在形状和相对长度上完全同ZB染色体。因此，ZA染色体和ZB染色体是相同的染色体。由于第二对即2A’和ZA染色体在形态上有差别，推测它们可能是性染色体。G、R、C显带结果表明，这对染色体的G、R、C带纹均显示有明显的差别，这进一步确证了它们是性染色体的观点。虽然Nogusa(1 957)认为B.belcheri存在XY型的性染色体，至于青岛文昌鱼的性染色体是XY型或ZW型尚待深入研究。更多还原

【Abstract】 Amphioxus or lancelet, a cephalochordate, is the extant invertebrate chordate considered to be the closest relationship to the vertebrates. It has been more than 100 years since the first description of the chromosomes of the amphioxus Branchiostoma lanceolatum by Stricht in 1895. However, only three species of the cephalochordate including B. lanceolatum, B. floridae, and B. belcheri, have so far been studied for their chromosome numbers, and the karyotypic analysis and banding study of amphioxus chromosomes remains largely untouched. Knowledge of the chromosomal karyotype and banding in amphioxus could be of particular use for the extensive study of comparative genome and cytogenetics. In this paper, the karyotype and banding pattern of amphioxus B. belcheri tsingtauense were reported for the first time. 1. Chromosome number (2n) and fundamental number (FN)The chromosome number and karyotype of amphioxus Branchiostoma belcheri tsingtauense were studied using embryonic cells of the animal. The counting of 150 well-spread samples revealed that the number of the chromosomes ranged from 18 to 38, but the modal number of the chromosome was 36, which was in proportion to 66%. This showed that the diploid chromosome number (2n) of amphioxus B. belcheri tsingtauense was 36.The chromosomes were extremely small, ranging from 1.4 to 3.7 um in length. The first pair of the 36 chromosomes of B. belcheri tsingtauense was subtelocentric and the other 17 pairs telocentric. Neither metacentric nor submetacentric chromosomes nor satellites were observed. Therefore, the karyotype of theamphioxus is 2n=36, 2st+34t, FN=36. This is the first report on the karyotype of the cephalochordata. It is likely that telocentric chromosomes with minute sizes may be ancestral types of the vertebrate chromosomes.2. G-bandingThe GTG banding (G-banding) was carried out by the standard trypsin method with slight modification, which works well for protochordate because a good number of reproducible G-bands are consistently obtained from the embryonic cells of late blastulae and early gastrulae of amphioxus B. belcheri tsingtauense. The trypsin treatment for G-banding is an important factor in obtaining well-banded chromosomes, while the slide aging is not. After G-banding, 65.5% of the chromosome surface is positively stained and the total number of G-banded is 149. There are 77 positive, 65 negative and 7 variable bands among the 149 G-bands.3. R-bandingThe RHG banding (R-banding) technique used was the method of Dutrillaux and Lejeune (1971) slightly modified. The total number of R-bands found in the chromosomes is 100, which is approximately 1/4 to 1/5 of that of the vertebrate metaphase chromosome R-bands. They consist of 57 positive, 33 negative and 10 variable bands. As the number of R-bands is associated with that of genes, and most genes that have been mapped to mammalian chromosomes with sufficient precision are usually distributed in R-bands rather than in G-bands. This is apparently in agreement with the hypothesis of two rounds of extensive gene duplication leading to the origin of the vertebrates. In addition, there exists 72.2% of the chromosome surface positively stained with R-banding.4. Complementarity of G- and R- banding patternIt is generally accepted that chromosome banding patterns appear closely related to differences in DNA base composition, and R-bands correspond to GC-rich DNA and G-bands to AT-rich DNA. Bands positively stained after G-banding are commonly negatively stained after R-banding and vice versa.The complementarity of G- and R-band patterns was evaluated by comparingthe features of each positive and negative chromosomal G- and R-band. It was found that the complementarity is only illustrated to some extent by the parts of some chromosomes. The G- and R-band patterns are found to be only about 38% complementary. The reason for this is unknown at the present. Possibly, CG-rich and AT-rich regions in amphioxus DNA are not as "rich" as those in the vertebrate DNA, that is, a更多还原