【作者】 黄军；

【导师】 陈国宏； 严美姣；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2007， 硕士

【摘要】 生长激素(Growth Hormone,GH)能使鱼增进食欲,提高饲料转换率,刺激鱼的体重和体长增长,促进发育,参与生长代谢调节。本研究基于畜禽、鱼类生长激素基因方面的研究,参照GenBank数据库中红鳍东方鲀和其它近缘物种的生长激素基因序列,设计了两对引物扩增出了暗纹东方鲀GH基因,构建了东方鲀生长激素基因的进化树,确证了该基因在进化史中的地位,另外以暗纹东方鲀(Takifugu obscurus)、红鳍东方鲀(Takifugu rubripes)、星点东方鲀(Takifugu niphobles)共82个个体为对象,运用PCR产物电泳检测、单链构向多态性(SSCP)技术和克隆测序技术检测到了GH基因的长度和序列多态性。研究结果如下:1、暗纹东方鲀GH基因含有6个外显子和5个内含子,编码196个氨基酸。2、从GenBank数据库中得到的24种脊椎动物的生长激素基因的mRNA序列以及氨基酸序列,通过Clustal W排列,用UPGMA法构建了GH基因的分子系统树。在UPGMA一致树中,25种脊椎动物分为两大类:第一大类包括野猪、狗、猫、褐鼠、小鼠、绵羊、牛、鸡、鸭、蝾螈、鳗鲡、鳝鱼和人;第二大类包括暗纹东方鲀、红鳍东方鲀、斑马鱼、草鱼、大比目鱼、大马哈鱼、斗鱼、杜父鱼、鲋鱼、金鲈、鲤鱼和鲶鱼。第一大类中,哺乳动物(猪、狗、猫、褐鼠、小鼠)先聚为一类,逐次与反刍动物(绵羊、牛)、家禽(鸡、鸭)、爬行动物(蝾螈)、鳝鱼、人以及鳗鲡聚为一大类。第二大类中,杜父鱼与金鲈首先聚为一类,然后逐次与斗鱼和东方鲀(暗纹东方鲀和红鳍东方鲀)聚为第1类;斑马鱼与草鱼,鲋鱼与鲤鱼各自先聚为一类,然后与大马哈鱼和鲶鱼聚为第2类。3、在GH基因内含子2中,存在9种长度类型,分别为A、B、C、D、E、F、G、H、I,变异频率达到24.22%。对这9种序列进行比对分析,发现9种长度类型A、G、T、C的平均百分比为17.15%、20.77%、37.38%、24.70%,其中G+C(45.47%)含量与A+T(54.53%)的含量差异不是很大;9种序列分别长351bp、327bp、319bp、303bp、295bp、291bp、287bp、283bp和271bp,引起长度变化的主要原因是短串联序列TCTG的重复次数(重复次数从20到40不等);发现4个突变位点,其中3个转换位点为83(C-T),101(A-G),296(G-A),一处颠换位点103(C-A)。用UPGMA法构建分子系统树,发现DD与II首先聚为一类,然后依次与GG、AA聚为一大类,BB与CC,EE与HH分别聚为一类,最后再与FF聚为一大类,由此可见GH基因内含子2在品种间的差异远大于品种内差异。4、在GH基因内含子5中,扩增的片断长度有差异,其中AA基因型长353bp,BB基因型长360bp,CC基因型长337bp,DD基因型长327bp,经Clustal W软件排列生成394个核苷酸位点,变异位点数为7个,占总位点数的1.77%。引起长度变化的主要原因是第28位A缺失,193-195位ATGATG缺失,244位G缺失,274-284位TGGTGAAGATG缺失,312-320位ATGATGATG缺失。发现7个突变位点,其中1处颠换即位点251(T-A),6处转换即位点181、190、268、251、262、271(C-T),颠换频率14.29%明显低于转换频率85.71%。5、在GH基因3′非翻译区存在两种长度多态性,分别为320bp和317bp,与GenBank (登录号为:FRU63807)的序列(316bp)有差异;共检测到6种基因型,分别命名为aa、bb、ab、bc、cd、dd,变异频率达4.36%,其中在暗纹东方鲀中检测到四种aa、bb、ab、bc,cd和dd基因型分别只在于星点东方鲀和红鳍东方鲀检测到;7个突变位点中有1处颠换即位点212(T-G),6处转换即位点120、180、227、265、287(C-T),和位点199(A-G)。6、在红鳍东方鲀、暗纹东方鲀和星点东方鲀这三个种中经过哈代-温伯格平衡鉴定,发现都不处于平衡状态;另外从基因杂和度、多态信息含量和有效等为基因数分析,在intron 2中具有较多的遗传学意义,其它位点的三个值都很低。更多还原

【Abstract】 Growth hormone (GH) has visible effect in promoting foodintake, boosting feedstuff conversion rate, incentiving and promoting growth, participating growth metabolic regulation in fish. The GH gene of Takifugu obscurus was amplificated with two pair of primers based on the study of GH gene in livestock and fish and the sequences of GH gene of other homology species in GenBank , and the phylogenetic tree of GH gene was constructed using UPGNA method. In addition, High size and nucleotide polymorphisms of GH gene was checked in 82 individuals from Takifugu obscurus, Takifugu rubripes and Takifugu niphobles with PAGE、SSCP and sequence analysis. The results showed that:1. There were 6 exons and 5 introns in GH gene in Takifugu obscurus, which codes 196 amino acids.2. The mRNA and amino acids sequences of GH gene of 24 vertebrate species were gained from GenBank., and the phylogenetic tree of GH gene was constructed with MEGA 3.1 using UPGMA method. In UPGMA consensus tree, 25 species were divided into two clusters. The first cluster, including Sus scrofa, Canis familiaris, Felis catus, Rattus norvegicus, Mus musculus, Ovis aries, Bos Taurus, Gallus gallus, Anas platyrhynchos, Ambystoma barbouri, Anguilla anguilla, Acipenser gueldenstaedtii and Homo sapiens, were subdivided as follows: mammalians(Sus scrofa, Canis familiaris, Felis catus, Rattus norvegicus, Mus musculus) constructed one group firstly, which were subsequently joined by ruminant(Ovis aries, Bos taurus), poultry(Gallus gallus, Anas platyrhynchos), reptiles(Ambystoma barbouri), Acipenser gueldenstaedtiihuman and Homo sapiens in order, Anguilla anguilla was joined last. The second cluster, including Takifugu obscurus, Takifugu rubripes, Danio rerio, Ctenopharyngodon idella, Verasper variegates, Oncorhynchus sp, Betta splendens, Cottus kazika, Carassius auratus gibelio, Perca flavescens, Cyprinus carpio and Pangasianodon gigas, were subdivided as follow: Cottus kazika clustered with Perca flavescens firstly, followed by Betta splendens and Takifugu in order, formed group 1; Danio rerio and Ctenopharyngodon idella, Carassius auratus gibelio and Cyprinus carpio were clustered together respectively, which was subsequently joined by Oncorhynchus sp and Pangasianodon gigas, formed group 2.3. There were nine size variants (A, B, C, D, E, F, G, H, I) in intron 2 of GH gene. Results showed that there were nine length variants (A~I) in these fishes, ranging from189 -209 bp, with a frequency of variation of 24.22%. Sequence analysis of the 9 length variants showed the average percentages of the four bases (A, T, G and C) were 17.15%, 20.77%, 37.38% and 24.70%, respectively, and the difference between GC content (45.47%) and AT content (54.53%) was not remarkable. Length variation was mainly due to the variable number of microsatellite TCTG repeats (N=20-40). Further sequence comparison revealed 4 substitution sites: a C-A transversion at nucleotide 103 and 3 transitions 83(C-T), 101(A-G) and 296(G-A), respectively. Phylogenetic analysis of these length variants placed DD and II into one group first, which was subsequently joined by GG and AA. BB and CC, EE and HH were then clustered together. FF was joined last. Thus, intraspecific variations were much smaller than interspecific variations for the intron 2 of the GH gene.4. There were four size variants AA(353bp), BB(360bp), CC(337bp), DD(327bp) in intron 5 of GH gene in three Takifugu, and the sequences analysis showed that there were 7 mutations. The frequency of variation was 1.77%. The length variants was mainly due to the deletion at 28(A), 193-198(ATGATG), 244(G), 274-284(TGGTGAAGATG), 312-320(ATGATGATG). Of the 7 mutations, 1 was transversion site, at 251(T-A), and 6 were transition sites, at 181, 190, 251, 262, 268, 271(C-T), and the frequency of transversion(14.29%) were lower than that of transition(85.71%).5. There were two size variants in 3′-UTR of Takifugu GH gene, 320bp and 317bp respectively, which was different from the sequence (316bp) in the GenBank (accession number: FRU63807). And 6 genotypes aa, bb, ab, bc, cd and dd were found, the frequency of variety was 4.36%; aa, bb, ab and bc was found in Takifugu obscurus , cd and dd can only be found in Takifugu niphobles and Takifugu rubripes respectively. Of the 7 mutations,1 was of transversion site, at 212(T-G), and 6 were of transition sites, at 120, 180, 227, 265, 287(C-T) and 199(A-G) respectively.更多还原