Prokaryotic Expression And Enzymatic Characteristics Of Maize Starch Synthase ? And ?

Starch is the final product of photosynthesis,and mainly stored in the endosperm of crop kernels.The content and structure of starch in grains determines the yield and quality of crops.Starch is formed by polycondensation of glucose to form polydextrose clusters,which further form regular semi-crystalline starch granules.The starch in plants is formed by stacking starch granules.The corn starch accounts for 70-80%of its dry weight.The starch in the grain is produced synthetically in the amyloplast.The synthesis of starch mainly depends on sucrose as a carbon source,and sucrose is hydrolyzed to form G-1-P.ADPG is formed through the catalysis of ADP-glucose pyrophosphorylase?EC 2.7.7.27;AGPase?,and then ADPG enters the amyloplast under the action of transporters.Starch Synthase?EC 2.4.1.21;SSs?,Starch Branching enzymes?EC 2.4.1.18;SBEs?,Starch-debranching enzymes?EC 3.2.1.68;DBEs?,Starch Phosphorylase?EC 2.4.1.1;SPs?and other proteins involved in the completion of the polysaccharide chain extension,polysaccharide chain branch and polysaccharide chain debranching,and ultimately biosynthetic starch.Starch synthase is involved in the extension of starch chain length in the process of starch synthesis.Previous studies on the activity and function of starch synthase I,Starch Synthase II and Starch Synthase III,the soluble starch synthase subfamily in starch synthase,have been conducted.It was confirmed that these three proteins occupy an important position in the process of starch synthesis for the extension of short and medium-long chains.Starch Synthase ? and Starch Synthase ? are newly discovered proteins belonging to this family in recent years.These two proteins are for starch granules,the initial formation and elongation of the starch chain have an essential role.In this study,ZmSS? and ZmSS? gene genes were cloned from maize plants;quantitative Real-time PCR was used to analyze the tissue-specific expression and circadian rhythm characteristics of the genes.use the prokaryotic expression system,heterologous expression of soluble protein with starch synthase activity was made,and the basic enzyme characteristics were analyzed;determination of chain length distribution by HPAEC-PAD;subcellular localization detection ZmSS? and ZmSS? located in the specific location of the cells was used to explore the functional areas in the plant,in order to analyze the specific functions of ZmSS? and ZmSS? in corn,an overexpression fusion vector was constructed and the target gene was over-expressed in maize,detection of positive transformed plants.The specific findings are as follows:1.The full-length ZmSS? coding region gene sequence was cloned from the grain of the 15-day pollinated with maize inbred line 18-599.The sequence of ZmSS? is 2730 bp and encodes 909 amino acids.The average molecular weight of the protein was 103.1 KD.Typical conserved domains of the GT5 and GT1 of the starch synthase family;the ZmSS? gene sequence is 2106 bp in length and has701 amino acids.The average molecular weight of the protein is approximately 78.6 KD,the conserved domains of the starch synthase family GT5 and GT1-like are analyzed using SMART software.2.Take 18-599 different maize tissues for RNA extraction and analyze the tissue expression specificity of genes.ZmSS? expression is relatively high in endosperm and leaves,and the expression in roots is lower;ZmSS? is mainly in endosperm and kernels.Expression is basically not expressed in the root.3.The ZmSS? and ZmSS? genes were constructed into the prokaryotic expression vector PGEX-6T-1.The E.coli BL21?DE3?strain was used for in vitro translation and prokaryotic expression.Soluble proteins were successfully expressed and the fusion tagged proteins were purified.Detection of fusion proteins by SDS-PAGE and Western-Blot,which can be used to analyze the starch synthase activity.4.The PGEX-6T-ZmSS? and PGEX-6T-ZmSS? fusion proteins can extend the starch chain length in the presence of ADPG,indicating that the purified protein has a starch synthase activity,and in the analysis of the enzymatic properties,ZmSS? was found to be Starch synthase activity was relatively high at pH=7.5 and 37?,and ZmSS? also had relatively high starch synthase activity at pH=7.5 and 37?;both of these enzymes have the highest activity in oyster glycogen type II substrates.The malttriose as the substrate has the lowest enzyme activity;the starch synthase activity of ZmSS? and ZmSS? was measured with different concentrations of ADPG,and the Michaelis constant and the maximum reaction rate were calculated using the double reciprocal mapping method.The Km_ADPG value of ZmSS? was 0.282±0.045 mmol/L and ?max value of 0.124±0.005nmol/min,ZmSS? with Km_ADPG value of 0.116±0.012 mmol/L and ?max value of 0.117±0.004nmol/min.5.Using oyster glycogen as primers,the chain length distribution of ZmSS? and ZmSS? was analyzed.It was found that ZmSS? was mainly involved in the elongation of DP11-15 and DP23-38,and ZmSS? performs functions in the DP11-15 and DP22-34 intervals.6.The 35S promoter was used to drive the fusion expression of ZmSS?,ZmSS? and eGFP,and transformed into protoplasts of maize yellow leaves.The observation of the luminescence of protoplast cells under different fluorescence,Yellow light in chloroplast cells in a mixture of GFP and Chlorophyll autofluorescence,ZmSS? and ZmSS? are considered to be located in the chloroplast of the leaf.