Prokaryotic Expression Of AtMYB2Transcription Factor And Interaction Analysis With S/CMO Promoter

AtMYB2transcription factor is a homolog of MYB family, it was induced by dehydration, high-salt condition and exogenous ABA. AtMYB2transcription factor can bind to the sequence of TGGTTAG (which is reverse and complementary to CTAACCA) and enhance of downstream genes expression. Choline Monooxygenase (CMO) is a ferredoxin-dependent stroma enzyme which catalyzes the first step of glycine betaine synthesis in plants. The transcription and expression of CMO gene are induced by salinity in plants.SICMO promoter was isolated from Suaeda liaotungensis and found the region-267～+1bp (named pC5) is a salt-induced promoter. pC5promoter contains AtMYB2transcription factor recognition sequence TAACCA (which is reverse and complementary to TGGTTA). AtMYB2transcription factor can interact with SICMO promoter in pC5-GUS/AtMYB2transgenic tobacco plants and activate the transcription of GUS reporter gene. We expressed AtMYB2fusion protein in Escherichia coli cells and get purified ATMYB2fusion protein. We analysis weather ATMYB2fusion protein can bind to the CMO promoter directly in electrophoretic mobility shift assay, thus analysis the expression of CMO genes regulated by AtMYB2transcription factor. The main contents and results are as follows:1. The expression vector pET30a-AtMYB2was constructed and transformed into E.BL21(DE3), E.BL21-pET30a-AtMYB2was obtained.2. AtMYB2fusion protein was expressed in Escherichia coli cells. The fusion protein both exist in the form of soluble protein, but also to the form of inclusion bodies. The induction condition was optimized from the IPTG concentration, induction temperature, induction time iri these factors. The optimal induction condition of soluble protein was induced at0.1mM of IPTG,30℃for10h.3. AtMYB2fusion protein and6×His vector protein were purified with Ni-NTA chromatography.4. The interaction between AtMYB2transcription factor and SICMO promoter was analysised by electrophoretic mobility shift assay. The result indicates that AtMYB2transcription factor bind to the sequence of TAACCA in SICMO promote directly in vitro, further to prove that AtMYB2protein may be a transcription factor that regulate the expression of SICMO promoter.