Study On The Rule Of The Laccase During The Whole Growth Of The Cultivated Ganoderma Lucidum And Its Induction

Ganoderma lucidum is an important medicinal fungus.It has a strong capacity of lignin degradation.Laccase is a kind of copper-containing polyphenol oxidase and one of the extracellular enzymes secreted by G.lucidum.It can provide nutrient by promoting the degradation of environmental lignin for the development of fungi during the growth and development of fungi.Study on the changes of laccase of G.lucidum is of great significance to the yield and quality of G.lucidum.At present,there are few studies on the mechanism of lignin degradation.The mechanism of the role of the laccase and the genes is not clear.Therefore,the laccase homologous gene and inferred amino acid sequence from bioinformatics analysis,the expression of laccase gene in the growth of G.lucidum and the induction by different concentrations of Cu2+ was studied by real-time quantitative PCR(RT-PCR).By understanding the changes of laccase gene in G.lucidum growth process,a theoretical basis was built on the expression mechanism of laccase in G.lucidum.The contents and results in this paper include the following:(1)Bioinformatics analysis showed that the composition of amino acid sequence of laccase was different.Predicting the signal peptides of 12 homologous genes of laccases by Signalp 4.1Server,it was found all N-terminal of amino acid sequences had signal peptide and the number of amino acid residues between 18-24 a.After comparison with NCBI,all laccase genes had laccase-specific sequences L1-L4,as well as three conserved copper oxidase domains.(2)With the PDA solid culture medium,the laccase activity and polysaccharide content of seven different strains of G.spp.were determined by ABTS and sulfuric acid-phenol method,respectively.The results show that the activity of laccase of G.spp.in the mycelium period is high.The growth rate and maximum laccase activity of the strains have emerged at the 4th or 6th day.From comprehensive analysis of the growth rate,biomass,laccase activity and polysaccharide and other factors,the activity of laccase and the growth rate of Ganoderma applanatum(Pers.)Pat.var.gibbosum was significantly higher than the others.It can be used as high-yield laccase strains.(3)The deduced amino acid sequence belongs to a class of MCO,at the same time,LCC1 and LCC2,LCC3 and LCC5 were 77%,80% and 77% similarity,and cluster analysis also showed that the four laccase genes belong to a group.Intron positions 1 and 2 are present in all laccase genes and may be the most conserved intron.In the cluster analysis,the consistency of the amino acid sequence laccase was predicted to be between 47-100%.LCC13 is closer to LCC4 and LCC7.It belongs to Group 3 in the intron subgroup,but it can also be considered to belong to Group A.While LCC8 is closer to group B.LCC10 and LCC11 is a relatively small number of intron genes which requires further experimental to explain its intron situation.(4)The transcription level of laccase was detected by RT-PCR.The transcriptional level of 12 laccase genes in different stages of G.lucidum S3 was analyzed,and the change of laccase gene of G.lucidum S3 was clarified.The results showed that LCC9 and LCC10 did not undergo transcription during different periods of G.lucidum S3.Compared with the relative expression of the control,the expression of LCC1,LCC3 and LCC5 genes was the highest in first stage and the highest expression of LCC2,LCC7,LCC8,LCC11,LCC12 and LCC13 appeared in the third stage.During the culture,the relative expression of LCC4 was higher than that of the control,and the maximum was in the fourth period.The relative abundance analysis of laccase showed that LCC8,LCC2,LCC12 and LCC4 were key laccase genes in the first,the second,the third and the fourth period,respectively.It is concluded that LCC8,LCC2 and LCC12 may participate in the process of lignin degradation,the formation of cap and the spore formation during the early growth of G.lucidum S3.The expression of LCC4 gene plays an important role in the synthesis of laccase.(5)The effects of different concentrations of copper ions on the growth rate,fresh weight,laccase activity and gene expression of G.lucidum S3 mycelium were studied.The results showed that the growth rate,quality,laccase activity and gene expression of 1.25 M,2.5M and 5M copper ions were significantly higher than those of the control in the process of mycelia growth of G.lucidum S3.The maximum activity of laccase was found on the 6th day and the best copper ion concentration was 5M.The results showed that LCC5,LCC10 and LCC13 genes were not detected and the other laccase genes were significantly up-regulated after 5M copper ion treatment.The most obvious increase of the LCC1 suggested that it may be the key synthetic laccase gene of G.lucidum S3 mycelia.