| 冯建海1，张玮2，李兴红2，燕继晔2*，黄金光1，3*.甘蓝枯萎病菌REMI转化体系的建立[J].植物保护,2013,39(3):101-104. |
| 甘蓝枯萎病菌REMI转化体系的建立 |
| Construction of transformation system in Fusarium oxysporum f.sp. conglutinans by REMI |
| 投稿时间：2014-04-18 修订日期：2014-09-11 |
| DOI： |
| 中文关键词: 甘蓝枯萎病菌 REMI 转化子库 |
| 英文关键词:Fusarium oxysporum f.sp. conglutinans REMI transformants library |
| 基金项目:公益性行业（农业）科研专项（201103022） |
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| 中文摘要: |
| 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pUCATPH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50 μg/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20 U。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。 |
| 英文摘要: |
| Establishment of an efficient and stable REMI transformation system in Fusarium 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 pUCATPH plasmid containing the hygromycin resistance gene was transformed into the protoplasts of F.oxysporum 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 transformation system. The Southern blot results proved the feasibility of the transformation system. |
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