四倍体鲫鲤及相关鱼线粒体DNA的限制性酶切分析
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摘要
用密度梯度离心法和DNase Ⅰ、RNase消化法从红鲫、湘江野鲤、鲫鲤F_1、鲫鲤F_2、异源四倍体鲫鲤、湘云鲫、白鲫和野鲫的肝组织和白鲫的卵巢中提取线粒体DNA(mitochondrial DNA，mtDNA)和纯化。纯化后的mtDNA用识别6个碱基序列的9种限制性内切酶，即EcoR Ⅰ、Hind Ⅲ、Pst Ⅰ、Bgl Ⅱ、BamH Ⅰ、Xho Ⅰ、Xba Ⅰ、Sal Ⅰ和Kpn Ⅰ进行单酶酶切，用凝胶图像分析仪(Gel-Pro Analyzer)检测，根据各酶切片段的长度，推算出红鲫、湘江野鲤、鲫鲤F_1、鲫鲤F_2、异源四倍体鲫鲤、湘云鲫、自鲫和野鲫的mtDNA大小分别为16.19±0.13kb、16.61±0.05kb、16.02±0.09kb、16.02±0.09kb、16.20±0.10kb、16.24±0.14kb、16.60±0.09kb和16.06±0.17kb。根据异源四倍体鲫鲤mtDNA单酶解、双酶酶解的片段数目和分子大小，构建了异源四倍体鲫鲤mtDNA的7种限制性内切酶酶切图谱，为进一步分析异源四倍体鲫鲤及其相关鱼mtDNA的遗传及进化关系和作用奠定了基础。同时对mtDNA提取和纯化的条件优化以及分子大小的估算方法进行了探讨。
根据Nei和Li(1979)建立的“利用限制性内切酶研究遗传差异的数学模型”，计算出不同实验鱼间的限制性酶切片段共享度(F)和遗传距离(P)。结果表明有直接亲缘关系的原始母本红鲫与鲫鲤F_1、鲫鲤F_2和异源四倍体鲫鲤之间均存在较小的遗传差异，而原始父本湘江野鲤与鲫鲤F_1、鲫鲤F_2和异源四倍体鲫鲤之间存在显著的遗传差异；鲫鲤F_1与鲫鲤F_2mtDNA酶切检测结果一致，mtDNA表现出完全的真实遗传。
同时还计算了三倍体湘云鲫与其亲本间的酶切片段共享度和遗传距离，从三个群体的遗传距离比较来看，湘云鲫与母本白鲫的遗传距离(0.62)最近，三倍体湘云鲫与异源四倍体鲫鲤mtDNA Ⅰ型、白鲫与
异源四倍体娜鲤mtDNAI型的遗传距离分别为1.97，2.76，存在较小
差异。根据检测到的野娜等几个细鱼品系mtDNA的酶切结果，分析了
几个群体间的遗传和进化关系·。
通过对几个种间或群体间限制性酶切片段的统计分析，表明
mtDNA在杂交育种形成的不同群体中遵循母系遗传的特性;培育的四
倍体和三倍体既可以作为研究mtDNA母系遗传的模式，同时可为具体
分析nltONA的父系渗漏及渗漏遗传问题提供准确的依据。从mtDNA的
进化角度，根据Brown等提出的mtDNA的进化速率，估算出异源四倍
体娜鲤和三倍体湘云鲡如果要从自然进化得到，分别需经过大约5百
万年和1百万年的时间。
The mitochondrial DNAs (mtDNAs) from livers of red crucian carp, common carp, F, hybrid of red crucian carp (♀) × common carp(♂), F2 hybrid, allotetroploid crucian carp, triploid crucian carp, Japanese crucian carp, C. auratus auratus&n& ovary of Japanese crucian carp were isolated by the means of differential centrifugation and nuclease digestion, then purifing them by means of phenol extraction. After having been digested by 9 restriction enzyme recogenizing 6 base pairs: Eco R I, Hind III, Pstl. Bglll, BamH I. Xho I, Xbal. Sal I and Kpn I,the mtDNAs were analysed by using agarose gel electrophoresis and detected by Gel-Pro Analyzer. The size of mtDNAs for red crucian carp, common carp, F, hybrid, F2hybrid, allotetroploid crucian carp, triploid crucian carp, Japanese crucian carp and C. auratus auratus were 16. 19kb ± 0. 13, 16. 61 ± 0.05kb, 16. 02 ± 0.09kb, 16. 02 ± 0. 09kb, 16. 20 ± 0.10kb, 16.24 ± 0. 14kb, 16. 60 ± 0. 09kb and 16. 06 ± 0. 17kb respectively. Based on the nu
m
ber and s
ize of the restriction fragments abtained by single or double enzyme digestion, the physical map of mtDNA from allotetraploid crucian carp of 7 kinds of restriction endonucleases (Pst I. Bgl II, Bam H I> Xho I, Sal I and Kpn I) was constructed, which laid a foundation for analysing further the heretidy and evolution of allotetraploid crucian carp and other correlated fishers. At the same time, the improvement of
mtDNAs extraction and purification conditions and the method estimating molecular size were also studied.
Using "Mathematical model for studying genetic variation in terms of restriction endonucleases", established by Nel and Li (1979), calculated the proportion of shared restriction fragments (F) and the genetic distances (P) between populations. The results manifested that there was minor genetic divergence between original maternal red crucian card and F1 hybrid, F2 hybricK allotetraploid crucian carp, but there was obvious genetic divergence between paternal common carp and Fi hybrid, F2 hybrid, allotetraploid crucian carp. The digestion result between F1 hybrid and F2 hybrid was unanimous, and mtDNA showed completely real heredity. .
With the same mathematical model, the proportion of shared restriction fragments (F) and the genetic distances (P) of triploid crucian carp and its parent were calculated. By comparing the genetic distance of three populations, the genetic distance between triploid crucian carp and maternal Japanese crucian carp was smallest (P=0. 62). The genetic distance between triploid crucian carp or Japanese crucian'carp and allotetraploid crucian carp(I-type) were 1.97 , 2.76 respectively. According to the endonucleases digestion results of mtDNAs from C. auralus auratus and other strains, the hereditary and evolutional relation of several populations was analysed.
The statistical analysis of restriction fragments of several inter- species or inter-populations showed that mtDNA followed the character of matrilinear inheritance in crossing breeding. The two different breeding ways(breeding allo- tetraploid and allotriploid) could not only be regarded as the model of
researching matrilinear inheritance of mtDNA, but could offered accurate testimony for concisely analysing mtDNA paternal leakage and leakage inheritance. From the point view of mtDNA evoltion and according to mtDNA the evolution rate being raised by Brown and his coleague,we can draw conclusions that allotetraploids and allotriploids gained from the natural evolution, they need 5 million years , 1 million years respect ively.
根据Nei和Li(1979)建立的“利用限制性内切酶研究遗传差异的数学模型”，计算出不同实验鱼间的限制性酶切片段共享度(F)和遗传距离(P)。结果表明有直接亲缘关系的原始母本红鲫与鲫鲤F_1、鲫鲤F_2和异源四倍体鲫鲤之间均存在较小的遗传差异，而原始父本湘江野鲤与鲫鲤F_1、鲫鲤F_2和异源四倍体鲫鲤之间存在显著的遗传差异；鲫鲤F_1与鲫鲤F_2mtDNA酶切检测结果一致，mtDNA表现出完全的真实遗传。
同时还计算了三倍体湘云鲫与其亲本间的酶切片段共享度和遗传距离，从三个群体的遗传距离比较来看，湘云鲫与母本白鲫的遗传距离(0.62)最近，三倍体湘云鲫与异源四倍体鲫鲤mtDNA Ⅰ型、白鲫与
异源四倍体娜鲤mtDNAI型的遗传距离分别为1.97，2.76，存在较小
差异。根据检测到的野娜等几个细鱼品系mtDNA的酶切结果，分析了
几个群体间的遗传和进化关系·。
通过对几个种间或群体间限制性酶切片段的统计分析，表明
mtDNA在杂交育种形成的不同群体中遵循母系遗传的特性;培育的四
倍体和三倍体既可以作为研究mtDNA母系遗传的模式，同时可为具体
分析nltONA的父系渗漏及渗漏遗传问题提供准确的依据。从mtDNA的
进化角度，根据Brown等提出的mtDNA的进化速率，估算出异源四倍
体娜鲤和三倍体湘云鲡如果要从自然进化得到，分别需经过大约5百
万年和1百万年的时间。
The mitochondrial DNAs (mtDNAs) from livers of red crucian carp, common carp, F, hybrid of red crucian carp (♀) × common carp(♂), F2 hybrid, allotetroploid crucian carp, triploid crucian carp, Japanese crucian carp, C. auratus auratus&n& ovary of Japanese crucian carp were isolated by the means of differential centrifugation and nuclease digestion, then purifing them by means of phenol extraction. After having been digested by 9 restriction enzyme recogenizing 6 base pairs: Eco R I, Hind III, Pstl. Bglll, BamH I. Xho I, Xbal. Sal I and Kpn I,the mtDNAs were analysed by using agarose gel electrophoresis and detected by Gel-Pro Analyzer. The size of mtDNAs for red crucian carp, common carp, F, hybrid, F2hybrid, allotetroploid crucian carp, triploid crucian carp, Japanese crucian carp and C. auratus auratus were 16. 19kb ± 0. 13, 16. 61 ± 0.05kb, 16. 02 ± 0.09kb, 16. 02 ± 0. 09kb, 16. 20 ± 0.10kb, 16.24 ± 0. 14kb, 16. 60 ± 0. 09kb and 16. 06 ± 0. 17kb respectively. Based on the nu
m
ber and s
ize of the restriction fragments abtained by single or double enzyme digestion, the physical map of mtDNA from allotetraploid crucian carp of 7 kinds of restriction endonucleases (Pst I. Bgl II, Bam H I> Xho I, Sal I and Kpn I) was constructed, which laid a foundation for analysing further the heretidy and evolution of allotetraploid crucian carp and other correlated fishers. At the same time, the improvement of
mtDNAs extraction and purification conditions and the method estimating molecular size were also studied.
Using "Mathematical model for studying genetic variation in terms of restriction endonucleases", established by Nel and Li (1979), calculated the proportion of shared restriction fragments (F) and the genetic distances (P) between populations. The results manifested that there was minor genetic divergence between original maternal red crucian card and F1 hybrid, F2 hybricK allotetraploid crucian carp, but there was obvious genetic divergence between paternal common carp and Fi hybrid, F2 hybrid, allotetraploid crucian carp. The digestion result between F1 hybrid and F2 hybrid was unanimous, and mtDNA showed completely real heredity. .
With the same mathematical model, the proportion of shared restriction fragments (F) and the genetic distances (P) of triploid crucian carp and its parent were calculated. By comparing the genetic distance of three populations, the genetic distance between triploid crucian carp and maternal Japanese crucian carp was smallest (P=0. 62). The genetic distance between triploid crucian carp or Japanese crucian'carp and allotetraploid crucian carp(I-type) were 1.97 , 2.76 respectively. According to the endonucleases digestion results of mtDNAs from C. auralus auratus and other strains, the hereditary and evolutional relation of several populations was analysed.
The statistical analysis of restriction fragments of several inter- species or inter-populations showed that mtDNA followed the character of matrilinear inheritance in crossing breeding. The two different breeding ways(breeding allo- tetraploid and allotriploid) could not only be regarded as the model of
researching matrilinear inheritance of mtDNA, but could offered accurate testimony for concisely analysing mtDNA paternal leakage and leakage inheritance. From the point view of mtDNA evoltion and according to mtDNA the evolution rate being raised by Brown and his coleague,we can draw conclusions that allotetraploids and allotriploids gained from the natural evolution, they need 5 million years , 1 million years respect ively.
引文
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73 K. W. P. Miller, J. L. Dawson and E. Hageelberg. A concordance of nucleotide substitutions in the first and second hypervariable segments of the human mtDNA control region. International Journal of Legal Medicine, 1996, 109: 107-113.
74 Lansman R A et al. The use of restriction endonucleases to measure mitochondrial DNA sequence relatedness in natural populations. Ⅲ. Techniques and potential applications. J Mol Evol, 1981, 17: 214-226.
75 Lee W J and Kocher T D. Complete sequence of a sea Lamprey (petromyzoa marinus) mitochondrial genome: Early establishment of the vertebrate genome organization Genetics, 1995, 139: 873-887.
76 Lee W J et al. Structure and evolution of fish mitochondrial control regions. Journal of Molecular Evolution, 1995, 41: 54-66.
77 Magoulas A and F Zouros. Restriction-site heteroplasmyin Anchovy (Engraulis encrasicolus) indicates incidental biparental inheritance of mitochondrial DNA. Mol Biol Evol, 1993, 10: 319-325.
78 Meyer A and A C Wilson. Oririn of tetraploids inferred from thcir mitochondrial DNA affiliation to lungfish. J Mol Evol, 1990, 31: 359-364.
79 Milinkovitch et al. Revised phylogeny of whales suggested by mitochondrial ribosomal DNA sequences. Nature, 1993, 361: 346-348.
80 M. V. Derenko and G. F. Shields. Mitochondrial DNA sequence divesity in three north asian aborginal population groups. Journal molecular biology, 1997, 31(5): 665-669.
81 Nei M and W H Li. Mathmatic model for studying genetic variation in terms of restriction endonuclease. Proc Natl Acad Sci USA, 1979, 76: 5296-5273.
82 Noack K et al. The complete mitochondrial DNA sequence of the Bichir(polypterus ornatipinnis), a basal ray-finned fish: ancient establishment of the consensus vertebrate gene order. Genetic, 1996, 144: 1165-1180.
83 Palva T K and Palva E T. Rapid isolation of fish mitochondrial DNA with a commericially available plasmid isolation kit. The progressive fish-culturist, 1993, 55: 125-127.
84 Perna, N T Kocher, T D Kocher. Mitochondrial DNA: Molecular fossils in the nucleus. Current Biology, 1996, 6: 128-129.
85 Sartou N. and M. Nei. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987, 4: 406-425.
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89 Ward R D and P Grewe. Appraisal of molecular genetics in fisheries. Rev Fish Biol Fish, 1994, 18: 7313-7318.
90 Wilson G M et al. Intra-and tnter-specific mitochondrial DNAsequences divergence in salmo: rainbow, steelhead, and cutthroat trouts. Can J Zool, 1985, 63: 2088-2094.
91 Zardoya R el al. The complete nucleotide sequence of the mitochondrial DNA genome of the Rainbow trout: Oncorhynchus mykiss. J. Mol. Evol., 1995, 41: 942-951.
92 Zardoya R and Meyer A. The complete nucleotide sequence of the mitochondrial genome of the lungfish(protopterus dolloi) support its phylogenetic position as a close relative of land vertebrates. Genetics, 1996, 142: 1249-1263.
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96 Susumu Ohno. Gene duplication and the uniqueness of vertebrate genomes circa 1970-1999. Cell & developmental biology, 1999, 10: 517-522.
97 Spring J. Vertebrate evolution by interspecific hybridization: Are we polyploid? FEBS Lett 400: 2-8.
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94 Zouros E et al. Mitochongdrial DNA inheritance. Nature, 1994, 368: 818.
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97 Spring J. Vertebrate evolution by interspecific hybridization: Are we polyploid? FEBS Lett 400: 2-8.