The Effect Of The Transcription Factor ZmMADS11 On Maize Starch Synthesis

Maize is one of the most important food crops in the world.A large number of experimental results showed that the starch content of grain was closely related to the enzyme activity of starch synthesis.Starch synthesis related enzymes include adenosine diphosphorylase,starch synthase,starch branching enzyme,starch debranching enzyme and starch phosphorylase.At present,the research on these key enzyme gene expression is more in-depth,but the research on the expression regulation mechanism of these key genes is relatively limited.Transcriptional regulation regulates the biosynthesis of starch by regulating the expression level of enzymes involved in starch biosynthesis.Therefore,it is of great significance to study the molecular regulation mechanism of maize starch biosynthesis.In the early stage of this study,some transcription factors were predicted to participate in the regulation of starch synthesis key enzyme genes.In this experiment,the predicted zmmads11 mutants were obtained by multiple generations of backhanded,self-handed and homozygous mutants,through PCR detection of mutated DNA mutants,semi-quantitative detection of RNA,combined with zmmads11 homozygous mutant test,identified zmmads11 homozygous mutant;Through real-time quantitative PCR analysis of the differences in the expression of amylase gene in wild type and zmmads11 homozygous mutant,Yeast single hybridization was used to verify the binding effect of starch-related enzyme gene promoter and ZmMADS11,and analyzed whether ZmMADS11 was directly or indirectly regulated by the enzyme genes related to starch synthesis;The differences in gene expression in 15 days after pollination of wild type and homozygous mutant were analyzed with high-throughput sequencing,the ZmMADS11 protein was expressed in escherichia coli,and the experiment was conducted in vitro.The specific experimental results were as follows:1?Mu transposon insertion zmmads11 mutant and wild type W22 after six generation of backcross selfing zmmads11 homozygous mutant,using SLS method proposed tender maize leaf DNA agarose gel electrophoresis to detect,identify homozygous mutant.2?Due to the pollen abortion of homozygous mutant,we detected homozygous mutant kernels and wild-type kernels through the detection of the mutation site of embryo DNA in the mature stage after the hybridization.After using Megazyme total starch assay kits to embryo grain total starch content,found in the wild type total starch content was70.83%,total starch content in zmmads11 homozygous mutant was 78.12,starch contentincreased by 7.29%.The results indicated that the mutation of ZmMADS11 gene had an effect on the synthesis of total starch;Using Megazyme determination of resistant starch resistant starch kit test found wild type W22 and zmmads11 mutant homozygous resistant starch content in grain difference was not significant,suggest ZmMADS11 gene mutation of resistant starch synthesis were not significantly affected.3?The total RNA was extracted from homozygous mutant and wild-type 15 DAP endosperm,and ZmMADS11 was not expressed in the mutant endosperm by semi-quantitative PCR.Real-time fluorescence quantitative PCR analysis of starch related enzyme genes: AGPISI(SH2),AGPSIA(BT2),SBEI,SBE2 B,ISAI,PUL,PHOL,BTI in the wild-type and zmmads11 mutant 15 DAP endosperm expression differences,The results showed that the expression of SSI and SSIII in zmmads11 mutant was significantly higher than that of wild type;The combination of transcription factor ZmMADS11 and starch synthesis related enzyme promoter was verified by yeast single hybridization experiment,The results showed that ZmMADS11 had no direct effect on SSI and SSIII promoter.4? Will be homozygous mutant and wild type 15 DAP endosperm transcriptome analysis for log2 threshold,found in 117 of zmmads11 homozygous mutant genes,62 genes,with starch and sugar with two genes increase in metabolic pathways,it is a beta-glucosidase and trehalose phosphatase,one gene was down-regulated to beta-uran glucosidase.There are ten genes up-regulated in the processing pathway of endoplasmic reticulum protein,and one gene is down-regulated,all of which are heat-induced proteins.It is speculated that the increase of starch content in zmmads11 homozygous mutant is related to the increase of related enzyme gene expression in the pathway of starch and sugar metabolism,and the increase of related enzyme protein stability in the process of endoplasmic reticulum protein.5?In the midst of e.coli expressed ZmMADS11 combination of protein and DNA M domain,the maize genome DNA after ultrasonic broken,trying to make a ChIP-Seq in vitro experiments,analyze ZmMADS11 regulation network way.