勒氏笛鲷微卫星位点的筛选及特征分析

采用PCR法快速筛选勒氏笛鲷(Lutjanus russelli)基因组文库, 以获得(CA)_n微卫星位点。勒氏笛鲷基因组DNA经限制性内切酶HaeⅢ+ DraⅠ双酶切后, 连接T-载体克隆, 构建基因组文库。以通用引物M13+/-与重复序列引物(CA)15对基因组文库进行筛选, 二次筛选后得到121个可能含有微卫星位点的阳性克隆。进行序列测定, 共获得53个CA(n≥7)重复序列, 重复次数主要分布于7~15(80.77%)。在所得微卫星序列中, 重复单元除CA外, 还观察到单碱基、三碱基、四碱基、五碱基重复单元。根据侧翼序列设计48对引物, 通过优化PCR反应条件, 可获得清晰可重复的目的条带。研究旨在为勒氏笛鲷遗传多样性研究及遗传图谱的构建等奠定基础, 为勒氏笛鲷资源的合理开发利用提供参考。

Rapid isolation and characteristics analysis of microsatellites from Lutjanus russelli

The (CA)n DNA sequences in Lutjanus russelli were isolated through PCR arrays, and the characteristics of the microsatellite were analyzed. DNA was extracted from a sample of Lutjanus russelli, and digested with Hae+Dra. The fragments were ligated to pUCm-T vector and transferred into DH5alpha to construct a genomic library. The positive clones were isolated with universal M13 reverse and forward primers, M13 forward primer and the simple sequence repeats (SSRs) probes (CA)15, M13 reverse primer and (CA)15. After twice isolation, 121 positive clones, whose PCR products with M13 forward primer and probes (CA)15 or M13 reverse primer and (CA)15 were smaller than those with universal M13 reverse and forward primers probably contained microsatellites, were obtained. Then, 53 (CA)n(n> or =7) microsatellites were obtained by sequencing. The repeat length mainly distributed in 7-15(80.77%). Besides (CA)n repeats, other repeat motifs, such as An, (CAC)n and (AACA)n, were also obtained. Scorable and constant amplification of DNA fragments were observed with 48 pairs of SSR primers designed from the flank sequences. This research makes a positive contribution to explorating genomes of Lutjanus russelli, offers genetic tools to examine the genetic variations and constructs genetic linkage map.