Studies On The Mechanism Of C-type Cytoplasmic Male Sterility And The Mirna Associated With Spikelet Development In Maize

The utilization of heterosis is prevalent in maize breeding,and the male sterile line is a useful system for their hybrid seed production.However,the occurrence and the mechanism of plant male sterility still remain mysteries that have not been unveiled thoroughly.Mi RNAs are small single stranded non-coding RNAs that range in length from roughly 21 to 24 nucleotides,which play a central rule in regulating plants growth,development,responsing to adverse environment and so on.Through directing the cleavage or repressing translation by interfering with expression of plant mi RNA,it is pressing to screen and study the role of the candidate mi RNA and its target genes in process of regulation,which involed in spikelet development and then identify its function,and ultimately help to understand the characteristic of spikelet development and the corresponding molecular mechanism.The mechanism of C-type cytoplasmic male sterility and the mi RNA associated with spikelet development in maize was studied,the main conclusion are as follows:1.Cytoplasmic male sterility?CMS?lines Cms-ES 87-1?rf4rf4?,its maintainer line N87-1?rf4rf4?and the corresponding restorer Cms-ES 87-1?rf4rf4?were considered as experimental materials in this study.10 orfs that had cytoplasmic mitochondria DNA differences in the sterile line and maintainer line were cloned,and were constructed into Escherichia coli through prokaryotic expression vector p ET28 a.The growth of the E.coli was influced by four orfs respectively.From Agrobecteria mediate transformation transgenic Arabidopsis plants were obtained with p CAMBIA1300-35s-orf415-GFP but abortive.We built a good transient expression vector by PEG mediated Arabidopsis protoplast instantaneous expression,found ORF415 and GFP fusion protein localization in the cell membrane and cytoplasm.Structure prediction of ORF415 across the membrane,it can code protein with four transmembrane regions respectively.By the method of chemical synthesis,the peptide sequence?RAHGRESEAFKYGVP?were conducted,after ELISA detection for western blot,polyclonal antibody in sterile lines and restorer meiotic prophase of materials Tetrad period and mononuclear period protein samples detected with a size between 40 k Da and specific hybridization between 55 k Da stripe,and in maintainer line anther without this hybrid bands appeared in the process of meiosis.2.In this study,in order to comprehensively understand the regulatory and functional differentiation between the CMS-C line?A4?and its restorer line?R4?of maize?Zea mays L.?,RNA-sequencing using Illumina technology was performed.The libraries were sequenced using a High-seq 2000 sequencing platform at BGI-Shenzhen,China,and two sets of reads were obtained.All high-quality short reads was aligned onto the maize genome?Zm B73_Ref Gen_v2?and the reference genes?Zm B73₅b?.After filtered out low quality data,a total of 27,618,604?A4?and 25,828,640?R4?clean reads were remained in the two libraries.Using gene structural optimization,1096 new transcripts were predicted.2369 genes were represented to be differentially expressed,with 1550 genes up-regulated and 819 genes down-regulated in the two libraries.All differentially expressed genes?DEGs?were searched against the Nr database,Cluster of Orthologous Groups?COG?and Kyoto Encyclopedia of Genes and Genome?KEGG?.826 DEGs were functionally classified into 25 COG categories which were shown to play important roles in transcription,signal transduction,duplication,recombination and gene repairing.Furthermore,all the DEGs can be categorized into 1,143 GO terms?126 cellular component terms,318 molecular function terms,and 699 biological process terms?.For further analysis,GO and KEGG functional enrichment were performed on all DEGs,and a group of differentially expressed candidate genes related to male sterility and fertility restoration were identified.The result of GO analysis indicated that there are 1793,1871 and 1668 genes were related to biological processes,constitute cellular components and molecule function,respectively.Phenylalanine metabolic pathway was significant enrichment by analysis of differentially expressed genes in KEGG pathways.Flavonoids have close relationship with plants development,including the fertility of anther.3.To detect micro RNAs?mi RNAs?involved in determining kernel row number in maize,next generation deep sequencing was performed on an elite inbred line Zong3 of maize in China and a single segment substitution line?SSSL?SSSL-10 derived from the same genetic background.28 mi RNAs belonging to 11 conserved mi RNA families in maize showed expression differences >2-fold in the two lines,among which 14 members from four mi RNA families were up-regulated and 14 members from seven mi RNA families were repressed in SSSL-10.A genome wide degradome was sequenced to validate the mi RNA target genes in solid experiment.In addition,novel mi RNAs associated with ear development were predicted using a series of strict criteria,and 29 mi RNAs representing eight families were predicted as novel mi RNAs.Among the novel mi RNAs,only one showed an expression difference >2-fold.The conserved and novel mi RNAs with >2-fold expression differences were treated as candidate mi RNAs involved in maize kernel row number determination.Based on the interaction of mi RNAs and their target genes,a possible mi RNA-dependent pathway leading to the given DNA fragment inducing a change in kernel row number was proposed.