Impact Of Transcription Factor ZmMADS11 On The Promoter Activity Of The Key Enzyme Genes Of Starch Synthesis In Maize

Starch is the most essential ingredient in many higher plant grains,and is closely associated with the yield and quality of crop.Starch is mainly catalyzed by ADP-glucose pyrophosphorylase(AGPase),starch synthase(SS),starch branching enzyme(SBE),starch-debranching enzyme(DBE)and starch phosphorylase(SP).Abundant researches indicated that grain weight is closely related to the key enzymes of starch synthesis during the grain filling process.At present,it has been very clear about the enzymatic properties of key enzymes of starch synthesis.MADS-box transcription factor is an important transcription factor in eukaryotes,and plays key role in plant growth and development,signal transduction and floral organ development.Thus,the study on the the transcription factors associated with starch biosynthesis has great significance for analysing the molecular regulation mechanisms of the key enzyme genes of starch synthesis,improving starch content of maize kernel and increasing the the maize yield.In our previous study,we have measured the starch accumulation dynamic of endosperm from the early filling phase to the middle filling phase using maize backbone inbred line Mo 17.According to the key time points of starch accumulation,performed high-throughput transcriptome sequencing of the key stage endosperms.Compared to the RNA expression changes and the starch accumulation dynamic,refer to the RNA-seq data in Arabidopsis,rice and maize,also combined with the cis-acting element database and transcription factor database,we screened some transcription factors possibly associated with expression regulation of the key enzyme genes of starch synthesis and predicted the key enzyme genes of starch synthesis,which were regulated by these transcription factors.In this study,we found ZmMADS11 also involved in the expression regulation of the key enzyme genes of starch synthesis by activating the starch synthesis related enzymes SSI and SBE2a.The results of our studies are as follows:(1)ZmMADSl 1 was cloned from maize backbone inbred line Mo 17.ZmMADS11,contained an open reading frame(ORF)of 684 bp,encoding a 227 amino-acid polypeptide.The encoded protein was predicted to be 25.841 kDa with an isoelectric point(pI)of 9.213.ZmMADS11 was a novel member of MADS-Box transcription factors family,contained MADS and K-box domain.Based the alignment of the amino acids of ZmMADS11 and MADS-Box subfamily MIKC in rice and Arabidopsis,the evolutionary tree was constructed by method of Neighbor-Joining(NJ).The results showed that the ZmMADS11 was highly homologous with OsMADS16 in rice.The OsMADS16 was the homologous gene of AP3 in Arabidopsis and ZmMADS11.(2)Real-time PCR analysis showed that the expression of ZmMADS11 was higher in endosperm and grain at 15 days after pollination(DAP),the expression was moderate in male tassel,filament and anther,the expression was lower in stem at seedling stage and female tassel,while the expression was almost undetectable in root and leaf at seedling stage,and embryo at 15 DAP.(3)Transcription activation in yeast revealed that the yeast cells with pGBKT7-ZmMADS11 was insensitive to the SD/-Trp+10mM X-a-gal culture medium and turned the substrate blue,which demonstrated that ZmMADS11 had transcription activation activity.Subcellular prediction localization by bioinformation software PSORT Prediction illuminated that ZmMADS11 was mainly located in the nucleus.The results of Particle Bombardment Transformation showed that ZmMADSll was primarily located in the nucleus and the cell membrane.(4)The result of transient expression in fresh corn endosperm indicated that ZmMADS11 had very significant promoting effect on the expression of starch synthesis enzyme gene SSI,and had significant promoting effect on the expression of starch-debranching enzyme gen SBE2a.Yeast one-hybrid assay suggested that ZmMADS11 protein specifically bounded to the promoter of SSI and SBE2a respectively.