| The content of starch in corn grain directly affects the yield of corn crop.Adenosine diphosphate glucose focal phosphorylase(AGPase)catalytic adenosine triphosphate(ATP)and 1-glucose phosphate(G-1-P)generating adenosine diphosphate glucose(ADPG)and pyrophosphoric acid,is a key limiting step of corn starch biosynthesis,AGPase in the corn is a small subunits by two Bt2 and two large subunit Sh2 four polymers of different source.Studies have shown that AGPase activity is regulated by redox regulation,temperature regulation and small molecule allosteric regulation,and there is a new mechanism of AGPase phosphorylation regulation.Protein phosphorylation occurs when the residues of serine(S),threonine(T),and tyrosine(Y)in a protein are dehydrated and a phosphate group is added,causing the protein to become negatively charged and change its properties.Aspartic acid(D)and glutamic acid(E)in the protein have a phosphate group,so the mutation of the amino acid residues in the protein to aspartic acid or glutamic acid with a negative charge can simulate the phosphorylation at this site.Previous identification found that the small AGPase subunit Bt2 was phosphorylated at S10,T451 and T462.In order to further study the effect of its phosphorylation on the function of AGPase,several biochemical and molecular biological methods were used for preliminary exploration,and the following results were obtained:1.The phosphorylation antibody at the Bt2S10 site was prepared,and the phosphorylation levels of the grains at different days after pollination at this site were detected.Firstly,the Phos-Bt2S10 peptide synthesized in vitro was immunized in rabbits to obtain the detection antibody against the phosphorylation of Bt2S10.Secondly,the results of immunoanalysis on the total eggs at 5,20,25 and 30 days after pollination showed that the small AGPase subunit Bt2 was phosphorylated at 20 to 30 days after pollination.2.The small subunit Bt2S10 of AGPase endosperm was mutated into different types of recombinant vectors.In order to explore the effect of phosphorylation at the small AGPase subunit Bt2 amino acid site on the function of maize,this study investigated the changes in the gene function of the small AGPase subunit Bt2 amino acid sites S10 and T451,which were mutated to alanine(A),aspartic acid(D)and glutamate(E),respectively.In order to simulate the phosphorylation state of AGPase endosperm small subunit Bt2S10,the negatively charged D and E were selected to replace the S10,thus we obtained the negatively charged small subunit Bt2 at this site.3.Six yeast vectors p GADT7 with small AGPase subunit amino acids S10 and T451 were mutated into A,D and E respectively,and the yeast two-hybrid experiment was conducted with Sh2-p GBBK7 with these small subunits,respectively.The results showed that Bt2 still had an interaction with Sh2 after mutation.In order to further verify whether the amino acid mutation affects the subcellular localization of Bt2,agrobacterium-infected method was used to observe the expression of Bt2 in benn’s tobacco leaves,and it was found that both Bt2-S10 E and Bt2-T451 E were located in the chloroplast,and the other sites were located in the nucleus and cell membrane after mutation.Furthermore,the interaction between Bt2 and its mutants and Sh2 in corn was further verified.In the experiment,the protoplasts of corn leaves were transfected with fluorescent bilayer complementary plasmid.The fluorescence signal of Bt2 and Sh2 fusion protein showed that different mutant types of Bt2 had protein interaction with Sh2,and only located in the chloroplast.4.Based on the above results,the enzyme activity of the protoplast was measured for the transient expression of E-type mutations with localized changes.The UG221-Bt2-S10 E,UG221-Bt2-S451 E and UG221-Bt2 vectors were co-overexpressed with UG221-Sh2 by protoplast transformation,and the AGPase activity was found to be decreased,but the T451 site was slightly higher than the S10 site.These results provide a basis for the study of important amino acid sites related to AGPase activity and phosphorylation. |
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