Expression And Regulation Of Non-coding RNA During The Formation Of Maize Callus

Non-coding RNAs?ncRNAs?are primary regulators of many biological processes in plants.microRNAs?miRNAs?and long intergenic non-coding RNAs?lincRNAs?are two important types of ncRNAs,which have significant regulatory roles in plants development.The formation of maize callus is an important factor that affects the efficiency of maize genetic transformation,and it is also a major developmental process.However,the changes in the expression of miRNAs and lincRNAs and their regulatory relationship during the maize callus formation are not yet clear.In this paper,we used small RNA sequencing data and degradome sequencing data of immature embryos of maize CAL inbred lines to identify miRNAs and their target genes in the formation of maize callus and study the changes of expression in the process.Meanwhile,combining with the transcriptome sequencing data,we also characterized related lincRNAs in callus formation,and explored the regulatory relationship between lincRNAs and miRNAs in callus formation.The main results are as follows:1.Through small RNA sequencing analysis on the maize immature embryos 10 days after pollination on medium with and without auxin analogue 2,4-dichlorophenoxyacetic acid?2,4-D?treatment for 2,4 and 8 days,174 known miRNAs and 175 newly discovered miRNAs were identified during the maize callus formation.Through differential expression analysis of samples at different time points,53 differentially expressed miRNAs were obtained.miR166,miR319 and miR156 were screened as differentially expressed miRNA families,which was speculated that these differentially expressed miRNAs may be involved in the formation of maize callus.2.To identify the target genes of miRNAs,the degradation libraries of 6 immature embryos were sequenced.A total of 138 target genes of 28 miRNA families were detected.It was found that most of the target genes are transcription factors.SPL18 and HB124 of the SPL and Homeobox transcription factor family members were the targets of miR156 and miR166,respectively.And the expression level changes were negatively correlated with the corresponding miRNA expression changes.These two pairs of miRNAs and their target transcription factors have been reported to be associated with the callus formation in model plants.Therefore,they may also be important candidate miRNAs and regulatory genes that affect the formation of maize callus.3.A total of 1,099 expressed lincRNAs were identified by analysing the transcriptome sequencing data.lincRNAs were lower than mRNAs in terms of transcript length,exon number,transcript number and expression level.Through differential expression analysis,431 specifically expressed lincRNAs were identified.The coexpression analysis of these lincRNAs discovered the presence of two lincRNAs XLOC₀22416 and XLOC₀30430,which coexpressed with the important candidate genes ZmWOX5 a and ZmLBD24,respectively,that can affect callus formation.4.By further analysis the relationship between miRNAs and lincRNAs during maize callus formation,totally,34 miRNA-lincRNA target pairs were detected.Combined with the results of miRNA targeted mRNA,it was found that the expression of three lincRNAs and mRNAs which simultaneously targeted by the same miRNAs showed opposite trends.Moreover,the expression of lincRNA XLOC₀32928 continued to rise at the initial time of callus formation on the first day and second day,which may be induced by auxin.It is speculated that this lincRNA might serve as ceRNA in the formation of maize callus.