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Light regulated gene expression vector for dunaliella salina and preparation method and application thereof

A technology of gene expression and light regulation, which is applied in the field of light regulation gene expression vectors of Salina salina, can solve the problems of high expression regulation and low expression efficiency of exogenous genes, and achieves high expression efficiency, fast expression, high efficiency and stability, and easy control. Effect

Inactive Publication Date: 2016-03-30
XINXIANG MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the low expression efficiency of exogenous genes and the inability to regulate expression are still the main bottlenecks restricting Salina salina as a host for efficient expression of exogenous genes.

Method used

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  • Light regulated gene expression vector for dunaliella salina and preparation method and application thereof
  • Light regulated gene expression vector for dunaliella salina and preparation method and application thereof
  • Light regulated gene expression vector for dunaliella salina and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 2

[0059] Construction of embodiment 2 intermediate carrier pU5-EGFP

[0060] The pEGFP-C1 and pU5-Cre vectors were digested with NheI and BamHI, respectively, and the EGFP gene DNA fragment and the linear pU5-Cre vector fragment were recovered and purified. The linear vector and the EGFP gene fragment were ligated at a ratio of 1:5 with a ligation reagent at 16°C, transformed, cultured, picked a single colony to expand culture, and extracted a plasmid according to the method in Example 1. The recombinant plasmid was identified by double digestion with NheI and BamHI (to obtain a linear vector and an EGFP fragment of about 800bp), and the result of the digestion was electrophoresed on a 1% agarose gel (such as Figure 4 shown). The correctly identified recombinant plasmids were then sequenced to confirm the correctness of the EGFP gene sequence. The recombinant plasmid vector pU5-EGFP ( image 3 ).

Embodiment 3

[0061] Example 3 Construction of eukaryotic expression vector pU5DSJ1

[0062] The cloning vector in Example 1 and the pU5-EGFP vector constructed in Example 2 were double-digested with NotI and NruI respectively, and the light-sensitive transcription factor GAVPO expression cassette and linear pU5-EGFP vector, linear vector and gene fragment were recovered and purified after digestion Ligate at 16°C with the ligation reagent at a ratio of 1:5, transform, culture, pick a single colony to expand culture, and extract the plasmid according to the method in Example 1. Recombinant plasmids were identified by NotI+SacI double digestion or HindIII single digestion, and the digestion results were electrophoresed on 1% agarose gel (such as Figure 6 shown). The correctly identified recombinant plasmids will be sequenced again to confirm the correctness of the linked gene sequences. The recombinant expression vector pU5DSJ1 ( Figure 5 ).

Embodiment 4

[0063] Embodiment 4 glass bead method transforms salina

[0064] The eukaryotic expression vector pU5DSJ1 plasmid successfully constructed in Example 3 was extracted with an endotoxin-free plasmid extraction kit, and the plasmid was introduced into Salina cells by glass bead method for expression verification. The specific operation method is as follows:

[0065] Salina cells were counted when they reached the logarithmic growth phase (the concentration was 10 6 cells / ml), take 800 μl of salina algae liquid, centrifuge at 500 rpm for 2 minutes, discard the supernatant, gently mix the salina cells with 1 ml of fresh salina liquid medium, and then centrifuge, repeat three times. Salina cells were resuspended in 800 μl liquid medium to a final concentration of 10 5 cells / ml. Add 150 μl PEG6000 to make the final concentration 3.5-5%, mix gently and add 90 μl plasmid DNA (0.55 μg / μl)), then add 300 μg high temperature and high pressure sterilized glass beads (pre-acid-treated, di...

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Abstract

The invention relates to a light regulated gene expression vector, and particularly, relates to a light regulated gene expression vector suitable for dunaliella salina and an application thereof. The vector includes two gene expression cassettes: 1) a photosensitive transcription factor GAVPO expression cassette including a dunaliella salina actin promoter and a photosensitive transcription factor GAVPO; and 2) a target gene expression cassette regulated by the photosensitive transcription factor GAVPO. The invention further provides the application of the light regulated gene expression vector in expression of a target gene in dunaliella salina through light regulation, dunaliella salina cells transfected with the light regulated gene expression vector can effectively regulate expression of the target gene through blue light, and the expression efficiency is high.

Description

technical field [0001] The invention relates to a light-regulated gene expression carrier, in particular to a light-regulated gene expression carrier suitable for salina. Background technique [0002] Gene expression systems are mainly divided into two types: constitutive and inducible. The former is used for gene expression that cannot be regulated continuously, while the inducible gene expression system can precisely control the on / off of gene expression. At present, there are a variety of gene expression systems induced by chemical substances, but these expression systems cannot specifically regulate specific cells or tissues, and have unavoidable potential toxicity. Wang Xue et al. developed a light-regulated gene expression system - LighOn system (WangX, ChengX, YangY. Spatiotemporal control of gene expression by alight-switchable transgene system. NatrueMethods, 2012, 9(3): 266-269). The system contains two expression vectors, and the expression of the target gene is ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/66C12N1/13C12R1/89
CPCC12N15/82C12N15/66
Inventor 贾岩龙高利洁崔柳苏郜惠苹郭潇王喜成邱乐乐武俊芳
Owner XINXIANG MEDICAL UNIV
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