聚乙二醇(PEG)介导的甜瓜蔓枯病菌的转化(英文)  

Polyethylene Glycol(PEG)mediated Transformation of the Phytopathogen Didymella bryoniae

作　　者：任海英[1] 李岗[2] 戚行江[1] 梁森苗[1] 郑锡良[1] 

机构地区：[1]浙江省农业科学院园艺研究所,杭州310021 [2]浙江省农业科学院农产品质量标准研究所,省部共建国家重点实验室培育基地浙江省植物有害生物防控重点实验室,杭州310021

出　　处：《农业生物技术学报》2013年第8期965-973,共9页Journal of Agricultural Biotechnology

基　　金：浙江省自然科学基金(No.Y307046);浙江省农业科学院创新项目(No.2007R17Y01E02);浙江省农业科学院国际合作项目(No.2009R17Y04E02)

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(200g/L马铃薯煎汁,20g/L葡萄糖)内震荡培养20h,然后收集产生的幼嫩菌丝,在10mg/mL lysing enzyme+5mg/mL driselase酶液内28℃酶解3h,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。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 bry oniae, 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 （NaCl2.34 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 �

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 

分 类 号：S513[农业科学—作物学]