摘要
应用磁珠富集法开发银鲫的三、四核苷酸重复的微卫星。生物素探针分别为Probe-(TGA)8,Probe-(AGAC)8,Probe-(GATT)8,结果得到4740个菌落。结合同位素放射法进行二次筛选,获得652个阳性克隆(阳性克隆率13.76%),利用PRIMER3PLUS在线软件设计引物163对,采用in silico方法,对微卫星序列进行BLASTN和BLASTX在线搜索。运用BLASTN搜索,首先将设计引物后的微卫星序列(侧翼序列长度大于50bp)与GEN-BANK中的斑马鱼的EST数据库进行比对,共匹配上44个同源序列。然后利用BLASTX搜索非冗余蛋白数据库,有31个微卫星位点成功的进行了功能注释,5个微卫星位点匹配上假设蛋白。本文利用模式生物斑马鱼的基因组资源对银鲫的的基因组进行比较研究,为以后银鲫种质资源的开发打下了良好的基础。
In this study,magnetic beads enriched method was used to isolated Polymorphic Tri-and Tetranucleotide Repeat microsatellite DNA from Fangzheng silver crucian carp（Carassius auratus gibelio（Bloch））.Three biotin-labeled microsatellite probes were Probe-（TGA）8,Probe-（AGAC）8 and Probe-（GATT）8.As a result,the 4740 colonies were obtained from the genome library.Combining the traditional radioisotope hybridization,six hundred and fifty-two positive clones were identified from above clones and the rate of positive clones was 13.76%.Eight hundred microsatellite sequences were isolated from these positive clones.681 repeat motifs（about 85%） were unique,while 542 sequences（about 68%） were perfect motifs.There were the remaining 119 compound motifs（about 15%） including 52 perfect motifs（about 7%）.One hundred and sixty-three pairs of primers from these microsatellite sequences were designed with the online software primer3plus.The in silico approach was used to identify homologies of microsatellite sequences by BLASTN and BLASTX searches.Microsatellte sequences after designed primers with flanking sequences over 50bp of silver crucian carp were compared with the zebrafish EST database,and 44 homologies were obtained using BLASTN.Except for 5 hypothetical proteins,31 microsatellites sites were found in the protein database through BLASTX searches.Using the genomic resources of zebrafish,this study was a pilot for comparison between zebrafish and the silver crucian carp,and laid a foundation for the development of germplasm resources.
出处
《水产学杂志》
CAS
2010年第4期30-36,59,共8页
Chinese Journal of Fisheries
基金
中国水产科学院黑龙江水产研究所基本科研专项(NO.2008HSYZX-SJ-05)
