| Rice(Oryza Sativa L.)serves as a staple food for more than half of the population in the world.It is also an important model plant for crop genomic research.Functional genomic studies in rice have not only identified key genes that regulate rice development and environmental responses,but also assisted in the rice breeding programs in agriculture.The rice genome is generally consisted of protein-coding genes as well as non-coding regulatory sequence.While the coding genes are extensively studied,the research on non-coding DNA sequence is in its infancy.It is also becoming an urgent task to investigate the interaction between these coding and non-coding DNA sequences,so as to uncover the novel regulatory mechanisms behind gene expression and the maintenance of genome integrity.Research in this field will greatly improve our knowledge of genome complexity and further direct practical use in the rice gene resource.In this study,the rice stress-responsive microRNAs were profiled for their transcriptional level under various stress conditions.Considering the microarray data of predicted target genes,we transformed the candidate genes into rice to evaluation their performance on drought resistance and yield improvement.Fortunately,a member of the CCT(CONSTANS [CO],CO-LIKE and TIMING OF CAB1)gene family was identified to regulate important agronomic trait,including grain number,plant height,heading date(named as Ghd2),as well as drought resistance.Meanwhile,Ghd2 3'-UTR,embedded a miniature inverted-repeat transposable element(MITE),modulate its protein synthesis,which was further investigated.The major results are as follows.1.Stress-responsive micro RNAs were profiled and analyzed by real-time quantitative polymerase chain reaction(RT-q PCR)and 35 micro RNAs were found to respond to stresses.Their target genes were predicted by bioinformatic analysis.Considering the microarray data of target genes,we chose the candidate genes for further investigation.2.Based on the analysis,the full length(Ghd2 FL)and coding region(Ghd2 FL?U)of candidate gene were constructed into p CAMBIA1301 H vector and transformed intoOryza sativa L.ssp.japonica var.Zhonghua11.Three independent single-copy transgenic lines for each transformation were generated for study.3.Ghd2 diurnal expression pattern was determined in wild type,indicating that the Ghd2 expression peaked at 20:30 under long-day and short conditions.Meanwhile,the flowering transition genes were profiled in the wild type and Ghd2 FL?U over-expression lines.The results showed that Ghd2 repressed the Ehd1 expression to suppress flowering transition in rice.The Ghd2 spatial expression pattern suggested that this gene highly expressed in leaves at the reproductive stage.4.The phenotypes of Ghd2 single-copy transgenic lines including Ghd2 FL and Ghd2 FL?U were examined in the field.Interestingly,the Ghd2 FL?U over-expression lines showed nearly 60-day delay in flowering time,whereas the Ghd2 FL over-expression lines exhibited approximately 15-day delay,which indicated that the Ghd2 3'-UTR would repress the Ghd2 expression.The Ghd2 transcripts and protein were quantified in the Ghd2 FL,Ghd2 FL?U lines,as well as their segregated non-transgenic lines,respectively.The results showed that the Ghd2 3'-UTR probably repress the Ghd2 expression at translational level.5.The Ghd2 3'-UTR deletion and substitute assay in the protoplasts showed a stowaway-like MITEs in the Ghd2 3'-UTR contributed the 3'-UTR mediated translational repression activity.Ghd2 without this MITE(Ghd2 FL?M)was overexpressed in the rice and the phenotype of transgenic lines were similar as that of Ghd2 FL?U over-expression lines.The quantification of Ghd2 transcripts and m RNA in the Ghd2 FL?U over-expression lines supported the MITE would repress the translation.6.The Ghd2 knockout lines were generated by CRISPR/Cas9 strategy.The Ghd2 reading-frame-shifted mutants flowered nearly 7-day earlier than wild type,which indicated that the Ghd2 is the suppressor in rice flowering time.7.The multi-gRNA-mediated genome editing was employed to excise the MITE from Ghd2 3'-UTR.Four g RNAs assembled into p RGEB32 were transformed in to rice.MITE excision without Cas9 transgenic lines were used to determine the MITEtranslational repression activity in the native plant.The stable m RNA level and accumulated protein of Ghd2,as well as the delayed flowering time suggested that the MITE repress Ghd2 translation in native.8.The bioinformatic analysis showed that two siRNA clusters were derived from the stowaway-like MITEs.These siRNAs were confirmed in the rice leaves by small RNA northern blotting,and over-expressed in the rice.The phenotype of transgenic lines and wild type showed similarity each other.Furthermore,the abundance of Ghd2 m RNA and proteins in them are at the same level.The evidence indicated that the 24-nt siRNAs were not involved in the translational repression.9.To investigate the mechanism of MITE-mediated translational repression,we employed the RNA ligase-mediated rapid amplification of 5' c DNA ends(5' RLM-RACE)for cleavage site in the Ghd2 3'-UTR.A cleavage site was mapped between two siRNAs.The MITEs with mutations of two bases beside the cleavage site were analyzed for translational repression activity in the rice protoplasts.The results indicated that the cleavage site is probably response for the translational repression.10.The Dicer-like genes RNAi lines or mutants were used to investigate which dicer-like protein was response for these siRNA biogenesis and MITE-mediated translational repression.These two siRNAs were determined in the Os DCL1,Os DCL3 a,and Os DCL3 b RNAi lines,as well as dcl4 mutant.The results showed that the Os DCL3 a processed the stowaway-like MITE into siRNAs.Meanwhile,the quantification of Ghd2 m RNA and proteins in these dicer-like genes RNAi lines and mutant showed that the Os DCL3 a was involved in the translational repression.Furthermore,the Os DCL3 a was knockdowned in the Ghd2 FL over-expression lines;result in the delayed flowering time.These lines of evidence implied that the MITE-mediated translational repression depended on the Os DCL3 a.11.The genes of RNA-dependent DNA methylation pathway had been identified in rice,including Os RDR2,Os AGOa,and Os AGOb.The Ghd2 m RNA and proteins wereanalyzed in the Os RDR2 and Os AGOab RNAi lines,indicating that the MITE-mediated translational repression was not related to these genes.12.Genome-wide analysis of MITE embedded 3' UTRs identified 1,182 3'-UTRs may repressed the host genes translation.Three randomly chosen 3'-UTRs were assayed in protoplasts.All of them were showed the translational repression activity for the reporter gene.In conclusion,we profiled the microRNAs in the responses to stresses and found 35 stress-responsive micro RNAs were induced or decreased in the stress treatments,which deepened our understanding to the mechanism of stress responses in rice.Meanwhile,translational repression of the Ghd2 mediated by a MITE was first investigated and identified in rice,indicating that the key role of MITE in gene regulation,as well as broadening our knowlodge of the DNA transposon regulatory roles in translation. |
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