AGO和RDRP基因参与黄曲霉压力应激反应

AGO and RDRP genes are involved in the stress response of Aspergillus flavus

目的探究RNA干扰(RNAi)机制中Argonaute(Ago)基因和RNA依赖的RNA聚合酶(RDRP)基因在黄曲霉生长发育中的作用。方法利用同源重组的方法构建黄曲霉Ago1、Ago2、RDRP1、RDRP3基因突变菌株,接种10^(6)CFU/mL孢子悬液3μl至马铃薯葡萄糖琼脂培养基(PDA)培养基上观察突变菌株的生长发育现象。在小量酵母提取物葡萄糖(YGM)培养基中加入200、400μg细胞壁压力试剂刚果红(CR),0.8 mol/L、1.6 mol/L渗透压药物氯化钠(NaCl),氧化压力试剂2 mmol/L、4 mmol/L过氧化氢(H_(2)O_(2)),基因组损伤剂0.01%、0.02%甲磺酸甲酯(MMS)试剂分析突变菌株的压力应激反应。结果成功构建黄曲霉突变菌株ΔAgo1、ΔAgo2、ΔRDRP1、ΔRDRP3且生长发育正常。ΔAgo1、ΔAgo2菌株相比对照可降低对细胞壁和渗透压的胁迫影响。Ago1基因缺失可降低H_(2)O_(2)的影响,相反RDRP3基因缺失,H_(2)O_(2)的抑制作用增加。ΔAgo2、ΔRDRP1菌株能降低对基因损伤剂的作用。此外,ΔRDRP1增加渗透压胁迫作用。结论黄曲霉Ago1、Ago2、RDRP1、RDRP3基因不参与生长速率和无性繁殖调控,可以参与调节宿主菌对环境的应激反应。

Objective To explore the role of Argonaute(Ago)gene and RNA-Dependent RNA Polymerase(RDRP)gene of Aspergillus flavus in the growth and development about the RNAi mechanism.Methods A.flavus Ago1,Ago2,RDRP1,RDRP3 gene mutant strains were constructed by homologous recombination.The growth and development of the mutant strains were observed on potato dextrose agar(PDA)+uracil uridine(UU)medium inoculated with 3μl 10^(6) CFU/mL spores.200,400μg cell wall pressure agent conidored(CR),0.8 mol/L,1.6 mol/L osmotic pressure agent NaCl,2 mmol/L,4 mmol/L oxidative pressure agent hydrogen peroxide(H_(2)O_(2))and 0.01%,0.02%genomic damage agent methyl mesylate(MMS)were added to the Yeast extract Glucose Minimum(YGM)+UU medium to analyze the stress response of the mutant strains.Results A.flavus mutant strains aboutΔAgo1,ΔAgo2,ΔRDRP1,ΔRDRP3 were successfully constructed and its growth and development were normal.TheΔAgo1 andΔAgo2 strains reduced the stress effects on cell wall and osmotic pressure compared to the control.Ago1 gene deletion reduced the effect of H_(2)O_(2),and conversely RDRP3 gene deletion increased the inhibition of H_(2)O_(2).The Ago2 and RDRP1 strains reduced the effect on genetic damage agent.In addition,ΔRDRP1 increased the effect of osmotic stress.Conclusion The Ago1,Ago2,RDRP1 and RDRP3 genes of A.flavus are not involved in the regulation of growth rate and asexual reproduction and can participate in the regulating of the host stress response to the environment.