The Mechanism Of Transcription Factor ZmMYB14 And ZmNAC126 Participated In The Regulation Of Maize Starch Synthesis

Maize(Zea mays L.)is one of the major crops in the world,and the storage starch in the grain is the mainly utilization products.Based on the yield and quality of maize starch,it is not only widely used as feed and industrial raw materials,but also has important application prospects in the fields of energy and chemical industry.Therefore,maize has certain effect to solve the world food crisis and the energy crisis.Starch biosynthesis in the maize endosperm mainly used the sucrose,which is the direct products of photosynthesis and transported into the amyloplasts.Starch biosynthesis process mainly includes ADPG biosynthesis and transport,extension of glycan chains,branching and debranching of glycan chains,and starch granule formation.The major functional proteins include Adenosine phosphate glucose pyrophosphorylase(AGPase),Starch synthase(SS),Starch branching enzyme(SBE),Starch debranching enzyme(DBE),Starch phosphorylase(SP)and ADPG transporter(BT1).Transcription regulation plays an important role in the starch biosynthesis in different species.It mainly depends on cis-acting elements and trans-acting factors to regulate the gene expression,and shows effect on the starch synthesis.However,there are still less researches on transcriptional regulation of starch biosynthesis in maize seeds.Therefore,the trans-acting factors and cis-elements identified can not only improve the regulatory pathway of starch biosynthesis in maize grain,but also provide new gene resources for molecular breeding to improve yield and quality of maize.In this study,maize inbred lines 18-599 were used to determine the relationship between starch accumulation and the expression pattern of starch synthesis related genes during maize grain development.The cis-acting elements of gene promoters related to starch synthesis in different species was predicted through Plant CARE conbined with the genome sequence.We cloned the promoter of Zm BT1 and identified the activity site through the maize endosperm transient conversion system.We obtained the candidate gene Zm MYB14 and Zm NAC126 based on cis-element,the expression pattern,and co-expression analysis.The RT-PCR was used for expression analysis.Different receptor systems were used to study the functional characteristics and transcriptional regulation mechanism of Zm MYB14 and Zm NAC126 involved in maize starch synthesis.The main conclusions are showed as follows:1.The results of starch determination and gene pattern analysis of maize inbred line 18-599 showed that the starch accumulation was detected after 10 DAP,and the trends in starch accumulation consistented with the expression patterns of starch synthesis related genes.The cis-element analysis of starch synthesis related gene in different species indicated that there were cis-acting elements responded to plant hormones,light signals,stress signals,and regulated gene expression in the gene promoter.2.The 1644 bp fragment of Zm BT1 promoter was cloned from maize inbred line 18-599 from the translation initiation site,and the activity of deletion and mutation fragment of promoter were detected through ?-glucuronidase and Luciferase reporter gene system.The fragment from-280 bp to-151 bp and-151 bp to +14bp were identified for the first time,and could significantly affect promoter activity.The MBSI locus was first time to be identified in-280 bp to-151 bp of Zm BT1 gene promoter as an important cis-element.3.Zm MYB14 was obtained through the gene expression pattern,MBSI cis-element and the effect on the promoter activity.According to a series of experiments,we found that Zm MYB14,a 2R-MYB transcription factor,located in the nucleus,and possessed transcriptional activation activity domain at the C terminal.Zm MYB14 showed a high expression in maize endosperm and induced by exogenous ABA and sucrose.The endosperm transient transformation and yeast one-hybrid analysis show that Zm MYB14 can significantly promote the activity of p Zm BT1 promoter through MBSI site by binding with-280 bp to-151 bp,but the binding effect is not directly affected by the mutation and deletion of MBSI site.These results indicate that Zm MYB14 involes in the transcriptional regulation of BT1 gene in maize.4.The transcriptional regulation mechanism of Zm MYB14 involved in the maize starch synthesis was studied by endosperm transient transformation,yeast one-hybrid,and exogenous expression Zm MYB14 in Zhonghua 11.The results indicated that Zm MYB14 could promote the activity of p Zm GBSSI,p Zm SSI,and p Zm SBE1 promoter through the directly binding,and significantly promote the activity of p Zm Sh2 and p Zm Bt2 promoters depend on the indirect effect.Zm MYB14 could bind to p Zm SSIIa,p Zm ISA1,and p Zm ISA2 directly,but did not show the effect on the promoter activity.The transcription levels of gene related to rice starch synthesis were detected in the T1 generation of Exogenous expression Zm MYB14 lines,Negative lines and Wild-type,and the results indicated that exogenous expression of Zm MYB14 gene could increase the transcription level of rice ADPG transporter encoding gene Os BT1,starch synthesis related genes Os AGPL2,Os SBEI,Os GBSSI,Os ISA1,Os SSIIa,Os SBE2 b and Os Pul.These results indicate that Zm MYB14 can participate in the transcriptional regulation of maize starch synthesis related gene,and simultaneously regulate the transcription of multiple genes.5.The Zm NAC126 was obtained by co-expression and analysis of the effect on p Zm BT1 promoter activity.Molecular biological experiments were conducted to study the transcriptional regulation mechanism in maize starch biosynthesis.The results showed that Zm NAC126 contained NAM binding domain,localized in the nucleus,C terminal possessed transcriptional activation activity,directly binded with the CACG repeats.Zm NAC126 possessed more transcripts in maize seeds and endosperm,and shared a similar expression pattern with starch synthesis related genes in development of maize grain.Yeast one-hybrid and endosperm transient transformation results indicated that Zm NAC126 could directly bind with promoter p Zm GBSSI and p Zm BT1,and promoted the activity of both.Meanwhile,Zm NAC126 could directly bind with promoter p Zm ISA1 and p Zm ISA2,and inhibited the activity.The Zm NAC126 could promote the activity of promoter p Zm Sh2,p Zm Bt2 and p SSIIIa without directly binding,and had no obvious effect on the activity of the p Zm SSI and p Zm SBEI promoter.These results demonstrate that Zm NAC126 involves in transcriptional regulation of maize starch synthesis related gene,and can participate in the transcriptional regulation of multiple genes in different ways.