亚洲棉5号染色体RGAs克隆与分析

Cloning and Analysis of RGAs from 5^(th) Chromosome in Gossypium arboreum

棉花是最主要的天然纤维作物,深入进行棉花基因组研究具有重要意义.采用酶解、前后低渗和轻压相结合的方法制备亚洲棉染色体中期膜载片,激光法分离亚洲棉第5号单染色体,建单染色体扩增池后克隆其抗病基因同源序列(RGAs),获得P7、P12、P19和P23等4个序列.序列比对和聚类分析表明,这4条序列均为NBS-LRR类RGAs,P7、P12、P19聚成一类,它们之间的同源性很高,P23聚成另一类,与黑松的RPS2和油菜的RGA30同源性较高.为该染色体分子标记开发、基因克隆乃至全序列测定奠定基础.

Cotton is one of the main natural fiber crops, it is important to accumulate basic data in cotton genome re- search. The high quality metaphase chromosome membrane preparations of GI arboreum were obtained integrated with method of pre-hypotonicity, enzymolysis, post- hypotonicity and squashed with cover slide. A 5th chromosome was microdissected. Amplified production was obtained after the sequential procedures of protein-removing, enzymolysis and linker adaptor PCR （LA-PCR）. A verified system was constructed after integrated the method of Southern blotting, SSR primer amplification and fluorescence in situ hybridization （FISH）. Four necleotide sequence P7, P12, P19 and P23 were obtained. The blast results showed that they are NBS-LRR-type resistant gene analog （RGA）. Clustering analysis indicated that the sequences of PT, P12, P19 were high homologous and in the same cluster, the F23 was in other cluster and homologous with RPS2 gene of B. nigra and RGA30 gene of B. napus.