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葡萄座腔菌是木本植物溃疡类病害的重要病原,研究该病菌的侵染和致病过程有助于揭示病原与寄主的互作机制。携带gfp基因并高效表达的病菌可有效地实时检测和分析病菌的侵染过程,但由于该病菌在致病和室内培养过程中均不易产孢,因此,制备高质量的原生质体是进行gfp基因转化和表达的首要步骤。通过对酶的种类、酶解液浓度、菌丝年龄、酶解时间、酶解温度和渗透压稳定剂6个可能影响原生质体制备效率的参数进行分析,结果表明:原生质体最大产量产生的条件是菌龄42 h,以1.5%崩溃酶、1.5%葡聚糖在0.7 mol·L-1NaCl的渗透压稳定剂中酶解3.5 h,最适酶解温度31℃,制备的原生质体在酵母蛋白胨蔗糖培养基(YPS)上再生率最高可达48.33%;通过PEG-CaCl2介导原生质体的遗传转化、gfp基因PCR检测、稳定性检测和荧光显微观察,实现了报告基因gfp在葡萄座腔菌转化子内的稳定遗传和高效表达。
Cabernet Sauvignon is an important pathogen of woody plant ulcer disease. Studying the infection and pathogenicity of this bacterium can help to reveal the mechanism of the interaction between pathogen and host. Bacteria carrying gfp gene and highly expressed can effectively detect and analyze the infection process of bacteria in real time. However, since the bacteria are not easy to produce spores during pathogenicity and indoor culture, the preparation of high quality protoplasts is carried out by gfp gene Transformation and expression of the first steps. Six parameters affecting the preparation efficiency of protoplasts, such as the type of enzyme, concentration of hyphae, age of mycelium, enzymolysis time, enzymolysis temperature and osmotic pressure stabilizer, were analyzed. The results showed that the conditions of protoplast production The bacterial strain was cultured for 42 h with 1.5% collapsing enzyme and 1.5% dextran in an osmotic stabilizer of 0.7 mol·L-1NaCl for 3.5 h. The optimal temperature for enzymolysis was 31 ° C. The prepared protoplast was digested with yeast peptone The highest regeneration rate was 48.33% on sucrose medium (YPS). Through the genetic transformation of protoplasts mediated by PEG-CaCl2, PCR detection of gfp gene, stability test and fluorescence microscope observation, Stable inheritance and high expression in bacterial transformants.