苎麻基因组微卫星的分离与鉴定(英文)  

Isolation and Characterization of Microsatellites from Ramie [Boehmeria nivea (L.) Gaud.]

作　　者：蒋彦波[1] 揭雨成[1] 周建林[2] 邢虎成[1] 佘玮[1] 

机构地区：[1]中国农业科学院麻类研究所,湖南长沙410205 [2]湖南师范大学生命科学院生物化学与分子生物学系,湖南长沙410081

出　　处：《作物学报》2007年第1期158-162,共5页Acta Agronomica Sinica

基　　金：National Natural Science Foundation of China(30270849),High-Tech Research and Development Program of China (2001AA241211) and Hunan Provincial Natural Science Foundation (04JJ3016)

摘　　要：以苎麻[Boehmeria nivea（L．）Gaud．]栽培品种芦竹青为材料，经Mse Ⅰ酶切并与相应接头连接，然后与生物素标记的探针（CT）15杂交，再用链亲和素包被磁珠（Dynabeads M-280）捕捉杂交产物，以21碱基接头寡核苷酸为引物经PCR扩增获得了双链目的片段，回收300—1000bp DNA片段，然后克隆到pMD18-T载体上，转化至DH5α中，这样就构建了苎麻富含（GA）n微卫星的部分基因组文库。随机挑取36个克隆，以锚定简单重复序列为引物对其进行PCR筛选。测序分析了22个阳性克隆，每个克隆都含有微卫星DNA，阳性克隆率为61．1％，微卫星种类以与探针互补的（GA／CT）n为主，占83．3％，同时还存在（TG／AC）n和三碱基、六碱基为单元构成的苎麻微卫星，如（GAC）n、（ACG）n、（TCT）n、（TCG）n、（GAA）n、（CCGACG）n、（GAGAAA）n等。最后以18个微卫星位点设计了18对苎麻微卫星特异引物。GMCT重复单元的长度从7到40范围内变化，平均为19．2，显示了它们将产生良好多态性，为一种强有力的遗传标记。苎麻微卫星DNA标记可广泛应用于苎麻基因型鉴定，基因和QTL分析，分子标记辅助育种，系谱分析等。Ramie [ Boehmeria nivea （ L. ） Gaud. ] is an important bast fiber crop. To study genetic background of this species, the microsatellite markers of ramie were isolated and characterized. Microsatellites were captured by using ramie cuhivar Luzhuqing digested with Mse Ⅰ and ligeted with adaptors and then linked to biotinylated oligeprobe （CT）15 attached to streptavidin-coated magnetic beads （Dynabeads M-280） . Double-stranded targeted fragments were obtained by PCR amplification using 21-mer adaptor oligonucleotide as primer, 300-1 000 bp DNA fragments were recovered from 1.5% ngarose gel electropheresis and then religated into pMD18-T vector and transformed into competent DH5α cell. Thus a genomic library containing inserts （GA）n repeats fragments was constructed and then screened by PCR using anchored simple sequence repeats as primers for the presence of （GA/CT） n repeats. A total of 22 clones were identified as positives after randomly picked 36 clones, all the 22 clones contained microsatellites and enrichment efficiency was 61.1%, among the existed microsatellites, the expected type of GA/CT is the most popular which accounted for 83.3% and is compatible to the （CT）15 probe, however some other types including （TG/AC）n and more complicated simple sequence repeats with 3 and 6 bases, such as （GAG）n, （ACG）n, （TCT）n, （TCG）n, （GAA）n, （CCGACG）n, （GAGAAA）n could also be detected in the microsatellite-enriched library. About 18 microsatellite loci were found after sequencing. Finally, 18 primer pairs were designed on the basis of DNA sequences flanking the repeat motif. The length of GA/CT repeat units varied between 7 and 40 with a mean value of 19.2, which suggests that they would be polymorphic and powerful genetic markers in ramie. These SSR markers are useful for ramie genetype identification, gene and QTL analysis, marker-assisted selection in breeding and pedigree analysis.

关 键 词：苎麻[Boehmeria nivea(L.)Gaud] 微卫星 磁珠 基因组文库 (CA)n重复序列 

分 类 号：S563.1[农业科学—作物学]