牙鲆基因组(CAG)_n微卫星DNA特征分析  

Characterization of (CAG)_n microsatellite in Paralichthys olivaceus genome

作　　者：王蕾[1,2] 刘继红[1,2] 张立冬[1] 孙效文[2] 

机构地区：[1]大连水产学院生命科学与技术学院,辽宁大连116023 [2]中国水产科学研究院黑龙江水产研究所,黑龙江哈尔滨150070

出　　处：《中国水产科学》2009年第6期807-815,共9页Journal of Fishery Sciences of China

基　　金：国家科技基础条件平台建设资助项目(2005DKA30470-005)

摘　　要：通过磁珠富集法筛选牙鲆(Paralichthys olivaceus)的微卫星分子标记,采用限制性内切酶Sau3A I对牙鲆完整基因组DNA进行酶切;通过蔗糖溶液梯度离心,收集400～900bp大小的片段,连接Brown接头,构建牙鲆基因组文库。用生物素标记的微卫星探针(CAG)15对基因组文库进行杂交,利用磁珠富集含有微卫星的DNA单链序列,并对其进行PCR扩增;将扩增产物连接到pMD18-T载体后转入感受态大肠杆菌DH5α中,得到微卫星序列文库。利用大量质粒检测法进行二次筛选,成功地从牙鲆基因组中分离出含有CAG重复的微卫星序列,测序其中的3000个单菌落,获得2805个(占93.5%)含有微卫星序列的克隆,其中含有微卫星座位3120个,完美型1808个,占57.97%;非完美型226个,占7.25%;混合型1085个,占34.78%。从中选出186个微卫星序列设计120对引物并合成,经过筛选,74对引物可扩增清晰条带,其中68对呈多态性。Magnetic beads enriched method was used to isolate microsatellite DNA from Japanese flounder（Paralichthys olivaceus）genome.Japanese flounder（Paralichthys olivaceus）genomic DNA was extracted and then digested with restriction enzyme Sau 3A I.Targeted segments of 400-900 base pairs were collected by centrifugation of sucrose density gradient and a whole genome PCR library was created.This genomic DNA was hybridized with a biotin-labeled microsatellite probe（CAG）15.The hybrid mixture was incubated with magnetic beads coated.After the non-microsatellite fragments were removed,the single-stranded DNA was obtained.The selected DNAs were then amplified using primers designed complementary to the linkers,cloned into the pMD18-T vector and transformed into competent Escherichia coli DH 5α,and finally a microsatellite library was obtained.The second screen was performed with plasmid detected method.By isolating（CAG）n microsatellite in Japanese flounder genomic,three thousands positive clones were obtained.From these positive clones,2 805 microsatellite sequence clone were isolated and 3 120 microsatellite loci were obtained.From these sequences,1 808 repeat motifs（about 57.97%）were perfect,226 repeat motifs（about 7.25%）were imperfect,and 1 085 repeat motifs（about 34.78%）were compound.This allowed us to design 120 pairs of primers from 186 microsatellite sequences with the software Primer Premier 3.0 and to compose them.As a result,74 pairs were screened and used successfully to amplify special fragments,among which 68 pairs were polymorphism.

关 键 词：牙鲆 微卫星 磁珠富集 

分 类 号：Q94[生物学—植物学]