Maize ZmRCL1 Gene Regulates Kernel Development By Participating In 18S Pre-rRNA Processing

The ribosome is an important organelle whose function is to synthesize proteins from amino acid monomers in accordance with the instructions of messengerRNA(m RNA).Ribosomes are complex structures composed of ribosomal RNA(rRNA)and various proteins.Plant ribosomes contain four types of rRNAs,5S,18 S,5.8S,and 25 S,the latter three coexist in a polycistronic transcript,and the processing and maturation of this transcript(pre-rRNA)is mainly in the nucleolus.For this to occur,multiple processing factors and nucleases are required to act cooperatively at multiple specific sites.In maize(Zea mays L.),these nucleases and related factors involved in the processing of pre-rRNA have rarely been reported.In this thesis,we obtained a Mu transposon insertion mutant with severe defects in maize kernels,named rcl1.The key gene causing the mutation,ZmRCL1,was identified by map-based cloning.Then,using cell biology and molecular biology methods,the mechanism of ZmRCL1 regulating maize kernel development was deeply analyzed,and the main results were as follows:1.A maize single-gene mutant,rcl1,was obtained with reduced grain shrinkage.The mature kernels of the rcl1 became smaller and shrunken,the hundred-grain weight was significantly reduced,and the mature seeds of homozygous mutants could not germinate.Paraffin section observation showed that the embryos of rcl1 mutants were arrested in differentiation.In situ hybridization analysis revealed abnormal differentiation of cells of the basal endosperm transfer layer and aleurone layer in rcl1 mutants.Biochemical analysis showed that the kernels of rcl1 had significantly reduced protein and starch accumulation.Subsequent genetic analysis indicated that the phenotype was a recessive trait controlled by a single nuclear gene.2.The gene,ZmRCL1 was identified by the method of map-based cloning combined with the reported RNA-seq data.Sequencing revealed a Mu1.7 transposon insertion in the first intron of the gene and Mu1.7 transposon insertion-tight linked to the phenotype of rcl1.The ZmRCL1 overexpression and knockout lines were created for functional complementation and allelic test analysis,and the results showed that the mutation of ZmRCL1 was the cause of the grain defect phenotype.3.The results of tissue expression analysis showed that ZmRCL1 was expressed in all maize tissues.In situ hybridization analysis revealed that ZmRCL1 was predominantly expressed in the embryo and endosperm during early kernel development.Through the construction of phylogenetic tree and analysis of amino acid sequence characteristics,it was found that ZmRCL1 encodes an RNA 3’-terminal phosphate cyclase-like protein that is highly conserved in plants.It is also found that ZmRCL1 ishomologous to yeast Rcl1 p protein.In addition,subcellular localization shows that ZmRCL1 is mainly localized to the nucleolus.These results suggest that ZmRCL1 and Rcl1 p are functional conservatism in pre-rRNA processing.4.Total RNA analysis of kernels,formaldehyde denaturing gel electrophoresis and RNA gel blot analysis,suggest that the level of 18 S rRNA in the rcl1 was significantly lower than that of the WT.RNA gel blot and Circular RT-PCR analysis found that the rRNA intermediates P-A3 and P’-A3 were accumulated in rcl1 mutant kernels,whereas 18S-A3 and 18S-A2 fragments were significantly reduced in rcl1 mutants.Interestingly,35 S and 33 S pre-rRNA were accumulated in rcl1 mutant grains,while 32 S pre-rRNA was reduced in rcl1 mutant kernels.These results suggest that ZmRCL1 promotes the maturation of 18 S rRNA by participating in the processing of P’,A1 and A2 sites on 35 S pre-rRNA.5.Polysome profile analysis showed that the levels of 40 S small subunit,80 S ribosome and polysome in kernels of rcl1 were significantly lower than those of WT.RT-q PCR analysis showed that the expression levels of multiple zein genes were reduced in rcl1 mutants.Subsequent analysis found that the transcription levels of the regulator of zein genes O2 and the starch synthesis-related genes PPDK,GBSS1,SS1,SS2 and Sh1 in the rcl1 mutant kernels were increased,while the protein levels were decreased.This inconsistency between protein and transcript levels suggests that rcl1 mutants have aberrant ribosomes resulting in impaired translation.In summary,we cloned a gene ZmRCL1 that affects the development of maize kernels and storage material synthesis.ZmRCL1 affects the maturation of 18 S rRNA and the formation of ribosome by participating in the processing of P’,A1 and A2 sites on 35 S prerRNA,thereby regulating the development of maize kernels and the accumulation of nutrients at the translational level.This study enriched the molecular regulatory network of maize kernels development and quality trait formation,and also promotes the understanding of how the formation of ribosome affects protein translation efficiency and participates in plant development.