摘要
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。
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.
出处
《林业科学》
EI
CAS
CSCD
北大核心
2014年第6期131-137,共7页
Scientia Silvae Sinicae
基金
Ministry of Science and Technology of the People’s Republic of China Project(2009FY210100)
World Bank Loans ProjectShandong Ecology Afforestation Program(SEAP-JC-2)
Natural Science Foundation of Zhejiang Province(Y3110028)
关键词
葡萄座腔菌
原生质体制备
再生
转化
GFP
Botryosphaeria dothidea
protoplast preparation
regeneration
transformants
gfp
