Sequencing Of Transcriptome And Screening Of Genes Involved In Enzymatic Browning In Agaricus Bisporus

Agaricus bisporus is delicious and nutritious,but it browned easily after harvest,causing serious economic losses.In order to study the mechanism of enzymatic browning of Agaricus bisporus from the molecular level,this study used Agaricus bisporus as the experimental material to sequence the genomicus of Agaricus bisporus in three storage time at 4 °C storage group and UV-C irradiation group.Transcriptome sequencing results are sequencing data evaluated,reference sequence alignment analyzed,gene expression level analyzed,correlation analyzed,differential expression analyzed,GO enrichment analyzed and KEGG enrichment analysed,and combined with RT-PCR.The genes related to enzymatic browning were screened to improve the reserve gene resources of Agaricus bisporus against browning,which provided a theoretical basis for improving the quality of Agaricus bisporus.The specific research results are as follows:(1)The total RNA of six Agaricus bisporus pillus samples was extracted by Trizol method,which could efficiently extract Agaricus bisporus RNA and detect total RNA by three detection methods.The results showed that RNA had high purity,good integrity and quality.Required,subsequent sequencing can be performed.After the total RNA was tested,the RNA-seq library was constructed and qualified.The constructed library was sequenced by Illumina HiSeqTM sequencing platform,and the obtained sequencing data was subjected to sequencing data quality evaluation,reference sequence alignment analysis,gene expression analysis and RNA-Seq correlation examination.The results showed that the number and quality of transcriptome sequencing data were compared.High gene expression levels,reliable and reasonable sample selection for subsequent analysis.(2)The browning degree of Agaricus bisporus increased gradually with the storage time during storage at 4 °C,and the transcriptome of Agaricus bisporus pillus was sequenced under this condition,and 176 329 392 raw reads and 168 607 212 high quality clean reads were obtained.According to the principle of?log2(the ratio of the expression of the two groups of samples)?> 1 and the q value < 0.005,727 differentially expressed genes were screened in the Pile_C7 and Pile_C1 groups,and 1 524 differentially expressed genes were screened in the Pile_C14 and Pile_C1 groups.GO functional enrichment results showed that the cytoplasmic,redox process and hydrolase activity were the most enriched in cell components,biological processes and molecular functions in the Pile_C7 and Pile_C1 groups,respectively,in the Pile_C14 and Pile_C1 groups,membrane,redox Process and redox activity are the most enriched items of cellular components,biological processes,and molecular functions,respectively.Pathway enrichment analysis found that 459 differentially expressed genes in the Pile_C7 and Pile_C1 groups were successfully annotated into 83 KEGG metabolic pathways;876 differentially expressed genes in the Pile_C14 and Pile_C1 groups were successfully annotated into 97 KEGG metabolic pathways,among which has ten significantly enriched pathways(P < 0.05).Five polyphenol oxidase-related genes(PPO3,PPO1,PPO5,LAC1 and LAC4)were screened from the obtained differentially expressed genes.Among them,PPO1,PPO3 and PPO5 may be involved in enzymatic browning,and the specific biological functions of different genes need further verification.The polyphenol oxidase activity of Agaricus bisporus increased gradually with the prolongation of storage time,which was consistent with the sequencing results of transcriptome.(3)The UV-C-treated Agaricus bisporus pillus were subjected to transcriptome sequencing,and 192 935 604 raw reads and 185 169 454 high-quality clean reads were obtained.According to the principle of?log2(the ratio of the expression of the two groups of samples)?> 1 and the q value < 0.005,42 differentially expressed genes were screened in the Pile_U1 and Pile_C1 groups,51 DEGs were screened in the Pile_U7 and Pile_C7 groups,and 37 DEGs were screened in the Pile_U14 and Pile_C14 group.The results of GO functional enrichment showed that the number of genes enriched in the biological regulation process was higher in the Pile_U1 and Pile_C1 groups;in the Pile_U7 and Pile_C7 groups,there were more DEGs in the single biological metabolic process and hydrolase activity entries;Pile_U14 Compared with the Pile_C14 group,the DEGs were less,and the DEGs enriched in the transcription factor activity entries were the most,and there was no significant enrichment.Pathway enrichment analysis revealed that 42 DEGs in the Pile_U1 and Pile_C1 groups were successfully annotated into 8 KEGG metabolic pathways;51 differentially expressed genes in the Pile_U7 and Pile_C7 groups were successfully annotated into 31 KEGG metabolic pathways;in the Pile_U14 and Pile_C14 group,37 differentially expressed genes were successfully annotated into 13 KEGG metabolic pathways.Two enzymatic browning-related genes(PPO3 and PAL1)were screened from the obtained differentially expressed genes.The differential gene was enriched by KEGG and enriched into five metabolic pathways,namely phenylalanine metabolism,riboflavin metabolism,tyrosine metabolism,biosynthesis and metabolic pathways of secondary metabolites.The expression levels of PPO3 and PAL1 in Agaricus bisporus in UV-C treatment group were lower than those in CK group in three different storage periods,and expression level of PAL1 increased first and then decreased during three storage periods in CK group.The results of real-time PCR analysis were consistent with the results of transcriptome sequencing.The results of the study provided a scientific basis for further analysis of the relationship between differential genes and browning,and for the improvement of Agaricus bisporus.