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A kind of rice cryptochrome site-directed mutein and its construction method

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

Active Publication Date: 2022-03-15
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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  • A kind of rice cryptochrome site-directed mutein and its construction method
  • A kind of rice cryptochrome site-directed mutein and its construction method
  • A kind of rice cryptochrome site-directed mutein and its construction method

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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 mutant protein and a construction method thereof. The invention obtains a gene sequence from a gene bank, performs artificial codon optimization and truncates the carboxyl terminal of the protein, and then obtains the target gene sequence and connects it to the pET22b plasmid. A recombinant expression plasmid was constructed. The truncated gene sequence is shown in SEQ ID NO:1. Design mutation primers, use recombinant plasmids for site-directed mutagenesis, and transform the mutant recombinant plasmids into Escherichia coli competent cells to construct engineering bacteria; compared with the prior art, the present invention has higher expression of mutant enzyme S392L, The aggregated form is more compact, which facilitates in vitro studies. The S393L mutant enzyme of the invention is more conducive to the regulation of rice characters and flowering cycle. After adding ATP, the peak near 425nm in the photoreduction process increases, which is beneficial to agricultural production and provides a direction for improving rice traits and increasing 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 Patents(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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