Molecular Characterization of a MYB Protein from Oryza sativa for its Role in Abiotic Stress Tolerance

ABSTRACT The MYB family represents one of the most abundant classes of transcriptional regulators that perform pivotal role under different developmental processes and abiotic stresses. In present study, a MYB gene from Oryza sativa was selected for functional characterization. Bioinformatics analysis revealed that OsMYB1 cDNA encodes R2-R3 type DNA binding domain consisting of 413 amino acids having size of 44 kDa and pI of 6.24. DNA binding domain containing region was cloned and over-expressed in E. coli. Then, the survival of pGEX-OsMYB1 transformed E. coli cells was compared with control plasmid under different concentrations of NaCl, mannitol, high and low temperature. pGEX-OsMYB1 enhanced the survival of cells at high temperature and salinity. Electrophoretic mobility shift assays (EMSAs) have shown that recombinant OsMYB1 protein was able to bind with DIG labeled probe containing MYB binding site. RT-qPCR analysis revealed high MYB1 expression under wounding, salt, drought and heat stresses in rice. Expression was 23 fold higher in response to wounding demonstrating the worth of OsMYB1 up-regulation in wounding. Intrinsic disorder profile predicted that OsMYB1 exhibits 60% degree of intrinsic disorder proposing that these regions might be involved in DNA binding specificity and protein-protein interaction. The positive response of OsMYB1 suggests that its over-expression in crop plants may help in providing protection to plants to grow under abiotic stresses.

Vector Construction,Protein Expression,Purification and Identification of Calmodulin Mg2+Binding Site Mutants / 中国医科大学学报

Objective To construct plasmid vectors of calmodulin(CaM)Mg2+binding site mutants,and to express,purify and identify the mutant proteins. Methods Three kinds of cDNAs coding for the mutated CaM were cloned into pGEX?6P?3 plasmid vectors. These recombinant plasmids were transfected into Escherichia coli BL21 to express GST fusion proteins of CaM mutants. The fusion proteins were purified with Glutathione?Sep?harose 4B beads and PreScission protease. Results Both enzyme digestion analysis and DNA sequence identification proved the successful con?struction of the CaM mutant plasmids. SDS?PAGE results showed the high purity of each CaM mutant protein. The concentrations of three CaM mu?tants were around 1.0 mg/mL. Conclusion Prokayotic expression vectors of CaM Mg2+binding site mutants were successfully developed,and the eli?gible CaM mutant proteins were obtained. This study provided an important basis for further study on CaM’s biological function.

Construction and identification of a recombinant Bifidobacteria (pGEX-TSO45W-4B) vaccine of Taenia solium / 中华地方病学杂志

Objective To construct and identify a recombinant Bifidobacteria(Bb) (pGEX-TSO45W-4B) vaccine of Taenia solium.Methods The TSO45W-4B gene of Taenia solium was synthesized.It was expected that the fragment length of the gene was 351 bp.The gene was cloned into Escherichia coli-Bifidobacteria(Bb) shutde vector pGEX-1λT to construct a recombinant plasmid pGEX-TSO45W-4B.The recombinant plasmid was electroporated into Bb to construct a recombinant Bb (pGEX-TSO45W-4B) vaccine.The vaccine was identified by restriction analysis,PCR and sequencing,which would demonstrate that the constructed recombinant Bb (pGEX-TSO45W-4B) vaccine of Taenia solium contained the gene of fragment length of 351 bp.Results The TSO45W-4B gene of 351 bp was synthesized.Restriction analysis,PCR and sequencing results showed that the recombinant Bb (pGEX-TSO45W-4B) vaccine of Taenia solium was successfully constructed and contained the gene fragment of 351 bp.Conclusion The recombinant Bb (pGEX-TSO45W-4B) vaccine of Taenia solium is successfully constructed,which lays a foundation for the study of expression and immunogenicity of the vaccine.

Dynamic observation on splenocyte in mice immunized with recombinant Bi f idobacterium bi f idum (pGEX-S j26GST) of Schistosoma j aponicum / 中国人兽共患病学报

To observe the dynamic changes of splenocyte proliferation ,subsets and apoptosis in mice immunized with re-combinantBifidobacteriumbifidum(pGEX-Sj26GST)ofSchistosomajaponicum,themiceweresubcutaneously(SCgroup) and intranasally (IN group) immunized ,respectively .Four mice from each group were sacrificed in every 2 wk during 0-20 wk after immunization .Splenocyte proliferation was investigated by MTT colorimetric assay ,subsets of CD+4 and CD+8 T cells and apoptosis of splenocytes by FACsort flow cytometry .In SC group ,unstimulated and stimulated with S jAWA ,the level of splenocyte proliferation significantly increased at 4-20 wk after vaccination and increased markedly at 4-18 wk stimulated with ConA ,both of which peaked at 8 wk;in IN group ,the proliferation level of splenocyte cultured with SjAWA and ConA signif-icantly increased during the 4-18 wk ,2-10 wk and 14-18 wk ,2-8 wk and 12-18 wk ,respectively ,and all reached the maximum at the 4 wk after immunization (P＜0 .01 or P＜0 .05) .CD+4 subsets increased obviously during 2-14 wk ,2 wk and 6-16 wk re-spectively ,and reached the peak at 8 wk (P＜0 .01 or P＜0 .05) in both group ,while CD8+ subsets rose lightly during 2-20 wk in both group ,and reached the maximum at 8 wk (SC group) and 6 wk respectively (P>0 .05) .Whether unstimulated or stimulated with ConA ,the level of splenocyte apoptosis of which remarkably increased at 2-4 wk and 2-6 wk separately in SC group ,and both peaked at 2 wk (P＜0 .01 or P＜0 .05 );in IN group ,the level of splenocyte apoptosis all increased at 4 wk and reached the maximum at the same time .In summary ,by inducing the proliferation of splenocytes ,increasing CD4 + T cells and decreasing splenocyte apoptosis ,the rBb (pGEX-Sj26GST ) vaccine plays a critical role in the protective immune response .

The expression and activity of Caspase 3 in E.coli BL21 / 医学研究生学报

Objective: To study the expression and activity of the apoptosis protease-Caspase 3 in(E.coli) BL21(DE3). Methods: The cDNA of Caspase 3 was amplified by PCR and inserted into the plasmid pCMV-Myc,it is then cloned to prokaryotic expression vector pGEX-6p,after which Caspase 3 was induced by IPTG.The protein induced was identified by SDS-PAGE and Western blot. Results: After induced by IPTG for 3 hours,the concentration of Caspase 3 reached the highest level. Conclusion: Active Caspase 3 can be induced within E.coli BL21(DE3),further research can be done about the role of Caspase 3 in apoptosis.