芦笋茎枯病菌原生质体的制备及再生  

Isolation and regeneration of Phomopsis asparagi protoplast

作　　者：张岳平[1] 罗绍春[1] 黄燕萍[1] 汤泳萍[1] 易克贤[2] 谢丙炎[3] 陈光宇[1] 

机构地区：[1]江西省农业科学院蔬菜花卉研究所,南昌330200 [2]中国热带农业科学院环境与植物保护研究所,海南海口571101 [3]中国农业科学院蔬菜花卉研究所,北京100081

出　　处：《植物保护学报》2014年第2期182-186,共5页Journal of Plant Protection

基　　金：国家公益性行业(农业)科研专项(201003074);中央级公益性科研院所基本科研业务费专项(1630042012004);国家自然科学基金(31360426);江西省自然科学基金(20132BAB214020);江西省博士后科研择优资助项目

摘　　要：原生质体的制备是真菌遗传转化的基础,为了解芦笋茎枯病菌Phomopsis asparagi的遗传转化体系,以芦笋茎枯病菌Pa1100为供试菌株,研究了细胞壁裂解酶、酶解时间、稳渗剂等对芦笋茎枯病菌原生质体制备的影响及稳渗剂对原生质体再生的影响。结果表明可制备芦笋茎枯病菌原生质体较为适宜的条件是:CM液体培养基中培养分生孢子3 d,以1.5%裂解酶、1%崩溃酶和1.5%蜗牛酶为组合酶解液,33℃水浴酶解4.5 h,以PBS(pH 6.98)与1 mol/L MgSO4混合液为酶解稳渗剂。含0.6 mol/L蔗糖的PDA培养基较适合于芦笋茎枯病菌原生质体的再生。Generation of fungal protoplast is an essential tool for genetic transformation system. To establish protoplast-mediated genetic transformation system of Phomopsis asparagi,conditions for the protoplast isolation and regeneration of Pa1100 mycelia,including various enzymes,digestion time,and osmotic stabilizers, were examined in this study. An efficient method for protoplast isolation and regeneration of P. asparagi was obtained. The results showed that 3-day-old mycelia cultured in liquid CM medium was adequate to protoplast isolation. The enzyme mixture of 1. 5% lyase,1% driselase and 1. 5% snailase was most beneficial for digesting the mycelia. The suitable incubation time with enzyme for the maximum release of protoplasts was 4. 5 h in a water bath at 33 ℃. The most effective osmotic stabilizer for the protoplast release was 1 mol / L MgSO4combined with phosphate buffer( pH 6. 98). P. asparagi protoplast regeneration results showed that the potato dextrose agar medium containing 0. 6 mol / L sucrose was the best medium for regeneration.

关 键 词：芦笋茎枯病菌 原生质体 制备 再生 

分 类 号：S436.44[农业科学—农业昆虫与害虫防治]