摘要
对标准的链霉菌接合转移方法进行优化建立起适用于吸水链霉菌NRRL5491的接合转移系统。40℃温浴20 min为孢子的最佳热激处理条件;整合型质粒pSET152的接合转移效率是穿梭型质粒pOJ446的3-6倍;当阿泊拉霉素覆盖浓度为1μg/mL时,pSET152的接合转移效率是50μg/mL时的4.6倍,而pOJ446接合转移效率相应提高了2.4倍;MS培养基上接合转移效率是SY琼脂培养基上的3.4倍。采用最适优化条件时,接合转移频率可达到2×10^-6,足够用于基因置换等遗传操作。
Streptomyces hygroscopicus NRRL5491 produces rapamycin which has potential antifungal,immunosuppressive and anticancer properties. An easy and efficient method of gene transfer is urgent in the genetic engineering of S. hygroscopicus. Here a genetic system was established. 40 ℃ heat shock for 20 min was the best condition for spore germination. The exconjugant frequency of pSET152 （an integrative plasmid） was 3 to 6 times more efficient than pOJ446 （a self-replicated vector） ; using overlay concentration of 1/μg/mL was 2.4 to 4. 6 times more efficient than using 50 μg/mL apramycin. The conjugation frequency on MS medium was 3.4 times higher than that on SY agar medium. The conjugation frequency is 2 ×10^-6 at optimal condition, which is sufficient for genetic manipulation such as gene replacement.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
2007年第5期637-641,共5页
Journal of Huazhong Agricultural University
基金
国家自然科学基金项目(30570030)资助
