CAJ | 학술논문

本文将含有潮霉素抗性基因(hph)的质粒 pAN7-1通过限制性内切酶介导法转入植病生防真菌粉红粘帚霉 Clonostachys rosea 67-1中,并由此建立该生防真菌的遗传转化体系.研究结果表明,PEG 介质含量、限制性内切酶种类和浓度、质粒形态和反应时间对转化效率具有显著的影响.当质粒经线性化后加入40% PEG3350和20 U HindⅢ,室温下转化10 min 时,转化效率可达到30~40 CFU·μg?1质粒 DNA.PDA平板上连续培养3代,再转接到潮霉素抗性平板中,获得遗传稳定的转化子.PCR 检测、Southern 杂交和Real-time PCR 检测等证明 hph 基因已整合到基因组中,且多为单拷贝插入.对随机挑取的50个转化子生长性状的测定结果表明,20%的转化子在 PDA 培养基中生长较快,12%的转化子产孢水平较高;另有16%~20%的转化子生长缓慢、产孢量减少;还有4%的转化子生长微弱,且无明显产孢现象.上述方法可以成功用于在粉红粘帚霉中引入外源基因,为今后生防粘帚霉高效工程菌株的构建提供了依据.
본문장함유조매소항성기인(hph)적질립 pAN7-1통과한제성내절매개도법전입식병생방진균분홍점추매 Clonostachys rosea 67-1중,병유차건립해생방진균적유전전화체계.연구결과표명,PEG 개질함량、한제성내절매충류화농도、질립형태화반응시간대전화효솔구유현저적영향.당질립경선성화후가입40% PEG3350화20 U HindⅢ,실온하전화10 min 시,전화효솔가체도30~40 CFU·μg?1질립 DNA.PDA평판상련속배양3대,재전접도조매소항성평판중,획득유전은정적전화자.PCR 검측、Southern 잡교화Real-time PCR 검측등증명 hph 기인이정합도기인조중,차다위단고패삽입.대수궤도취적50개전화자생장성상적측정결과표명,20%적전화자재 PDA 배양기중생장교쾌,12%적전화자산포수평교고;령유16%~20%적전화자생장완만、산포량감소;환유4%적전화자생장미약,차무명현산포현상.상술방법가이성공용우재분홍점추매중인입외원기인,위금후생방점추매고효공정균주적구건제공료의거.
@@@@Authors tried to genetically transform mycoparasitic fungus Clonostachys rosea 67-1 with hygromycin B resistance gene hph vectored in plasmid pAN7-1 via restriction enzyme-mediated integration. Results showed that content of polyethylene glycol (PEG), type and concentration of restriction endonuclease, plasmid structure, and reaction time showed significant effects on the transformation. When linear pAN7-1, 40% PEG3350 solution, 20 U HindⅢ and 10 min for reaction were adopted, efficiency of the transformation was 30—40 CFU·μg?1 plasmid DNA. Continuous subcultures for 3 times on PDA plates, followed by culturing on hygromycin resistance plates indicated that the transformants obtained were genetically stable. PCR, Southern blotting, and Real-time PCR analyses showed that hph gene was successfully integrated into the genomic DNA of the transformants,in most cases by single copy insertion. Observation on fifty randomly selected transformants on PDA plates demonstrated that 20% transformants grew faster and 12% sporulated more in comparison with their parent strains. This may provide an effective protocol for integration of exogenous genes into C. rosea 67-1 and related fungal species.