坛紫菜微卫星DNA序列的筛选

自坛紫菜(Porphyrahaitanensis)丝状体中提取的DNA,经Sau3AI限制性内切酶消化后,将300~900bp之间的DNA片段回收,并连接到经BamHI酶切并去磷酸化的pUC18载体上,最后转化至大肠杆菌JM109感受态细胞中,构建坛紫菜小片段DNA质粒文库。选用pUC18质粒的通用引物进行PCR反应,对文库进一步筛选并检测插入片段的大小,在384个阳性克隆中,有278个含有大小合适的插入片段。经测序及序列分析,在103个克隆中获得172个微卫星序列,其中完美型107个,占62.2%,非完美型53个,占30.8%。(GC)n与(CG)n在坛紫菜DNA中非常丰富,分别占25%及17%,但重复频率相对较低。

Isolation of microsatellite DNA in Porphyra haitanensis

DNA fragments isolated from Porphyra haitanensis between 300 bp and 900 bp were recovered after treatment with restriction endonuclease of Sau3AI, and then were recombined to vector of pUC18 previously digested by BamHI and dephosphorylated. The partial DNA libraries were constructed after transmitting these recombinant vectors into Escherichia coli JM109 competent cells. Polymerase chain reactions with the general primers of pUC18 vector were used to examine whether there was target DNA inserted in the libraries or not and to detect the size of inserted DNA fragments. There were 278 clones containing suitable size of target DNA in 384 tested clones. One hundred and seventy-two microsatellites were screened from 103 clones after the analysis of sequence of the 278 clones. The perfect and imperfect microsatellites were 107 and 53, respectively, which accounted for 62.2% and 30.8%. The microsatellites of P. haitanensis were characterized as the higher abundance of (GC)n and (CG)n, which accounted for 25% and 17%, respectively, but the relatively lower repeat frequency.