Profiling Mirna Expression In Anther Of Thermo-Sensitive Sterile Rice Pa64S Under High And Low Temperature

The fertility conversion of PA64 S of photothermally sensitive genic male sterile line was closely related to sunshine and temperature,which was sterile under long days and high temperatures,and fertile under short days and low temperatures.The identification of photosensitive male sterile gene pms3,pms1 is revealed the molecular mechanism of PA64 S rice regulating pollen fertility under the influence of photocycle,but the internal mechanism of how temperature participates in the regulation of PA64 S fertility is still unclear.The purpose of this study was to investigate the differences in miRNA expression profiles at high and low temperatures of PA64 S at booting stage,and to screen miRNAs and their target genes associated with PA64 S and temperature-sensitivities,so as to lay a foundation for further analysis of the molecular mechanism of temperature-sensitivities regulation of PA64 S.In this study,two cDNA libraries(PA64S-H and PA64S-L)were established with the anthers of early mononuclear stage of PA64 S rice treated at 30?(male sterility)and 22?(male fertility),respectively.MicroRNA sequencing was performed on the two libraries by means of high-throughput sequencing technology.The main results are as follow:1.This research material are two weeks booting stage PA64 S rice anther under the temperature higher(30 ?)and lower(22 ?)criticaltemperature of sterility,than used the method of Trizol to extracted the RNA.Agarose GEL electrophoresis detection shows the 28 SRNA and18SRNA stripe is clear.It means samples of RNA degradation is less,Sample OD260 / OD280 ratio between 1.8 and 2.2.It suggests that RNA is pure to used for the next experiment.Two cDNA libraries,PA64S-H and PA64S-L,were constructed by RT-PCR.2.After high-throughput sequencing for two libraries,it was found that there were 14030132 raw sequences in PA64S-H library,of which6788401 readable effective sequences(54.14%)was found which passed through background interference filtering such as low-quality sequences and pollutants.4289438 sequences(63.18%)could be recognized at miRbase.There were 16844884 raw sequences in PA63S-L,of which12484929 sequences(89.71%)were readable and effective after background filtering and 7743506 sequences(62%)could be cognized at miRbase.The small RNA length analysis of the two libraries showed the highest frequency length is 24nt(mi RNA standard length),it indicating that the analysis results ware effective.These raw data that were preliminary processed will be analyzed in next analysis.3.According to the analysis of miRBase,GeneBank database and prediction of PatMatch software,584 miRNAs were found in the two cDNA libraries,including 263 known miRNAs(45%)and 321 candidate new miRNAs(55%).The analysis of the first nucleotide bias in the twolibraries revealed that the known miRNA and the new candidate miRNA were both U(uracil)in the majority,and both formed the typical stem ring structure of the miRNA,indicating that the structure of the two miRNA was consistent.According to the mi RNA expression level of the sequencing library,151 known miRNAs were expressed in both libraries,and 56 known miRNAs were specifically expressed in PA64S-H and PA64S-L.63 new candidate mi RNAs were expressed in both libraries,144 new candidate miRNAs were only expressed in PA64S-H,and 114 new candidate miRNAs were only expressed in PA64S-L.Both known miRNAs and new candidate miRNAs had significant specific expressions in two libraries.Through the above analysis and related expression level analysis,this study found that 133 known mi RNA expressions in PA64S-H and PA64-L were significantly different.These significantly different known miRNA might be related to fertility of sterile line rice PA64 S.4.In order to further verify whether the data of differentially expressed mi RNA obtained from high-throughput sequencing are reliable,we randomly selected 6 known mi RNA with differentially expressed miRNA and detected their expression levels through real-time fluorescence quantitative PCR analysis.The results shows that,compared with PA64S-L,the expression levels of mi R531,miR6300 and mi R9773 in PA64S-H were down-regulated,while the expression levels ofmiR1118,mi R2275 and miR9473 were up-regulated.The results of RTFQ-PCR were consistent with those of high-throughput sequencing,indicated that the high-throughput sequencing results were reliable.5.We focused on the miRNAs and target genes that might be related to the fertility of rice at different temperatures when we predicting these differentially expressed miRNA and their target genes functions.5944 target genes corresponding to differentially expressed mi RNAs were predicted based on miRBase and GeneBank databases.After remove duplication miRNAs 2226 known miRNAs target genes and 233 new candidate mi RNAs target genes were predicted.The number of predicted target genes of the new candidate mi RNA is significantly less than that of the known miRNA.The new candidate mi RNA is similar to the known mi RNA in the structure and target genes function.Therefore,this research focus on the analysis of known miRNA.Many of the target genes predicted by these signifinicant different known mi RNAs include MYB transcription factors gene and TCP transcription factors gene,auxin response factor(ARFs)gene and so on,which have been reported to be related to the response of external temperature signal and anther pollen development in rice.This suggests these miRNAs and its target genes may be related to rice fertility.6.To further illuminate the function of the differentially expressed miRNAs,gene ontology(GO)analysis and KEGG pathway annotationwere used to enrich and classify the given target genes.GO term analysis found the biological processes in which the target genes of these significantly differentially expressed miRNAs are found to be mainly concentrated in metabolic processes,intracellular processes and external stimuli.The components of the cells are mainly concentrated in cells,partial areas of cells and organelles.The molecular functions are mainly concentrated in binding,catalysis and transport.This results are consistent with the molecular functions and functional cell components of rice pollen anther development genes.KEGG analysis found the metabolic pathways involved included starch and sucrose metabolic pathways,sphingomyelin metabolic pathways,arginine and proline metabolic pathways,which have been reported to be related to anther pollen development in rice.In addition,the involvement of target genes in plant hormone signal transduction pathways also suggests that miRNA may influence the development of rice pollen anthers by regulating plant hormone signal pathways.7.Cluster analysis of metabolic pathways of differentially expressed miRNA target genes was conducted according to the physiological and biochemical characteristics of photothermophilic male sterile rice,which can be divided into plant hormone and signal transduction related pathways,energy and substance metabolism related pathways,protein synthesis and transport and degradation pathways,and DNA RNAsynthesis and transport related pathways.Most of the significantly differentially expressed miRNA target gene metabolic pathways are in these four categories,suggesting that miRNA is closely related to photothermally sensitive male sterile rice.In summary,we analyzed the miRNA expression profile of photothermally-sensitive sterile rice PA64 S anther under the conditions of sterility temperature and fertile temperature by high-throughput sequencing technology,and found that miRNA was involved in the fertility regulation of PA64 S in sterile rice under different temperatures.