CAJ | 학술논문

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 -1 NaCl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。
포도좌강균시목본식물궤양류병해적중요병원，연구해병균적침염화치병과정유조우게시병원여기주적호작궤제。휴대 gfp기인병고효표체적병균가유효지실시검측화분석병균적침염과정，단유우해병균재치병화실내배양과정중균불역산포，인차，제비고질량적원생질체시진행 gfp기인전화화표체적수요보취。통과대매적충류、매해액농도、균사년령、매해시간、매해온도화삼투압은정제6개가능영향원생질체제비효솔적삼수진행분석，결과표명：원생질체최대산양산생적조건시균령42 h，이1.5%붕궤매、1.5%포취당재0.7 mol·L -1 NaCl적삼투압은정제중매해3.5 h，최괄매해온도31℃，제비적원생질체재효모단백동자당배양기( YPS)상재생솔최고가체48.33%；통과 PEG-CaCl2개도원생질체적유전전화、gfp 기인 PCR 검측、은정성검측화형광현미관찰，실현료보고기인 gfp 재포도좌강균전화자내적은정유전화고효표체。
Botryosphaeria dothidea is a major important pathogen infecting a wide range of woody plant species. Understanding infection and pathogenic processes of the pathogen could help reveal the interaction mechanism between the pathogen and host better. Pathogen with expressed gfp gene can be used as an effective approach to detect and analyze the infection process. B. dothidea is difficult to produce spores during naturally infecting process and in vitro culture process, therefore,preparation of high quality protoplasts is essential for gfp gene transformation and expression. In this study,six parameters influencing protoplast preparation were analyzed,including enzyme species,enzyme concentration,mycelial age,time and temperature of enzymolysis and osmotic stabilizer. The results showed that optimal condition for gaining maximum yields of viable protoplasts was of 42-hour-old mycelia age incubated in 0. 7 mol·L -1 NaCl solution with 1. 5%driselase and 1. 5% glucanase at 31℃ for 3. 5 h. The prepared protoplasts showed a regeneration efficiency of 48. 33% in yeast extract peptone sucrose (YPS) medium. A reporter gene gfp conferring green fluorescent protein was transformed successfully to B. dothidea mediated by PEG-CaCl2. Polymerase chain reaction (PCR) analysis,fluorescent microscope observation and stability test of transformants indicated that the gfp gene was stable in heredity and effective expression. This protocol was the first report for protoplast preparation and gfp transformation of B. dothidea.