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Isozyme Patterns in Various Tissues, Phylogenetic Analysis in Scorpaeniformes Base on Mitochondrial cyt B、COⅡof Inimicus Japonicus and Isolation and Characterization of High Polymorphic Microsatellite Markers in It

Author: DongXuHui
Tutor: LiMingYun
School: Ningbo University
Course: Aquaculture
Keywords: Inimicus japonicas isozyme tissue specific expression cyt b COⅡ phylogenetic analysis microsatellite polymorphism
CLC: S917.4
Type: Master's thesis
Year: 2011
Downloads: 21
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Abstract

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.

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