Analyzing The Regulatory Network Of LL Gene Regulating Cucumber Organ Size And The Number Of Side Branches Based On MicroRNA-mRNA Joint Analysis

The number of cucumber lateral branches and organ size are very important agronomic traits.The occurrence and development of lateral branches will not only weaken the growth potential of the main cucumber stems,cause nutrient depletion,affect the main vine melon,but also directly affect the ventilation and light penetration of the cultivation group,accelerate the occurrence and spread of diseases,not conducive to light and simplified cultivation.Fruit size and shape are not only important quality traits,but also important commercial traits,as well as important agronomic traits affecting cucumber yield.Our group has identified a gene,Cs LL（Little Leaf）,that controls the number of lateral branches and organ size and constructed a near-isogenic line,HAUC106,in the background of 9930 North China cucumber.In this paper,we investigated the genetic regulation of Cs LL by phenotyping 9930 and HAUC106 and combining RNA-seq and micro RNA-seq transcriptome In this paper,we explored the genetic regulatory network of Cs LL by phenotypic investigation of 9930 and HAUC106,and combined RNA-seq with micro RNA-seq transcriptome analysis to explore the regulatory mechanism of lateral branch development and organ size of cucumber,which provides a theoretical basis for further research on the molecular mechanism and regulatory network of lateral branch development and organ size development of cucumber.The main findings are as follows.1.Investigation of multiple phenotypes of North China-type cucumber 9930 and the ll-gene near-isogenic line HAUC106 throughout the reproductive period revealed that,the Cs LL gene regulates the developmental processes of several traits.HAUC106 seeds germinate and form tendrils at a faster rate than 9930 However,the average height of the hypocotyl of is 2.75 cm,which is significantly lower than that of 9930.which is significantly lower than the hypocotyl height of 9930 at 4.00 cm.The average hypocotyl height of 9930 is2.75 cm,which is significantly lower than that of 9930 at 4.00 cm.In addition The introduction of the ll gene into the near-isogenic line HAUC106 resulted in a significant increase in the number of lateral branches.The average number of lateral branches in it was11 at 30 days after planting,while the number of lateral branches in 9930 was only 3-4.Comparison of internode numbers revealed that The number of internodes of HAUC106 was significantly higher than that of 9930 throughout the reproductive period.The number of internodes of HAUC106 was 27-28 at 45 days,while the number of internodes of 9930 was3-4.The number of internodes of HAUC106 was 27-28 at 45 days,while the number of internodes of 9930 was 22-23.The number of internodes of HAUC106 was 27-28 at 45 days,while that of 9930 was 22-23.For both materials leaf blade,and 9930 were found to be significantly smaller in HAUC106 compared to 9930,with HAUC106 and 9930 having 5.2cm2 and 20 cm2 of male flower area on flowering day,respectively.The leaf length of and leaf width of were analyzed and found that the smaller leaves in HAUC106 were mainly due to the smaller leaf width of The comparison of ovary and fruit of the two materials revealed that there was almost no significant difference in ovary width on the day of flowering and fruit width at different times,but the length of 9930 ovary and fruit was about 1.7 times longer than that of HAUC106 ovary and fruit..These results suggest that Cs LL is a positive factor regulating organ size and a negative factor regulating the number of lateral branches.2.By sequencing the transcriptomes of the apical buds and axillary budsof 9930 and HAUC106,we obtained an average of 8.25 GB of raw data for each of the 12 samples sequenced at.We obtained an average of 8.25 GB of raw data per sample from the 12 samples sequenced in the apical and axillary buds,of which 97.34% of the sequences could be matched to the reference genome.A total of 544 differentially expressed genes（DEGs）were obtained in the comparison of the acinar transcriptomes of the two materials,of which262 DEGs were significantly up-regulated and 182 DEGs were significantly down-regulated.In two materials,4542 differentially expressed genes were detected in the axillary bud transcriptome,of which 2218 DEGs were significantly up-regulated and 2384 DEGs were significantly down-regulated.KEGG enrichment analysis of the differentially expressed gene in terminal and axillary buds revealed,the phytohormone signaling pathway and the transcription factor pathway as pathways enriched in both terminal and axillary bud transcriptome data.This coincides with our endogenous hormone assays for both materials in HAUC106 with a significant increase in growth hormone content in the terminal and axillary buds and a significant decrease in cytokinin levels.In addition In addition,we found a large number of genes associated with lateral branch development in the enriched transcription factor pathway,and quantitative fluorescence analysis of some of these genes showed that was significantly higher than 9930 in its near-equivalent line HAUC106,such as TCP2（Csa V3₁G004660）expression is down-regulated and SPL9（Csa V3₃G0038950）expression is up-regulated.This result suggests that the Cs LL gene may synergistically regulate the number of lateral branches in cucumber by regulating the transcription factors TCP2,SPL13 and growth factors and cytokinin.3.In sequencing the transcriptome of the cotyledons of both 9930 and HAUC106 material 0 DAP,yielded an average of 7.05 GB of raw data per sample,of which 97.35% of the sequences could be compared to the reference genome.A total of 4261 differentially expressed genes were obtained by transcriptome comparison between the two materials,of which 2378 DEGs were significantly up-regulated and 1883 DEGs were significantly down-regulated.KEGG enrichment analysis revealed that these differentially expressed genes were also significantly enriched in the phytohormone signaling pathway and transcription factor pathway.A large number of genes related to organ size were also found in the enriched transcription factor pathway In addition,the transcriptome analysis and fluorescence quantification of the apical and axillary buds were combined to identify a large number of genes related to organ size.The enriched transcription factor pathway also identified a large number of genes related to organ size.,SPL9（Csa V3₃G0038950）,GRF3（Csa V3₃G036970）,BB（Csa V3₁G042670）,DA1（Csa V3₀28370）and other genes were further validated by fluorescence quantitative PCR in the daughter house The results showed that The results showed that In the near-isogenic line HAUC106 TCP2 expression was up-regulated,SPL 9 expression was downregulated,GRF3 expression was downregulated,and Big brother gene expression up-regulated,DA1 gene expression was downregulated..This suggests that Cs LL gene may affect multiple transcription factor pathways to regulate organ size.4.We also performed microRNA-seq sequencing and comparison of the acrosomes of both 9930 and HAUC106 materials A total of 1.2 GB of raw data was obtained from,of which 96.79% of micro RNAs could be compared to the reference mi RNA sequences.91 known micro RNAs were successfully identified in micro RNA-seq,and 50 new micro RNAs were predicted.22 of these 141 micro RNAs were differentially expressed,of which 11 micro RNAs were significantly up-regulated,and 11 micro RNAs were significantly down-regulated.A total of 2027 target genes were detected using bioinformatics software and online prediction website to predict the target genes of the 22 differential micro RNAs,and GO and KEGG enrichment analysis was performed on 2027 target genes,and 2027 target genes were significantly enriched in metabolic,transport and cytokinesis pathways.We performed quantitative analysis of some of the differential micro RNAs and their target genes to verify The results were consistent with the transcriptome sequencing results.The results are consistent with the transcriptome sequencing results.Among them The mi R396 family member ath-mi R396b-5p was most significantly expressed in the differential analysis,and GRF3（Csa V3₃G036970）was predicted to be one of the highest scoring target genes for ath-mi R396b-5p by bioinformatics software and online website.We further validated Csa V3₃G036970 as a target gene downstream of ath-mi R396b-5p by tobacco transient transfection assay.These results suggest that ath-mi R396b-5p and the target gene Csa V3₃G036970 module may also be a pathway involved in Cs LL gene regulation to control organ size.