粗糙脉孢菌中几丁质合酶1缺失突变体的构建及表型分析

在丝状真菌中,几丁质是真菌细胞壁的主要成分之一,对维持细胞形态和结构起到了重要的作用.几丁质是由几丁质合酶(chitin synthase,CHS)催化合成的,几丁质合酶参与生物钟调节的生理活动.本文以丝状真菌粗糙脉孢菌几丁质合酶1(CHS-1)为研究对象,通过同源重组基因敲除技术、电转化、分生孢子过膜以及PCR鉴定的方法,获得了chs-1缺失突变菌株CHS-1KO.通过竞争性生长管(racetube)培养分析发现:对照Ku70RIP和突变菌株CHS-1KO(Ku70RIP背景)均具有明显的分生孢子带,但分生孢子带昼夜节律周期缩短,直线生长速率显著减慢,ku70RIP菌丝生长长度是3.4±0.31 cm/24 h,而突变菌株CHS-1KO(Ku70RIP背景)菌丝生长长度是2.07±0.19 cm/24 h.并进一步结合细胞壁染色对细胞形态分析发现:变菌株CHS-1KO菌丝沿生长方向膨胀、分支变短.这些结果表明:几丁质合酶1在粗糙脉孢菌分生孢子带昼夜节律形成和菌丝生长中发挥重要的作用.

Construction and Phenotype Analysis of CHS-1 Knockout Mutant in Neurospora crassa

In filamentous fungi,chitin is a structural component of cell wall and is necessary for cell morphology.Chitin biosynthesis is catalyzed by chitin synthase and chitin synthase perhaps involve in physical activities regulated by circadian clock.In this paper,Neurospora crassa is used as materials and chs-1 was selected as target gene.chs-1 deletion mutants （CHS-1KO） is obtained with the aid of electro-transformation,filtration of spore suspension and PCR based on the homologous recombination knockout principle.Using the racetube assay,the circadian conidiation rhythm and linear growth rate of the CHS-1KO strain and wild type were assayed.The double strains displayed remarkable circadian conidiation rhythm in constant darkness,but short-period circadian conidiation rhythm in CHS-1KOstrain.The CHS-1KOstrain grown at 3.4 ± 0.30 cm/24 h,which was significantly reduced relative to the wild type growth rate of 2.07 ± 0.19 cm/24 h.In addition,calcofluor white staining revealed hyphal swelling and short branches in the CHS-1KOstrain.These results suggest that chs-1 plays an important role in circadian clock regulation and hyphal development.