摘要
日本鬼鲉是一种有较高经济价值的的鱼类。本文从日本鬼鲉不同组织同工酶差异,基于Cyt b,COII基因序列的系统发育地位分析和多态性微卫星引物的筛选等三方面进行了研究。
采用垂直聚丙烯酰胺平板电泳技术,对象山港野生日本鬼鲉的9种组织器官的8种同工酶进行研究,并分析了其酶谱表型。结果表明,琥珀酸脱氢酶在所有组织中均存在,组织间差异较小;乳酸脱氢酶、苹果酸脱氢酶、苹果酸酶、酯酶、醇脱氢酶、过氧化物酶、超氧化物歧化酶则具有明显的组织特异性,且这些酶在表型、分布和活性上均表现出高度的组织特异性。
根据鲉行目鱼类cyt b和COⅡ上下游保守序列各自设计引物,对日本鬼鲉的线粒体DNA进行PCR扩增,克隆并测定了cyt b和COⅡ因的全序列, COⅡ基因为691 bp和cyt b为1141 bp。从GenBank下载鲉形目28种鱼类的cytb部分或全序列和10种鱼类的COⅡ部分或全序列,然后进行比对分析,用NJ法和ML法构建分子系统发育树。系统发育树结果显示:线粒体cytb和COⅡ基因序列构建的分子系统树,和传统分类方法的结果基本一致;毒鲉科的日本鬼鲉和鲉科的关系最近;在飞角鱼科(Dactylopteridae)是否属于鲉形目还是单独成飞角鱼目的问题上,本研究比较支持飞角鱼科(Dactylopteridae)独立归成飞角鱼目(Dactylopteriformes)。鲉形目的COⅡ与昆虫类的COⅡ不同,鲉形目的COⅡ基因比cyt b基因保守,即COⅡ基因比cyt b基因进化慢。COⅡ基因进化树相应的节点上数值稍微较高。
用磁珠富集法构建的日本鬼鲉基因组微卫星文库中共有近1000个单菌落。随机挑选352个克隆用载体引物进行第二次筛选,获得阳性候选克隆262个并随机选择了174个克隆进行测序。测序结果表明,156个克隆含有明显(重复次数大于5次)的微卫星重复单元,共含有181个微卫星结构域。两碱基重复次数大于12或三碱基重复次数大于8的克隆共67个。所获得含微卫星的序列总长度范围是152-854bp,平均长度在330 bp左右。其中两碱基重复单元出现的次数最多,占所有分离的微卫星数目的79.0%,三碱基重复单元出现的次数居次,10.5%,此外还发现一碱基重复,四碱基重复,五碱基重复等多种重复类型,共占分离微卫星数目的10.5%,在获得的181个微卫星位点中,完美型微卫星序列共有98个,占54.1% ;非完美型的66个,占36.5% ;复合型的17个,占9.4%。对两碱基复次数大于12或三碱基重复次数大于8的克隆的67个克隆,用Primer premier 5.0引物设计软件设计65对引物,并随机挑选32个合成。用32对引物在厦门日本鬼鲉群28个个体进行PCR扩增,筛选出25对有效扩增的微卫星引物,其中19对微卫星引物在此群体有高度多态性。每个位点等位基因个数(A)在4-14之间,观察杂合度在0.500–0.892之间,期望杂合度0.521–0.914之间。有两个位点(J004, IJ014)严重偏离Hardy-Weinberg平衡。本文首次报道了日本鬼鲉微卫星DNA标记的多态性筛选,在毒鲉科鱼类中也尚属第一次。
The devil stinger, Inimicus japonicus, was a species with higher economic value. Three aspects coverd by this study are the isozyme patterns in various tissues of Inimicus japonicus, the phylogenetic analysis of it in Scorpaeniformes base on mitochondrial cyt b、COⅡand the isolation and characterization of high polymorphic microsatellite markers in it.
Eight isozymes in 9 tissues or organs of Inimicus japonicus were studied by the vertical polyacrylamide gel electrophoresis, then the phenotypes and the expressions of the isozymes were analyzed. The results showed that the isozyme of Succinatedehydrogenase(SuDH) existed in all these tissues, showing little differences in various tissues. The isozymes of Lactic dehydrogenase(LDH)、Malate dehydrogenase(MDH)、Malic enzyme(ME)、Esterase(EST)、Alcoholdehydrogenase (ADH)、Peroxidase(POD) and Superoxide dismutase(SOD) showed clearly tissue-specific bands. And these enzymes in thier phenotype, distribution and activity all showed high degree of tissue specificity.
The entire mitochondrial cyt b and COⅡs equence of Inimicus japonicus, 691 Bp for COⅡand 1141 bp for cytb,was cloned, whose primers was desiged by the upstream and downstream sequences of the mitochondrial cyt b and COⅡconserved sequences in Scorpaeniformes.They was compared respectly with the mitochondrial cyt b sequences of 28 other fish in Scorpaeniformes and the mitochondrial COⅡs equences of 10 other fish in Scorpaeniformes downloaded from GenBank, building molecular phylogenetic tree by NJ method and ML method. Phylogenetic tree showed that: phylogenetic trees constructed by mitochondrial cyt b and COⅡs equences was consistent with traditional classification methods;the relations of Inimicus japonicus (Synanceiidae) and Scorpaenidae was mostly close; Dactylopteridae to build up Dactylopteriformes was more reasonable than to be Scorpaeniformes in this paper. Differently with the mitochondrial COⅡin the insects, the mitochondrial COⅡgene was conservative than he mitochondrial cyt b gene in Scorpaeniformes, that is, the mitochondrial COⅡgene evolved slower than the cyt b gene in Scorpaeniformes. Therefore, the value of the nodes in the phylogenetic tree of the mitochondrial COⅡgene was corresponding slightly higher.
There are almost 1000 colonies in the enriched library. 352 colonies were selected randomly by the second PCR screening. The second PCR screening was performed using the primers of T vector, and 262 positive clones were obtained in which 174 positive clones were Sequenced. Sequencing results showed that 156 clones contained microsatellite repeat unit (repeat number > 5), including 181 microsatellite doman. There were 67 clones contained two basic repeat whose repeat number > 12 or three basic repeat whose repeat number >8. The length of the sequence contained microsatellite doman ranged from 152 to 854bp, with an average of 330bp. The two basic repeat was the most abundant motifs with a percentage of 79%, and three basic repeat was the second abundant motifs with a percentage of 10.5% .Several other repeat types were also detected , accounting for 10.5%. Among all the 181 microsatellites, 98(54.1%) sequences were perfect type, 66(36.5%) were imperfect type and 17 (9.4%) were compound type. The 65 pairs of primers were designed using Primer premier 5.0 according to the 67 clones and 32 pairs selected randomly were synthized. The PCR amplification were performed in 28 individuals from the xiamen population with the 32 pairs primers. As a result, 25 pairs of primers were screened effective and 19 loci showed polymorphism in this population. The number of alleles per locus ranged from 4 to 14. The ranges of observed and expected heterozygosity were 0.500–0.892 and 0.521–0.914, respectively. Significant deviations from Hardy–Weinberg equilibrium were detected at two loci. These were the first microsatellite loci characterized from I. japonicas, even the first microsatellite loci developmented from Synanceiidae.
引文
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