草鱼羧肽酶A1基因(CPA1)部分片段的单核苷酸多态性(SNP)多态性及其与生长性状的关联分析

摘要

Carboxypeptidase A (EC 3.4.16) includes a type of digestive enzymes that hydrolyzes the C-terminal aromatic or aliphatic amino acids of a protein. Single nucleotide polymorphism (SNP) analysis of genes functionally related to the growth traits of grass carp (Ctenopharyngodon idella) can provide useful information for grass carp molecular breeding. In this study, genomic fragments of carboxypeptidase Al (CPA1) gene were amplified based on the sequences of two available contigs in the EST-SNP database of grass carp. Two SNP mutations were identified after sequencing the PCR prodcts, C+412A and A36C, which were located in exon 5 and intron 3 of the CPA 1 gene, respectively. Regarding the two SNPs, the genotype and gene frequency of 296 grass carps were further assayed with the method of SnaPshot. The frequencies of AA, AC and CC were 26.7%, 52.0% and 21.3% at the A36C site, but 15.5%, 40.5% and 43.9% at the C+412A site, respectively. A general linear model was used to do the correlation analysis between the CPA 1 gene SNPs and growth traits. The three different genotypes of the A36C site were significantly associated with body weight and eye distance (P<0.05). At this site, the genotype of AA significantly showed a higher value on body weight, body length, body width and eye distance compared to the genotype of CC (P<0.05). Although the C+412A site showed no significant correlation with body weight (P<0.05), the genotypes of CC and AC differed significantly for body weight and length before anus (P<0.05). Moreover, the genotype of CC always showed a greater values than that of AC or AA for six growth characters. Five diplotypes consisted of the two SNPs were different on body weight. For example, the body weight of diplotypes D3 and D5 differed significantly (P<0.05). Diplotype D3 showed a significant higher value of body weight, body length, body width and the ratio of body length/head length differences compared with genotype D8 (P<0.05).These results indicate that the CPA 1 gene can be a candidate gene that can be useful for grass carp molecular breeding.%羧肽酶A (EC 3.4.16)是一类水解蛋白和多肽底物C端芳香族氨基酸或脂肪族氨基酸残基的消化酶.在草鱼生长相关的功能基因上研究单核苷酸多态性(SNP)与生长的相关性可为草鱼的分子辅助育种提供依据.本研究根据草鱼(Ctenopharyngodon idella)EST-SNP库的羧肽酶A1基因(CPA1)重叠群的2个Contig扩增该基因的序列片段,采用直接测序法,经过序列比对,共筛到2个颠换SNP位点:C+412A和A36C,分别位于CPA 1基因外显子5的34bp和内含子3的36 bp处,前者为错义突变.然后用一个群体的296尾草鱼对这2个位点用SnaPshot的方法进行检测和分型,统计基因型频率:A36C位点的AA基因型占26.7％,AC基因型占52.0％,CC基因型占21.3％.C+412A位点的AA基因型占15.5％,AC基因型占40.5％,CC基因型占43.9％.利用一般线性模型分析2个SNP位点与草鱼体质量、体长等重要生长性状的关系.关联分析结果显示,C+36A位点不同基因型在体质量、眼间距均值上存在显著差异(P＜0.05).并且AA基因型和CC基因型在体质量、体长、体宽和眼间距上存在显著差异(P＜0.05).AA基因型各项指标均值显著高于CC基因型.C+412A位点在体质量等生长性状上差异不显著(P＞0.05),该位点和生长不相关,但是CC基因型和AC基因型在体重和肛前距上差异显著(P＜0.05),该位点CC基因型的6个生长性状均值都高于AA基因型.由两个位点组成的5种双倍型在体质量上存在显著差异(P＜0.05).双倍型D3和D5在体质量上存在差异显著(P＜0.05).双倍型D3和D8在体质量、体宽、体长、体长/头长等生长性状上都存在显著差异(P＜0.05).D3的各项指标均值最高,D8的各项指标均值最低.本研究结果显示,可以考虑将草鱼CPA1基因作为候选基因,用于草鱼的分子辅助育种.