PEG介导的苹果果生刺盘孢Colletotrichum fructicola原生质体转化
PEG-Mediated Transformation of Colletotrichum fructicola

为建立PEG介导的苹果果生刺盘孢Colletotrichum fructicola的遗传转化体系，以果生刺盘孢SQ06-E的幼嫩菌丝为受体，通过PEG介导的原生质体转化法，将含有潮霉素标记的质粒pCB1003转入果生刺盘孢菌丝的原生质体中；对获得的转化子进行遗传稳定性检测、PCR检测和Southern杂交分析。试验获得果生刺盘孢的最优转化体系为：密度为1×106 mL-1分生孢子在M3S培养基中，28 ℃、200 r/min震荡培养 8 h ；过滤收集菌丝，在质量浓度为0.25 g/mL崩溃酶+0.05 g/mL溶壁酶的10 mL酶解液中加入0.5 g湿菌体酶解3 h；整个试验的渗透压稳定剂为0.8 mol/L KCl。试验共得到76个转化子，转化率为1 μg DNA获得 3~4 个转化子。对转化子的PCR检测和Southern杂交分析都表明hph基因已整合入果生刺盘孢的基因组中。所有转化子在PDA培养基上继代5次后仍能正常生长，说明外源基因hph能在果生刺盘孢中稳定遗传。
To establish the PEG-mediated transformation technology system of Colletotrichum fructicola,we used the fresh hypha of Colletotrichum fructicola strain SQ06-E as recipient and then transformed a plasmid pCB1003 harboring the hygromycin B phosphotransferase gene (hph) into the protoplast of SQ06-E mediated by PEG (polyethylene glycol).The obtained transformants were screened and identified by hygromycin B resistance,PCR and method of Southern Blot analysis.The optimal transformation conditions were that 1×106 spores per milliliter of Colletotrichum fructicola spore suspension were shocked in M3S medium for 8 h in 200 r/min at 28 ℃; collecting 0.5 g hypha and hydrolyzing them in 10 mL enzyme solution which contained 0.25 g/mL Driselase and 0.05 g/mL Lysing enzyme for 3 h;using 0.8 mol/L KCl as the osmotic pressure stabilizer in all experiments.76 transformants have been get and the efficiency reached to 3-4 transformants per microgramme.DNA.Analysis of the transformants by PCR and Southern Blot showed that the selectable marker gene hph was integrated effectively into the genome of Colletotrichum fructicola.After 5 subculturing on PDA,all transformants could grow.This stability test of transformants suggested that the foreign gene hph was stable in heredity.