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Recombinant vector CTE528, and construction method and application thereof

A technology of CTE527 and recombinant vector, which is applied in the field of genetic engineering, can solve the problems of limited effect and limit the development of microalgae genetic engineering, and achieve the effect of convenient and efficient screening

Pending Publication Date: 2022-01-04
GUANGZHOU INST OF ENERGY CONVERSION - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, promoters commonly used in plant genetic engineering, such as CaMV35S / SV40, have limited effects in microalgae
At present, most of the studies on microalgae transgenics focus on the model algae Chlamydomonas reinhardtii, and there are few studies on other microalgae, which limits the development of microalgae genetic engineering.

Method used

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  • Recombinant vector CTE528, and construction method and application thereof
  • Recombinant vector CTE528, and construction method and application thereof
  • Recombinant vector CTE528, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Construction of CTE527 carrier system

[0022] (1) Making templates for PCR amplification

[0023] After the plasmid pCAT-Control was digested with HincⅡ, two DNA fragments of about 4.1kb and 350bp were recovered by using TaKaRa Agarose GelDNA Purification Kit Ver.2.0 (Code No.DV805A) respectively, and named as CTE527template-1 and CTE527template-2.

[0024] (2) Production of target fragments

[0025] 1. PCR amplification

[0026] 1-1. Using PrimeSTAR HS DNA Polymerase (Code No. DR010S), perform PCR amplification on CTE527template-1 and CTE527template-2.

[0027] Reaction system (50μl): Template *1 (about 10ng / μl) 1μl, Primer *2 (20pmol / μl) 0.5μl, Primer *3 (20pmol / μl) 0.5μl, dNTP Mixture (2.5mM each) 4μl, 5×PrimeSTAR Buffer (Mg 2 +plus) 10μl, PrimeSTAR HS DNA Polymerase (2.5U / μl) 0.5μl, dH 2 O to 50 μl, where, *1 Represent templates CTE527template-1 and CTE527template-2, *2 Representative primers CTE527 F01, CTE527 F02, *3 Representative primers CT...

Embodiment 2

[0043] Example 2 Construction of CTE528 carrier system

[0044] (1). Purpose fragment production

[0045] 1. PCR amplification

[0046] 1-1. PCR amplification was performed on pGA1611-Ubil and pUC57-Q using PrimeSTAR HS DNA Polymerase (Code No. DR010S).

[0047] Reaction system (50μl): Template *1 (about 10ng / μl) 1μl, Primer *2 (20pmol / μl) 0.5μl, Primer *3 (20pmol / μl) 0.5μl, dNTP Mixture (2.5mM each) 4μl, 5×PrimeSTAR Buffer (Mg 2+ plus) 10μl, PrimeSTAR HS DNA Polymerase (2.5U / μl) 0.5μl, dH 2 O to make up to 50 μl. in, *1 Representative template pGA1611-Ubil, pUC57-Q, *2 Representative primers CTE528 F01, CTE528 F02, *3 Representative primers CTE528 R01, CTE528 R02.

[0048] Reaction conditions: 98°C 10sec; 98°C 10sec, 55°C 10sec, 72°C 1.5min, 30Cycles; 72°C 10min.

[0049] 1-2. Take 5 μl of the amplified products of primers CTE528 F01 / CTE528 R01 and CTE528 F02 / CTE528 R02 for 1% agarose gel electrophoresis, the results are as follows image 3 shown.

[0050] 2.PCR ...

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Abstract

The invention provides a recombinant vector CTE528, and a construction method and application thereof, and belongs to the field of gene engineering. The invention provides a vector system construction method capable of being efficiently used in dunaliella salina gene engineering by aiming at the defect that research of microalgae transgenosis mostly focuses on mode algae Chlamydomonas reininhardtii. The vector system construction method comprises the following steps of: after NdeI is added to two sides of a CAT gene, cloning the CAT gene to a pBI221-GFP vector to serve as an intermediate vector CTE527, splicing Ubi and Omega, adding Sse8387I / BamH I to two sides, and cloning the Sse8387I / BamH I to the CTE527.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a recombinant vector CTE528 and its preparation method and application. Background technique [0002] Dunaliella parva is a single-cell salina, which can grow normally in the medium containing 0.05-5M NaCl, and has strong anti-pollution ability in large-scale outdoor cultivation. It has the following advantages: photoautotrophy, strong stress resistance and simple culture conditions; no cell wall, which is conducive to genetic transformation; rich in β-carotene and glycerol, with high nutritional value. [0003] In transgenic algae, the use of selectable marker genes is necessary because only a small fraction of algal cells are successfully transformed. Selectable marker genes, usually antibiotic resistance genes, are a class of dominant markers that confer a novel phenotype on transformed algal cells. In addition, promoters commonly used in plant genetic engineer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N15/65C12N15/113C12N1/13C12R1/89
CPCC12N15/79C12N15/65C07K14/405
Inventor 秦磊尚常花朱顺妮王忠铭
Owner GUANGZHOU INST OF ENERGY CONVERSION - CHINESE ACAD OF SCI
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