巴氏蘑菇微卫星序列的分离及其对Co^60辐射诱变菌株的鉴别

Microsatellite sequences for identification of Agaricus blazei mutants induced by Co^(60) irradiation

根据链霉素磁珠和生物素特异结合的特性,用生物素标记的二聚核苷酸重复序列探针从巴氏蘑菇的基因组中分离微卫星序列。将结合于链霉素磁珠上的标记探针同两端连接已知序列人工接头的巴氏蘑菇DNA酶切片段杂交。洗脱未杂交DNA片段后,用磁珠富集的片段建立微卫星文库。挑取522个菌落用对应重复序列为引物进行PCR筛选,得到48个阳性克隆,经测序有32个菌落含微卫星序列。微卫星富集效率为阳性克隆数的67%,总克隆数的6%。除去重复或无效的微卫星序列,在设计出的12对用于鉴别85个巴氏蘑菇的Co60辐射变异株微卫星引物中,有4对引物总共扩增出明显的变异菌株17个。证明有些微卫星位点可用于巴氏蘑菇辐射变异品种的指纹筛选与鉴别。

Based on the specifical combination between biotin and streptavidin coated magnetic beads, biotinylated probes composed of di-nucleotide repeat were used to isolate microsatellite sequences from genome of Agaricus blazei. DNA digested fragments of Agaricus blazei ligated with artificial adapters in both ends were hybridized with the labeled probes absorbed in magnetic beads. After eluting the unhybridized DNA, the enriched fragments in beads were released to establish the microsatellite library. Of 522 clones detected by PCR with corresponding repeat primers, 48 clones have shown to be positive reaction. After sequencing, 32 clones were proved to contain microsatellite sequences （SSR）, with a microsatellite enrichment efficiency of 67% as against positive clones and of 6% as against the total detected clones. By deleting the duplicated and unavailable sequences, 12 microsatellite loci were selected to design primers for identifing 85 candidate mutants of Agaricus blazei induced by Co^60 irradiation. The total 17 mutants could be determined by 4 pairs of primer. It demonstrates that some microsatellite loci can be applied to screen and distinguish the mutants ofAgaricus blazei induced by Co^60 irradiation.