香梨果萼黑斑病菌Alternaria alternata遗传转化体系的建立及GFP标记菌株的获得

 为建立香梨果萼黑斑病菌链格孢(Alternaria alternata)的原生质体遗传转化体系,本实验以香梨果萼黑斑病菌强致病性菌株LI1为供试材料,研究菌龄、酶系统、酶解时间等对链格孢菌原生质体制备的影响。链格孢菌菌丝在CM液体培养基中培养20 h,以0.7 mol·L^-1 NaCl为稳渗剂,1%裂解酶+1%崩溃酶+1%蜗牛酶的酶液组合下,28 ℃酶解4 h,原生质体制备效率最高。通过PEG/CaCl₂介导法将含有潮霉素B抗性基因和绿色荧光蛋白基因的质粒转入链格孢菌LI1,转化子生长表型及外源基因的PCR鉴定结果表明抗性基因已成功整合到香梨果萼黑斑病菌中。成功建立了香梨果萼黑斑病菌链格孢菌的原生质体遗传转化体系,并成功获得GFP标记菌株,为病原菌侵染定殖过程及致病机制研究奠定了基础。

Development of efficient genetic transformation system in Alternaria alternata and its application for strain labeled with GFP

In this study, we aimed to develop an efficient protoplast transformation system in Alternaria alternata. Several main parameters for isolation of protoplasts, such as the mycelial age, enzyme system, and digesting time, were analyzed and optimized for the strain LI1 of A. alternata with strong pathogenicity. The combined conditions for highest efficiency of protoplast isolation for LI1 strain were as below: the mycelia cultured in CM liquid medium for 20 hours, 0.7 mol·L^-1 NaCl as the osmotic stabilizer, enzyme mixture including 1% Driselase, 1% Lysing enzyme and 1% Snailase for 4 hours of incubation with the mycelia at 28 ℃ at 110 r·min^-1. Moreover, the regeneration of protoplasts was tested with transformation of the plasmid pCT74 DNA containing GFP reporter and the Hyg B resistant genes into LI1 by using PEG/CaCl₂-mediated method. The following confirmation of transformants by PCR and growth phenotypes indicated that the GFP gene was successfully integrated into the LI1 strain genome. In summary, we successfully developed the protoplast transformation system and generated the GFP transgenic strain of A. alternata, which would benefited for understanding of the infection, host colonization, and pathogenic mechanism of this fungus.