The Study Of The Construction Of The T Vector And Expression Vector With Maize CobA Gene As The Red Fluorescent Reporter

Uroporphyrinogen III methyltransferase (UPMT), a key regulatory enzyme in the branches synthesis of vitamin B12, is a novel red fluorescence indicator. There are a few reports on the application. In this study, we studied the inactive insertion of the maize UPMT and the soluble protein expression using the UPMT as the reporter. The results as followed:1. The background expression for cobA gene encoding maize UPMT under different promoters in E.coli was analyed. The cobA gene with the background expression was detected under Lac, T5, Trc and Tac promoter respectively, based on the red fluorescence appearance of the recombiniant colonies, whereas it has not background expression under T7 and araBAD promoter.2. The expression level of different strains for cob A gene was analyzed, encoding maize UPMT under Lac promoter. The results showed that colonies emitting red fluorescence under long UV light had not obvious difference for DH5α, JM109, XL-Bluel and TG1 transformation strains.3. T vector was constructed basing on maize UPMT insertional inactivation, which was the effect of different lengths PCR products insertion and the cloning efficiency of inserting into a 500 bp fragment was assayed. The results showed that the fragments of different lengths (250 bp,370 bp,420 bp,1000 bp and 1800 bp) can insert into the T vector. The T vector inserted into trinucleotide (TCA) led to red fluorescence disappearance of recombinant colonies. The TCA encodes a Ser residue which is the smallest molecular weight amino acids residue of TXA encoding in T vector. Argrose electrophoresis results showed that the cloning effiency of 500 bp insertion fragment is up to 92%, similarly to the cloning efficiency of traditional T vector based onα-fragment insertional inactivation.4. The insertional inactivation mechanism was preliminarily analyed. T vector inserted into a Ser residue led to UPMT protein expressed as inclusion bodies, indicating that this region involved in the folding.5. Three maltose binding protein (MBP) mutants was constructed by using the method of site-directed mutagenesis:folding-and solubility-defective (G32D) and two type of inclusion bodies (respectively A264D and G32D/I33P). Fusions of theα'-fragment to C terminus of wild type and mutant MBP construct fusion expression vector. C terminus of wild type and there MBP mutants individually fusing with theα-fragment construected fusion expression vector. Electrophoresis analysis showed that A264D, G32D/I33P expressed as inclusion bodies, while G32D expressed in both supernatant and precipitate.6.β-galactosidase activity belonging to a peptide of C terminus of wild-type and there MBP mutants detected level of protein soluble and folding. The results showed that there was high positive correlation between the wild and three mutants.7. UPMT were replaced with a peptide of C terminus of wild-type and mutant MBP. Fluorescence analysis showed that there are positive correlation with the level of soluble and folding of wild-type and mutant MBP, being consistent with results of detection activity of a peptide.In summary, The constructed resultant of the T vector had high sensitivity using maize UPMT as a selectable marker. An amino acid insertion led to the disappearance of red fluorescent recombinant colonies. As a screening marker, it can be sensitive to detect the level of folding and soluble of the fusion proteins, which layed the foundation for the rapid screening heterologous protein of solube expression in E. coli.