勒氏笛鲷微卫星位点的筛选及特征分析

doi:10.1360/yc-007-0355

遗传 ›› 2007, Vol. 29 ›› Issue (3): 355-355―359.doi: 10.1360/yc-007-0355

勒氏笛鲷微卫星位点的筛选及特征分析

郭昱嵩¹, 王中铎², 刘楚吾^1,², 刘筠¹

1. 湖南师范大学生命科学院, 长沙 410081;
2. 广东海洋大学水产学院, 湛江 524025

收稿日期:2006-06-12 修回日期:2006-09-25 出版日期:2007-03-01 发布日期:2007-03-01
通讯作者: 刘楚吾

Rapid isolation and characteristics analysis of microsatellites from Lutjanus russelli

GUO Yu-Song¹, WANG Zhong-Duo², LIU Chu-Wu^1,2, LIU Yun¹

1. College of Life Science, Hunan Normal University, Changsha 410081, China;
2. Fisheries College, Guangdong Ocean University, Zhanjiang 524025, China

Received:2006-06-12 Revised:2006-09-25 Online:2007-03-01 Published:2007-03-01
Contact: LIU Chu-Wu

摘要/Abstract

摘要：

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。

关键词: PCR, 勒氏笛鲷, 微卫星

Abstract:

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 HaeⅢ+DraⅠ. The fragments were ligated to pUCm-T vector and transferred into DH5α 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≥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 A_n, (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 ge-nomes of Lutjanus russelli, offers genetic tools to examine the genetic variations and constructs genetic linkage map.
Keywords: microsatellite; Lutjanus russelli; PCR

郭昱嵩，王中铎，刘楚吾，刘筠. 勒氏笛鲷微卫星位点的筛选及特征分析[J]. 遗传, 2007, 29(3): 355-355―359.

GUO Yu-Song, WANG Zhong-Duo, LIU Chu-Wu, LIU Yun. Rapid isolation and characteristics analysis of microsatellites from Lutjanus russelli[J]. HEREDITAS, 2007, 29(3): 355-355―359.

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勒氏笛鲷微卫星位点的筛选及特征分析

Rapid isolation and characteristics analysis of microsatellites from Lutjanus russelli

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