| 摘要: |
| 【目的】建立深色有隔内生真菌（dark septate endophyte，DSE）的遗传转化体系，获得绿色荧光蛋白(GFP)标记的DSE转化子，为研究DSE在植物根系的侵染定殖行为和侵染定殖规律奠定基础。【方法】以前期从健康枸杞根系分离的1株DSE菌株枝状枝孢菌（Cladosporium cladosporioides）S12菌株为供试材料，确定潮霉素B对其的最低抑制质量浓度，然后研究菌龄（13，15，17，19和21 h）、酶解时间（1.0，1.5，2.0，2.5，3.0和3.5 h）、崩溃酶质量浓度（10，15，20，25，30和35 mg/mL）和渗透压稳定剂种类（NaCl、KCl、CaCl2和MgSO4）对S12菌株原生质体制备的影响。采用聚乙二醇（PEG）介导原生质体转化法将PDL2质粒（含hph和gfp基因）转入S12菌株，对所获转化子进行表型、GFP荧光、PCR及拮抗活性（以尖孢镰刀菌枸杞专化型Fusarium oxysporum f. sp.Lycium barbarum LR-1菌株为靶标菌）检测，筛选与野生型S12菌株无明显差异且带有GFP标记的转化子。【结果】S12菌株在YPD液体培养基中培养17 h，以0.7 mol/L NaCl为稳渗剂，用30 mg/mL的崩溃酶酶解2.5 h，原生质体制备数量最多，为4.53×106 mL-1。通过PEG介导，将PDL2质粒（含hph和gfp基因）转入S12菌株，可得47株转化子，转化效率2.35株/μg，从中筛选出10株生长速度和产孢量与野生型S12菌株无明显差异的转化子。荧光观察、PCR和拮抗活性检测结果表明，外源基因已成功整合到S12菌株中，成功建立了S12菌株的遗传转化体系，并获得了GFP标记菌株，有6株转化子对LR-1菌株的抑菌率与野生型S12菌株无明显差异。【结论】成功建立了遗传稳定性良好的S12菌株遗传转化体系，筛选出了6株抑菌效果与野生型菌株相当的转化子。 |
| 关键词: 深色有隔内生真菌 枝状枝孢菌S12菌株 原生质体 遗传转化 |
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| 基金项目:国家自然科学基金项目（32260697）；国家重点研发计划子课题“酿酒葡萄病虫害早期多元化预警与防控技术研究与示范”（2019YFD1002502） |
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| PEG-mediated transformation system of dark septate endophyte Cladosporium cladosporioides S12 with GFP gene for strain visualization |
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ZHAO Nan,YAN Siyuan,PEI Ruirui,et al
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| Abstract: |
| 【Objective】The PEG-mediated transformation system of dark septate endophyte (DSE) was established and DSE transformants with GFP labeling were obtained to provide basis for studying the infection colonization behavior tracing and patterns of DSE in plant roots.【Method】The DSE strain (Cladosporium cladosporioides) S12 was isolated previously from roots of healthy Lycium barbarum.The minimum inhibitory concentration of hygromycin B was determined.The effects of strain age (13，15，17，19 and 21 h),enzymatic hydrolysis time (1.0，1.5，2.0，2.5，3.0 and 3.5 h),enzyme mass concentration (10，15，20，25，30 and 35 mg/mL) and osmotic stabilizer (NaCl，KCl，CaCl2 and MgSO4) on the preparation of DSE strain protoplasts were investigated.The expression vector PDL2 containing gfp and hph gene was then transformed into the protoplasts of the DSE strain.The phenotype,fluorescence intensity,PCR and antagonistic activity (Fusarium oxysporum f. sp.Lycium barbarum LR-1 strain as indicative fungi) of the obtained transformants were examined, and the transformants with GFP labelling without significant difference from the wild type S12 strain were selected.【Result】The optimal concentration of the DSE protoplast,approximately 4.53×106 mL-1,was obtained under the growth for 17 h in YPD medium with 0.7 mol/L NaCl as osmotic stabilizer and digestion with 30 mg/mL of Driselase for 2.5 h.After transformation of obtained protoplasts with expression vector PDL2 containing gfp and hph genes,47 transformants were obtained with the transformation efficiency of 2.35 transformants per μg DNA.Ten transformants without significant difference in growth rate and sporulation from wild type S12 strain were also obtained.Fluorescence,PCR and antagonistic activity showed that the exogenous GFP genes were integrated into the genome of the DSE strain.The PEG-mediated transformation system of the DSE strain was successfully established and GFP-labeled transformants were successfully obtained.The inhibition rate of 6 transformants on strain LR-1 was not significantly different from that of wild type S12 strain.【Conclusion】The genetic transformation system of S12 strain with good genetic stability was successfully established,and 6 transformants with comparable antibacterial effect to wild type strains were screened. |
| Key words: dark septate endophyte Cladosporium cladosporioides S12 protoplast PEG-mediated transformation |
