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【目的】通过分析不同酶解条件对金黄壳囊孢菌[Cytospora chrysosperma(Pers.)Fr.]原生质体释放的影响,建立高效制备原生质体及其遗传转化体系的方法,为开展杨树腐烂病菌的致病分子机制研究奠定基础。【方法】以杨树腐烂病菌菌株CFCC 89981为受体,在细胞壁降解酶作用下产生用于转化所需的原生质体,通过PEG(Polyethylene glycol)介导将g GFP DNA导入杨树腐烂病菌的原生质体中获得转化子。经PCR扩增、Southern blot和荧光观察验证g GFP DNA插入到杨树腐烂病菌基因组中并表达出GFP(Green fluorescent protein)蛋白。【结果】以p H 5.5的1.2 mol/L KCl为稳渗剂,杨树腐烂病菌菌丝经Driselase和Lysing enzymes共同酶解4 h可获得1.2×108个/m L原生质体,再生率可达63.74%±9.73%,FDA(Fluorescein diacetate)溶液染色结果显示98%左右的原生质体具有较高的活力。利用PEG介导的遗传转化方法,转化效率可达76个/μg DNA。PCR检测和Southern blot均可在转化子基因组中检测到GFP基因片段,且荧光检测转化子的菌丝均呈绿色荧光,表明GFP基因在杨树腐烂病菌中表达。此外,GFP转化子在无潮霉素抗性的PDA培养基中多代转接后仍稳定遗传并表达GFP蛋白。【结论】通过筛选酶解条件,获得高质量、高活性的杨树腐烂病菌原生质体,并利用PEG介导的转化方法建立了高效稳定的原生质体遗传转化体系。该体系的建立为杨树腐烂病菌的后续研究奠定了技术基础。
【Objective】 The objective of this study was to establish a method for efficient preparation of protoplasts and their genetic transformation systems by analyzing the effects of different enzymatic hydrolysis conditions on the release of protoplasts from Cytospora chrysosperma (Pers.) Fr. Pathogenesis of molecular mechanisms laid the foundation for the study. 【Method】 The protoplasts required for transformation were produced by the cell-wall degrading enzyme (CFCC 89981) and introduced into the protoplasts of B. poplar by PEG (Polyethylene glycol) Transformants are obtained in the body. The GFP DNA was inserted into the genome of Poplar tree and the green fluorescent protein (GFP) was expressed by PCR amplification, Southern blot and fluorescence observation. 【Result】 With the 1.2 mol / L KCl of p H 5.5 as the stabilizing agent, 1.2 × 108 protoplasts of protoplast 1.2 × 108 per m L were obtained by enzymatic digestion of Driselase and Lysing enzymes with the regeneration rate of up to 63.74% ± 9.73%. Fluorescein diacetate (FDA) solution staining showed that about 98% of protoplasts had higher viability. Using PEG-mediated genetic transformation, the transformation efficiency of up to 76 / μg DNA. The GFP gene fragment was detected in the transformant genome by both PCR and Southern blot. The mycelia of the fluorescent transformants were all green fluorescence, which indicated that the GFP gene was expressed in the poplar rot bacteria. In addition, GFP transformants remained stably inherited and expressed GFP protein after multiple passages in hygromycin-resistant PDA medium. 【Conclusion】 Protoplasts of P. populari of high quality and high activity were screened by enzymatic hydrolysis conditions, and a stable and efficient protoplast genetic transformation system was established by PEG-mediated transformation. The establishment of this system laid the technical foundation for the follow-up study of poplar rot bacteria.