FIP-fve基因沉默载体构建和金针菇的遗传转化  

Construction of Silencing Vector for FIP-fve Gene and Genetic Transformation of Enoki Mushroom(Flammulina velutipes)

作　　者：张昕 夏雪 段作文 关山越[1] 李浩戈[1] 陈丽静[1] 张丽[1] 范文丽[2] 林景卫[1,2] 

机构地区：[1]沈阳农业大学生物科学技术学院,沈阳110866 [2]沈阳农业大学园艺学院,沈阳110866

出　　处：《分子植物育种》2017年第11期4491-4497,共7页Molecular Plant Breeding

基　　金：辽宁省农业领域青年科技创新人才培养计划项目(2015043)资助

摘　　要：为了探究金针菇FIP-fve基因对金针菇本身的生物学功能,本研究克隆FIP-fve基因正反片段并构建其RNAi载体,并将其转化农杆菌LBA4404,建立以农杆菌介导的金针菇遗传转化体系。双酶切琼脂糖电泳检测结果表明成功构建了FIP-fve基因的RNAi载体:p TCK303-fve(F)-fve(R),并用液氮转化法获得重组农杆菌转化子。金针菇遗传转化以菌丝体为外植体,潮霉素B(Hygromycin B)和头孢霉素(Cephalosporins)使用浓度分别为9μg/m L和600μg/m L。重组农杆菌浓度OD600为0.5,侵染金针菇菌丝体30 min,共培养培养基AS使用浓度为200μg/m L,共培养25℃3 d,抗性筛选10 d。最终通过潮霉素抗性筛选和PCR检测从60个外植体中筛选得到5个金针菇遗传转化子,转化率为8.33%。经过连续培养,对比观测转化子和野生型金针菇,FIP-fve基因沉默的金针菇转化子菌丝的生长在第3天、第6天和第10天明显慢于野生型金针菇菌丝。初步说明FIP-fve基因对金针菇菌丝体时期的生长发育具有一定调节作用,这为研究金针菇FIP-fve以及FIP对真菌本身生物学功能提供一定研究基础和直接证据。In order to explore the biological functions of enoki mushroom（Flammulina velutipes） FIP-fve gene on enoki mushroom itself. In this research we cloned the pros and cons of FIP-fve gene and constructed its RNAi vector, and the recombinant RNAi vector was transformed into Agrobacterium LBA4404, the genetic transformation system of enoki mushroom（Flammulina velutipes） was established by Agrobacterium mediated. Double enzymes digestion agarose electrophoresis showed that the RNAi vector, p TCK303-fve（F）-fve（R） was successfully constructed, the recombinant Agrobacterium transformants were obtained by using liquid-nitrogen transformation method. The mycelium was used as explant in the genetic transformation of enoki mushroom（Flammulina velutipes）, with the application of Hyg and Cef concentrations were 9 μg/m L and 600 μg/m L, respectively. The recombinant Agrobacterium OD600 concentration of 0.5 infected mycelia of enoki mushroom（Flammulina velutipes）for 30 min, the AS concentration of 200 μmol/L in the co-medium cultured for 25℃ 3 d and resistance screened for 10 d. Finally, we obtained 5 positive genetic transformants of enoki mushroom（Flammulina velutipes） from 60 explants by Hyg resistance screening and PCR, and the transformation rate was 8.33%. After continuous culture,by comparative observation between transformant and wild type of enoki mushroom（Flammulina velutipes）, which found that the growth of enoki mushroom（Flammulina velutipes） transformant mycelia of FIP-fve silencing gene was apparently slower than the wild type mycelia of enoki mushroom（Flammulina velutipes） in 3 th, 6 th and 10 th.This preliminarily showed FIP-fve gene had a certain adjustment function on growth and development of enoki mushroom（Flammulina velutipes） mycelium, which also can provided a research foundation and directly evidence to research the biological functions of FIP-fve and FIP on fungil themselves.

关 键 词：金针菇免疫调节蛋白 RNAI载体 遗传转化 

分 类 号：S646.15[农业科学—蔬菜学]