Cytological Observation And Different Express Genes Analysis On Anther Abortion Of The Mutant K305ms In Maize

Plant male sterility mutant was the valuable germplasm resource,it has been widely studied for both its biological significance to explore the reproductive processes in flowering plants and commercial use in hybrid seed production.To better understand the mechanism of male sterility in maize K305ms,cytological observation and transcriptome analysis were carried out in the anther at three development stages(pollen mother cell,dyad and tetrad stage,A,B and C representative,respectively)between K305ms(ms/ms,M for short)and K305F(ms/Ms,F for short),which are fertile siblings.Differentially expressed genes(DEGs)and the metabolic pathways related to K305ms abortion were analyzed by bioinformatics methods.The relative expression levels of candidate genes were detected by qRT-PCR.The main results were as follows:1.Observation found the K305ms exhibited elliptical shape of pollen mother cells that could form distorted dyads and tetrads by carbol fuchsin or 1%aceto carmine staining.However,the young K305ms microspores were completely collapsed after they were released from tetrads,failed to form functional pollens before maturity.The abortion of K305ms persistently occurred at the microspore stage.2.From the paraffin section and scanning abservation,the K305ms anther locules had normal four-layer wall cells but the distinguishable abortion characteristics appeared at the microspore stage.Tapetum was disintegrated with the abnormal microspores,the anther locules were subsequently shrivelled,the locule wall layers without complete nucleus structure,the anthers epidermis surface were smaller than K305F and lacked a normal reticulate pattern of wax structure.Its abortion characteristics are significantly different from the reported ms32 and other maize male sterile mutants.3.The three developmental stages of fertile and sterile anther were sequenced by Illumina HiSeqTM2000 sequencing platform.About 30,000 transcripts(nearly 10%of novel transcripts),50-60 thousands alternative splicings and 100-140 thousands SNPs were identified for each sample.Utilizing NOIseq to integrate the data of the three biological repeats,based on FDR?0.001 and log2| ratio | ? 1,genes were identified as DEGs.The results showed that 451 up-regulated and no down-regulated in FA vs MA,66 up-regulated and 16 down-regulated in FB vs MB,the most obvious number of DEGs in FC vs MC,up 1173 and down 262.Among them,34 DEGs were co-expressed in the three periods,only GRMZM2G133718(serine carboxy peptidase)expression quantity in K305F was lower than K305ms,and the other 33 genes were higher expression in K305F.4.The Gene Ontology analysis showed that DEGs in the three stages between K305F and K305ms,respectively.There were 219,42 and 817 DEGs annotated into the cellular component,the dominant categories including cell,cell part,organelle,cell membrane and macromolecular complex;216,48 and 856 DEGs annotated into the biological process,the dominant categories including metabolic process,cellular process,response to stimulus and regulation of biological process;221,45 and 832 DEGs annotated into the molecular function,the dominant categories including binding,catalytic activity and transporter activity.5.KEGG pathway enrichment analysis showed that a total of 18788 genes were successfully mapped to the reference canonical metabolic pathways in the three periods.Among them,165 DEGs were targeted to 75 metabolic pathways in the A stage,mainly including biosynthesis of secondary metabolites,metabolic pathway,phenylpropanin synthesis,starch and sucrose metabolism,etc;31 DEGs were targeted to 31 metabolic pathways in the B stage,mainly including biosynthesis of secondary metabolites,metabolic pathway,phenylpropanin synthesis and plant hormone signal transduction;685 DEGs were targeted to 97 metabolic pathways in the C stage,mainly including phenylpropanyl synthesis,plant hormone signal transduction,amino acid synthesis or degradation,fatty acid synthesis,secondary metabolite synthesis,unsaturated fatty acid synthesis,starch and sucrose metabolism.Indicating that phenylpropanine synthesis,biosynthesis of secondary metabolites,starch and sucrose metabolism and lipid metabolism are closely related to K305ms abortion.6.Further analysis of DEGs enriched in the lipid metabolism,including biosynthesis of unsaturated fatty acids,fatty acid synthesis,fatty acid metabolism,fatty acid elongation,cutin,suberine and wax biosynthesis.These DEGs were annotation as putative cytochrome P450 superfamily protein,carbonyl reductase,transferase activity,acyl desaturase,aldehyde dehydrogenase and 3-ketoacyl-CoA synthase.The expression patterns of these genes were different in the three periods,the relative expression levels of some candidate DEGs was detected by real-time quantitative PCR(qRT-PCR).The expression pattern was consistent with the result of sequencing.7.The results of cytological observation and transcriptome analysis showed that the expression level of genes related to K305ms anther development was insufficient,leading to the deregulation in biosynthesis of metabolites,biochemical metabolism and energy transmission,result in the abnormal male gametophyte morphogenesis and development process,Meanwhile,the abnormal lipid metabolism resulting in the disorder degradation of tapetum,microspores fail to form pollen wall and then disintegrate maybe the main cause of the K305ms abortion.