顶头孢霉菌转化平台的优化及其Ku70同源基因敲除质粒的构建

发布时间：2018-01-26 02:00

  本文关键词： 顶头孢霉 原生质体 转化 Ku70同源基因 基因敲除　出处：《华东理工大学》2016年硕士论文　论文类型：学位论文

【摘要】：半合成头孢菌素是国际上重要的抗微生物感染类药物之一,销量占所有抗感染类药物的65%,且这个比例仍在近年来一直持续稳定上升。因此,对于顶头孢霉的基因工程领域的研究具有重要的价值。但是丝状真菌的遗传改造很困难,原因主要有两个：一,丝状真菌的转化效率极低。二,外源基因进入受体细胞后,插入到基因组中的位点往往是随机的。基因打靶的同源重组成功率极低,想要对丝状真菌基因进行定点改造的难度极大。敲除Ku70同源基因以提高真核细胞基因打靶效率的策略,已经在多种真核菌类中得以实现。因此构建顶头孢霉菌的高效外源基因转化平台,并且构建该菌种Ku70基因缺陷型,是顶头孢霉工业菌菌种遗传改造的重要基础。本文阐述了一种基于聚乙二醇介导的原生质体转化方法,并且尝试构建Ku70基因敲除质粒,为构建Ku70敲除株提供基础。我们首先对顶头孢霉菌原生质体的制备和再生条件进行了系统优化。通过对酶种类和复配比、酶解时间、菌丝形态、再生培养基种类和涂布方式等考察了顶头孢霉原生质体的制备和再生条件。最终优化的条件是选择中度膨大初期的菌丝体,使用1%溶壁酶,30℃酶解2h后采用双层平板法接种于原生质体再生培养基I (HCM)中。原生质体的释放数可以达2×108个/ml,再生率为20%。其次,对顶头孢霉原生质体进行转化和验证。利用质粒pYG233转化制备的原生质体,验证转化效率为10个阳性转化子/μg质粒。构建出了将CPC转化成为7-ACA潜在生产菌株。此外,我们还开展了敲除质粒pAcKu70的构建工作。该质粒含有顶头孢霉Ku70同源基因的上、下游片段,腐草霉素标记基因以及pcbAB启动子。上述研究为实现顶头孢霉的代谢工程改造奠定了基础。
[Abstract]:Semi-synthetic cephalosporins are one of the most important antimicrobial drugs in the world. The sales volume accounts for 65% of all anti-infective drugs, and this proportion has been increasing steadily in recent years. The research on genetic engineering of Cephalosporium acrocephalus is of great value, but the genetic transformation of filamentous fungi is very difficult for two reasons: first, the transformation efficiency of filamentous fungi is very low; second, the transformation efficiency of filamentous fungi is very low. After the foreign gene enters the receptor cells, the sites inserted into the genome are often random. The success rate of homologous recombination of the gene targeting is very low. In order to improve the efficiency of gene targeting in eukaryotic cells, it is very difficult to modify the filamentous fungal genes by knockout Ku70 homologous genes. It has been realized in many eukaryotes. Therefore, the high efficient exogenous gene transformation platform of Cephalocephalus was constructed, and the Ku70 gene deficient type of this strain was constructed. It is an important basis for genetic transformation of industrial strains of Cephalosporium acrocephalus. In this paper, a method of protoplast transformation based on polyethylene glycol (PEG) was introduced, and Ku70 gene knockout plasmid was constructed. In order to construct Ku70 knockout plant, we first optimized the preparation and regeneration conditions of protoplasts of Cephalosporium acuminata. Through the enzyme species and composition ratio, enzymatic hydrolysis time, mycelium morphology. The preparation and regeneration conditions of protoplasts of Cephalocephalus were investigated. The optimal conditions were to select the mycelia of medium expansion stage and use 1% lysozyme. After enzymatic hydrolysis at 30 鈩，

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