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Rice cryptochrome site-directed mutagenesis protein and construction method thereof

A site-directed mutagenesis, cryptochrome technology, applied in the fields of gene, bioengineering, protein engineering, can solve the problem of scarcity of cryptochrome

Active Publication Date: 2021-06-04
ANHUI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on rice cryptochrome is relatively scarce.

Method used

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  • Rice cryptochrome site-directed mutagenesis protein and construction method thereof
  • Rice cryptochrome site-directed mutagenesis protein and construction method thereof
  • Rice cryptochrome site-directed mutagenesis protein and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: the acquisition of S392L mutant enzyme gene and the construction of expression vector

[0039] 1.1 Rice cryptochrome 2 gene oscry2

[0040] Find the rice cryptochrome gene sequence from 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 send it to Nanjing GenScript Company 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.Involvement of rice cryptochromes in de-etiolation responses and flowering[J].Silicon Valley,2009(01):23-24.)(Fumiaki Hirose.Involvement of rice cryptochromes in de-etiolation responses and flowering[J].Plant CellPhysiol,2006 , 47(7):915-25.).

[0041] 1.2 Construction of mutant strains

[0042] Design a pair of mutation primers, the...

Embodiment 2

[0059] Example 2: Expression and purification of mutant rice cryptochrome S392L protein

[0060] 2.1 Protein expression

[0061] (1) Inoculation: pick engineering bacteria Rosetta(DE3) / pET22b-S392L and inoculate it in 5ml LB (Amp + ,Cam + ) overnight cultivation in liquid medium at 37°C and 225rpm;

[0062] (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;

[0063] (3) Induction: Add 500 μl of 1MIPTG to a final concentration of 1 mM, 20°C, 180 rpm, induce expression for 20 h;

[0064] (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.

[0065] 2.2 Protein purification

[0066] Start Buffer (500ml)

[0067]

[0068] Elution Buffer (500ml):

[0069]

[0070] Protein Buffer (500ml):

[0071]

...

Embodiment 3

[0081] Example 3: Expression and purification of WT (wild type) rice cryptochrome protein

[0082] Except: Inoculation: Pick engineering bacteria Rosetta(DE3) / pET22b-OsCRY2 and inoculate it in 5ml LB (Amp + ,Cam + ) in liquid medium at 37°C, 225rpm for overnight culture;

[0083] All the other are identical with embodiment 2.

[0084] Such as figure 1 , the purified protein was identified by 12% SDS-PAGE, M is a low molecular weight standard protein, and lanes 1 and 2 are wild-type and S392L mutant enzyme purification proteins respectively to collect samples. 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 enzyme band of the S392L mutant was thicker than that of WT, indicating that the expression level of the S392L mutant enzyme was increased.

[0085] Such as figure 2 , the elution volume of WT rice cryptochrome...

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Abstract

The invention discloses a rice cryptochrome site-directed mutagenesis protein and a construction method thereof. The construction method comprises the following steps: obtaining a gene sequence from a gene pool, carrying out artificial codon optimization and protein carboxyl terminal truncation to obtain a target gene sequence, and connecting the target gene sequence with a pET22b plasmid to construct a recombinant expression plasmid. The sequence of the truncated gene is as shown in SEQ ID NO:1. The construction method further comprises the following steps: designing mutation primers, carrying out site-directed mutagenesis by using the recombinant plasmid, and transforming the mutant recombinant plasmid into escherichia coli competent cells to construct genetically engineered bacteria. Compared with the prior art, the expression quantity of the expressed mutant enzyme S392L is higher, the polymerization form is tighter, and convenience is provided for in-vitro research. The S393L mutant enzyme provided by the invention is more beneficial to regulation and control of rice characters and flowering period. After ATP is added, the peak value near 425 nm in the photoreduction process is increased, agricultural production is facilitated, and a direction is provided for improving rice characters and increasing the rice yield.

Description

Technical field: [0001] The invention belongs to the technical field of bioengineering, relates to the technical field of gene and protein engineering, in particular to a rice cryptochrome gene and a construction method thereof. Background technique: [0002] The cryptochrome / photolyase family (CPF) consists of two types of proteins: cryptochrome and photolyase. Members of this family are ubiquitous in organisms and have great differences in functions such as DNA damage repair, circadian clock regulation and transcription regulation. This family of proteins has high sequence homology and similar spatial structure, but their functions are very different. [0003] The mechanism of action of photorepair enzymes and animal cryptochrome has been studied very clearly, but there are currently two different models for the light reaction mechanism of plant cryptochrome. One of the models believes that the plant cryptochrome binds to the oxidized FAD coenzyme in the ground state, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/70C12N15/10C12N1/21C12R1/19
CPCC07K14/415C12N15/70C12N15/1031C12N2800/22C12Q2531/113
Inventor 朱国萍刘莉王鹏文斌徐蕾卞命杰陈雪霏胡德港王孟黎赵家鑫信晓蕊闫子木
Owner ANHUI NORMAL UNIV
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