Functional Studies Of Maize DYW88 And Arabidopsis Emb105 Genes In Seed Development

Maize is an important cereal crop in China.Seed development is a key factor for maize yield.Many genes were involve in maize seed development.Therefore,cloning and functional analysis of key genes of maize seed development has a great significance on the maize yield.1.Studies of the maize gene DYW88PPR proteins play key roles in maize seed development.As RNA binding proteins,PPR proteins are often involved in the post-transcriptional processing of organelle RNAs,including intron splicing,RNA editing,RNA maturation and stabilization,and the initiation of translation.In this work,the DYW88 gene was identified by bioinformatic analysis,and the dyw88 mutant was isolated from the UniformMu population.The following studies were carried out to uncover the functional mechanism of DYW88 in maize seed development:1)Phenotype investigation of dyw88 and linkage analysis between dyw88 and the emb phenotype;2)Subcellular localization analysis of DYW88;3)editing and Intron splicing analysis of plastid genes of WT and dyw88.Followed are the major results:1)Mutation of DYW88 severely arrests maize embryogenesisEmbryogenesis was seriously inhibited in dyw88 mutant,but the endosperm development was barely affected.Paraffin section confirmed that loss-of-function of DYW88 aborts the embryogenesis,and development of embryo is arrested at proembryo stage.The ration of wild type to emb kernels of the selfed ear dyw88 heterozygous mutant was about 3:1.The linkage between the emb phenotype and dyw88 was verified in the F2 population.2)DYW88 encodes a PPR-DYW protein localizing in plastidDYW88 encodes a-PPR-DYW protein,which consists of 16 PPR motifs and one E1,E2 and DYW domain.Subcellular localization analysis showed that DYW88 is localized in chloroplasts.3)DYW88 mainly functions in the intron splicing of plastid transcriptsThe plastid transcripts in the embryo and endosperm of wild-type and the dyw88 mutant were analyzed by RT-PCR and qRT-PCR.The results showed that,splicing of rp12,atpF,ycf3,ndhA,ndhB,petB and petD was affected,and the splicing efficiency of rps12 intron 1,rps12 intron 2,ycf3 intron 2,rpl2 and atpF was decreased in the dyw88 embryo in comparison with wild type.These results suggested that DYW88 is a key factor for the intron splicing of maize plastid genes.2.Studies of the Arabidopsis gene Emb105AAA-ATPase proteins form a large family widely spreading across prokaryotes and eukaryotes.They participate in numerous cell biological processes,including protein degradation,26S proteasome activity regulation,vesicle trafficking,peroxisome biogenesis,and hypersensitive responses of plants.In this work,it has been found that Emb105 encods an AAA-ATPase.A T-DNA insertion mutant of Emb105 was isolated from ABRC mutant library.The following researches were carried out:1)Phenotype analysis of the emb105 mutant and linkage analysis between Emb105 and the emb phenotype;2)Subcellular localization analysis of EMB105;3)Functional mechanism analysis of Emb105.Followed are the major results:1)Emb 105 encodes a mitochondrial-targeted AAA-ATPaseGenomic sequence of Emb105 consisted of two introns and three exons encoding an AAA-ATPase with a DnaX domain.Subcellular localization analysis showed that EMB 105 was targeted to mitochondria.2)Loss function of EMB 105 leads to seed abortionDevelopment of the embryo of emb 105 mutant was aborted,showing a emb phenotype.The ration of normal seeds to emb seeds of selfed emb105 heterozygous mutant was about 3:1,and the linkage between emb phenotype and the emb105 mutation was confirmed in the F2 population.3)EMB105-GFP and EMB 105-HA partially complemented the emb phenotype of emb 105The transgenic complementation experiment showed that the growth and development of Emb105-GFP::emb105 or Emb105-HA::emb105 plants were significantly slower than wild type,showing a later flowering and slower growth of root.This result suggests that Emb105-GFP or Emb 105-HA partially complemented the phenotype of emb105.