核盘菌热休克蛋白Hsp70基因的克隆及功能分析

Cloning and functional analysis of heat shock protein Hsp70 from Sclerotinia sclerotiorum

摘要:

为明确核盘菌热休克蛋白70(heat shock protein 70,Hsp70)基因在菌核形成过程中的功能及其与病菌致病力的关系，本研究采用逆转录PCR (reverse transcription PCR,RT-PCR)方法克隆核盘菌Hsp70基因并进行序列分析，采用实时荧光定量PCR (quantitative real-time PCR,qRT-PCR)方法测定基因在病菌不同生长阶段、环磷酸腺苷(cyclic adenosine monophosphate,cAMP)及低温和高温胁迫下的相对表达量；对Hsp70蛋白进行耐热性检测；通过农杆菌介导法构建Hsp70基因沉默转化菌株，采用离体油菜叶片接种法进行致病力检测，在含腐霉利和甲基托布津药物培养基中进行耐药性检测。结果表明，核盘菌Hsp70基因全长1 890 bp，与叶杯菌属(Ciborinia)的Hsp70亲缘关系较高；Hsp70在菌核中表达量最高，为菌丝中的30倍以上，cAMP胁迫显著诱导Hsp70的表达，40℃下，Hsp70表达量呈先升后降再升高的趋势，4℃处理无显著影响；高表达Hsp70的重组菌株耐热性显著增加；核盘菌Hsp70沉默转化子不能形成菌核，其致病力和耐药性均下降。本研究揭示了Hsp70在核盘菌菌核形成及对抗不良环境中发挥重要作用，为下一步深入解析核盘菌Hsp70的生物学功能提供参考。

Abstract:

To clarify the roles of the heat shock protein gene Hsp70 in the sclerotial formation and pathogenicity of Sclerotinia sclerotiorum, we employed reverse transcription PCR (RT-PCR) to clone Hsp70 from S. sclerotiorum and performed sequence analysis. Quantitative real-time PCR (qRT-PCR) was employed to determine the relative expression levels of Hsp70 at different growth stages and under the stress induced by cyclic adenosine monophosphate (cAMP) and low and high temperatures. The thermal stability of Hsp70 was measured. The Agrobacterium-mediated method was employed to construct the Hsp70-silenced strain. The pathogenicity and fungicide resistance of strains were tested by inoculation in detached rapeseed leaves and cultivation in the media containing procymidone and thiophanate-methyl, respectively. The results showed that the cloned Hsp70 had a total length of 1 890 bp and close relationship with the Hsp70 gene of Ciborinia. Hsp70 showcased the highest expression level in sclerotia, which was more than 30 times higher than that in hyphae. The cAMP stress significantly induced the expression of Hsp70. The expression level of Hsp70 showed an increasing-decreasing-increasing trend at 40 ℃ and no significant change at 4 ℃. Recombinant strain with high expression of Hsp70 showed good thermal stability. The Hsp70-silenced transformant did not form sclerotia, with decreased pathogenicity and fungicide resistance. This study reveals that Hsp70 plays an important role in the sclerotial formation and stress resistance of S. sclerotium, providing reference for further in-depth research on the biological roles of Hsp70 in S. sclerotium.