摘要
建立高效、稳定的甘蓝枯萎病菌REMI转化体系,为进一步获得特定表型突变体及基因功能研究建立技术储备。利用REMI(restriction enzyme mediate intergration)转化方法,将线性化的含有潮霉素抗性基因的pUCAT-PH质粒转化甘蓝枯萎病菌A6菌株的原生质体,摸索获得转化子最适的潮霉素筛选浓度以及不同限制性内切酶和酶量对转化效率的影响;利用PCR(polymerase chain reaction)技术对潮霉素抗性转化子进行验证。结果表明转化子的最适潮霉素筛选浓度为50μg/mL;转化效率较高的限制性内切酶为HindⅢ,并且转化效率最高时的酶量为20U。利用该转化体系构建了含1 050个转化子的甘蓝枯萎病菌转化子库,对转化子进行Southern验证,证明该转化体系是可行的。
Establishment of an efficient and stable REMI transformation system in Fusariurn oxysporum f. sp. conglutinans by restriction enzyme-mediated integration is a key step to build a transformants library for further studying mutants with specific phenotype and gene function. By REMI transformation method, the linearized pU- CATPH plasmid containing the hygromycin resistance gene was transformed into the protoplasts of F. oxysporurn strain A6. The hygromycin concentration to transformants was optimized, and different restriction enzymes were tested on the transformation efficiency. Hygromycin-resistant transformants were checked by PCR. Hygromycin at 50/μg/mL was optimal for screening transformants. Transformation efficiency by HindⅢ was higher than other enzymes, and reached to the highest at 20 U. A library with 1 050 transformants was built by the REMI transfor- mation system. The Southern blot results proved the feasibility of the transformation system.
出处
《植物保护》
CAS
CSCD
北大核心
2013年第3期108-111,115,共5页
Plant Protection
基金
公益性行业(农业)科研专项(2000903049)
北京市农林科学院植保环保所创新基金(CXJJ200901)
山东省“泰山学者”建设工程专项经费
青岛农业大学高层次人才科研基金(631217)
关键词
甘蓝枯萎病菌
REMI
转化子库
Fusarium oxysporum f. sp. conglutinans
REMI
transformants library
