Exploration Of Degradome-based Small RNA-mediated Regulatory Networks Of OsSPL Gene Family

The SQUAMOSA promoter-binding-like(SPL)gene encodes a specific family of transcription factors in plants and is a key factor in regulating plant growth and development and resistance to stress and disease.Nineteen SPL transcription factors have been found in rice,which play key roles in regulating rice tillering,seed development,panicle architecture,stress response and other life processes.The conservative regulation of OsSPL by miRNA156 reveals the important role of small RNA in OsSPL's regulatory networks.In recent years,the cleavage activity of other small RNAs on OsSPL has been detected one after another,and people are paying more and more attention to the regulatory complexity of OsSPL upstream.In this study,a total of 162,412 small RNAs with different mature sequences were detected from 127 small RNA sequencing libraries.Using 16 public degradation libraries and 1 laboratory sequencing degradation library,3,173 different small RNA pairs were detected.OsSPL's cleavage activity,through statistical analysis of these small RNAs and their cleavage sites,and further construct and explore the network after verification of the degradation group with small RNA and OsSPL as the core,in order to obtain an available regulatory module and discover OsSPL The breeding potential of traits.The main research contents are:1.To get avoidance of the false negatives caused by differential expression analysis of miRNAs,we explored the strategy of degradome-dependent analysis of small RNA,which screened functional small RNA with differential cleavage abundance rather than the expression.We designed the parameter false positives,which are the miRNAs with differential expression but no targets,and false positives which are the miRNAs have targets but without differential expression features.Based on that,we caluated the area under ROC curver of expression-dependent methods in all three species,which are all below 0.5,showing poor efficiency on the discovery of functional miRNAs.While degradome-dependent methods provide scientist identical functional miRNAs and specific cleavage information such as slicing sites and degradation abundance.2.Perform data cleaning on 127 public small RNA sequencing data from different experiments,retain sequences that can be compared to more than 80%of the rice genome,and count the basic information such as the tissue source,processing method,and sequencing depth of each library.Through classification,a total of small RNA sequencing data from 7 tissues such as roots,stems,leaves,seedlings,seeds,young flowers,and ears were sorted out.Since this study mainly discusses the regulation activities of small RNA based on the verification results of the degradation group,the verification cut rating of the degradation group is above Cat₂,and any small RNA with a reading greater than 1 detected in any data set is retained.After the preliminary screening,the small RNA is de-redundant and the expression level is calculated.Anything that can detect the reading value is retained,and it is named simply and not repeatedly in the form of "RNA#n",where n is a non-repeating integer of 1?162,412.Upload the complete non-redundant small RNA and OsSPL complete transcript sequence(downloaded from RAP-DB)to the psRNATarget platform to predict the small RNA-OsSPL targeting relationship(Small RNA-Target Interaction,STI)to obtain the predicted regulation Relationship information,and then use all existing degradation group libraries to verify these prediction relationships,and retain the regulatory relationship of the first and second class of cleavage ratings after the degradation group verification,including a total of 3,173 small RNAs with different sequences and 3460 pairs of different cleavages Relationship,build a network and analyze on this basis.3.By comparing the diversity of small RNA cleavage sites and cleavage activities of different OsSPL family members,it was found that different OsSPLs have different degradation abundances and differences in small RNA cleavage sites in different tissues,and the number of degradation groups based on the detected abundance To classify STIs and sites that are conservative and specific among different members,and organization-specific and conservative.From this,it was found that the miRNA156-based microRNA can detect cleavage activity in more than 2 tissues.Although not all OsSPLs can be cleaved,it can still target a large amount of OsSPL with poor specificity;other small RNAs have High tissue specificity and OsSPL specificity,such as phasiRNA,most of which are more actively cleaved in reproductive organs such as flowers and ears;OsSPL15 is specifically regulated by a large number of redundant small RNAs.4.Taking OsSPL3 and OsSPL8 as examples,we found that some OsSPL have differences in the regulation mode between tissues,that is,a combination of different slicing sites,such as OsSPL3;while some do not show the regulation modes always cut near the same sites,such as OsSPL8.5.In addition,we also verified the co-expression of the small RNA-OsSPL relationships based on the degradation group:using bowtie to re-align these small RNAs to the genome,and organize them into small RNA position annotation files,and then small RNA position annotation files For indexing,use ShortStack to calculate the expression of small RNAs in specific samples,and calculate the correlation coefficients of their co-expression with OsSPL.From the aspect of expression,it is necessary to explain the complex and varied regulation of OsSPL by small RNAs:Top 50 nodes selected by cytoHubba For example,in expression,miRNA expression is mostly negatively correlated with OsSPL,while other siRNAs are mostly positively correlated with OsSPL.6.In addition,find the SNPs at the cleavage site of small RNA from the three thousand rice resequencing data platform(RFGB),and obtain the phenotype information of the corresponding haplotype from the RFGB,including grain size,ear length,thousand kernel weight,etc.11 Two different rice traits,taking OsSPL14/IPA1 and OsSPL18 as examples.Among them,an SNP that has not been utilized in the vicinity of the small RNA cleavage site has been detected in IPA1,and it has traits such as thousand-grain weight,grain length,seedling height,etc.It has an impact;in OsSPL18,a SNP that has been reported to be related to grain weight has also been detected to occur near the location of small RNA cleavage,and there is a large difference of nucleotides between indica and japonica.