DNA Methylation And Transcriptome Analysis Of Mulberry Fruits At Different Development Stages And Functional Study Of BZIP60 Gene

DNA methylation,as an epigenetic mechanism,plays important roles in gene expression regulation,and it was reported that DNA methylation is also associated with fruit development,but the function and regulation mechanism of DNA methylation in fruit development is still poorly understood.In this study,an integrative methylome and transcriptome analysis of mulberry fruits in different developmental stages was performed,and the genes both different in DNA methylation and expression levels in mulberry fruits at different developmental stages were screened.Moreover,the expression characteristics and biological functions of the MulbZIP60 gene which was screened by the integrative analysis were explored.The information provided will facilitate to elucidate the molecular mechanism in the development of mulberry fruits and also provided candidate genes for the genetic improvement of mulberry.The main results of this study are as follows:(1)Transcriptome analysis of mulberry fruits at different developmental stagesThe transcriptome sequencing libraries of mulberry fruits at fruit expanding period(MG),color changing period(MR),and maturity period(MP)were constructed and sequenced.In the MR and MG,MP and MG,MP and MR comparison sample groups,there were 2836,8760 and 6072 differentially expressed genes were identified,respectively,and these differentially expressed genes were mainly enriched in carbon metabolism,starch and sucrose metabolism,amino acid biosynthesis pathways.In addition,there were more genes are enriched in the metabolic pathways of phenylpropionic acid and flavonoid secondary metabolism pathways.According to the expression patterns of these differentially expressed genes,they are divided into 7 different clusters.Among these 7 clusters,there are 194 transcription factors,93 genes associated with hormone metabolism,41 genes involved in anthocyanin synthesis,and 26 genes related to DNA methylation.(2)Differential analysis of DNA methylation of mulberry fruits at different developmental stagesMethyl RAD-Seq technology was used to analyze the DNA methylation levels of mulberry fruits at different developmental stages,and there were 191,465 and 934 genes,which were different methylation at CCGG sites,were identified in the MR and MG,MP and MG,MP and MR comparative sample groups,respectively.Meanwhile,there were 166,421 and 668 genes,which were different methylation at CWCGG sites,were identified in those sample groups.The methylation differences sites of these methylation differential genes were mostly distributed in the intergenic and exon regions of these genes.According to the change trend of methylation level of these genes at different developmental stages,the genes which were different methylated at CCGG sites can be divided into 8 clusters,and the genes which were different methylated at CCWGG sites can be divided into 7 clusters.The genes in different cluster are mainly enriched in MAPK signaling,mRNA monitoring,purine metabolism and other pathways.The genes in these clusters contain 868 transcription factors,218 genes associated with hormone metabolism,74 genes involved in anthocyanin synthesis,and 86 genes related to DNA methylation.(3)Integrative analysis of transcriptome and DNA methylation levels of mulberry fruits at different developmental stagesThe screened DNA methylation differential genes and mRNA differential expression genes were analyzed integratively.Among the differentially expressed genes,there were 101,827 and 1057 genes which were differentially methylated at CCGG sites,in the MR and MG,MP and MG,MP and MR comparison sample groups,respectively;In addition,there are 35,445 and 512 genes which were differentially methylated at CCWGG sites in those groups,respectively.The methylation difference sites of these genes are mainly distributed in the intergenic region and exon regions,and their functions are mainly enriched in transcription,glucose transport,ABA response,and flavonoid synthesis pathways.(4)Functional study of Mul-bZIP60 geneThe Mul-bZIP60 gene of mulberry was cloned for the first time,and the protein encoded by this gene contains 330 amino acids and is located on the cell membrane.The Mul-bZIP60 gene does not have tissue expression specificity in mulberry,but the expression levels of the gene were significant differences in different tissues.Meanwhile,the promoter of Mul-bZIP60 gene was also coned,and it was found that there were various response elements such as hormones,light,drought,low temperature,etc.in the sequence of the promoter cloned.Furthermore,its inducible expression activity was analyzed with Agrobacterium-mediated transient expression system combined with GUS histochemical staining,and it was found that the activity of the promoter of Mul-bZIP60 can be induced by ABA,SA and light.In addition,it was showed that the expression of Mul-bZIP60 gene in Arabidopsis can promote the synthesis of anthocyanin in transgenic plants and enhance the accumulation of anthocyanin induced by ABA and drought.(5)Screening and identification of the interacting proteins of Mul-bZIP60Using yeast two-hybrid technology,15 proteins that may interact with Mul-bZIP60 were selected.These proteins identified are including glyceraldehyde-3-phosphate dehydrogenase,F-box protein PP2-A13,probable histone-arginine methyltransferase 1.4,subtilisin-like protease SBT1.4,calvin cycle protein CP12-1,chaperone protein dnaJ A6,alpha-glucosidase,bZIP transcription factor 17,luminal-binding protein 5,60 S ribosomal protein L17-2,protein iojap-related,BTB/POZ domain-containing protein,heme-binding protein 2,aldehyde dehydrogenase family 3 member F1 and Barwin.Furthermore,the genes of the luminal-binding protein 5 were cloned,and the interaction between Mul-bZIP60 with luminal-binding protein 5 was verified,with yeast two-hybrid and bimolecular fluorescence complementary experiments.