FIP-fve基因沉默载体构建和金针菇的遗传转化

发布时间：2018-06-06 04:39

  本文选题：金针菇免疫调节蛋白 + RNAi载体　； 参考：《分子植物育种》2017年11期

【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。
[Abstract]:In order to investigate the biological function of Flammulina velutipes (Flammulina velutipes) FIP-fve gene on Flammulina velutipes, the positive and negative fragments of FIP-fve gene were cloned and its RNAi vector was constructed, and the Agrobacterium tumefaciens LBA4404 was transformed into Flammulina velutipes genetic transformation system mediated by Agrobacterium tumefaciens. The results of double enzyme digestion agarose electrophoresis showed that the RNAi vector of FIP-fve gene was successfully constructed, and the recombinant Agrobacterium tumefaciens transformant was obtained by liquid nitrogen transformation. The genetic transformation of Flammulina velutipes (Flammulina velutipes) with hygromycin (B(Hygromycin B) and ceftomycin Cephalosporins (Cephalosporins) concentration was 9 渭 g / mL and 600 渭 g / mL, respectively. The concentration of recombinant Agrobacterium tumefaciens was 0.5, infecting the mycelium of Flammulina velutipes for 30 min, the concentration of co-culture medium was 200 渭 g / mL, co-cultured at 25 鈩，

本文编号：1985138