玉木耳原生质体制备条件的优化及再生菌株的变异检测  

Optimization of Protoplast Preparation of Auricularia cornea (Yumuer) and Variation Detection of Regenerated Strain

作　　者：崔玮洁 赵一橦 宋冰[1] 代月婷 孙磊[1] 付永平[1] 李玉[1] Cui Weijie;Zhao Yitong;Song Bing;Dai Yueting;Sun Lei;Fu Yongping;Li Yu(Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi,Jilin Agricultural University,Changchun,130118)

机构地区：[1]吉林农业大学食药用菌教育部工程研究中心

出　　处：《分子植物育种》2019年第18期6006-6012,共7页Molecular Plant Breeding

基　　金：公益性行业(农业)科研专项(201503137);吉林省科技厅项目(20170101053JC);吉林省地方标准项目(DBXM028-2018)共同资助

摘　　要：玉木耳是毛木耳的天然白色变异菌株。为研究玉木耳天然突变的分子机制,本研究分别从菌丝培养时间、溶壁酶浓度、稳渗剂种类、酶解温度和酶解时间对玉木耳原生质体制备条件进行筛选及优化,并利用显微观察鉴定再生单核菌株。结果表明:玉木耳原生质体制备的适宜条件是菌丝培养时间为15 d,溶壁酶(Lywallzyme)浓度为2.0%,甘露醇作为稳渗剂且浓度为0.6 mol/L,27℃酶解5 h。通过对再生菌株的显微镜观察、荧光染色以及分子鉴定,鉴定出共有13个玉木耳单核菌株。与双核菌株相比,单核菌丝生长速度慢但更浓密。在基因组水平发现大量变异,共产生1 432 175个SNPs (Single nucleotide polymorphisms)和251 663个InDel (Insertion-deletion)位点。本研究为玉木耳的全基因组测序、基因功能验证及分子育种提供了良好的参考基础。Auricularia cornea(Yumuer) is a white mutant strain of Auricularia cornea. In order to study the molecular mechanism of natural mutation of A. cornea, this study screened the preparation conditions of protoplasts of A. cornea from mycelial culture time, concentration of lywallzyme, type of osmotic stabilizer, enzymatic hydrolysis temperature and enzymatic hydrolysis time. The regenerated mononuclear strains were identified by microscopic observation. The results showed that the conditions of protoplast preparation for optimal were mycelia culture time of 15 d, using 2% digestion enzyme(Lywallzyme) with 0.6 mol/L mannitol, incubated for 5 h at 27℃. A total of 13 monokaryons of A. cornea were identified by microscopic observation of the regenerated strain, DAPI fluorescence staining and molecular identification. Compared with the dikaryon strain, the mononuclear hyphae grew slowly but more strong. A large number of mutations were found at the genome level, resulting in a total of 1 432 175 SNPs and 251 663 InDel sites. The above results would provide a good foundation for the whole genome sequencing, gene function verification and molecular breeding of A. cornea.

关 键 词：玉木耳(Auricularia cornea) 原生质体 单核菌株 变异 

分 类 号：S64[农业科学—蔬菜学]