Map-based Cloning And Function Research Of ZmSWEET4a Related To Maize Seed Development

Maize is an important food,feed and industrial raw material,which plays an important role in the national economy and food security system.With the improvement of people’s living standards and the development of social economy,the demand for maize in our country continues to increase.Maize seed development is closely related to its yield and quality,and has always been a key character in breeding.Maize seed development is a complex character regulated by many genes.Through the study of maize seed development mutants,some maize seed development related genes have been cloned,which promotes our understanding for the regulation mechanism of maize seed genetic development.Screening and identifying new maize seed development mutants,isolating their mutant genes and carrying out systematic functional research will be beneficial to the discovery of key genes in maize seed development and the analysis of genetic regulatory networks.So as to provide genetic resources and theoretical support for maize genetic improvement.In the early study,we screened and obtained a seed development mutant induced by EMS,named dek502f-9.Through the map-based cloning,we isolated the candidate gene ZmSWEET4a and conducted preliminary functional research on it.The specific contents are as follows:1.Phenotypic identification of dek502f-9.Morphological observation showed that the seeds of dek502f-9 were withered and the 100-seed weight was only about 21%of wild-type(WT).The observation of paraffin sections of seeds at different developmental stages showed that the development of dek502f-9 embryos was relatively slower than that of WT,the accumulation of dry matter in endosperm was relatively less,BETL was obviously thinner,and there were some defects in the inward growth of cell wall.Under scanning electron microscope,it was found that the starch grains in dek502f-9 were significantly smaller than those in WT.Therefore,the mutation of Dek502F-9 seriously affected the seed development of maize.2.Map-based cloning of ZmSWEET4aGenetic analysis showed that the seed development defect phenotype of dek502f-9 was a recessive trait controlled by a single nuclear gene.The mutant seeds in the ear separated by W64A/dek502f-9 F2 were selected to extract genomic DNA,and the candidate genes were located in the 1.47 Mb region on chromosome 5 by Indel markers.Sequence comparison of 15 predetermined genes in this region showed that only one gene had base sequence difference between dek502f-9 and WT,and the difference in base sequence resulted in amino acid substitution.This gene encodes ZmSWEET4a sugar transporter protein.At present,its transgenic function is being verified.3.Expression pattern and subcellular localization of ZmSWEET4aThe plant expression vector of GUS driven by ZmSWEET4a promoter was constructed and introduced into maize.And GUS staining showed that ZmSWEET4a was mainly expressed in the pedicels of developing maize kernels.Subcellular localization analysis showed that ZmSWEET4a was located on the cell membrane.4.The mutation of ZmSWEET4a affected the distribution of carbohydrate in kernels,leading to developmental defects in maize seeds.The results of soluble sugar detection showed that the total soluble sugar content of dek502f-9 was higher than that of WT at the same development stage,and the total soluble sugar content of dek502f-9 mutant kernel was more than 2 times of WT at 1624 days after pollination(DAP).Thin layer chromatography showed that the contents of sucrose and monosaccharides(glucose and fructose)of dek502f-9 were higher than those of WT,especially the content of sucrose was significantly higher than that of WT.The starch content of kernels at different developmental stages showed that the starch accumulation of dek502f-9 seeds was significantly lower than that of WT from the 14 DAP.The above results showed that the functional deletion mutation of ZmSWEET4a affected the distribution of carbohydrates in developing kernels.More carbohydrates existed in the form of soluble sugars while less carbohydrates flowed to the starch synthesis pathway resulting in abnormal starch grain development,significantly reduced starch accumulation and kernel development defects in dek502f-9.