重寄生枝孢菌的原生质体制备条件优化及抗性筛选  

Optimization of Protoplast Preparation Conditions and Resistance Screening of Mycoparasitic Cladosporium sp.

作　　者：李明娇 范世昌 严梦玲 陈晨 李靖[1] LI Mingjiao;FAN Shichang;YAN Mengling;CHEN Chen;LI Jing(Southwest Forestry University,Kunming Yunnan 650224,P.R.China)

机构地区：[1]西南林业大学生物与食品工程学院,云南昆明650224

出　　处：《西部林业科学》2024年第6期97-104,共8页Journal of West China Forestry Science

基　　金：云南省农业基础研究专项重点项目(202301BD07000-159)。

摘　　要：为探究重寄生枝孢菌最佳原生质体的制备条件,摸索枝孢菌原生质体遗传转化体系。采用单因素试验和响应面法对枝孢菌的原生质体制备条件,包括菌龄、酶解浓度、酶解时间和渗透压稳定剂进行筛选与优化。结果显示:在菌龄为17 h,崩溃酶浓度为25 mg·mL^(-1),酶解时间为2.5 h以及使用0.7 mol·L^(-1)NaCl作为渗透压缓冲液的条件下,其原生质体产量最高。响应面分析进一步确定了最佳条件为菌龄17.51 h,酶解液浓度26.74 mg·mL^(-1),酶解时间2.26 h,预测原生质体数量为3.52×10^(6)mL^(-1)。复测实验结果得到原生质体产量约为4.01×10^(6)mL^(-1),比预测产量高出12.2%。敏感性实验发现重寄生枝孢菌对潮霉素敏感,其生长在20 mg·L^(-1)潮霉素浓度下受到抑制。研究结果成功优化了重寄生枝孢菌原生质体的制备条件,并筛选出20 mg·L^(-1)潮霉素作为后续分子遗传转化的条件,为重寄生枝孢菌的生物防治研究提供了理论基础。In order to explore the optimal protoplast preparation conditions of mycoparasitic Cladosporium sp.and explore the genetic transformation system.Single factor experiment and response surface method were used to screen and optimize the protoplast preparation conditions including mycelial age,enzyme hydrolysis concentration,enzyme hydrolysis time,and osmotic pressure stabilizer of C.sp.The results showed that under the conditions of a mycelial age of 17 hours,a concentration of Driselase of 25 mg·mL^(-1),an enzyme hydrolysis time of 2.5 hours,and using 0.7 mol·L^(-1) NaCl as the optimal osmotic pressure buffer,the protoplast yield was the highest.Response surface analysis further determined the protoplast preparation conditions as mycelial age of 17.51 hours,enzymatic hydrolysis solution concentration of 26.74 mg·mL^(-1),and enzymatic hydrolysis time of 2.26 h,with the predicted protoplast number being 3.52×106 mL^(-1).The results of the repeated measurement experiment showed that the protoplast yield was approximately 4.01×10^(6) mL^(-1),which was 12.2%higher than the predicted yield.The sensitivity experiment showed that the parasitic C.sp.was sensitive to hygromycin,and its growth was inhibited under the hygromycin concentration of 20 mg·L^(-1).The research results successfully optimized the preparation conditions of protoplasts of Mycoparasitic C.sp.and screened out 20 mg·L^(-1) hygromycin as the screening condition for subsequent molecular genetic transformation,providing a theoretical basis for the biological control research of mycoparasitic C.sp.

关 键 词：重寄生菌 枝孢菌 原生质体制备 敏感性测试 响应面法 

分 类 号：S763.15[农业科学—森林保护学]