摘要
为构建播娘蒿SSR文库，通过Dynal磁珠富集法富集生物素标记的（AC）8，（AG）8和（ATG）12探针与播娘蒿基因组的酶切片段杂交的片段，捕获含有SSR位点的DNA序列，连接到pMD18-T载体上，并转入感受态细胞DH5d，共获得778个克隆．挑选阳性克隆，利用SuperSNX24一F引物对茵液克隆进行PCR检测．利用M13F，M13R载体引物和探针结合的方法对文库进行2次筛选，获得93个长度为400～1000bp的克隆并测序．结果表明，82条序列舍有SSR位点，占测序条数的88．17％．共有SSR位点数127个．其中完全型SSR位点102个，不完全型SSR位点10个．混合型SSR位点15个，分别占SSR位点总数的80．31％，7．87％，11．81％．SSR重复基元中，以二核苷酸（CT）n，（AC）n，（GT）n，三核苷酸（GAT）n最为常见，得率分别为20．47％，26．77％，21．26％和15．75％．
In order to construct enriched microsatellite library of Descuminia sophia, Dynal magnetic beads were used to enrich biotin-labeled （AC）s, （AG）s and （ATG）12 probes hybridized with genomic fragments that have SSR of Descuminia sophia. The enriched DNA fragments were connected to pMD 18-T easy vector and then were transformed into competent DH 5oL cell, 778 clones were obtained. U- sing SuperSNX 24-F primers to detect the positive clones and using the M13F, M13R vector primers and probes for the second screening, 93 clones containing 400 - 1 000 bp DNA fragments were sent to sequencing. The results showed that 82 sequences contained SSR locus, accounting for 88.17% of the number of sequencing. In total, 127 SSR locus were obtained, including 102 perfect type SSR locus, 10 imperfect type SSR locus and 15 mixed type SSR locus, accounting for 80. 31% , 7. 87% , 11.81%, respectively. Dinucleotide （CT） n, （AC）., （ GT）,, and trinucleotide （GAT） n were com- mon, and the percentage was 20.47% , 26.77% , 21.26% and 15.75% respectively.
出处
《河南农业大学学报》
CAS
CSCD
北大核心
2014年第2期186-190,共5页
Journal of Henan Agricultural University
基金
国家自然科学基金项目(31170351)
河南省教育厅自然科学基金项目(2008A180012)
关键词
播娘蒿
磁珠富集法
SSR文库
Descuminia sophia
magnetic beads enriched method
SSR library
