Preliminary Study On The Regulatory Mechanism Of ZmMYBR19 Participating In Development Of Endosperm Transfer Cells And Starch Synthesis In Maize

In recent years,maize(Zea may 1.)has become the first major grain crops in the world and our country and holds a significant position in global food security,economic trade and economy of our country.In maize kernels,the content of starch is as high as 70%.Transfer cells is a specialized type which involved in the short-distance of material transport and exist widely in various group of plant kingdom.The development of transfer cell in grain crop is closely related to output.Transfer cells in endosperm provide nutrients for it to influence the accumulation of starch.At present,There are no studies on the transcription factors participating in development of transfer cell and starch synthesis in maize at the same time.Our group analyzed the expression spectra data of maize kernels in different stages of development and screened 15 transcription factors with high expression on the twelfth day of after pollination.ZmMYBR19 is homologous with the identified ZmMRP-1.Relevant sequencing data show that ZmMYBR19 has expression in the endosperm.This thesis mainly adopted RT-PCR,subcellular localization,transient expression in maize endosperm mediated by gene gun or PEG-mediated transformation of protoplast,transformation of yeast,yeast one-hybrid,yeast two-hybrid,prokaryotic expression and a series of techniques,in order to study the characteristics of the transcription factor and analyze its expression pattern,cis element and regulatory function on the expression of high expression gene in endosperm transfer cells and starch synthase gene.We can complete the function identification of this gene preliminarily and reveal the molecular pathway on development of transfer cell and starch synthesis further.Then the concrete results are as follows:1.The phylogenetic tree(NJ tree)was builted with the homology protein of ZmMYBR19 from other species,the results demonstrated that ZmMYBR19 shared closely phylogenetic relationship with ZmMRP-1 which regulates the differentiation and function of maize endosperm transfer cells.Excellent maize inbred lines B73 as experimental materials,we has successfully cloned the transcription factor from maize endosperm,this gene has an open reading frame for encoding a protein of 290 amino acids.Analysis the amino acid sequence and senior structure of ZmMYBR19 protein,those results indicated that ZmMYBR19 wasatypical plant MYB transcription factor.2.Real Time PCR analysis indicated that ZmMYBR19 has hardly no expression in filaments,tassel,seedling root,seedling leaf,mature embryo axis and maize embryo.ZmMYBR19 gene expresses highest on the 14 day after pollination in the maize seed and endosperm and expresses in low at other times.Utilizing gege gun transformed construction plamid of ZmMYBR19 into the onion cells,the result indicated that ZmMYBR 19 was localized in the nucleus.Transactivation activity assays showed the total ZmMYBR19 protein had no transcriptional activation.Yeast one-hybrid experiment showed that ZmMYBR19 can specifically bind to GAGATA.3.Yeast one-hybrid experiment and transient expression experiments were used to study the interaction between ZmMYBR19 and the associated gene promoters.Starch synthesis enzyme gene promoters SSI,ISA1,Bt2 and Sh2 were in combination with ZmMYBR19 directly.Some gene promoters related with the development of endosperm transfer cells could also combine with ZmMYBR19 in yeast.The transient expression analysis in leaf protoplast showed that ZmMYBR19 promoted expression of Tc8403 which has high expression in the maize endosperm transfer cells.The transient expression in maize endosperm showed that ZmMYBR19 could upregulate the expression of BT1,Bt2,Sh2,SSI,SBEIIb and ISA1.The up-regulation to Bt2,Sh2,SSI and ISA1 may be through the direct combination with ZmMYBR19 and promoters on the basis of Yeast one-hybrid experiment results.4.Yeast two-hybrid experiment on maize endosperm library showed that protein HP2 and ZmMADS11 can interact with ZmMYBR19 in yeast.Cloning the coding sequence of ZmMYBR19 protein in maize to insert into the pGEX-6P-1 vector,in the strain of escherichia coli RosettaTM(DE3)to conduct prokaryotic expression,Then ZmMYBR19 fusion protein was purified by GenStar column..