The Cloning Of A Nuclear Sterile Gene Ms305 And Lipidomics Analysis In Maize

Plant male sterile material is a precious germplasm resource.It is of great significance to explore the male sterile gene and study its metabolic regulation mechanism for corn breeding and production applicatioN n.The maize sibling population K305 ms is a single recessive nuclear male sterility mutant obtained by radiation mutagenesis of inbred line K305.It consists of sterile plant K305 S and fertile plant K305 F,and has good application potential.Preliminary studies have been carried out on the cytological characteristics of the material and the location of the sterile gene.In this study,the sterile gene ms305 was cloned by PCR amplification technology,and the regulation model was defined by gene expression analysis.The ultrastructure of anthers was observed by transmission electron microscopy,and the abortion characteristics were further clarified.The lipidomics analysis of sterile and fertile plants were performed to probe the relationship between key fatty acid metabolism and fertility,futher to lay the foundation for the subsequent abortion mechanism research and breeding utilization.The main findings are as follows:1.The specific primers were designed with reference to the ms33 gene sequence,and the ms305 gene was amplified by PCR,and the sequences were spliced by DNAMAN software.The results revealed that the ms305 gene consisted of two exons and one intron,with a total length of 2161 bp and encoding 523 amino acids.Compared with K305 and K305 F,the ms305 gene in K305 S lacks 6 bases at the first exon of 177 bp,Which encodes valine(V)and aspartic acid(D),and located at amino acid positions 59 and 60.Bioinformatics predicts showed that the secondary structure of the ms305 encoded protein has changed,two amino acid deletions may result in binding or transport disruption to the substrate,which may affect protein transport function.RT-PCR analysis showed that the expression level of ms305 increased gradually in K305 and K305 F with the progress of anther development,while that decreased sharply in K305 S during the tetrad period and the early vacuolate microspore stages,which indicates that the tetrad period may be a crucial period of abortion.2.Observation by ultrathin sectioning technique and transmission electron microscopy showed that the anthers and microspores of K305 F developed normally and could form mature pollen.Compared with K305 F,K305S had no significant difference in anther tissue structure at pollen mother cell stage;however,at the tetrad stage,the anther wall was not normally degraded and thinned,and it existed until the vacuolated pollen stage;at early vacuolate microspore stage,anther tapetum was abnormally degraded and microspore was completely degraded,and it cannot continue to develop into mature pollen.Hence,K305 S has been aborted since the tetrad period,and the final abortion period is at early vacuolate microspore stage.3.Lipidomics analysis was performed by liquid chromatography-mass spectrometry(LC-MS).A total of 1010 lipid molecules were identified in the K305 ms anther during the tetrad period,which were classified into 25 lipid subclasses.By using VIP>1 and Pvalue<0.05 as the standard,124 kinds of significantly different lipid molecules were screened between K305 S and K305 F,mainly including glycerolipids(TG,DG),sphingolipids(Cer,CerGI,SM)and glycerophospholipids(PA,PG,PC,etc.).Compared with K305 F,most of differentially expressed lipids were down-regulated,and only 27 Cer and 24 TG and a few other lipids were up-regulated in K305 S.The hierarchical clustering results were consistent with those of different lipid molecules.Correlation analysis revealed that the up-regulated expression of lipids DG(33:5),DG(34:3P),PG(47:3)and PC(32:0)in K305 S was negatively correlated with other lipids,and most of the correlation coefficients tend to-1;and there is a positive correlation among the above four lipids,and among other residual lipids,and most of the correlation coefficients tend to 1,Which further clarified the mutual exclusion and synergy among different lipids.4.The gas chromatography-mass spectrometry(GC-MS)method was used to determine the fatty acid content of anthers.From the pollen mother cell stage to the early vacuolate microspore stage,the total contents of fatty acids in K305 F anthers increased gradually,while that of those gradually decreased in K305 S.There were no significant difference in the contents between two materials before the tetrad stages,but the difference reached a very significant level at the early vacuolate microspore stage.During this stage,16 fatty acids were detected in the anthers.Compared with K305 F,there was no significant difference in the content of all five medium-chain fatty acids in K305 S,and the contents of 8 long-chain fatty acids were significantly different.Among them,long-chain fatty acid palmitoleic acid(C16:1),linoleic acid(C18:2)and ?-linolenic acid(C18:3N6)were down-regulated by 43.97%,94.68% and 55.29% in K305 S,respectively.which reached an extremely significant level.It is speculated that ms305 gene may play a regulatory role in the polyester biosynthesis and metabolism pathway,affecting the prolonged metabolic process of medium-chain fatty acids,resulting in insufficient fatty acid content,which cannot meet the developmental needs of pollen outer wall,anther cuticle and tapetum.It may be the main reasons for the abortion of K305S.