The Cloning And Characterization Of A Small Kernel Mutant Gene ZmPPR278 In Maize

Maize(Zea mays L.)is an important food,economic and feed crop in the world,and an important raw material for industrial production.It plays an indispensable role in human consumption,livestock feeding and industrial consumption.So how to improve the yield and quality of maize has become one of the core issues concerned by breeders.The growth and development of maize seeds is closely related to this problem.Therefore,the research on maize seed development will enrich this research and provide a way and solution for the improvement of maize yield and quality.The mutant used in this study was from the inbred line B73 which pollen treated by EMS.In M2 generation of identification to the mutation phenotype of kernel,while in the generation of M3,an ear with phenotypic separation,we insolated 10-12 wild and mutant kernel,mixed pool,constructed RNA-Seq library,analyzed the data,then got the candidate gene Zm00001d015156.Next through co-seperation and allele test confirmed this gene is the causal gene for the small kernel phenotype.Finally,in this article,the study of necleotide sequence and protein sequence alignment,the ananlysis of the patten of gene expression,the subcellular localization of this protein and the analysis of C-to-U editing had in hand.The main results are as follows:1.We got two mutant,CY16-225 and CY16-82,by Ethylmethane sulfonate.Both are monogenic and recessive,caused by candidate gene Zm00001d015156 that have nonsynonymous mutant in coding region by easy bulked sagregation RNA-seq mapping.Through the verification of co-isolation and allele test,it was found that the phenotype was regulated by Zm00001d015156 and the two mutants are alleles that cotrolled by ZmPPR278.2.ZmPPR278,which is located on chromosome 5 of maize,encodes a P subclass PPR protein that contains 11 p motifs.The genome length is 36,347 bp,including 16 exons and 15 introns.While coding sequence is 2,676 bp in length only.The sequence similarity of its homologous proteins in sorghum,rice and arabidopsis is 79.75%,61.77% and 31.36%,respectively.3.The expression analysis of the gene showed that the gene was constitutively expressed,with the highest expression in 6 days after pollinationin kernel,followed by 12 DAP kernel,and the lowest expression in stem.The ZmPPR278 was coexpressed with the nuclear marker AHL22-RFP.This result is different from most PPR proteins that are located in mitochondria or/or plasmids.4.Through the comparion of mitochondrial genes’ sequence between the wild type and mutant type,it found that ZmPPR278 involved in mitochondrial genes C-to-U RNA editing.The protein is required for the editing of ~22% of mitochondrial target Cs,which is different from the common function belonging to P subclass in intron splicing in transcripts in mitochondria and/or plastid.