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

作　　者：韩小路 白静科 张玮[1] 张荣[1] 孙广宇[1] 

机构地区：[1]西北农林科技大学植物保护学院,陕西杨凌712100

出　　处：《西北农业学报》2016年第3期442-449,共8页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：国家自然科学基金(31171797)~~

摘　　要：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1×10^6 mL-1分生孢子在M3S培养基中,28℃、200r/min震荡培养8h;过滤收集菌丝,在质量浓度为0.25g/mL崩溃酶＋0.05g/mL溶壁酶的10mL酶解液中加入0.5g湿菌体酶解3h;整个试验的渗透压稳定剂为0.8mol/L KCl。试验共得到76个转化子,转化率为1μg 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 1×10^6 spores per milliliter of Colletotrichum fructicola spore suspension were shocked in M3 Smedium for 8hin 200r/min at 28℃;collecting 0.5g hypha and hydrolyzing them in 10 mL enzyme solution which contained 0.25g/mL Driselase and 0.05g/mL Lysing enzyme for 3h;using 0.8mol/L KCl as the osmotic pressure stabilizer in all experiments.76 transformants have been get and the efficiency reached to 3-4transformants 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 5subculturing on PDA,all transformants could grow.This stability test of transformants suggested that the foreign gene hph was stable in heredity.

关 键 词：苹果炭疽叶枯病 遗传转化 转化子鉴定 

分 类 号：S436.611.1[农业科学—农业昆虫与害虫防治]