冠突曲霉Ac-nsdD基因鉴定分析及抗盐胁迫功能分析  

Appraisal analysis and salt stress resistance function analysis of Ac-nsdD gene in Aspergillus cristatus

作　　者：余春芳 曹广鑫 张亭笛 吴炅臻 杨靖 谭玉梅[5,6] 刘永翔 刘作易[5,6] 位秀丽 YU Chun-fang;CAO Guang-xin;ZHANG Ting-di;WU Jiong-zhen;YANG Jing;TAN Yu-mei;LIU Yong-xiang;LIU Zuo-yi;WEI Xiu-li(Department of Microbiology,Faculty of Basic Medical Sciences,Hubei University of Medicine,Shiyan,Hubei 442000,China;Institute of Medicine and Nursing,Hubei University of Medicine,Shiyan,Hubei 442000,China;The First Clinical Medical College,Hubei University of Medicine,Shiyan,Hubei 442000,China;The Third Clinical Medical College,Hubei University of Medicine,Shiyan,Hubei 442000,China;Guizhou Key laboratory for Agricultural Biotechnology,Guiyang 550006,China;Guizhou Institute of Biotechnology,Guiyang 550006,China)

机构地区：[1]湖北医药学院基础医学院微生物学教研室,湖北十堰442000 [2]湖北医药学院药护学院,湖北十堰442000 [3]湖北医药学院第一临床学院,湖北十堰442000 [4]湖北医药学院第三临床学院,湖北十堰442000 [5]贵州省农业生物技术重点实验室,贵阳550006 [6]贵州省生物技术研究所,贵阳550006

出　　处：《西南农业学报》2022年第6期1282-1288,共7页Southwest China Journal of Agricultural Sciences

基　　金：湖北医药学院药护学院2021年大学生创新创业计划项目(S202113249006);湖北医药学院2021年大学生创新创业计划项目(S202110929004);湖北医药学院2020年大学生创新创业计划项目(S202010929012)。

摘　　要：【目的】进一步探讨GATA转录因子Ac-nsdD在茯砖茶的冠突曲霉中的调控作用。【方法】根据基因组注释结果及构建系统进化对冠突曲霉Ac-NsdD蛋白序列分析。运用同源重组和根癌农杆菌介导的方法构建Ac-nsdD基因敲除的突变体,对突变体进行表型分析。【结果】根据基因组注释结果分析冠突曲霉Ac-nsdD编码一个假定的具有保守DNA结合结构域ZnF GATA的转录因子。系统进化分析表明,Ac-nsdD与其它真菌同源的NsdD有较为紧密的亲缘关系。进一步运用同源重组的方法,获得一个该基因敲除的突变体。与野生型菌株比较,突变体在M3Y培养基培养,表现为产闭囊壳量明显减少;然而在5%NaCl M3Y培养基培养,突变体表现产闭囊壳量变少,产分生孢子量显著变多,中间培养物黑色色素形成显著增加,生长速度变小;在17%NaCl M3Y培养基培养,突变体表现闭囊壳数目几乎为零,分生孢子量变少,生长速度变小。【结论】Ac-nsdD基因在无盐环境下可能激活有性发育。该基因在应答低渗透压盐胁迫条件下,可能激活有性发育和抑制无性发育,平衡有性和无性发育,且参与色素的生物合成;在应答高渗透压盐胁迫条件下,可能激活有性发育、无性发育和营养生长。本试验为进一步研究与Ac-nsdD基因相偶联的一些信号通路及其下游基因,为丝状真菌的有性及无性发育的一些共有的和独特的信号转导途径提供实验室数据。【Objective】The regulation of GATA transcription factor Ac-nsdD in Aspergillus cristatus of Fu brick tea was further researched.【Method】The The characteristic of Ac-NsdD protein sequence of A.cristatus was analyzed by genome sequencing and annotation and phylogenetic construction.The Ac-nsdD deletion mutant was constructed by homologous recombination via Agrobacterium tumefaciens-mediated transformation,and the phenotype of the A.cristatus mutant was analyzed.【Result】Based on the genome sequencing and annotation,A.cristatus Ac-nsdD encoded a putative transcription factor with a conserved DNA-binding domain,ZnF GATA.Phylogenetic analysis showed that A.cristatus Ac-nsdD gene was closely related to other homologs from filamentous fungal species.The Ac-nsdD gene deletion mutant strain during in vitro cultivation in M3 Y medium displayed the number of cleistothecium decreased significantly when compared with their wild type.Interestingly,during in vitro cultivation in M3 Y medium containing 5%NaCl,the mutant strain showed the amount of cleistothecium was decreased,the amount of conidiation was significantly increased,growth rate slower,pigment formation in the middle of the medium was significantly increased.Furthermore,during in vitro cultivation in M3 Y medium containing 17%NaCl,the Ac-nsdD gene mutant showed the amount of cleistothecium was reduced to nearly zero,the amount of conidiation was decreased,growth rate slower.【Conclusion】The Ac-nsdD gene in a salt-free environment might have a positive effect on sexual development in A.cristatus.In response to low salt-induced osmotic stress,Ac-nsdD gene might positively regulate sexual reproduction,negatively regulate asexual development,maintain a developmental balance between asexual and sexual sporulation,be involved in pigment biosynthesis.The Ac-nsdD gene might negatively regulate sexual development,asexual development and vegetative growth in response to high salt-induced osmotic stress.This study provides laboratory generated data support for furt

关 键 词：冠突曲霉 Ac-NsdD 同源重组 基因敲除 盐胁迫 

分 类 号：Q933[生物学—微生物学]