Molecular Pathways Of The Key Enzyme Genes' Expression Regulated By MiR159a In Maize Starch Synthesis

Maize(Zea mays L.)is one of the most important cereals for human all over the world.And the storage starch of the kernel is its cardinal product.The biosynthesis of starch is catalyzed by several enzymes.The functional proteins involved in the starch biosynthesis include AGPase,SS,SBE,DBE and SP.According to the recent studies,transcription regulation plays a significant role in the starch biosynthesis.Moreover,miRNA can target transcription factors to regulate its expression.According to the high-throughput transcriptome sequencing of miRNA,we find miR159 a and we predict the target transcription factors of miR159 a.According to the eFP browser and RNA-seq from maizeGDB,target genes ZmMYB138 and ZmMYB115 specificly and highly express in maize endorsperm and may regulate the biosynthesis of starch.Based on the previous analysis,this study aims to test and verify the regulation of ZmMYB138 and ZmMYB115 by miR159 a and the regulation of the key enzyme genes of starch biosynthesis by ZmMYB138 and ZmMYB115.The results are as follows:1.To verify the the regulation of the two transcription factors ZmMYB138 and ZmMYB115 by miR159 a,we transfer the constructed vectors into the fresh endosperms of maize to get the activity of hRLuc/Gus by the transient transcription.The results of the transient transcription say that miR159 a can very significantly decrease the mRNA of ZmMYB115 and significantly decrease the mRNA of ZmMYB138.2.In order to test and verify the influence of the promoters' activity of the key starch syntesis enzyme genes by ZmMYB138,the vectors are transferred into the fresh endosperm of maize to measure the activity of Luc/Gus by the transient transcription assay.The results indicate that ZmMYB138 very significantly promotes the promoter's activity of starch-branching enzyme gene ZmSBE2 b,and has significant promoting effect on the promoter's activity of AGPase gene ZmBt2.But ZmMYB138 has no significant effect on the promoters of soluble starch synthase gene ZmSSIIa and AGPase gene ZmSh2.3.In order to makesure the direct binding of the transcription factor ZmMYB138 with the promoters of the key starch synthesis enzyme genes,we transfer the vectors into the yeast Y187 by LiAc-PEG.The results suggest that ZmMYB138 protein can specifically and directly bind to the promoters of AGPase gene ZmBt2 and starch-branching enzyme gene ZmSBE2 b.4.In order to test and verify the influence of the promoters' activity of the key starch syntesis enzyme genes by ZmMYB115,the vectors are transferred into the fresh endosperm of maize by the transient transcription assay to measure the activity of Luc/Gus.The results indicate that ZmMYB115 very significantly promotes the activity of granule-bound starch synthase gene ZmGBSS' promoter,and has no significant effect on the promoters' activity of soluble starch synthase genes ZmSSIIIa and ZmSSI,and AGPase gene ZmSh2.5.In order to verify the direct binding of the transcription factor ZmMYB115 with the promoters of the key starch synthesis enzyme genes ZmGBSS,ZmSSIIIa and ZmSSI,we transfer the vectors into the yeast Y187 by LiAc-PEG.The results suggest that ZmMYB115 protein can specifically and directly bind to the promoters of granule-bound starch synthase gene ZmGBSS,soluble starch synthase genes,ZmSSIIIa and ZmSSI.Considering the results of this study,we can draw a conclusion that miR159 a could indirectly regulate the key starch synthesis enzyme genes' expression,incluing ZmSBE2 b,ZmBt2 and ZmGBSS,by decreasing the mRNA of the two transcription factors ZmMYB138 and ZmMYB115.