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Chlamydomonas reinhardtii instability domain gene, protein expression regulation method based on instability domain and application of chlamydomonas reinhardtii instability domain gene

A rhine coat and gene technology, applied in the field of genetic engineering, can solve the problems of low expression, silence, limited means, etc.

Active Publication Date: 2020-05-15
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although there have been reports on the expression of foreign genes in Chlamydomonas reinhardtii, the expression level is difficult to control, and the expression level is usually low or even "gene silencing", which seriously affects the in-depth research on Chlamydomonas reinhardtii
At present, there are limited means to regulate the expression of exogenous genes in Chlamydomonas reinhardtii, and most of them are controlled by strong promoters, which belong to transcriptional level regulation

Method used

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  • Chlamydomonas reinhardtii instability domain gene, protein expression regulation method based on instability domain and application of chlamydomonas reinhardtii instability domain gene
  • Chlamydomonas reinhardtii instability domain gene, protein expression regulation method based on instability domain and application of chlamydomonas reinhardtii instability domain gene
  • Chlamydomonas reinhardtii instability domain gene, protein expression regulation method based on instability domain and application of chlamydomonas reinhardtii instability domain gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1 Design and synthesis of Chlamydomonas reinhardtii instability domain gene crDi

[0099] Take the mutant rapamycin-binding protein containing point mutation sites F36V and L106P as the optimization object, select the codon with the highest frequency according to the codon preference of Chlamydomonas reinhardtii, and redesign the coding sequence; use ChlamySequence Optimizer (https: / / www.mathworks.com / matlabcentral / fileexchange / 65416-iddoweiner-coding-sequence-optimization-for-chlamydomonas-reinhardtii) software to further optimize the coding sequence, optimize the secondary structure of 5'UTR, and remove redundant potential content Containing the splicing site, obtaining the mutant type rapamycin binding protein gene shown in SEQ ID NO:2;

[0100] Calculated from the translation initiation site of the gene shown in SEQ ID NO: 2, the Chlamydomonas reinhardtii RBCS2 intron 1 (SEQ ID NO: 3 ), obtain the final optimized Chlamydomonas reinhardtii instability domai...

Embodiment 2

[0102] Example 2 Regulating the Expression of Chlamydomonas reinhardtii Aminoglycoside Phosphotransferase Using the Destabilizing Domain

[0103] (1) Construction of destabilizing domain-aminoglycoside phosphotransferase fusion gene recombinant vector

[0104] Using the synthesized crDi gene as a template, primers DParF (SEQ ID NO: 5) and DParR (SEQ ID NO: 6) were amplified at 62°C to obtain the destabilizing domain gene; the plasmid pOPt-mVenus-paro (source Based on the literature Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with aversatile, modular vector toolkit) as a template, primers ParF (SEQ ID NO: 7) and ParR (SEQ ID NO: 8) were used to amplify at 62°C to obtain aminoglycoside phosphotransfer Enzyme gene: using a mixture of 50ng of destabilizing domain gene and aminoglycoside phosphotransferase gene as a template, using primers DParF and ParR to amplify at 63°C to obtain the fusion gene crDi-pa;

[0105] After digesting the fusion gene crDi-p...

Embodiment 3

[0131] Example 3 Regulating the expression of Chlamydomonas reinhardtii luciferase by destabilizing domain

[0132] (1) Design and synthesis of Chlamydomonas reinhardtii luciferase gene

[0133] In this embodiment, the luciferase gene is first optimized, and Gaussia princeps luciferase (amino acid sequence shown in SEQ ID NO: 9) is used as the optimization object, and the codon with the highest frequency of use is selected according to the codon preference of Chlamydomonas reinhardtii ; Use Chlamy Sequence Optimizer (https: / / www.mathworks.com / matlabcentral / fileexchange / 65416-iddoweiner-coding-sequence-optimization-for-chlamydomonas-reinhardtii) software to further optimize the coding sequence and optimize the secondary of 5'UTR structure, remove redundant potential intron splicing sites; insert Chlamydomonas reinhardtii RBCS2 intron 1 (SEQ ID NO: 10 ), insert Chlamydomonas reinhardtii RBCS2 intron 2 (SEQ ID NO: 11), which has the effect of enhancing the expression of exogenou...

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Abstract

The invention provides a chlamydomonas reinhardtii instability domain gene, a protein expression regulation method based on an instability domain and application of the chlamydomonas reinhardtii instability domain gene. The chlamydomonas reinhardtii instability domain gene includes a mutant rapamycin binding protein gene and chlamydomonas reinhardtii RBCS2 intron 1; and the instability domain geneincludes a nucleic acid sequence shown in SEQ ID NO:1. Expression product instability domain protein of the instability domain gene constructed by binding the mutant rapamycin binding protein gene and the chlamydomonas reinhardtii RBCS2 intron 1 in chlamydomonas reinhardtii cells is extremely unstable and can be rapidly degraded by intracellular proteases to enable fusion protein containing an instability domain to be degraded; and the stability of the instability domain can be improved by small molecule inducer Shield-1, so that the fusion protein is stably and continuously expressed, and the purpose of regulating protein expression in chlamydomonas reinhardtii is realized.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a Chlamydomonas reinhardtii destabilization domain gene, a destabilization domain-based protein expression regulation method and an application thereof. Background technique [0002] The unicellular eukaryotic microalga Chlamydomonas reinhardtii is called "green yeast" or "photosynthetic yeast". It has the characteristics of special structure, easy culture, fast growth, clear genetic background and easy transformation. model organisms. Chlamydomonas reinhardtii has three sets of genetic systems including nuclear chromosomes, mitochondria and chloroplasts, all of which can undergo genetic transformation (Boynton J E, Gillham N W, Harris E H, Hosler J P, Johnson A M, Jones A R, Randolph-Anderson B L, Robertson D, Klein T M , Shark K B, Et A.1988.Chloroplast transformation in Chlamydomonas with high velocity microprojectiles.Science, 240(4858):1534-1538), is an ideal tran...

Claims

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Application Information

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IPC IPC(8): C07K14/405C12N15/31C12N15/62C12N1/13C12N15/79
CPCC07K14/405C12N15/79C12N9/0071C12N9/0069C12Y114/14003C12Y113/12C12N9/12C07K2319/00
Inventor 贾彬胡章立黄瑛李欣怡陈家颖
Owner SHENZHEN UNIV
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