黄曲霉ste20-like基因功能鉴定及与ste20基因功能的比较

Identification of ste20-like and comparison with the function of ste20 in Aspergillus flavus

为了鉴定ste20-like基因在黄曲霉生长、分生孢子形成、菌核形成中的作用以及其侵染性,并比较其与ste20基因功能的差异,以A.flavus NRRL3357为研究对象,对两个基因进行相似度比对和生物信息学分析,根据同源重组策略获得单基因缺失菌株Δste20、Δste20-like和双基因缺失菌株Δdouble(Δste20-like&ste20),并分析基因敲除对菌株生长、分生孢子形成、菌核形成、黄曲霉毒素B1(AFB1)生物合成和侵染功能的影响。应用pyrG的双向遗传筛选成功获得Δste20、Δste20-like和Δdouble;与野生型相比,单基因缺失和双基因缺失突变株的生长速率、分生孢子量、菌核数量以及侵染能力都显著降低,在毒素合成方面,Δste20、Δste20-like和Δdouble菌株分别降低约76%、93%和72%;突变株之间比较发现,ste20-like缺失对生长速率的影响大于ste20基因;突变株之间的分生孢子产生量无显著差异;Δste20无菌核产生而Δste20-like还有少量菌核产生;Δste20-like菌株产生的毒素显著低于Δste20和Δdouble菌株。ste20-like基因正调控黄曲霉生长、分生孢子生成、菌核形成、AFB1生物合成,并影响黄曲霉分生孢子对粮油种子的侵染能力。与ste20基因功能相比,ste20-like基因在调控黄曲霉生长、毒素合成方面对菌株的影响方面较显著,而在菌核形成方面ste20的基因调控作用更显著。

The function of ste20-like in the growth,conidia formation,nucleus formation,and invasiveness was examined,and the differences in these functions with those of ste20 in Aspergillus flavus were determined.Aspergillus flavus NRRL3357 was used in this study.The two genes were compared to identify their similarities and subjected to bioinformatics analysis using NCBI.The target genes were disrupted using homologous recombination.The Δste20,Δste20-like,and double-gene deletion strain(Δdouble)were constructed to examine the effects of gene deletion on the fungal phenotype,sporulation,sclerotia formation,and aflatoxin production.Compared with the wild-type,the growth rate,conidial number,sclerotia number,and pathogenicity were significantly reduced in Δste20,Δste20-like,and theΔdouble strains,showing approximately 76%,93%,and 72% reductions in aflatoxin B1(AFB_(1))synthesis,respectively.Comparison of the mutant strains revealed that ste20-like deletion more significantly decreased the growth rate compared to that in Δste20.Conidia and sclerotia production did not significantly differ between the mutant strains.No sclerotia were detected in theΔste20 strain,whereas very few sclerotia were observed in the Δste20-like strain.AFB1production in Δste20-like was significantly lower than that in Δste20 and the double-gene deletion strain.ste20-like plays important roles in positively regulating mycelia growth,conidia production,sclerotia formation,and AFB1biosynthesis in A.flavus.Compared with ste20,ste20-like regulates the growth of A.flavus and has a more significant effect on AFB1synthesis in this species,whereas ste20 has stronger regulatory effects on nucleus formation.