鲫鱼生长激素Ⅰ基因内含子2的多态性分析

Polymorphism in Intron 2 of Crucian Carp GHⅠ Gene

利用变性聚丙烯酰胺凝胶电泳 (Denaturing PolyacrylamidGelElectrophoresis,DPAGE)和单链构象多态性(Single StrandConformationPolymorphism ,SSCP)分析技术 ,在普通鲫鱼种群 (7个野生鲫鱼群体和 2个金鱼群体 )和银鲫种群 (1个方正银鲫群体 )共 16 2个个体生长激素Ⅰ (GrowthHormoneⅠ ,GHⅠ )基因的内含子 2中检测到丰富的长度和序列多态性。在所有群体中 ,GHⅠ基因内含子 2的长度存在 7种类型 ,分别为A、B、C、D、E、F和G型 ,变异频率为 4 32 % ;其序列存在 15种单倍型 ,分别为A1、A2、A3、B、C1、C2、D1、D2、D3、D4、D5、E1、E2、F和G型 ,变异频率为 9 2 6 %。对这 15种单倍型的DNA序列进行分析 ,结果表明 :1)鲫鱼GHⅠ基因内含子 2的扩增DNA片段长为 2 4 3～ 2 6 3bp ,其中包括部分外显子 2 (3′端 ,33bp)、内含子 2和部分外显子 3(5′端 ,2 1bp)。 15种单倍型A、T、G、C碱基平均百分比组成分别为 34 13%、37 36 %、15 13%、13 38% ,其中G +C(2 8 5 1% )含量明显小于A +T(71 4 9% )含量。内含子 2与外显子 2、3的接头区存在GT—AG保守序列 ,此为真核基因中mRNA剪接的识别信号。内含子 2的 5′端存在有一序列为GTAAGAT的保守区域 ,在其近 3′端分布有一高丰度嘧啶区 ;2 )内含子 2的

High length and nucleotide polymorphisms in intron2 of GHⅠ gene were detected in 162 individuals,which were from seven wild crucian carp colonies,two goldfish colonies and one Fangzheng crucian carp colony.Using denaturating polyareylamide gel electrophoresis (DPAGE) and single-strand conformation polymorphism (SSCP),seven length variants and 15 haplotypes were identified in these fishes.The length types and haplotypes diversity was 4.32% and 9.26%,respectively.Sequence analysis of the 15 haplotypes indicated the following results:(1) The size of intron2 varied from 243 to 263 bp.In the 15 haplotypes,the average percentages of the four bases (A,T,G and C) were 34.13%,37.36%,15.13% and 13.38%,respectively;the frequency of G+C(28.51%) was much lower than that of A+T (71.49%).The GT/AG domain was found in exon-intron junctions,which was the 5′ and 3′ splice donor and acceptor sites in higher eukaryotic gene introns.The similarity sequence of GTAAGTA was located on the junction between exon2 and intron2.And there existed a richer pyrimidine region (TTTGCCTTTTGTTATC) near the 3′ end of intron2.(2) The seven length variants(A,B,C,D,E,F and G) were determined to be 189,196,204,205,206,207 and 209 bp,respectively.The polymorphism resulted from the variable repeat number of T (N=0,8,9,10,11 and 13) and the difference in one or two motifs deletions of TGAAAAC,TT and GAGTG.(3) Compared the sequences of the 15 haplotypes,17 substitution sites were observed,of which two were of transversion sites and 15 were of transition sites.Obviously,the transition mutations (88.24%) were more frequent than transversion mutations (11.76%).Analysis of the distributions of the length types,haplotypes and composite genotypes suggested that genetic diversity was varied in different colonies.In the goldfish colonies,only one length type (A),two haplotypes (A1 and A2) and one composite genotype (A1A2) were observed.Two length types (C and D),four haplotypes (C1,C2,D2 and D5) and one composite genotype (C1C2D2D5) presented in the Fangzheng crucian carp colonies.The highest level of genetic diversity was exhibited in the seven wild crucian carp colonies:seven length types (A,B,C,D,E,F and G),14 haplotypes (A1,A2,A3,B,C1,C2,D1,D2,D3,D4,E1,E2,F and G) and 14 composite genotypes (A1A2A3,A1A2A3B,A1C1C2D1D2D3,A1C1C2D2,A1C1C2D2D3,A1C1C2D3E2,BC1C2D2,BC1C2D3D4,C1C2D2,C1C2D2D3,C1C2D3D4,C1C2D3D4F,C1C2D4,D2E1G) were shared by the seven wild colonies.The numbers of observed length types,haplotypes and genotypes within the wild colonies ranged from 3 to 6,6 to 10 and 2 to 6,respectively.Whether the length and nucleotide polymorphisms in the intron2 of crucian carp GHⅠ gene were associated with gene expression and gene regulation remained unsolved and required further investigations.