摘要
以日本扁柏 4个种源的DNA样品为材料 ,经NdeⅡ酶切并电泳后回收 30 0～ 10 0 0bpDNA片段 ,与含有生物素标记的 (CT) 15探针杂交 ,再用磁珠 (Dynabeads M2 80streptavitinbeads)固定杂交产物 ,成功地实现了日本扁柏核基因组微卫星的高效分离。以pUC118BamHⅠ /BAP为载体 ,E .coli为寄主 ,构建了日本扁柏 2个富含微卫星的基因组文库 ,共获得 2 2 0 0多个阳性克隆。从构建的基因组文库中 ,随机挑选 4 80个阳性克隆进行测序 ,发现含有微卫星的克隆比例高达 6 5 6 % ,其中 2 15个非同源性克隆共含有 380个微卫星位点 ,平均每个克隆含 1 7个。微卫星种类以与探针 (CT) 15互补的 (GA/CT) n 为主 ,占 86 1% ,同时还存在 (TG/AC) n和 3～ 4个碱基为单元构成的微卫星 ,如 (ATAG) n、(CAGA) n、(TGA) n、(ACG) n、(TCA) n、(TCT) n 等。利用Oligo 5 1软件为 114个含有微卫星的克隆共 12 8个微卫星位点设计出了PCR扩增引物。
Using 300～1 000 bp DNA fragments retrieved from 2% agarose gel electrophoresis on nuclear DNA samples of Chamaecypress obtusa with NdeⅡ restricted digestion,microsatellites were efficiently isolated by using Dynabeads M280 streptavitin beads.These beads could immobilize hybrid products between biotin-labeled (CT)_(15) probe,and 300～1 000 bp DNA fragments previously linkaged to Sau3AⅠcassette.Two enriched microsatellite libraries with about 2 200 positive white clones were constructed by inserting potential microsatellite fragments into vector pUC118 BamHⅠ/BAP and culturing with host E.coli.After sequence analysis on 480 randomly picked out positive clones,it was revealed that the number of SSR-bearing clones accounts for 65.6%,and 215 clones were identified as non-homology.Furthermore,380 microsatellite locus were found in 215 non-homology clones indicating 1.7 loci per clone.Among the existed microsatellites,the expected type of (GA/CT)_n is the most popular which accounts for 86.1% and were compatible to the (CT)_(15) probe,however some other types including (TG/AC)_n and more complicated simple sequence repeats with 3～4 bases,such as (ATAG)_n ,((CAGA)_n ,)(TGA)_n ,(ACG)_n,(TCA)_n,(TCT)_n,could be also detected in these two libraries.Finally,primers for amplifying 128 microsatellite locus from 114 SSR-bearing clones were designed by Oligo 5.1 (http:∥lxg.users4.50megs.com/ssr.html).
基金
日本科技厅STA特别研究员项目资助 (编号 :3 0 0 0 3 7)~~
关键词
日本扁柏
微卫星
基因组
测序
Chamaecypress obtuse
microsatellites
genomics
sequencing
