NirA1基因对球孢白僵菌生长、抗逆及毒力的影响

Effects of NirA1 gene on growth,stress resistance and virulence of Beauveria bassiana

【目的】探究参与硝酸盐同化的一个特异转录因子NirA1对球孢白僵菌(Beauveria bassiana)生长、抗逆及毒力的影响。【方法】利用RT-PCR检测球孢白僵菌NirA1基因在不同培养基中的表达特征。通过构建敲除突变株ΔNirA1、超量表达OENirA1及回复互补菌株ComNirA1,解析NirA1在球孢白僵菌生长发育、逆境胁迫反应和致病昆虫中的功能。【结果】NirA1基因在营养较贫瘠的CZB培养基中表达量高于丰富培养基PDB和SDB。敲除NirA1导致了菌株在人工培养基上的生长缓慢,对NaNO_(2)和Urea的利用效率降低。RT-PCR显示,ΔNirA1中硝酸盐转运蛋白基因NrtA的表达较野生型显著下调。在CZA、PDA和1/4 SDAY培养基中,其分生孢子产量分别较野生型降低了21.6%、16.2%和25.6%。逆境胁迫分析发现,在高温(32℃),高渗(1mol/LNaCl)和H2O_(2)处理后,ΔNirA1菌落生长抑制率较野生型分别降低29.0%、25.2%及49.0%;但在添加SDS和刚果红(CR)的处理中,突变体菌落的生长抑制率分别较野生型升高34.1%和96.2%。因此,缺失NirA后,菌株对SDS和CR更加敏感,但对高温、H2O_(2)和NaCl的耐受性提高。以3龄的大蜡螟为试虫的毒力测定表明,敲除NirA1基因导致菌株的毒力提高,半致死时间较野生型提前17.4%。【结论】NirA1在球孢白僵菌生长过程中参与了氮源的利用,并在球孢白僵菌的菌落生长、分生孢子生产、胁迫反应和致病宿主过程中发挥了重要作用。

[Objective]We investigated the effects of the nitrate-specific activator NirA1 on the growth and stress resistance of Beauveria bassiana.[Methods]The expression of NirA1 was detected by RT-PCR in different culture mediums.Several NirA1 strains,including gene knockout(ΔNirA1),complementary(ComNirA1),and over-expression(OENirA1),were constructed and the functions of NirA1 involved in fungal developmental differentiation,stress reactions and virulence were analyzed.[Results]Expression level of NirA1 was higher in CZB medium than that in PDB or SDB.The growth ofΔNirA1 in mediums with different nitrogen sources was significantly slower compared to wild type.The expression of nitrate transporter NrtA was down-regulated in NirA1 deletion mutant.Compared to wild type,conidial yield in CZA,PDA and 1/4 DAY mediums decreased by 21.6%,16.2% and 25.6% respectively.The growth inhibition rate ofΔNirA1 in 32℃,1 mol/L NaCl and H2O_(2) mediums was reduced by 29.0%,25.2% and 49.0%,but increased by 34.1% and 96.2% in SDS and Congo red mediums respectively.Bioassay of fungal strains were performed using 3 rd-instar larvae of G.mellonella.Compared to wild type,deletion of NirA1 increased fungal virulence,decreasing mean lethal time(LT50)by 17.4%.[Conclusion]These results indicated NirA1 is involved in the utilization of nitrogen sources and play crucial roles in colony growth,conidiation,stress responses,and pathogenesis of B.bassiana.