大口黑鲈IGF-I基因内含子1、3和4序列多态性研究

根据大口黑鲈IGF-I基因的cDNA序列设计引物，克隆IGFI基因内含子核苷酸序列，采用PCR产物直接测序方法，在中国养殖群体和美国野生群体中筛选IGF-I基因内含子上的多态位点。应用RFLP、CRS—RFLP和SSCP技术建立多态位点的检测方法，同时比较分析其中4个多态位点在两个群体中基因频率分布。结果表明：（1）IGF-I基因内含子1、3和4序列长分别为1317bp、712bp和1941bp；（2）在3个内含子上共发现7个多态位点，其中在内含子1的208和1070位为G—A突变；内含子3的第40个碱基为一个“A”的插入-缺失突变，第307位为C—T突变，683位是G—A突变；内含子4上的696碱基处有一个20bp的插入-缺失突变，在1563位为G—A突变，表明大口黑鲈IGF-I基因序列上存在较多的SNPs；（3）内含子1上SNPG1070A的A等位基因能为HindIII限制性内切酶识别，采用RFLP技术分型。根据内含子1上SNPG208A侧翼序列，设计错配引物，使错配碱基和A等位基因共同形成TaqI酶切位点，建立CRS—RFLP检测方法。内含子4上SNPG1563A采用SSCP检测方法，同时对内含子4上的插入-缺失突变进行PAGE电泳分型。试验结果显示，RFLP、CRS—RFLP和SSCP三种SNP检测方法简单易行，适于在水产动物SNP标记研究中推广应用；（4）在中国养殖群体中，只有内含子1上的SNPG1070A具有多态性，其它3个多态位点只在美国野生群体中存在多态，证实中国养殖群体的遗传多样性相对较低。

Polymorohisms of intron 1,3 and 4 of insulin-like growth factor I gene in largemouth bass

The sequences of intron 1, 3 and 4 of the largemouth bass IGF-I gene were isolated using primers that designed from IGF-I gene cDNA. PCR products of three introns were sequenced for detecting polymorphisms. The methods were developed for detecting polymorphisms. Moreover, the alleles frequencies of four polymorphisms were calculated in Chinese cultured population and American wild population. The results indicated： 1） The lengths of intronl, 3 and 4 were 1 317bp,712bp and 1 941bp, respectively. 2） Seven polymorphisms that included six SNPs and one insertion/deletion mutation were found in Chinese cultured population and American wild population. The polymorphisms in intron 1 were SNP G208A and SNP G1070A （number meant relative position in each intron） ; The polymorphisms in intron 3 were ＂A＂ nucleotide insertion/deletion mutation at 40 basepair and SNP G208A and SNPG1070A; The polymorphisms in intron 4 contained 20bp insertion/deletion mutation and SNP G1070A. The result indicated that there were abundant polymorphisms in largemouth bass IGF-I gene. 3 ） The polymorphisms in introns 1 and 4 were genotyped by RFLP,CRS-RFLP and SSCP methods. 4） In Chinese cultured population, only SNP G1070A in intron 1 was identified. The frequencies of gdnotypes were not significantly different between two populations. Other three polymorphisms did not exist in Chinese cultured population, which suggested that genetic diversity was very low in Chinese cultured population.