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

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.

Key words:Sclerotinia sclerotiorum;heat shock protein;expression analysis;gene silencing;pathogenicity