| In this study, the great O. argus var Kimnra and Channa argus were taken as research materials. A comparative study on the methods of morphology, biology, histology, molecular biology was done. Comprehensive discusses the classification, characteristics and mechanism of skin albinism and the the anti stress ability of introduction and breeding process etc.. 1. Phylogenetic relationship among Channa species using morphological methodThe fish bodies of five Channa species were measured using traditional morphometric and frame analysis. In terms of 11 countable properties and 29 measurable characters of a total of 89 individuals, principal component analysis showed that the cumulative contribution of all five main components was 78.928%. Cluster analysis showed that the species could be clearly distinguished from each other. The O. argus var Kimnra had a close phylogenetic relationship with C. argus. Morphometric and frame data analysis revealed no obvious difference between them. Global materials of the five Channa species were photographed by X-ray, the O. argus var Kimnra and C. argus shared a similar result. From above, we can know that the O. argus var Kimnra is similar to C. argus, and has no significant difference. 2. Genetic diversity and intergeneric relationships among eight Channa species based on AFLP-capillary electrophoresisTen Eco R I/Mse I primer pairs were selected out and used for further practical AFLP analysis. The result showing that the total amplified bands, consistency bands and polymorphic bands ranged from 114 to 206, from 0 to 12 and from 114 to 196, respectively. The dendrogram generated with NTSYS software indicated that the eight Channa species could be divided into four major clusters according to the genetic similarity coefficient of 0.58. The O. argus var Kimnra was closest to C. argus. These results could be useful for conserving the eigth Channa species and guiding genetic breeding research. 3. Phylogenetic relationship among Channa species based on complete sequence of mt DNA and its 12 s RNA, Cytb, CO1 gene and its D-Loop sequencesThe Mitochondrial DNA genome sequence of the O. argus var Kimnra was first measured and consistent with C. argus. The genetic distance between O. argus var Kimnra and C. argus is 0.002, which has not yet reach up to subspecies differentiation level. Molecular phylogenetic tree was built using NJ, ME and ML method, the O. argus var Kimnra, C. argus and C. maculata x C. argus gather together. At the same time, 12 s r RNA, CO1 and Cytb gene, as well as D-Loop control region sequences were analyzed. We found that the largest genetic distance between O. argus var Kimnra and C. argus were less than 0.010, which are 0.006, 0.002, 0.002 and 0.004, respectively. Molecular phylogenetic tree showed that all the haploids gather together as a branch and crossing each other. These indicate that the O. argus var Kimnra should be regard as a albino species of C. argus. 4. Compare and analysis of basic nutrient components, Skin melanin content and its histologic in O. argus var Kimnra and C. argusBy comparing the nutrition value, the O. argus var Kimnra had a higher nutritional value than C. argus. Meanwhile, the melanin content of O. argus var Kimnra skin significantly lower than C. argus. The distribution, density, shape, size and different growth state have obvious differences with each other. The results showed that the pigment layer tends to degradation, no obvious melanin cells in O. argus var Kimnra skin by H&E and Masson Fontana technology. Then the ultrastructure was observed using transmission electron microscopy. The results showed that the melanocytes melanosomes of the O. argus var Kimnra skin is lighter, disorganized and showing a variety of shapes, as well the irregular arrangement of cells. The lack of melanin cells lead to the histological characteristics of its albinism. 5. Molecular cloning, characterization and expression analysis of TYR gene O. argus var KimnraThe full-length c DNA of O. argus var Kimnra and C. argus TYR were cloned and characterized, BLAST analysis showed that the sequences between them are consistent. Homology analysis indicated that it has a highest similarity with teleost, but a low similarity with bacterias. Relative quantitative real-time PCR(q RT-PCR) revealed tissue specific TYR expression. The highest expression is in eyes of O. argus var Kimnra, while in skin of C. argus. Compare the same tissues, the expression in skin of C. argus is significantly higher than O. argus var Kimnra. Then the immunofluorescence observation of subcellular localization was mainly distributed in the endoplasmic reticulum and Golgi membrane, cytoplasm, consistented with the prediction results. From these results we can know that the TYR may be involved in the development, differentiation and functional regulation of the skin melanoma cells. Its low expression is a one of the key factor leading to the skin albinotic characteristics. 6. Molecular cloning, characterization and expression analysis of MITF gene in O. argus var KimnraThe full-length c DNA of O. argus var Kimnra and C. argus MITF were cloned and characterized, BLAST analysis showed that the sequences between them are consistent. Homology analysis indicated that it has a highest similarity with teleost, but a lowest similarity with insects. q RT-PCR revealed tissue specific MITF expression. The highest expression of MITF is in heart of O. argus var Kimnra, while in muscle of C. argus. Compare the same tissues, the expression in skin of C. argus is significantly higher than O. argus var Kimnra. Then the immunofluorescence observation of subcellular localization was mainly distributed in the nucleus, and weak fluorescence was also detected in cytoplasm and mitochondria, which consistented with the prediction results. From these results we can know that the MITF may be involved in the regulation of skin color, and its low expression may be closely related to the occurrence of skin albinotic characteristics. 7. Molecular cloning, characterization and expression analysis of SOX10 gene in O. argus var KimnraThe full-length c DNA of O. argus var Kimnra and C. argus SOX10 were cloned and characterized, BLAST analysis showed that the sequences between them are consistent. Homology analysis indicated that it has a highest similarity with teleost, but a lowest similarity with vertebrates. q RT-PCR revealed tissue specific SOX10 expression. The highest expression of SOX10 is in eyes of O. argus var Kimnra, while in skin of C. argus. Compare the same tissue, the expression in skin of C. argus are significantly higher than O. argus var Kimnra. Then the immunofluorescence observation of subcellular localization was mainly distributed in the nucleus, consistented with the prediction results. From these results we can know that the SOX10 may be involved in the regulation of the body color. Its low expression may be one of the important factor of skin albinotic characteristics. 8. Molecular cloning, characterization and expression analysis of heat shock protein 60 gene in O. argus var KimnraThe full-length c DNA of O. argus var Kimnra HSP60 gene were cloned and characterized. Homology analysis indicated that its amino acid sequence has a highest similarity with teleost, and low similarity with bacterias. q RT-PCR revealed tissue specific HSP60 expression. Compared to the normal temperature treatment group(26°C), expression was significantly increased after heat shock(37°C) in brain, kidney, liver and spleen, but showed no significant differences after cold treatment(8.5°C) except the brain. Then Edwardsiella tarda(strain NO. DL1476) injection in different time points(0-, 6-, 12-, 24-, 36-, 48-, 72h) were investigated. HSP60 m RNA levels were significantly increased after different post injection time. These results indicate that HSP60 expression might be sensitive to pathogen infection, but more related to thermal resistance. 9. Molecular cloning, characterization and expression analysis of heat shock protein 70 gene in O. argus var KimnraThe full-length c DNA of O. argus var Kimnra heat shock protein 70 gene were cloned and characterized. Homology analysis indicated that its amino acid sequence has a high similarity with other species. q RT-PCR revealed tissue specific HSP70 expression. Compared to 26°C, expression was significantly increased in brain, kidney, liver and spleen after heat shock, but showed no significant differences after cold treatment. HSP70 m RNA levels were significantly increased after different post injection time of E. tarda. These results indicate that HSP70 expression plays a very important role in regulating the immune response and protecting the pathogenic process. 10. Molecular cloning, characterization and expression analysis of heat shock protein 90 gene in O. argus var KimnraThe full-length c DNA of O. argus var Kimnra heat shock protein 90 gene were cloned and characterized. Homology analysis indicated that its amino acid sequence has a highest similarity with teleost, but a low similarity with bacterias. q RT-PCR revealed tissue specific HSP90 expression. Compared to 26°C, expression was significantly increased after heat shock in brain, kidney, liver and spleen, but no significant differences after cold treatment. HSP90 m RNA levels were significantly increased after different post injection time of E. tarda. These results indicate that HSP90 involved in the body by adverse external stimulation of immune response. |
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