Correlation Analysis Of Differential Expression Protein RAB17 In Maize Seed Drying And After-ripening

Crop seed development need to be mature through the stage of dehydration, and dehydration is the bridge of crop seed from dormancy to germination. There are differences with some protein changes of seed embryo and endosperm in the dehydration process, and they play an important influence in the process. The study about proteome changes of the embryo and endosperm after ripening in the drying process will help us to further understand the process of corn seeds.RAB17 (Responsive to ABA, RAB) belongs to the family dehydrins (DHNs), also belongs to LEA II (late embryogenesis abundant protein, LEA). It is rich in the mature seeds and degradation in the germination to play an important role on the acquisition of seed desiccation tolerance and the process of after-ripening, but also affects seed germination. The studies also shown that, RAB17 is with a variety of physiological functions in cell and expressed in the drought, low temperature, and salinity etc. And it can stabilize biological macromolecules and cellular structures. There are a lot of researches about RAB17, and RAB17 is regulating by the kinase CK2 in cells. Marta Riera, etc. (2004) found that there is other protein kinase involved in the phosphorylation of RAB17 through the extracts of corn embryo with phosphorylation in vitro, in addition to CK2. In adversity stress conditions, Goday, etc. (1994) found that the phosphorylation of RAB17 homologous proteins TAS14 in tomato is through CK2 and the cAMP-dependent protein kinase in vitro. However, there are no further reports.The proteomics of the seed of inbred lines 08-641 before and after dehydration suggests that RAB17 differentially expressed. The ear of corn inbred lines S37 before and after natural drying process, we analyzed the differences of the proteomics of embryo and endosperm in the ear by two-dimensional electrophoresis, and also found the up-regulated RAB17 protein. According to NCBI database, using nested PCR cloned rab17 gene from embryo of the seed maize inbred 08-641, and constructed prokaryotic expression vector of RAB17 to transform into E. coli to expression. The protein is purification and chelating with affinity chromatography column, interacting proteins from reactive protein of corn embryo by pull-down from the whole corn germ protein to identify by two-dimensional electrophoresis mass spectrometry. Using bioinformatics methods, we completed some characteristic analysis of rab17 nucleic acids and proteins, and also the interacting protein LOC100276911 about its molecular weight and isoelectric point, the relationship between the structure and the evolution relationship. According the analytical results, we cloned its gene and expressed through the prokaryotic vector for the further research. The main results are as follows:(1) Using two-dimensional electrophoresis and mass spectrometry, we totally identified 46 protein spots in the inbred S37 embryo and endosperm between after and before the natural drying:30 differentially expressed proteins in the embryo; 16 in the endosperm. It was detected the same protein up-regulated RAB17 in embryos with inbred 08-641 after drying, but with RAB17 protein expression polymorphism.(2) The results of the analysis based on protein RAB17 from seeds of inbred 08-641 after drying, we cloned the CDS area of rab17 with the length of 507bp from its mature embryos. Bioinformatics showed that the expression existed allele polymorphism.(3) By successfully prokaryotic expressing rab17 prokaryotic protein with tag of 6xHis and extraction of corn active proteins, we identified the protein LOC100276911 by pull-down and 2-D.(4) Bioinformatics analysis showed that there were certain conserved proteins with the interaction in grasses. We cloned its CDS region from maize embryo and expressed prokaryotic expression protein successfully.In summary, we have successfully identified the different expression protein RAB17 between before and after drying from the embryo of seed in inbred S37. We also cloned rab17 from mature embryo of maize inbred lines 08-641 and got its interacting protein LOC100276911, and demonstrated RAB17 is with expression polymorphism in corn inbred lines, which plays a key role in the process of maize after-ripening and dehydration to further test to verification with yeast two-hybrid system and lay the foundation about the analysis of RAB17 protein function.