Biochemistry Analysis Of The Maize Starch Synthase I And Starch Phosphorylase

Endosperm is one of the most critical storage tissue of maize（Zea may.L）.Maize endosperm is also an essential starch source and used for food,industry and medicine.Amylopectin is approximate 70%of total maize kernel starch.Therefore improving the yield and quality of maize starch by genetic and biochemistry method has tremendous economic and civil meaning.The synthesis of starch is achieved by multi-enzymes,Starch synthase（SS）catalyzes the chain length extension of glucans.SSI is one of the crucial SS isoform in amylopectin synthesis and SSI could directly affect the shape of granule starch.Plastid Starch Phosphorylase or Starch Phosphorylase Low-affinity type（PHOL）catalyzes the only reversible reaction in starch synthesis pathway.PHOL is known for its multi-stage role through starch synthesis and modification,including the initial synthesis of amylopectin.The biochemistry characteristic of SS isoform in barley,rice and maize have been re-understood during the development and improvement of technical stratagies.The newest reports about PHOL in rice and barley support that PHOL has a strong impact on amylopectin initial synthesis.Therefore,further studying the biochemistry characteristics of SSI and PHOL could contribute a well understanding of the bio-synthesis of amylopectin.And it’s great meaningful in explain starch synthesis pathway both in theory and practical.This research is focused on two biochemistry characters of ZmSSI,the substrate saturation and affinity,and the feature of chain elongation.For ZmPHOL,this research plans to identify the impact of L80 on its stability and protein interaction.The main achievement of this research will be listed as following:1.In order to study the novol kinetic of SSI,this research use different concetration of maltotrise,maltotetraose and maltopentaose as acceptor substrate to measure the kinetic activity curve and Michaelis constant.The kinetic curve of ZmSSI shows no plateau effect which indicates ZmSSI is not saturated with acceptor substrate.The Km（ADP-G）of ZmSSI with different acceptor shows some difference which indicates ZmSSI has affinity on acceptor substrate.2.The chain length and ditribution of products from ZmSSI catalyzing variable acceptor reaction were detected by Thin Layer Chromatography（TLC）and High Performance Liquid Chromatography（HPLC）.It is prooved that ZmSSI can elongate long chain（DP25）dependently and the chain distribution is similar as reported,mainly short and middle chain.In order to detect the polarity of chain elongation of ZmSSI,[¹³C]ADP-Glucose is used as glucose unit donner.According to the result of ESI-MS/MS,ZmSSI only extends chain length on the non-reducing end of MOS.A preliminary study of SSIII-HD-SSI indicates that the binding of SSIII-HD has no obvious impact on SSI kinetic activity,affinity to acceptor or chain elongation.3.According to the bioinformatics analysis,ZmPHOL prefer to form homo-dimer,L80 region is a flexible loop and far away from the dimerization surface;L80 has 7 phosphorylated Serine residues and a PEST region,phosphorylated Ser430 and Ser431 are in PEST;6 unreported interacted protein are identified from ZmPHOL protein interaction prediction.The bioinformatics prediction suggested that L80could regulate the degradation of ZmPHOL and affects protein interaction,specific phosphorylated Ser430 and Ser431 could have impact on L80 regulation of ZmPHOL.4.An in vitro degradation experiment is processed with recombinant protein PHOL,PHOM,PHOL-S430A and PHOL-SA,then Western blotting will detect the products by using special primary antibody Anti-1 and Anti-2.The results illustrate that PHOL,PHOL-S430A and PHOL-SA could occur degradation,only PHOM without L80 remains a good stability.PHOL could show good stability in the absence of ATP and amyloplast lysates.It suggests that the degradation of PHOL requires ATP and enzymes from amyloplast.5.PHOL has been reported that it could form multi-enzyme complex with SBEIIb and SSIIa through protein phosphorylation.In order to detect the difference of protein binding from amyloplast lysates,Pull-down and protein gel MS were used to detect the bond proteins.According to the result,6 unreported protein were detected and their gene expression have overlapped with PHOL in stages and tissues.The difference of bond protein among PHOL,PHOM,PHOL-S430A and PHOL-SA suggests that protein interaction of PHOL with other protein occuring in L80 and Ser 430,Ser 431 could affect PHOL protein interaction directly.PHOL protein interaction could be regulated by phosphorylation.In this research,some un-reported or novol chemical characteristics of ZmSSI and ZmPHOL were identified.It is prooved that SSI has preference to substrate acceptors and isn’t saturated with substrate acceptors.SSI only elongates glycosyl units in the non-reducing end and can extend long glucans chain up to DP25 dependently.The thermolstablity and protein interaction of PHOL was affected by L80directly and the specific phosphorylated Ser430 and Ser431 had impact on these two process.The new kinetic charateristics demonstrated in this research could inspire a further understanding of the efficient maize starch synthesis pathway and provide new challenge of explain the function of SS\SBE complex.The bond protein of PHOL through this preliminary selection requires further research such as Y2C,BiFC et al in the future.Explaining the biological machanism of the protein-protein interaction among PHOL and 14-3-3,BT1 could indicate the real role of PHOL playing in starch metabolism.