Site-directed mutagenesis rice cryptochrome and construction method thereof
A technology of site-directed mutation and cryptochrome, applied in the fields of bioengineering, gene and protein engineering, can solve the problem of scarcity of cryptochrome and achieve the effect of increasing yield
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Embodiment 1
[0030] Embodiment 1: the acquisition of mutant enzyme D393N gene and the construction of expression vector
[0031] 1.1 Obtaining WT rice cryptochrome gene oscry2
[0032] Find the cryptochrome gene sequence of rice in NCBI (National Center for Biotechnology, AB103094), optimize and truncate it based on the codon preference in Escherichia coli (E.coli), design a gene suitable for expression in E. coli, and submit it to Nanjing GenScript artificially synthesized the gene and loaded it into the pET22b plasmid vector. After successful sequencing by the company, the successfully constructed recombinant plasmid pET22b-OsCRY2 was sent back. (Zheng Binqiong. Escherichia coli synonymous codon preference overview [J]. Silicon Valley, 2009 (01): 23-24.) (Fumiaki Hirose. Involvement of ricecryptochromes in de-etiolation responses and flowering [J]. Plant Cell Physiol, 2006, 47(7):915-25.).
[0033] 1.2 Construction of mutant strains
[0034] Design a pair of mutation primers, the muta...
Embodiment 2
[0051] Example 2: Expression and purification of mutant rice cryptochrome D393N protein
[0052] 2.1 Protein expression
[0053] (1) Inoculation: pick engineering bacteria Rosetta(DE3) / pET22b-D393N, inoculate in 5mlLB (Amp + ,Cam + ) overnight cultivation in liquid medium at 37°C and 225rpm;
[0054] (2) Expansion culture: Take 5ml of the overnight cultured bacterial solution and transfer it to 500ml of liquid LB containing the corresponding antibiotics, culture at 37°C, 225rpm for 4-5h, until OD 600 is about 1;
[0055] (3) Induction: Add 500 μl of 1MIPTG to a final concentration of 1 mM, 20°C, 180 rpm, induce expression for 20 h;
[0056] (4) Bacteria collection: After induction of expression, centrifuge at 5500 rpm for 5 min at 4°C to collect the cells, and store them in a -80°C refrigerator.
[0057] 2.2 Protein purification
[0058] Start Buffer (500ml)
[0059]
[0060] Elution Buffer (500ml):
[0061]
[0062] Protein Buffer (500ml):
[0063]
[0064] ...
Embodiment 3
[0072] Example 3: Expression and purification of WT (wildtype) rice cryptochrome protein
[0073] Except: Inoculation: Pick engineering bacteria Rosetta(DE3) / pET22b-OsCRY2 and inoculate it in 5ml LB (Amp + ,Cam + ) overnight cultivation in liquid medium at 37°C and 225rpm;
[0074] All the other are identical with embodiment 2.
[0075] Such as figure 1 Shown: there is a single highly specific band at 56kDa, indicating that the purified protein has a high purity (above 90%). And it is consistent with the molecular weight calculated by the amino acid sequence. The D393N mutant enzyme band was thicker than that of WT, indicating that the expression level of the D393N mutant enzyme was increased.
[0076] Such as figure 2 As shown, the elution volume of the WT rice cryptochrome prepared in Example 3 is 14.75 ml, and the calculated molecular weight is 57 kDa, indicating that the WT rice cryptochrome is a monomer. The highest protein absorption peak is 254mAU.
[0077] Suc...
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