3种鳜属鱼生长激素基因差异的检测与分析

Diversity of growth hormone gene in three Siniperca fishes

为探索鳜(Siniperca chuatsi)、大眼鳜(S.kneri)、斑鳜(S.scherzeri)生长性状差异与基因差异间的联系,以3种鳜野生群体为材料,采用PCR-SSCP分析及测序方法,对3种鳜野生群体的生长发育相关基因——生长激素(GH)基因的差异进行了检测。结果表明,在生长激素基因5′侧翼区至第2外显子区域检测到4种多态型,均为群体特有多态型;在第2内含子3′段至第3内含子5′段区域检测到5种多态型,其中3种为群体特有多态型;在第4外显子至第5外显子5′段区域及第5外显子3′段至3′侧翼区均检测到4种多态型,均有3种为群体特有多态型。对各多态型编码区的分析表明,斑鳜与鳜、大眼鳜在GH第150位氨基酸位点存在差异。在该位点,斑鳜编码蛋氨酸(M),鳜、大眼鳜均编码亮氨酸(L)。各区域多态型共组合成9种GH基因型,3种鳜间无共有基因型。GH基因遗传分化指数(Fst)为0.8598,分化程度较高。遗传多样性分析显示,斑鳜GH基因遗传多样性高于鳜和大眼鳜。3种鳜属鱼GH基因相似度比较结果显示,鳜与大眼鳜的相似度最高,鳜与斑鳜的相似度最低。研究结果可为进一步利用GH基因差异进行鳜分子育种研究提供依据。

Siniperca chuatsi, Siniperca kneri and Siniperca scherzeri are three specific Siniperca fishes in East Asia. They are cultured as edible fishes in China and have similar habits and body configuration. They can intercross, but there are significant differences on grouth traits among them. S. chuatsi grows the fastest while S. scherzeri the slowest. GH gene as one of the most important functional genes that determine animal growth trait, is generally related to growth trait in live stocks and fowls and mutation of GH gene often leads to variation of growth traits. To find out the relationship between growth trait difference and diversity of genes, genetic diversity detection ofGH gene was performed in wild populations orS. chuatsi, S. scherzeri and S. kneri by PCR- SSCP analysis and sequencing method. 45,45 and 42 individuals were sampled respectively from S. chuatsi, S. kneri and S. scherzeri population for DNA extracting. Four fragments of GH gene were amplified by PCR. PCR products were then detected by SSCP and sequencing. Sequence comparation was performed by Clustalw 1.83 software. POPGENE1.32 software was adopted to calculate heterozygosity, polymorphic type frequency, genotype frequency, Shannon diversity index and genetic distance. Arlequin 3.11 software was adopted to analyze genetic differentiation index （Fst）and AMOVA： and PHILIP 3.66 software was adopted to construct UPGAM phyletic evolution tree based on genetic distance. Four polymorphic types are identified in the region from 5＇ -flanking region to exon Ⅱ, which are all species-specific. Five polymorphic types are identified in the region from 3＇ part of intron Ⅱ to 5＇ part of intron Ⅲ, among which 3 polymorphic types are species-specific. Four polymorphic types are identified in both the region from 3p part of intron V to 3＇ -flanking region V and the region from exon IV to 5＇ part of exon V respectively, both of which have 3 species-specific polymorphic types. Analysis of the coding region indicates diversity of the 150th coded amino acid, on which is methionine in S. scherzeri, but leucine in S. chuatsi and S. kneri. All polymorphic types form 9 GH genotypes without one shared between the three fish species. Fst is found to be 0.859 8, which indicates a high level of differentiation. Analysis of genetic diversity indicates that diversity in S. scherzeri is higher than those in S. chuatsi and S. kneri. Among the three Siniperca fishes, genetic similarity between S. chuatsi and S. kneri is the highest, while that between S. chuatsi and S. scherzeri is the lowest. The result of phyletic evolution analysis according to GH gene on chromosome is identical to that according to mitochondrion sequences, which confirms the phyletic evolution relationship between these fishes at population level. Abundant diversity in GH gene is also found in the three fishes. Many polymorphic types are species-specific, which can be of use in identifying hybrids when it is difficult to distinguish them from their parents by phenotype. Though it is still unclear if these diversities lead to growth trait differences, these findings have laid foundation for further study of the relationship between gene diversity and trait difference as well as further utilization of the diversity of GH gene in molecular breeding of Siniperca fishes.