Research On LeSKP1 Gene Function And Auxin Rapid Response Genes Of Lentinula Edodes Under Heat Stress

Lentinula edodes is one of the renowned and delicious edible fungi,which was first semi-cultivated in China.In recent years,with the global climate warming,the adverse effects of heat stress on L.edodes production have become increasingly serious,even causing a large amount of rot in L.edodes spawn,often resulting in significant economic losses.In plant heat stress research,it has been found that auxin signal transduction plays a key role in responding to heat stress,and the auxin signal is transmitted to the nucleus through the SCF^TIR1 complex（SKP1,Cullin and F-box complex）.However,there are no reports on the study of the auxin signal transduction pathway in fungal stress response so far.Previous studies in our laboratory have found that auxin（IAA）is related to the heat tolerance of L.edodes mycelium.Exogenous addition of IAA or its analogs NAA and 2,4-D can significantly improve the heat tolerance of L.edodes mycelium,and the LeSKP1protein that interacts with the auxin responsive element"TGTCGG"has been screened.This study used yeast two-hybrid technology to analyze the interaction between LeSKP1 protein and F-box protein and Cullin protein.q RT-PCR was used to determine the expression pattern of LeSKP1 gene in response to IAA signal,and RNAi technology was used to analyze the function of LeSKP1 gene in the heat-sensitive strain YS48 and the heat-tolerant strain YS3334 of L.edodes.Systematic research was carried out on LeSKP1 gene.The strains YS48,YS3334,YS3355,and Z60 were selected,among which YS48 and YS3334 were insensitive to IAA,and YS3355 and Z60 were sensitive to IAA.They were treated with 0.01 m M IAA for 5 min at room temperature,and transcriptome sequencing technology was used to analyze and mine the genes that responded rapidly to auxin.The main research results are as follows:1.Analysis of LeSKP1 protein revealed that it is highly homologous to Arabidopsis ASK1 protein,with a sequence similarity of 51.90%and a sequence matching degree of96%.L.edodes has only one LeSKP1 gene.Yeast two-hybrid and pull-down techniques were used to demonstrate that both LeSKP1 protein and ASK1 protein interact with the L.edodes F-box protein Le01Gene01446,indicating that LeSKP1 protein and ASK1 protein may have similar functions.2.The exogenous 0.01 m M IAA was applied to YS48 and YS3334 strains separately for 5 min,30 min,1 h,2 h,and 6 h.q RT-PCR analysis of LeSKP1 gene expression revealed that the expression of LeSKP1 gene in YS48 strain was significantly down-regulated after1 h of IAA treatment,while the expression of LeSKP1 gene in YS3334 strain was slightly up-regulated after 5 min of IAA treatment.The study results indicated that LeSKP1 gene did not respond to exogenous IAA in a short period of time.3.Using Agrobacterium-mediated genetic transformation method to silence the LeSKP1 gene in L.edodes mycelium,positive transformants were screened,with the strains transformed with p CAMBIA1300-RNAi empty vector serving as controls.The results showed that 11 positive transformants were screened from YS48 strain,among which the expression level of LeSKP1 gene in three positive transformants was down-regulated to 70%of the control strain;and 12 positive transformants were screened from YS3334 strain,and the expression level of LeSKP1 gene in all positive transformants was down-regulated to57%of the control strain.Further measurement of mycelial growth rate showed that the down-regulation of LeSKP1 gene expression had no significant effect on the mycelial growth rate of L.edodes YS48 and YS3334 strains.Measurement of heat tolerance of L.edodes mycelium showed that the heat-tolerant phenotypes of different transformants varied in L.edodes YS48 and YS3334 strains.4.Exogenous addition of IAA was used to test the mycelial growth rate of YS48,YS3334,YS3355,and ZP60 strains,and it was found that YS48 and YS3334 strains were insensitive to 0.01 m M IAA at room temperature,while YS3355 and ZP60 strains were sensitive to 0.01 m M IAA at room temperature.Exogenous addition of Yucasin,a synthetic inhibitor of auxin synthesis,was used to test the mycelial growth rate and endogenous IAA content of the four tested strains.The results showed that low concentration of IAA promoted mycelial growth,while high concentration of IAA inhibited mycelial growth,similar to the effect of IAA on plants.5.Transcriptome sequencing technology was used to identify genes in L.edodes that respond rapidly to auxin.64 differentially expressed genes were identified in YS3355 strain,and 47 differentially expressed genes were identified in ZP60 strain,but no differentially expressed genes were identified in YS3334 and YS48 strains.GO enrichment analysis of differentially expressed genes showed that DEGs in YS3355 strain were enriched in functions related to protease activity and pyridoxal phosphate binding,while DEGs in ZP60strain were enriched in functions related to oxidoreductase activity,peroxidase activity,antioxidant activity,and transcription regulation activity.This study has preliminarily clarified the function of LeSKP1 gene in response to heat stress,determined the effect of auxin on L.edodes mycelial growth,and identified auxin-responsive genes,laying a foundation for studying the L.edodes auxin signal transduction pathway and revealing the molecular mechanism of auxin regulating L.edodes heat tolerance.It is conducive to further research on high-temperature tolerant germplasm innovation of L.edodes.