Screening And Effect Of Heat Shock Proteins On AGPase Activity In Maize Endosperm

Maize is one of the most important grain crops in the world.Starch is an important part of corn grain,which influence the yield and quality of corn.The biosynthesis of starch involves a series of enzymes,including adenosine diphosphate glucose pyrophosphorylase(AGPase),starch synthase(SS),starch branching enzyme(SBE),starch debranching enzyme(DBE)and starch phosphorylase(SP).AGPase is generally considered to be the rate-limiting enzyme affecting starch content.In the previous study,we found that the content of endosperm starch of a maize zmmads11 mutant was significantly increased,but the activity of AGPase in the mid-filling mutant was reduced to about 30% compared with that of the wild type.In this paper,the main factors affecting AGPase activity in the mutant were analyzed as the objective of a series of experiments.Two heat shock proteins affecting AGPase protein content and activity were identified by enzyme activity assay,high-throughput sequencing and protoplast transformation,and a new pathway affecting AGPase activity was found.The main findings were as follows:1.The maize zmmads11 mutant inserted by Mu transposon was backcrossed with wild-type W22 for 8 generations,and the homozygous mutant was obtained by self-crossing.The total starch content and protein content of zmmads11 and wild-type W22 endosperm were determined.The results showed that the total starch content of zmmads11 homozygous mutant was significantly higher than that of wild-type W22,but the protein content of zmmads11 homozygous mutant was not significantly different.2.Extracting total RNA from endosperm of zmmads11 homozygous mutant and that of wild-type W22 15 DAP(Days after pollination).The main coding genes AGPase,AGPL1(SH2),AGPS1(BT2)and ADPG transporter encoding gene BT1 in maize endosperm were analyzed by real-time quantitative fluorescence PCR.The results showed that there was no significant difference between the three genes.3.The activity of AGPase was determined by extraction crude enzymes of 15 DAP endosperm from zmmads11 homozygous mutant and W22 wild type.The results showed that the activity of AGPase of zmmads11 homozygous mutant decreased to about 30% of wild type compared with that of W22 wild type.4.The content of AGPase protein SH2,BT2 and ADPG transporter BT1 in 15 DAP crude enzymes of endosperms were determined by Western Blot.The results showed that the amount of SH2 and BT2 protein in zmmads11 homozygous mutants was significantly decreased compared with wild-type W22 and BT1 protein was also decreased.5.The 15 DAP endosperm RNA of wild-type W22 and zmmads11 homozygous mutants were analyzed by RNA-seq.Ten up-regulated heat shock protein genes in zmmads11 homozygous mutants were found and classified into the protein process pathway.Two heat shock protein genes HSP1,HSP22 containing introns and expressed highly at mid-grain-filling period were screened by gene structure and expression analysis.The two heat shock protein genes,HSP1,HSP22 and AGPL1(SH2),AGPS1(BT2)were constructed as over expression vectors and transiently transformed into maize leaf protoplasts.The results showed that AGPase activity in leaf protoplast was significantly reduced by the two heat shock protein HSP1 and HSP22.