Study On The Regulatory Mechanism Of CeRNA Network Associated With ATAC-seq In Mulberry Leaf Morphological Differentiation

China has a long history of mulberry cultivation and silkworm rearing,both of which have had a great impact on the nation’s economic and social development.The leaf shape of the Mulberry（Morus alba’Pendula’）is a significant agronomic characteristic that can be used to measure yield,growth,development,and environmental fluctuations.M.alba’Pendula’has a rich leaf morphology,including full split,one split,two splits,and up to 6-7 splits.Therefore,Morus alba’Pendula’is the most suitable research material for studying the mechanism of leaf shape regulation in mulberry.This study comprehensively identified transcription factors,miRNAs,and target genes related to mulberry leaf shape regulation through ATAC-seq combined with ceRNA sequencing technology.Screening was conducted for target genes related to leaf shape regulation and key miRNAs,and effective transformation was achieved by combining target gene VIGS silencing and overexpression to explore the molecular mechanism of mulberry leaf shape differentiation.Clarifying the molecular mechanism of leaf shape differentiation differences is of great significance in agricultural production,genetic breeding,and other aspects.The main research results are as follows:1.Identification of transcription factors associated with mulberry leaf shape changes using ATAC-seqATAC-seq is a recently developed technology for genome-wide analysis of chromatin accessibility.The samples used for ATAC sequencing in this study were divided into two groups:whole leaf（CK-1,CK-2）and split leaf（HL-1,HL-2）.Bioinformatics was used to screen for differentially expressed motifs and motifs related to mulberry leaf shape,enabling the identification of transcription factors responsible for the differential changes in the leaf shape.These transcription factors mainly include the common TCP family in plants,which has been proven to be related to leaf development and growth in other woody plants.The majority of the TCP family was enriched in the CK group,indicating that the transcription of TCP in the HL group may decrease,leading to changes in leaf shape（from whole to split leaves）.In addition,the TCP family analyzed by downregulating peak from the HL group showed a similar pattern,further supporting the possibility of reduced expression of the TCP transcription factor family in the split leaves.The results indicate that the TCP family is an important transcription factor that leads to different leaf shapes.2.Molecular regulatory patterns associated with leaf shape differences in mulberry using ceRNA sequencing to construct regulatory networksCompetitive endogenous RNA（ceRNA）does not refer to a new RNA molecule,but rather to theRNA contained in a complex type of transcriptional regulatory network in organisms,including m RNA,miRNA,lnc RNA,and circ RNA.ceRNA regulation plays an important role in the growth and development of mulberry leaf shape.The samples used for ceRNA sequencing in this study were divided into two groups:whole leaves（CK-1,CK-2,CK-3）and split leaves（HL-1,HL-2,HL-3）.A ceRNA differential network was constructed for split leaves and whole leaves,and further analysis of the ceRNA network showed that ceRNA regulation plays an important role in the occurrence of abnormal leaves.The common core ceRNA network identified through comparative analysis,and functional analysis showed that these ceRNAs were related to heteromorphosis.Moreover,differential expression analysis showed that ceRNAs in the obtained ceRNA interaction network significantly enriched with differentially expressed genes,suggesting that the miR156x/MaSPL3/MSTRG.25812.1 regulatory mode may be one of the main reasons for the differences in mulberry leaf shape.3.Identification of leaf shape regulation function of mulberry miR156x by transient transformation overexpressionIsolation,cloning,and identification of pre miR156x,the precursor of miR156x,from the young leaves of the weeping mulberry was achieved.The length of the pre miR156x was107 bp,with a minimum folding free energy of 50.27 kcal/mol,which was derived from a lnc RNA（Gen Bank No:MSTRG.25812.1）.A binary overexpression vector p CAMBIA-35S-GUS-miR156x was constructed and a transient transformation system for mulberry was established.The GUS gene in mulberry leaves displayed an extremely significant high expression level（P≤0.01）on the fourth day after transformation of the transformant solution(OD₆₀₀=0.7),while the target gene of miR156x showed lower expression levels.A series of physiological and biochemical indicators changes indicated that plant miRNAs can serve as negative regulatory factors for gene expression in response to changes in mulberry leaf shape regulation.The experimental results further confirm that the miR156x/MaSPL3/MSTRG.25812.1 regulatory mode may be involved in the regulatory mechanism of differences in mulberry leaf shape.4.Prokaryotic expression of MaSPL3 in mulberry tree and its targeting relationship with miR156x and study on its interaction proteinThe complete MaSPL3 gene was cloned,with a c DNA sequence of 1503 bp,encoding500 amino acid residues,a protein molecular weight of 55.04 k Dd,and an isoelectric point of8.83.Through multiple sequence alignment and phylogenetic tree analysis,it was found that the amino acid sequence of MaSPL3 gene had high homology with other alignment species.The prokaryotic expression vector p ET-20b（+）-MaSPL3 with MaSPL3 as the target gene was constructed.In addition,in order to clarify the targeting relationship between miR156x and MaSPL3,miR156x can inhibit the expression of MaSPL3 through the double Luciferase reporting experiment.Forty seven MaSPL3 interacting proteins were obtained through GST Pull experiment.It has been reported that these proteins are mainly related to the growth and development of mulberry.5.Functional Identification of MaSPL3 Gene in MulberryA virus induce gene silencing（VIGS）system for MaSPL3 was established using the PDS gene as the reporter gene.The expression level of MaSPL3 was detected by RT-q PCR at 0-45days.The gene expression level gradually decreased at 0-30 days,and the expression level of MaSPL3 reached its lowest value on the 30th day.Subsequently,the expression level of MaSPL3 gradually increased,and the silenced plants showed significant phenotypic changes such as dwarfing and leaf yellowing,indicating that this system can be effectively used for silencing the MaSPL3 gene in mulberry.By heterologous overexpression of MaSPL3 in Honji tobacco,compared with wild-type tobacco plants,the T1 generation resistant seedlings showed phenotypic changes such as curling,yellowing,and thinning in their leaves.The experimental results showed that overexpression of MaSPL3 indeed affected leaf morphology and development,further suggesting that MaSPL3 can cause changes in mulberry leaf shape.This study systematically and comprehensively identified the mulberry leaf shape regulation associated miRNA and its target gene MaSPL3 through ATAC seq combined with ceRNA sequencing technology.The functions of the selected miR156x and target gene MaSPL3 in mulberry leaf shape regulation were verified through the transient transformation of miR156x,double Luciferase reporting experiment,GST pull down experiment,VIGS and tobacco heterologous overexpression and other experimental methods.The regulation of the target miR156x on MaSPL3 gene expression at the intact plant level was revealed.By exploring the molecular mechanism of mulberry leaf shape changes,the research outcomes have highlighted the molecular regulatory network responsible for the variations in mulberry leaf shape.This work provides an important theoretical basis and exploration pathways for the study of mulberry leaf shape change mechanism and the selection of resistant varieties.It is an important way to protect mulberry germplasm resources,improve mulberry yield,and promote the sustainable development of sericulture.