Functional Analysis For ZmDof3 Gene To Regulates Starch Synthesis In Maize Seed

Maize(Zea mays L.)is an important food,feed and industrial raw material,as well as corn is one of the major crops all over the world.In recently years,corn planting area and output more than rice,became the first major food crops.Corn starch accounts for about 80% of the world's annual starch output.Thus,it is of great significance to the genetic improvement of crop quality and cultivating high yield and perfect quality of corn,through carrying out the research of corn starch biosynthesis pathways and digging starch synthesesis-related genes.Based on the result of screening the endosperm-specific expression transcription factors by genome-wide association analysis and the bioinformatics analysis of the Dof transcription factor,we cloned the maize candidate gene ZmDof3,and detected its function in starch synthesis metabolic pathways through the overexpression technology.The major results are as follows:(1)Bioinformatics analysis and forecast for the candidate gene ZmDof3 indicates that it belongs to the Dof transcription factor family,with the cDNA sequence length of 1057 bp,isoelectric point 5.81 and molecular weight of 36.73 KD.ZmDof3 contains the nuclear export sequence.(2)An analysis of the subcellular localization showed that ZmDof3 has a nuclear localization signal.The results of experimental about transcription activity suggested that ZmDof3 possesses transcriptional activation activity,and the minimal active region is 227-285 amino acids.(3)The RT-PCR was been used to analysis of ZmDof3 tissue-expression pattern,which showed that the ZmDof3 was highly expression in 12 d,14d,16 d,18d and 20 d endosperm after pollination,But other tissues nearly had no expression level.So,we can further determind that ZmDof3 belongs to endosperm specificity transcription factor.(4)Compared with the wild type,we find the transgenic line seed size,thousand seed weight,total starch content,amylase content,amylopection blue value were all increasing,But the souble sugar and reducing sugar were decreasing.We also use the scanning electron microscopy to observe the grain internal structure,we found that starch grains present a polygon,closely packed between starch granules.These result showed ZmDof3 made an influence on the size and starch content of transgenic line seed.(5)The results of yeast single hybrid showed that ZmDof3 interact with known starch synthesis genes of maize,including ZmAGPL1?ZmAGPS1a?ZmGBSSI?ZmSSIIa?ZmISA1and ZmPUL.By analysis of the starch synthesis gene expression level in transgenic line seed from different periods of pollination,showed that the expression levels of seed in the transgenic line have changed comparing with wild type.Related to the amylose synthesis ZmGBSSI gene showed higher expression lever in transgenic maize than in wild type.On the contrary,hydrolysis of starch branching process related ZmISA3 expression level is declining.These results revealed the ZmDof3 was involved in the regulation of starch synthesis pathways in maize.