| Rice blast is caused by the Magnaporthe grisea. It occurs widely in various rice growing regions in the world and it’s one of the main factors limiting the yield of rice. Sterol 14a-demethylase (CYP51)is an important drug target enzyme that is widely distributed in nature and can be found from prokaryotes to the human. This enzyme is involved in the biosynthesis of ergosterol which is key to the forming of fungal cell membrane. It catalyzes sterol 14a-demethylation reaction. The reduction of ergosterol will affect the membrane fluidity of fungal cell, destroy the membrane structure of cell and cause fungal to death ultimately.14a-demethylase inhibitors (DMIs) which are widely used in agriculture and play a role in inhibiting the synthesis of the fungal CYP51. CYP51 of eukaryotic microorganism are membrane protein. When the fungal CYP51 is expressed in prokaryotic systems, the recombinant protein usually can not be correct expressed or be active. It often needs to modify the CYP51 gene and optimize the expression.In this study, we have investigated the expression of CYP51 from M. grisea and analyzed the secondary structure of mRNA translation initiation region about MgCYP51. The CYP51 gene was amplified by PCR, and molecular cloning techniques were used to construct a recombinant plasmid. The results are listed as below:1. CYP51A and CYP51B gene from M.grisea were cloned. The recombinant expression plasmids (pET28-Mg and pET32-Mg) were constructed and expresssed in E. coli BL21 (DE3) or BL21 (DE3) pLysS. After analyzed by SDS-PAGE, the obvious expression bands were not detected. Despite the change of inducible temperature, inducible time, IPTG concentration, the specific expression bands still could not be detected. These phenomena illustrated that the full length of rice blast fungus CYP51 gene could not be expressed in E. coli.2.MgCYP51A and MgCYP51B are trans-membrance proteins through the analysis and prediction of their transmembrane structure. Two truncated mutants of MgCYP51A were gained through truncation of N-terminal sequence and predictive analysis. The N-terminal sequences of one truncated mutant was truncated by 35 amino acid residues named as MgA-35, and the other one was named as MgA-53 similarly. Two truncated mutants of MgCYP51B denoted by MgB-83 and MgB-105 were obtained like them (The N-terminal 83 amino acid residues was truncated in MgB-83; and 105 amino acid residues was truncated in MgB-105).3. The MgCYP51A genes encoding the transmembrane regions in N-terminal were truncated by genetic engineering techniques. The truncated mutants of MgCYP51A were cloned into pET-28 to construct recombinant expression plasmids. After optimizing the host strain, temperature, time, IPTG concentration, etc. it was found that the recombinant protein was expressed as inclusion bodies. In order to increase the solubility of recombinant protein, a squense encoding the soluble short peptides(LPPGP)was added to the 5’end of the truncated mutant gene of MgCYP51A. It can be found that about 20% of the recombinant protein pET28-MgA-53m was presented in the supernatant and the soluble expression of the protein was improved. Different expression vectors were applied to construct new kinds of recombinant plasmids. The recombinant plasmids pET32-MgA-53m, pET42-MgA-53m, pMEK-MgA-53m and pCW-MgA-53m were constructed and the expression product was analyzed by SDS-PAGE. It was found that the expression of label gene could be detected, but the specific expression of recombinant proteins could not be detected. After optimization of the expression conditions, it can be concluded that when induced by 0.5 mM IPTG at 20℃, the specific expression strip pMAL-MgA-53 could be detected. It was about of 102KD and about 70% of recombinant proteins were soluble.4. When the full-length and truncated genes of MgCYP51B were subcloned into three different expression vectors, respectively, nine kinds of recombinant expression vectors were constructed. Only the pET28-MgB-83 could achieve the expression of recombined proteins after exploring a variety of expression conditions, which could lay the foundation for further study of the structure and function of MgCYP51B.The mRNA secondary structure of these mutant genes was analyzed; the free energy of secondary structure about mRNA translation initiation region (TIR) in pET28-MgB-83 (including 30bp from vector and 70bp from 5’-MgCYP51B-83 gene) was proved to be least. It indicated that secondary structure of mRNA TIR could affect the binding of ribosome binding site with initiation codes as well as ribosome; which in turn affect the expression of recombination protein.More stable the secondary structure is, more difficult the expression is. |
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