【作者】 颜勤；

【导师】 罗琛；

【作者基本信息】 湖南师范大学 ， 动物学， 2004， 硕士

【摘要】 超氧化物歧化酶(SOD)是一种对生物细胞保护至关重要，在进化上比较保守的酶。因此，超氧化物歧化酶作为分子钟或分子标记已被广泛应用于生物进化研究、群体遗传结构分析以及品系鉴定。但目前人们对鱼类超氧化物歧化酶的生物化学和遗传学特性都尚未进行过系统和深入的研究。为使这一重要的分子标记能更好地应用于鱼类遗传育种、种质资源保护以及进化研究，本实验采用聚丙烯酰胺梯度凝胶垂直电泳法，研究了草鱼线粒体型超氧化物歧化酶(fm-SOD)的同功酶形式、生化遗传表型、在不同组织中的分布、亚基组成以及金属类型。所得实验结果如下： (1)草鱼线粒体型超氧化物歧化酶有三种不同的同功酶形式；按其在酶谱上迁移率的不同，从正极到负极分别命名为fm-SOD 1，fm-SOD 2，fm-SOD 3，其中fm—SOD 1迁移率最快，而fm-SOD 3迁移率最慢。每种不同的fm—SOD均由两条相关的酶带组成，分别命名为主带和亚带。主带相对于亚带而言，迁移率较小而酶活力较高。 (2)在草鱼群体中三种fm-SOD的不同组合可构成三种不同的草鱼线粒体型超氧化物歧化酶生化遗传学表型，即表型1、表型2与表型3。表型1个体只含有迁移率最快的fm-SOD 1同功酶；表型3个体只含有迁移率最慢的fm-SOD 3同功酶；而表型2个体中含有所有三种不同形式的同功酶。在表型2中，当这三种不同形式的同功酶均存在的时候，fm-SOD 2活力比fm-SOD 1和fm-SOD 3高，而fm-SOD 1与fm-SOD 3活力相当。 (3)在草鱼心脏、肝脏、脾脏、肾脏、肌肉、尾鳍六种不同的组织中，草鱼线粒体型超氧化物歧化酶存在三种不同的同功酶形式和三种不同的生化遗传学表型。同一草鱼个体的线粒体型超氧化物歧化酶在六种不同组织中表现出相同的酶谱类型，但在不同组织中，酶活性有所不同。其中肝脏中活性最高，而其他组织活性相当。 (4)在野生草鱼群体中，存在所有三种表现型；而在基因纯合型的雌核发育草鱼群体中只检测到表型1和表型3个体。野生草鱼群体中三种表现型的个体数之比为15：51：28。经过X~2测试，发现其比值符合常染色体上一个基哪座位~对等位基因1:2:1的孟德尔遮传分离比例。筑麟摧燕巍;基霉、(封草鱼f二s0D由两个亚基组成。更多还原

【Abstract】 Superoxide dismutase is an evolutionarily conserved enzyme with an important role in protecting organisms and cells from the damages caused by oxygen free radicals. As a molecular clock and genetic marker, superoxide dismutase has been widely used in evolutionary studies, population genetics, and identification of different strains in the same species. However, the biochemical and genetic features of the mitochondrial superoxide dismutase have not been well characterized in fishes. In order to make this molecular marker applied in fish breeding, species protecting and evolutionary research further, we investigated the isozyme forms, biochemical genetic phenotypes. And we also study the distribution of different tissues and subunit construction of Ctenopharyngodon idellus (grass carp) mitochondrial superoxide dismutase (fm-SOD) with polyacrylamide gradient gel electrophoresis in this study. Our data revealed as follows:(1) There are three isoforms of fm-SOD which were named as fm-SOD 1, fm-SOD 2 and fm-SOD 3 according to their positions from the positive pole to the negative pole. Fm-SOD 1 is the fastest, while fm-SOD 3 is the slowest. In every different fm-SOD there are two bands, which is named as major band and sub-band. The major band migrates a little more slowly, while at the same time shows the higher activity.(2) The combination of three isoforms of fm-SOD constitutes three distinct biochemical phenotypes: phenotype 1, phenotype 2 and phenotype 3. Phenotype 1 was only associated with the fastest migratory isoform fm-SOD 1 and phenotype 3 with the slowest migratory isoform fm-SOD 3, while phenotype 2 was associated with all three isoforms. In phenotype 2, whileall of the three isoforrs all exist, fm-SOD 2 shows higher activity than that of fm-SOD 1 and fm-SOD 3, but fm-SOD 1 and fm-SOD 3 seem to be the same.(3) In the six tissues, such as heart, liver, spleen, kidney, muscle and tailfin, the three isoforms and the three phenotypes all exist. Six tissues of the same individual show the same zymogram while they have different activity: the activity of liver is highest and that of other tissues seems to be the same.(4) In wild group of grass carp, all the three phenotypes were observed, whereas in mito-gynogenetic group, only phenotypes 1 and 3 were observed. The ratio of the individual numbers of the three phenotypes is 15:51:28. After X2 test for probability, we find that the ratio of the three phenotypes in the wild group was consistent with the 1:2:1 ratio of Mendelian inheritance for 2 alleles of a single locus in the autosomal chromosomes.(5) In addition, all of the three fm-SODs were sensitive to the mixture of 15% ethanol and 25% chloroform but resistant to H2O2, indicating that the fm-SOD in grass carp is Mn-SOD.These results suggested that: (i) the mitochondrial superoxide dismutase gene in grass carp resided in chromosomes instead of mitochondrial DNA; (ii) mitochondrial superoxide dismutase was encoded by a single locus; (iii) there were two variant alleles at least in grass carp and (iv) the fm-SQD is composed of two subunits.更多还原