| Ganoderma lucidum is a widely cultivated edible and medicinal fungus,which contains many active ingredients with pharmacological functions.With the completion of genome sequencing and the construction of genetic transformation system,it provides a solid foundation for the study of secondary metabolism and mycelial growth of G.lucidum.Heme oxygenase（HO）decomposes heme into biliverdin（BV）and releases one molecule of carbon monoxide（CO）and one molecule of Fe2+.Heme oxygenase is widespread in eukaryotes and plays a role in many cell processes.In this study,heme oxygenase was cloned from G lucidum for the first time.It was found that heme oxygenase gene was involved in the physiological process of mycelial growth,polysaccharide synthesis,heat stress response and triterpenoid（GA）synthesis of Ganoderma lucidum.Firstly,HMX1 gene was cloned from G.lucidum.The length of HMX1 open reading frame was 1092 bp,encoding 363 amino acids.Then,HMX1 protein was expressed in prokaryote and the enzyme activity of the purified protein was determined.The Km value of the protein was 0.699 μM,Vm was 81.9 nmol BV h-1 nmol-1 protein,the optimal pH and temperature were 7.2 and 37℃.By constructing the silencing vector pAN7-dual-HMX1,the transformants HMX1i1 and HMX1i2 with silencing efficiency of 91%and 85%,respectively,were obtained.Compared with wild type（WT）,the average growth rate of HMXlil and HMX1i2 decreased by 31%and 23%,respectively.Compared with WT strain,the biomass of HMX1i1 and HMX1i2 decreased by 53%and 48%,respectively.When HMX1 gene was silenced,extracellular polysaccharides（EPS）and intracellular polysaccharides（IPS）also increased in varying degrees.Compared with the EPS of WT,HMXli1 and HMX1i2 increased 59%and 51%respectively.Compared with WT,the IPS of HMX1i1 and HMX1i2 increased by 24%and 22%,respectively.The transcription levels of four key enzyme genes for polysaccharide synthesis were also measured,namely,phosphoglucose isomerase（PGI）,α-phosphoglucose mutase（PGM）,UDP glucose pyrophosphorylase（UGP）and phosphomannose isomerase（PMI）showed that the expression level of PGI,UGP and two PMI genes increased significantly,while the transcription level of PGM did not change significantly.These results indicate that HMX1 gene affects the growth and polysaccharide synthesis of G.lucidum.Then,this study examined the transcription level of HMX1 under different stress conditions.It was found that under 30 min of heat shock,the transcription level of HMX1 changed most significantly,and the activity of G.lucidum heme oxygenase and carbon monoxide content changed significantly under heat stress.Under heat stress,HMX1 silencing inhibited the growth of G.lucidum mycelium and increased GA content,in addition,exogenous changes in heme oxygenase activity can indeed affect the content of GA.When exogenous CO donors and scavengers change the content of intracellular CO under heat stress,the content of GA will also change,but exogenous bilirubin has no significant effect on the synthesis of GA.In order to further explore the mechanism of CO response to heat stress and change of GA,we found that the activity of slt2 and Hogl would change under heat stress,and the change trend of slt2 activity in HMX1 silenced strain was inconsistent with that of WT strain.Because slt2 is a member of the cell wall integrity（CWI）pathway,further detection showed that silencing HMX1 would reduce the sensitivity to cell wall stress.Furthermore,it was found that the content of chitin and glucan in cell wall of G.lucidum increased significantly when HMX1 was silenced,and the content of chitin and glucan increased further under heat stress.These results suggest that HMX1/CO may respond to heat stress by slt2 affecting cell wall components.In conclusion,these results show that HMX1/CO in G.lucidum plays an important role in influencing mycelial growth,polysaccharide synthesis,heat stress response and GA synthesis,which lays a foundation for further research. |
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