亚洲棉7号染色体分离及文库构建

摘要

采用前低渗、酶解、后低渗和盖片轻压相结合的方法,建立了激光法分离二倍体亚洲棉石系亚1号单条染色体技术.单染色体经去蛋白、酶切和PCR扩增后,产物以Southern杂交、简单重复序列(Simple sequence repeats,SSR)引物扩增和荧光原位杂交技术(Fluorescence in situ hybridization,FISH)进行验证,构建了棉花单条染色体文库.克隆片段长度在150～1000 bp,平均为550 bp;文库包含1.38×105个克隆,覆盖染色体长度1倍左右,空载率为1％,滴度为1.3×106 pfu· mL-1,单一拷贝和低拷贝比例达到59％以上,为该条染色体分子标记的筛选、重要基因克隆和定位、遗传图谱的饱和奠定基础.%In order to obtained the high-quality well-differentiated mitotic metaphase chromosomes with clear background for laser microdissection in cotton, integrated mothed of pre-hypotonicity, enzymolysis, post-hypotonicity and squashed on mitotic metaphase root tip cell was adopted, the high quality preparations of Gossypium arboreum were obtained, and the single chrom-some laser cutting technique in cotton was constructed. Amplified single chromosome production was verified by the integrated method of simple sequence repeats (SSR) primer amplification, Southern blotting and fluorescence in situ hybridization (FISH). The 7* chromosome library of Shixiya No. 1, a variety of G. Arboreum was constructed. The library contains 1.38xl05 clones, which almost cover 1 time of the chromosome length. The inserts averaged 550 bp, varied from 150 bp to 1000 bp. And the titer was 1.3×106 pfu·mL-1, rate of no-load was 1%, and the ration of single and low copy sequences were over 59%. The library will facilitate the specific probe screening, gene cloning and sitting, genetic map saturation on this chromosome.