| Maize(Zea mays L.)is one of the most important grain crops in China,and plays an important role in agricultural production and the growth of the national economy and kernel is a primary source for humans and animals.Therefore,cloning of genes associated kernel development and dissecting the molecular mechanisms may contribute to understanding the regulatory network that affect yield and quality,and it plays an important role in cultivating high yield and nutritional quality varieties in a faster speed.In the process of breeding,we characterized a naturally occurring maize mdh4-1 mutant,which produces small with a deformed endosperm crown as compared with wide-type(WT)and is controlled by a single recessive gene.The main findings of this study are as follows:1.The mutant mdh4-1 kernel is small,coupled with opaque phenotype,reduced starch content and increased in lysine: Compared with WT,the 100-kernel weight of homozygous mutant kernels was only 55.1% of the WT,and the length,width,and thickness were 10.8%,16.5%,and 29.8% lower than those of the WT,respectively,and the lysine is increased by approximately 1.4-fold,mdh4-1 exhibited a 50% lower germination,the plant heights of seedings and adult plants both were reduced.2.The cytological analysis showed that: the mdh4-1 mutant exhibits delayed endosperm and embryo development,reduced ingrowth in the cells of the basal endosperm transfer layer(BETL)in mdh4-1 kernels,the starchy granules became exhibited irregularly shaped,smaller and less.3.Using map-based cloning approach,the interval was further narrowed down to a ~224-kb region,which contains two protein-encoding genes(Zm00001d032695 and Zm00001d032699),between markers C362 and Indel98 on bin1.08 of chromosome 1.By the analysis of genomic sequences,we found that there was a 3-bp deletion in the seventh exon of Zm00001d032695 and the deletion showed exclusively in the mutant.An association panel consisting of 540 inbred lines were used to identify the functional polymorphisms associated with kernel characteristics.Three SNPs in the 5’ UTR were found to associate with lysine content and/or kernel thickness.Functional complementary experiment showed that Zm Mdh4 can rescue the mutant phenotype and confirmed that Zm Mdh4 was the causal gene of the mdh4-1 mutant.Meanwhile,using the CRISPR/Cas9 knockout mutants to verify its function.4.The gene encodes a cytosolic malate dehydrogenase 4(Zm MDH4)and its protein is localized to the cytoplasm and it’s a constitutively gene and its transcript level was higher in the endosperms of mutant than the WT.In vitro enzyme activity analysis,Zm MDH4 predominantly catalyzes the conversion from OAA to malate and the mutant catalytic activity almost completely lost.5.The results of the targeted metabolomics analysis are shown that the metabolites are changed involved in glycolysis and cellular energetics,for example,lactate and the aspartate pathway related metabolites increased,alpha-ketoglutaric acid reduced,all of these result in significant impairment of mitochondrial complex I and II.Furthermore,transcriptome analysis revealed that the expression levels of genes related to starch and protein biosynthesis were significantly down-regulated in the mdh4-1 mutant,suggesting that Zm Mdh4 plays an important role in storage reserve accumulation.6.We screened an endosperm c DNA library in yeast two-hybrid screening and acquired nine possible interacting proteins.Among these proteins,Zm00001d042453 and Zm00001d051001 encode fructose-diphosphate aldolase and glyceraldehyde triphosphate dehydrogenase,respectively.Both of them are belong to cytoplasmic enzymes and participate in the process of glycolysis.7.We used EMS(ethylmethane sulfonate)technology to screen the suppressor of mdh4-1 and one ear that had normal kernels was achieved.With the help of whole-genome resequencing,we will clone the suppressor and carry out some functional researches. |
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