Identification Of PhasiRNAs And Indications For A Role Of CMS-C In Maize(zea Mays L)

phasiRNA（phased,secondary,small interfering RNA）is a special kind of non-coding small RNA,which has been found to play an important role in nuclear male sterility in plants.However,its related research in cytoplasmic male sterility has not been reported yet.In this study,anthers of pollen mother cells（PMC）stage and uninucleate microspores（UM）stage in C48-2 and 48-2 were carried by RNA sequencing.PHAS loci and phasiRNA were identified by using Phase Tank software,and the adjacent genes of PHAS loci were searched to investigate the regulatory relationship between phasiRNA and adjacent genes.In addition,through PHAS loci reads abundance analysis,differentially expressed phasiRNAs were screened,and then target genes were predicted for differentially expressed phasiRNA.To screen possible target genes related to flower development,we carried out target genes GO and KEGG analysis.The q RT-PCR analysis of the screened flower development-related target genes and corresponding phasiRNAs were performed to investigate the regulatory relationship between phasiRNA and target genes.Finally,we also analyzed whether phasiRNA and maize transposon sequences had a complementary relationship to lay the foundation for further exploration of the function of phasiRNA.These results were as follows:1)Through the identification of PHAS loci and phasiRNA in C48-2 and 48-2 PMC and UM2 stages,found that the number of 21 nt PHAS loci/phasiRNAs decreased during the abortive phase of C48-2,and the number of 24 nt PHAS loci/phasiRNAs increased,suggesting that the production of 21 nt,24nt PHAS loci is possible related to the abortion of CMS-C in maize.2)To increase the sensitive identification of PHAS loci,we combined a total of 12 Small RNA libraries from PMC and UM of C48-2 and 48-2.We identified 253 21 nt PHAS loci and 150 24 nt PHAS loci by using Phase Tank.We found that novel 21 nt and 24 nt PHAS locis were 30 and 57,respectively.Moreover,through searching for adjacent gene to PAHS loci,we found most PHAS locis located in intergenic region and function were unkown.3)According to the PHAS loci reads abundance analysis,compared with 48-2,during the PMC phase,differentially expressed 21 nt and 24 nt phasiRNAs were 14 and 11 in C48-2,respectively.Besides,they were both 15 differentially expressed during the UM period in C48-2.4)Based on target gene prediction of these differentially expressed phasiRNAs and conducted target genes GO（Gene Ontology）and KEGG（Kyoto Encyclopedia of Genes and Genomes）analysis.The results showed that differentially expressed 21 nt phasiRNA target genes GO and KEGG analysis were relatively more transcripts in the intracellular carbon metabolism and energy metabolism both in the PMC and UM stages.Moreover,we filtrated18 target genes of differentially expressed 21 nt phasiRNA related to flower development both in the PMC and UM phases.However,the 24 nt phasiRNA target genes GO analysis results showed more transcripts involvement in galactose metabolism,ATP biosynthesis and phosphoinositide metabolism signal transduction system.We filtrated 3 target genes of differentially expressed 24 nt phasiRNA related to flower development both in the PMC and UM stages.5)Through analyzing adjacent genes of differentially expressed PHAS loci,a total of12 21 nt PHAS loci and 2 24 nt PHAS loci were identified.Their corresponding phasiRNAs target genes was related to flower development.Five of the PHAS loci’s adjacent gene functions were annotated.Interestingly,the adjacent genes of PHAS loci 7₁7,4₇9 and10₆4 participated in the same metabolic process as the target genes of their phasiRNAs.6)Not only selected 4 21 nt and 2 24 nt phasiRNAs of these differentially expressed phasiRNAs but also 6 their target gens were analyzed by q RT-PCR at different periods in anthers.The results showed that the expressions of 21 nt phasiRNA 4₉6₃59（+）、7₅0₂35（-）were down-regulated and were consistent with the predicted results in C48-2 at the PMC stage.21 nt phasiRNA 4₂2₈4（+）and 24 nt phasiRNA 4₇9₄63（-）,10₆4₁03（-）were down-regulated and were consistent with the predicted results in C48-2 at the UM stage.The expression level of 21 nt phasiRNA 7₄3₃10（-）was down-regulated expression in C48-2 at UM stage,which was contrary to the prediction.At the same time,we found that the target genes GRMZM2G326643,GRMZM5G867882 and GRMZM2G117240 were almost highly expressed at different stages in C48-2 anther,and their biological functions involved in pollen development,ATP hydrolysis coupled proton transport and DNA methylation,respectively.In addition,the experimental results also found that 21 nt phasiRNA4₉6₃59（+）,7₅0₂35（-）and 6₂57₈1（+）had possible positive regulation to target genes as well as the 24 nt phasiRNA 4₇9₄63（-）,10₆4₁03 were possible negative regulation to target genes.7)Complementation analysis of phasiRNA and maize transposon sequence,found 31 sequences of 24 nt phasiRNA complementary to maize transposon,but these the complementary 24 nt phasiRNAs were not included in the differentially expressed phasiRNA data（P value < 0.05）.