Genome And Transcriptome Analysis Of Tricholoma Giganteum Anti-browning Mechanism

Browning is an important factor influencing the postharvest mushroom qualit y during storage period.Tricholoma giganteum can keep color under 8¹2 degree centigrade for 30 days,which had the anti-browning characteristic.The T.giganteum mononuclear strain A6 genomesequencing was obtained by Illumina HisSeq and PaciBio method.Combining with resequencing transcriptome of T.giganteum,analysis the browning mechanism of ferrous ion stress in the molecular level using PC R and real time RT-PCR and the anti-browning inherent possible factor with the method of bioinformatics analysis.The results are as follows:1.The T.giganteum A6 genome was 27.9Mb through sequencing and assembly,and genome feature was presented by circo,including scaffold,GC%,sequencing depth,gene expression quantity and co-line paralogous genes.The quality of the data assembly was high.2.Comparing with the genomes of A.bisporus,H.marmoreusj,L.bicolor,it showed that the colinearity of orthologous gene by circo,which revealed the similarityamong T.giganteum and A.bisporusas the theoretical basis for the study of the characteristicof storage.3.Analysis different expression genes from the transcriptome of CK and FK(Fe²⁺treated)refering to A6 genome and transcriptome sequenced by BGISEQ-500,KEGG enrichment were conducted to screen the melanin biosynthesis pathway related to browning for illuminating the process of T.giganteum browning stressed by Fe²⁺.4.Based on the results of our previous study on the activity of tyrosinase and ferrous ion stress,tyrosinase was the key enzyme for T.giganteum browning stressed by Fe²⁺,three tyrosinases sequences GME158?GME6843 and GME7523 were obtained by local blast in A6 genome.5.Six reference genes were selected from eight normal fungal reference genes by PCR,including?-Tubulin,?-Actin,60s,NADH,RPB2 and GAPDH in T.giganteum.Through quantitative real-time PCR,the expression levels of these candidate genes were evaluated under different experimental conditions,including different tissues,different growth stages and differe nt density of T.giganteum as treatment by using three common statistical softwares,GeNorm,NormFinder and Bestkeeper.The result showed that?-Tubulin was the appropriate reference gene for T.giganteum.6.Analysis the thirty-three associative genes includ ing tyrosinase from seven metabolic process related to browning.Combined with the pathway from transcriptome,it indicated that twenty-four genes have the similar expression patterns.The reason of T.giganteum browning stressed by Fe²⁺was the up regulated expression of tyrosinase,which is the only DEG related to melanin biosynthsis7.The three tyrosinase genes GME158,GME6843 and GME7523 were cloned and sequenced,and the three genes were analyzed by their predicted protein?GME158p,GME6843p and GME7523p?structures.The result showed that GME6843p was a secretory protein possessed signal peptide and GME158p and GME6843p both have transmembrane structure.Three different sequences of GME7523?GME7523-1,GME7523-2 and GME7523-3?from the c DNA of T.giganteum were amplifiedby PCR,,which can prove GME7523 exist varied transcript by alternative splicing.The results above indicated that the key factor of T.giganteum browning stressed by ferrous ion was caused by up-regulated expression of tyrsinase,and the reason T.giganteum anti-browning might be the deficiency of the functional site of the TYR.