Polyadenylation And DNA Methylation Regulate The Sink-source Relationship In Cucumber（Cucumis Sativus L.）

The source and sink of plants can interact with each other,and the coordinated source-sink relationship can effectively promote leaf photosynthesis and improve assimilate transport efficiency,which is crucial for improving crop yield.Raffinose family oligosaccharide（RFOs）is the main transport form of assimilates in cucumber,and its source-sink regulation mechanism is significantly different from that of other sucrose transport plants.Alternative polyadenylation（APA）and DNA methylation are important ways to regulate gene expression in higher plants.Previous studies have shown that they are widely involved in the regulation of source-sink in sucrose transporters,but their role in the regulation of source-sink in RFOs transporters such as cucumber remains unclear.In this study,different sink strengths were achieved by retaining or not retaining fruit on the plant.The roles of APA and DNA methylation in source-sink regulation of cucumber and the important genes involved were analyzed by leaf APA sequencing and whole genome DNA methylation sequencing.After treatment with methylation inhibitor 5-aza,DNA methylation related gene expression,assimilate loading gene expression,enzyme activity change and fruit growth rate change were observed to further verify the role of DNA methylation in fruit growth.In order to provide clues for revealing the source-sink regulation mechanism of RFOs transport crops such as melon,and provide theoretical basis for high-yield cucumber cultivation.1、Three generations of Nanopore sequencing were used to screen 11144 genes at FNL（Nodes with fruit leaves）and NFNL（Nodes without fruit leaves）.Among the differential APA genes,3770 APA differential genes tended to use the proximal Ploy（A）locus,of which 89 genes were down-regulated and 304 genes were up-regulated,compared with NFNL;The proximal Ploy（A）locus was used in 3948 APA differential genes,of which 192 were down-regulated and 121 up-regulated.These results indicated that the gene expression could be affected by regulating APA during cucumber assimilate transport.Combined analysis of differential APA and differential lncRNA showed that genes related to differential genes were mainly enriched in DNA replication,starch and sucrose metabolism,and plant hormone signaling pathways.Sucrose phosphate synthase（CsaV3₂G033300）,starch synthase（CsaV3₅G001560）and ethylene receptor（CsaV3₇G029600）may be involved in cucumber source-sink regulation.The expression of sucrose phosphate synthase（CsaV3₂G033300）gene in FNL increased compared with that in NFNL,which indicated that the FNL needs to produce more sucrose for further synthesis of RFOs output.Combined analysis of differential APA and differential mRNA indicated that genes related to differential genes were mainly concentrated in the pathways of secondary metabolite biosynthesis,plant hormone signal transduction and DNA replication.Among them,trehalose 6-phosphatase（CsaV3₃G016150）,sucrose synthase（CsaV3₅G020420）,auxin reactive protein（CsaV3₅G004700）and other genes may be involved in cucumber source-sink regulation.The gene expression of sucrose phosphosynthase（CsaV3₂G033300）using the proximal poly-A site increased in fruit plants compared with NFNL,suggesting that the leaves of FNL using the proximal poly-A site may produce more sucrose for further synthesis of RFOs output.2、WGBS-seq sequencing technology was used to construct genome-wide DNA methylation sequencing libraries from cucumber at different source-sink levels（FNL and NFNL）.Among mCG,mCHG and mCHH methylation types,forty,seven and six thousand 4 hundred and eighty-seven differential genes were identified,which were mainly enriched in photosynthesis,redox and transmembrane transport pathways.Among the three types of DNA methylation differential genes,22 genes related to carbohydrate metabolism and 8 genes related to plant hormone signal transduction were screened.The methylation levels and gene expression of the above genes were increased in 3 FNL compared with those in NFNL.Methylation level increased but gene expression decreased in 4 gene.19 had decreased methylation levels and genes;4 showed decreased methylation levels but increased gene expression.The methylation level and gene expression of the above genes were increased in three FNL compared with those in NFNL.Methylation level increased but gene expression decreased in 4 gene.19 had decreased methylation levels and genes;4 showed decreased methylation levels but increased gene expression.In the study of source-sink interaction related genes screened from different pool strength mRNA transcriptomes and methylated groups,22 genes were found to be enriched in carbohydrate metabolism pathway,and eight genes were enriched in plant hormone signaling pathway.Combined analysis of differential mRNA and DNA methylation with different sink strengths showed that it was mainly enriched in carbohydrate metabolism and plant hormone signal transduction pathways.The raffinose synthase gene CsRS（CsaV3₃G042690）showed higher methylation level and increased gene expression in the leaves of FNL.The methylation level of sucrose synthase（CsaV3₄G000970）in leaves of FNL was low and the gene expression was increased.This suggests that more sucrose needs to be synthesized in FNL,and reducing the methylation level of sucrose synthase can promote sucrose synthesis for further synthesis of RFOs output.3、The expression levels of CsDMR1,CsMETl and CsCMT3,which regulate three different types of DNA methylation,were significantly decreased in cucumber leaves treated with 200 μmol/L methylation inhibitor 5-aza,while the expression levels of CsCMT2 were increased.These results indicated that 5-aza treatment could change partial methyltransferase activity.After 5-aza treatment,the total expression level of stachyose synthase（CsSTS）and raffinose synthase（CsRS）genes,which were key enzymes of assimilate loading,were significantly increased,and CsSTS were found in three transcripts formed by APA,There was no significant change in CsSTS2 and CsSTS3 expression,These results suggest that DNA methylation can promote the selection of the proximal poly-A site for the key gene CsSTS in cucumber isogenate loading,and improve the expression of CsSTS1 with high stability,thus promoting the overall expression of CsSTS.After 5-aza treatment,both fruit length and dry weight were significantly increased,suggesting that the change of DNA methylation level could promote the transport of assimilates from leaves to fruits.4、The CRISPR-Cas9 gene double target editing vector suitable for cucumber was constructed to provide transgenic plant materials for the further study of the mechanism of DNA methylation regulating cucumber sink source interaction.In conclusion,APA and DNA methylation are widely involved in the source-sink regulation of RFOs transport crop cucumbers,and play important roles in carbohydrate metabolism,plant hormone signaling and biosynthetic metabolic pathways of secondary metabolites.The change of DNA methylation status can promote the transport of cucumber assimilates to fruit,which has potential application value in high-yield breeding and cultivation of cucumber.