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聚乙二醇(PEG)介导的甜瓜蔓枯病菌的转化
任海英1,3,李岗2,戚行江1,3,梁森苗1,3,郑锡良1,3
1. 浙江省农业科学院园艺研究所
2. 浙江省农业科学院
3. 浙江省农业科学院园艺研究所
Polyethylene Glycol(PEG)-mediated Transformation of the Phytopathogen Didymella bryoniae
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摘要 蔓枯病是当前危害瓜类的主要病害,但是蔓枯病菌(Didymella bryoniae)病原学研究还非常落后,关于该菌功能基因的研究报道还较少。为了建立聚乙二醇(polyethylene glycol, PEG)介导的甜瓜蔓枯病菌原生质体遗传转化体系,本研究利用带有潮霉素B磷酸转移酶(hph)基因的质粒pSGate1为载体,通过PEG(分子量3350)介导的融合法转化蔓枯病菌株ZJDB32。将病菌分生孢子于PDB培养液(200 g/L马铃薯煎汁, 20 g/L葡萄糖)内震荡培养20 h,然后收集产生的幼嫩菌丝,在10 mg/mL lysing enzyme+5 mg/mL driselase 酶液内28℃酶解3 h,每克湿菌丝能产生3.8× 107 个原生质体。PEG介导融合转化pSGate1至D. bryoniae原生质体,每毫克ZJDB32的DNA转化效率最高能得到1230个转化子。结果表明,20个代表性的ZJDB32的转化子继代4次后其潮霉素抗性、生长速率、产孢量和致病性与野生型都没有明显变异,这些转化子可以进一步用于相关功能研究。因此,该转化体系的建立为甜瓜蔓枯病菌功能基因的深入研究提供了基础资料。
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任海英
李岗
戚行江
梁森苗
郑锡良
关键词 甜瓜蔓枯病菌原生质体转化聚乙二醇转化效率稳定性    
Abstract:Gummy stem blight, a plant disease caused by Didymella bryoniae, is one of the major diseases in melon. However, little information is available on the genetics and functional genomics of the fungal pathogen. In order to develope a polyethylene glycol (PEG)-mediated transformation system of Didymella bryoniae, it was transformed by PEG-induced fusion of protoplasts. The plasmid pSGate1 carrying hygromycin B phosphotransferase gene (hph) gene was used and D. bryoniae ZJDB32 isolate was used as the host strain. After 1010 conidia were incubated for 20 h in PDB medium (200 g/L potato extract, 20 g/L dextrose) by shaking at 150 r/min, the mycelia were collected and enzymatically hydrolyzed for 3 h at 28℃ by shaking at 90 r/min at 40 mL of enzyme solution (NaCl 2.34 g, 1 mol/L MgCl2 0.4 mL, 100 mmol/L K3PO4 4 mL, 400 mg lysing enzyme, 200 mg driselase, and dH2O), the most protoplasts (3.8×107 /g fresh mycelia) were generated. The pelleted protoplasts were suspended in a 4∶1 mixture of STC (sorbitol, 1.2 mol/L; Tris-HCl, 10 mmol/L at pH 7.5; CaCl2, 10 mmol/L)∶PTC (PEG moleculor weight 3 350, 50 g/100 mL; Tris-HCl, 10 mmol/L at pH 7.5; CaCl2, 10 mmol/L) and adjusted to a concentration of 2×108 /mL. Twenty micrograms of the plasmid in less than 20 μL STC∶PTC (4∶1) were added to 100 μL of the above protoplast suspension, mixed, and incubated on ice for 20 min. The protoplast: plasmid suspensions were amended with 100, 300, or 600 μL PEG∶STC solution (25 g PEG molecular weight 3 350 with STC in a total volume of 50 mL) and incubated for 20 min at 25℃. Finally, the mixtures were amended 1, 3, and 4 mL of STC, respectively, and mixed gently. Protoplasts were pelleted by centrifugation at 3500 g for 10 min, re-suspended in 1.6 mL recovery medium (RM) (sucrose, 1 mol/L; yeast extract, 0.1%; tryptone, 0.1%) and incubated at 25℃ for 2~4 h with gentle shaking at 75 r/min. Each protoplast suspension was then mixed gently with 20 mL of recovery agar medium (RAM) (sucrose, 1 mol/L; yeast extract, 0.1%; tryptone, 0.1%; agar, 0.8%) containing hygromycin B at 100 μg/mL at 50℃. Each mixture was poured into a Petri plate and incubated at 28℃. After 3~5 d, transformants were transferred to fresh potato dextrose agar (PDA) (200 g/L potato extract, 20 g/L dextrose, 15 g/L agar) with hygromycin B at 100 μg/mL and incubated at 25℃. The transformants were purified by single spore isolation. About 1230 hygromycin resistant transformants were generated per mg plasmid DNA. All tested twenty transformants had stable resistance to hygromycin B after four serial passages. The hph gene was confirmed by PCR with hph-specific primers and integrated into the genomes of all 20 tested transformants. Transformants had similar colony morphology, growth rate, sporulation, and pathogenicity compared to the wild type. The transformants can be used in the subsequent functional research of D. bryoniae strain ZJDB32. In conclusion, we have demonstrated and optimized a PEG-mediated protoplast transformation system for D. bryoniae strain ZJDB32 with pSGate1. Therefore, this paper establishes an alternative genetic transformation system for D. bryoniae for functional genomics studies.
Key wordsDidymella bryoniae    Protoplast transformation    Polyethylene glycol    Transformation efficiency    Stability test
收稿日期: 2013-03-29     
通讯作者: 戚行江   
引用本文:   
任海英1,3,李岗2,戚行江1,3,梁森苗1,3,郑锡良1,3. 聚乙二醇(PEG)介导的甜瓜蔓枯病菌的转化[J]. , 2013, 21(8): 965-973.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2013/V21/I8/965
 
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