The Purification And Polymer Analysis Of BoSEP3and AtSEP3Protein

Bamboo, the biggest grass, a resourceful perennial lignified plant, has considerable economicand scientific significance. However, limited genetic research has focused on bamboo partiallybecause of the lack of genomic resources. The key factor for the bamboo flowering physiologicalprocess is particularly important. SEPALLATA3(hereinafter referred to as SEP3) proteinsassemble into polymer composites. However, it is not known if binding of DNA is required for thepolymer formation and how the polymer is formed, but it plays an important role in plant growth.To counter these questions, the expression and interaction regulation and its highconservation were analyzed. The mian points are listed as follow:1. SEP3was firstly constructed as a fusion protein and expressed in prokaryotic cells. Byscreening of different structure domains and expression conditions, an optimal expression strategyby inducing at22℃for15hours with0.1%IPTG and by pET15b vector was achieved. Here, theM domain SEP3, IK domain SEP3, IKC domain SEP3protein and the inclusion body proteinAtSEP3protein were purified to get the soluble protein.2. Through analytical matrix assisted laser ionization time of flight mass spectrometry(MALDI TOF MS) fingerprint analysis, Mascot program get polypeptide fragments andSwissProt database information, which validated of SEP3protein.3. N terminer of SEP3protein is M domain, which is specificed by binding to DNA. Aftermixing the M domain SEP3purified protein and CArG box sequences and the gel shiftexperiment, we verified the activity of combining with specific DNA.4. It displayed as a polymer in the size exclusion chromatography（SEC）, and the polymershowed containing of dimer unit in the glutaraldehyde crosslinking assay. Yeast two hybrid testsfurther suggested that SEP3protein forms homologous polymer in vivo. It surmise that K domainplay the main role in the homodimers.Arabidopsis thaliana and Bambusa oldhamii belongs to dicotyledon and monocotyledon respectively, the sequences of AtSEP3and BoSEP3are not only highly homologous, but also hassimilar protein structure. Being capable to isolate SEP3domain proteins paved a foundation forthe further study of flowering of plant.