转录因子Afndt6对黄曲霉毒力和侵染力的影响

Effect of the transcription factor Afndt6 on the virulence and infection of Aspergillus flavus

为探究转录因子Afndt6对黄曲霉生长发育、毒素生物合成及侵染粮食籽粒能力的影响,通过同源重组构建Afndt6基因缺失的黄曲霉菌株,采用体视显微镜和切片观察手段研究Afndt6基因缺失对黄曲霉生长发育和孢子产生的影响,采用薄层色谱层析测定黄曲霉毒素生物合成量,以花生和玉米籽粒为基质评价基因缺失菌株的侵染力。结果表明:与对照菌株相比,ΔAfndt6菌株的生长速度、分生孢子的产量没有显著变化,但菌落分生孢子颜色变为白色;基因缺失菌株对细胞壁胁迫试剂刚果红和氧化胁迫压力不敏感,200μg/mL刚果红处理后对照菌株和ΔAfndt6菌株的生长抑制率分别为31.10%和25.81%,15 mmol/L H_(2)O_(2)处理后对照菌株和ΔAfndt6菌株的生长抑制率分别为47.94%和17.05%;ΔAfndt6菌株的黄曲霉毒素产量显著升高,对花生、玉米种子的侵染能力增强。Afndt6基因编码的转录因子能够影响黄曲霉生长发育,负调控黄曲霉毒素的生物合成,可为储粮过程中防控黄曲霉及黄曲霉毒素污染提供参考。

The objective of this study was to investigate the impact of the transcription factor Afndt6 on the growth,development,aflatoxin biosynthesis,and pathogenicity of Aspergillus flavus.In this study,A.flavus strain with Afndt6 gene deletion was constructed by homologous recombination using the selection gene pyrG and identified via diagnosis PCR analysis.The regulatory roles of Afndt6 on cell growth and development,including spore production and conidiophore formation,were investigated by means of stereomicroscope observation.Aflatoxin B 1 production was determined via thin layer chromatography,and the transcriptional level of genes participated in aflatoxin biosynthetic pathway was profiled with qRT-PCR.The medium containing reagents such as congo red(CR)and hydrogen peroxide(H_(2)O_(2))was used to investigated the response ability of A.flavusΔAfndt6 strain to external stress.Additionally,the pathogenicity of A.flavusΔAfndt6 strain on peanut and maize seeds was also evaluated.The results showed that compared with the control strains,the growth rate and conidial production ofΔAfndt6 strain had no significant change,but the conidial color of colony became white.The gene deletion strain was insensitive to cell wall stress agent CR and oxidative stress.The growth inhibition rates of control strain andΔAfndt6 strain were 31.10%and 25.81%,respectively,when treated with 200μg/mL CR.The growth inhibition rates of control strain andΔAfndt6 strain were 47.94%and 17.05%,respectively,when treated with 15 mmol/L H_(2)O_(2).The aflatoxin yield ofΔAfndt6 strain was significantly increased,and the infection ability on peanut and corn seeds was enhanced.This study showed that the transcription factor encoded by Afndt6 gene can affect the growth and development of A.flavus and negatively regulate the biosynthesis of aflatoxin,which can provide reference value and theoretical basis for the prevention and control of A.flavus and aflatoxin contamination in the process of grain storage.