人参灰葡萄孢原生质体制备条件的优化及再生菌株致病性研究

Condition optimization for protoplast preparation of Botrytis cinerea and the patho-genicity of regenerated strains

为了获得人参灰葡萄孢(Botrytis cinerea)高质量和足够数量的可用于遗传转化的原生质体,本文研究了菌龄、酶系组成、渗透压稳定剂种类及酶解温度、时间等因素对其原生质体制备和再生的影响。结果表明人参灰葡萄孢JA-6在PDA培养基上25℃培养36 h,崩溃酶、蜗牛酶、细胞溶壁酶的浓度分别为1%、0.1%、1%,28℃,120 r·min^(-1)酶解3 h,渗透压稳定剂为0.6 mol·L^(-1)KCl+50 mmol·L^(-1)CaCl_(2)缓冲液的条件下,酶解5 g·mL^(-1)的人参灰葡萄孢菌丝可以获得1.06×107个·mL^(-1)原生质体。将原生质体涂布于再生培养基RM后,得到再生菌株的菌落形态、生长速率、分生孢子产量和致病力与野生型无明显差异。PEG介导法将绿色荧光蛋白基因(Green fluorescent protein,GFP)转入制备好的原生质体中,其后代荧光信号能稳定遗传。本研究优化并确定了人参灰葡萄孢原生质体制备的条件,在此条件下制备的原生质体的质量和数量能够满足其遗传转化的要求。

In order to obtain high-quality and sufficient protoplasts of Botrytis cinerea that can be used for ge-netic transformation,effects of multiple parameters including mycelial age,combinations of lytic enzymes,types of osmotic stabilizers,enzymatic hydrolysis temperature,and time of enzyme digestion on protoplasts prepara-tion were studied.The optimal lytic enzyme was determined to be the combination of driselase,snailase,and ly-sing enzyme at an active ingredient of 1%,0.1%and 1%,respectively,and the mycelial age,composition and concentration of osmotic stabilizer,enzyme digestion temperature,and enzyme digestion time were as follows:mycelia of JA-6 was cultivated on PDA at 25°C for 36 h,osmotic stabilizer contained 0.6 mol·L^(-1)KCl and 50 mmol·L^(-1)CaCl_(2),and enzyme digestion time was 3 h at 120 r·min^(-1)at 28°C.Enzymatic hydrolysis of 5 g·mL^(-1)of B.cinerea mycelium can yield 1.06×107 protoplasts·mL^(-1)under above optimal protoplast preparation condi-tions.No significant differences of the colony morphology,growth rate,conidial production and pathogenicity were observed between the regenerated strain and the wild-type strain.The GFP(green fluorescent protein)gene was subsequently transformed into B.cinerea JA-6 by PEG mediated transformation.The fluorescence signal of the transformants can be stably inherited.The established protoplast preparation method in the present research would meet the requirements of genetic transformation of B.cinerea for further study.