【作者】 孔令锋；

【导师】 王如才； 王昭萍； 李琪；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2004， 博士

【摘要】 本研究以我国重要的经济贝类太平洋牡蛎(Crassostrea gigas)作为研究对象，为探讨二倍体和三倍体繁殖期间能量来源、能量在体内的流动、繁殖同糖原代谢的关键酶-糖原磷酸化酶和糖原合成酶的关系、糖原酶在繁殖季节糖原分解和合成中的作用，通过生化方法对二倍体和三倍体牡蛎2003-2004年度性腺和闭壳肌生化成分的周年变化、繁殖期糖原磷酸化酶和糖原合成酶活力的季节变化作了详细的研究。另外，通过组织切片、扫描电镜和透射电镜等方法比较了二倍体和三倍体太平洋牡蛎的几种组织器官的细胞大小、鳃的表面结构和鳃的超微结构，二倍体和四倍体精于的超微结构上的差异，旨在为更好的发展多倍体育种技术、探索多倍体的快速生长机制提供理论依据。研究结果如下： 一、三倍体牡蛎鳃上皮细胞核长轴、外套膜与唇辦结缔组织中血淋巴核径的大小明显大于二倍体(P＜0.001)，二倍体牡蛎直肠上皮细胞的高度与三倍体差异不明显(0.05＜P＜0.1)。对鳃丝的超微结构和表面结构的观察结果显示，二倍体和三倍体鳃丝的粘液细胞有明显的差别，三倍体中粘液细胞中的分泌颗粒较二倍体小，染色较浅；三倍体牡蛎鳃丝间的距离较二倍体大，而且三倍体牡蛎鳃丝间通过丝间连接形成的进出水流的孔洞也较二倍体大，三倍体牡蛎鳃丝的内部结构比二倍体更发达，表明二倍体和三倍体可能在鳃的功能上存在差异。 二、对四倍体牡蛎精子的透射电镜观察结果表明，四倍体太平洋牡蛎的精子的形态结构与其二倍体基本相同，都是由头部、中段和鞭毛三个部分组成。四倍体顶体长平均1.02±0.06μm，二倍体顶体长平均0.88±0.08μm，四倍体核径长为1.93±0.08／μm，二倍体核径长为1.87±0.11μm，四倍体顶体和精核明显大于二倍体(P＜0.05)．四倍体太平洋牡蛎的部分精于至少存在5个以上的线粒体，而且这些线粒体并不位于一个层面上。四倍体精子比二倍体更多的线粒体，可能是由于四倍体精于更大的体积需要更多的线粒体来提供授精时所需的能量。 三、对二倍体与三倍体太平洋牡蛎的性腺和闭壳肌生化组分含量2003．4-2004．3的年度变化的分析结果显示：二倍体牡蛎性腺糖原含量在配子发生过程中明显降低，至性腺成熟时降低了78.57％(P＜0.01)，繁殖期维持在较低水平上，繁殖期过后逐渐升高，10月份至翌年3月份都保持较高的水平。而性多倍体太平洋牡蝠组织学与生化研究腺蛋白质和甘油三脂的含量在配子发生季节则显著增高(P<0.001)，至性腺成熟时分别增高110.8%和104.5%，繁殖期逐渐下降，可能是由于性腺排放所致，冬季水温较低时，蛋白质含量趋于逐渐上升趋势，而甘油三脂含量趋于稳定。三倍体牡蜘性腺的糖原含量除在7月份略有下降外，在整个年度变化均保持较高水平(21.7%一29.2%)，三倍体蛋白质和甘油三脂含量年度变化趋势与二倍体类似，也随着配子发生逐渐上升，在上升到顶点后逐渐下降，但从时间上推迟了约1个月。以上研究结果表明在太平洋牡蝠的性腺发育过程中，糖原大量分解提供了性腺发育所需的主要能量，三倍体由于不育导致糖原利用模式与二倍体存在巨大差异。二倍体和三倍体闭壳肌糖原含量在配子发生期间都存在下降的现象，二倍体下降了67.9%，三倍体下降了392%，下降幅度小于二倍体，繁殖期后闭壳肌糖原含量都有所回升，冬季(10月一翌年2月)二倍体和三倍体闭壳肌糖原含量水平较低。二倍体和三倍体闭壳肌蛋白质和甘油三脂含量在配子发生期逐渐上升，在繁殖期出现下降现象，表明闭壳肌在繁殖过程中是重要的能量贮存和转化器官，在配子发生过程中闭壳肌主要靠分解糖原来提供繁殖所需能量，在繁殖期也消耗蛋白质和甘油三脂供应能量。在非繁殖期二倍体和三倍体性腺和闭壳肌生化成分含量受倍性影响不大。 四、对糖原磷酸化酶和糖原合成酶活力的季节变化研究结果显示，在配子发生期间，二倍体性腺总糖原磷酸化酶(GPT)和糖原磷酸化酶活性形式(GPa)的活力随着糖原含量的下降而下降，繁殖期间，糖原含量保持在较低的水平上，GPT和GPa活力有上升趋势，但无统计学差异;繁殖期后，糖原水平逐渐回升，GPT和GPa有下降趋势。在性腺成熟过程中，闭壳肌GPT和GPa活力随着糖原含量逐渐下降而下降，繁殖期后，闭壳肌GPT和GPa各月份活力无统计学差异。 二倍体牡蝠性腺总糖原合成酶(GST)在配子发生过程中活力呈增高趋势，而糖原合成酶活性形式(GSD活力在这一时期呈下降趋势，表明性腺在配子发生时期对糖原合成酶的调控主要是控制非活性形式到活性形式间的转化，二倍体性腺GST和GSI活力在8一9月份出现明显的升高伊<0.05)，这时性腺糖原含量也明显的回升，表明在繁殖期结束后牡蜘通过提高总合成酶的活力和非活性形式的转化两种手段来在体内重新积累糖原，而糖原水平的提高又反过来抑制了GSI的活力，使得9一10月份性腺Gsl活力下降(P<0.05)。二倍体牡蜗闭壳肌GST、GSI活力和GSI%(酶活性形式与总酶的活力比)在配子发生季节表现出逐渐下降的趋势，表明在糖原分解的过程中闭壳肌中糖原合成酶活力是受到抑制的，二倍体闭壳中国海洋大学博士论文肌GST、Gsl活力9一10月份存在一个明显的上升(P更多还原

【Abstract】 For determining the energy resource, energy mobilization of reproduction, and the relationship of reproduction and glycogen metabolism, biochemical composition during a annual period, glycogen phosphorylase and glycogen synthetase during sexual maturation were compared in diploid and triploid Pacific oyster (Crassostrea gigas), and cell size of several organs, ultrastructure and surface structure of the gill in diploid and triploid Pacific oyster, ultrastructure of spermatozoa in diploid and tetraploid Pacific oyster was also compared with histology method in this study to examining the mechanism of fast-growing in polyploidy. The result showed that:1 Gill epithelium nucleus major-axis size, hemolymph nucleus diameter in the connective tissue of mantle and labial palps were significantly (P0.001) larger in triploids than in diploids, no significantly differences were found in rectum epithelium height between diploids and triploids. The results support the hypothesis of Polyploid gigantism and show the effect of polyploidy on nuclear size. The ultrastructure of gill filament show significant difference in the mucus cells of diploid and triploid oyster, and the surface structure of the gill of diploid and triploid oyster show that filament width and inter-filament distance are larger in triploids than in diploids, the minuteness structure of filament are more delicate in triploids than in diploids, and the dimension of holes which formed by connection of inter-filament are greater in triploids than in diploids. That indicates there might be differences in respiring and feeding between diploid and triploid Pacific oysters.2 The ultrastructure of spermatozoa in diploid and tetraploid showed that ultrastructure of tetraploid oyster was similar to diploids. The mean size of acrosomes (1.02 0.06m vs. 0.88 ?0.08m), nuclear (1.93 0.08m vs. 1.87 0.11m) were larger than that from diploids, and number of mitochondria were more than diploids (5 or more than 5 to only 4 in diploid); moreover, these mitochondria were not in one layer in tetraploids. The increased number of mitochondria in tetraploids seemed to adapt the demanding of more energy for fecundation in tetraploids because of larger size.3 Biochemical compositions in gonad and adductor muscle between diploids and triploids in the Pacific oyster, Crassostrea gigas, during an annual period wereexamined. The results indicate that biochemical composition in oysters is closely related to its sexual maturation. As sexual maturity progressed, the glycogen content of gonad in diploids decreased (decreased by 78.57%, P<0.01), and maintained low level during spawning, then increasing, in winter (October to February of next year) glycogen content keep higher level than that of spawning. However, protein and triglyceride content increased with gametogenesis (increased 110.8% and 104.5%, respectively) and decreased in spawning. Glycogen levels of gonad in triploids also declined, but at a slower rate, and after spawning glycogen content of triploid maintained in a high level (21.7%~29.2%), protein and triglyceride content in gonad showed similar p attern of utilization as diploid. The results from above suggested that glycogen was the major energy resource for reproduction, triploid showed a different pattern of glycogen utilization. Glycogen content of adductor muscle in diploid and triploid oysters both decreased during reproductive period, but in triploid decreased slightly (67.9% in diploids vs. 37.2% in triploids), then increased after spawning in both diploids and triploids, in winter glycogen content of adductor muscle maintained lower level. Protein and triglyceride content in adductor muscle increased during gametogenesis, and then deceased in spawning. The results from adductor muscle suggesting adductor muscle is an important organ for energy storage and mobilization, and during gametogenesis, glycogen break down in adductor muscle provided energy for gonad maturity, protein and triglycerides also break down to provided energy during spawning.4 Seasonal ch更多还原