本文關(guān)鍵詞： 頂頭孢霉 原生質(zhì)體 轉(zhuǎn)化 Ku70同源基因 基因敲除　出處：《華東理工大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：半合成頭孢菌素是國(guó)際上重要的抗微生物感染類藥物之一,銷量占所有抗感染類藥物的65%,且這個(gè)比例仍在近年來(lái)一直持續(xù)穩(wěn)定上升。因此,對(duì)于頂頭孢霉的基因工程領(lǐng)域的研究具有重要的價(jià)值。但是絲狀真菌的遺傳改造很困難,原因主要有兩個(gè)：一,絲狀真菌的轉(zhuǎn)化效率極低。二,外源基因進(jìn)入受體細(xì)胞后,插入到基因組中的位點(diǎn)往往是隨機(jī)的�；虼虬械耐粗亟M成功率極低,想要對(duì)絲狀真菌基因進(jìn)行定點(diǎn)改造的難度極大。敲除Ku70同源基因以提高真核細(xì)胞基因打靶效率的策略,已經(jīng)在多種真核菌類中得以實(shí)現(xiàn)。因此構(gòu)建頂頭孢霉菌的高效外源基因轉(zhuǎn)化平臺(tái),并且構(gòu)建該菌種Ku70基因缺陷型,是頂頭孢霉工業(yè)菌菌種遺傳改造的重要基礎(chǔ)。本文闡述了一種基于聚乙二醇介導(dǎo)的原生質(zhì)體轉(zhuǎn)化方法,并且嘗試構(gòu)建Ku70基因敲除質(zhì)粒,為構(gòu)建Ku70敲除株提供基礎(chǔ)。我們首先對(duì)頂頭孢霉菌原生質(zhì)體的制備和再生條件進(jìn)行了系統(tǒng)優(yōu)化。通過(guò)對(duì)酶種類和復(fù)配比、酶解時(shí)間、菌絲形態(tài)、再生培養(yǎng)基種類和涂布方式等考察了頂頭孢霉原生質(zhì)體的制備和再生條件。最終優(yōu)化的條件是選擇中度膨大初期的菌絲體,使用1%溶壁酶,30℃酶解2h后采用雙層平板法接種于原生質(zhì)體再生培養(yǎng)基I (HCM)中。原生質(zhì)體的釋放數(shù)可以達(dá)2×108個(gè)/ml,再生率為20%。其次,對(duì)頂頭孢霉原生質(zhì)體進(jìn)行轉(zhuǎn)化和驗(yàn)證。利用質(zhì)粒pYG233轉(zhuǎn)化制備的原生質(zhì)體,驗(yàn)證轉(zhuǎn)化效率為10個(gè)陽(yáng)性轉(zhuǎn)化子/μg質(zhì)粒。構(gòu)建出了將CPC轉(zhuǎn)化成為7-ACA潛在生產(chǎn)菌株。此外,我們還開展了敲除質(zhì)粒pAcKu70的構(gòu)建工作。該質(zhì)粒含有頂頭孢霉Ku70同源基因的上、下游片段,腐草霉素標(biāo)記基因以及pcbAB啟動(dòng)子。上述研究為實(shí)現(xiàn)頂頭孢霉的代謝工程改造奠定了基礎(chǔ)。
[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，

本文編號(hào)：1464332