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Recombinant expression vector of Chlamydomonas reinhardtii Dof (DNA binding with one finger) gene as well as construction method and application of recombinant expression vector

An expression vector, the technology of Chlamydomonas reinhardtii, which is applied in the field of recombinant expression vector and construction of the Dof gene of Chlamydomonas reinhardtii, can solve the problem that the method of regulating oil metabolism is not known, the transformation of the Dof protein of Chlamydomonas reinhardtii has not been found, and the CrDof has not yet existed. Genes and other issues, to achieve the effect of transforming algae metabolic pathways, reducing human harm and increasing yield

Inactive Publication Date: 2016-07-13
SHENZHEN UNIV
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Problems solved by technology

[0006] At present, no research on the transformation and expression of Chlamydomonas reinhardtii's own Dof protein has been found, and the way of regulating lipid metabolism is not yet known. In the prior art, there is no technical solution or method for expressing CrDof gene using Chlamydomonas reinhardtii as a host. Related reports

Method used

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  • Recombinant expression vector of Chlamydomonas reinhardtii Dof (DNA binding with one finger) gene as well as construction method and application of recombinant expression vector
  • Recombinant expression vector of Chlamydomonas reinhardtii Dof (DNA binding with one finger) gene as well as construction method and application of recombinant expression vector
  • Recombinant expression vector of Chlamydomonas reinhardtii Dof (DNA binding with one finger) gene as well as construction method and application of recombinant expression vector

Examples

Experimental program
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Effect test

Embodiment 1

[0058] [Example 1] Selection and cultivation of transgenic recipient algal strains

[0059]The CrDof gene-transformed recipient algal strain selected in the present invention is: cell wall-deficient Chlamydomonas reinhardtii (Chlamydomonas reinhardtii, cc-849, purchased from the Chlamydomonas Genetic Center of Duck University, USA). TAP was selected as the medium for the transgenic algae strain, and its preparation method was as follows: 25mL 4×Beijerincksalts (4gMgSO4.7H2O, 2gCaCl2.2H2O, 16gNH4Cl dissolved in deionized water, dilute to 1L), 2.42gTris, 1mL1M(K)PO4, 1mLTrace Trace element mixed solution (50gNa2EDTA, 1.1g(NH4)6Mo7O24, 1.57gCuSO4.5H2O, 1.61gCoCl2.6H2O, 4.99gFeSO4.7H2O, 22gZnSO4.7H2O, 5.6gMnCl2.4H2O, 11.4gH3BO3 to 1L deionized water), the The above solutions were mixed and dissolved in 975 mL of deionized water, and the pH value was adjusted to 6.98-7.02 with glacial acetic acid. The culture conditions of Chlamydomonas reinhardtii should be at a temperature of 22...

Embodiment 2

[0060] [Example 2] Obtaining the target fragment of CrDof gene

[0061] 1. Extraction of total RNA from Chlamydomonas reinhardtii and acquisition of full-length cDNA

[0062] The total RNA of Chlamydomonas reinhardtii cc-849 was extracted with the Takara total RNA extraction kit, and the extraction steps are detailed in the instructions of the RNA extraction kit.

[0063] Using the total RNA as a template, reverse transcribe the total RNA of Chlamydomonas reinhardtii with the ReverseTranscriptaseM-MLV (RNaseH-) enzyme of Dalian Baobiology to obtain the first strand of cDNA. For specific steps, see the instructions for use of the enzyme.

[0064] 2. Cloning of the target fragment of CrDof gene

[0065] Using the cDNA obtained by reverse transcription of Chlamydomonas reinhardtii cc-849 total RNA as a template, according to the known CrDof gene (Genbank accession number: XM_001696866.1), design specific primers and add PmacI at both ends of the upstream and downstream primers ...

Embodiment 3

[0072] [Example 3] Construction of the CrDof gene Chlamydomonas reinhardtii genetic expression vector

[0073] The nuclear genetic transformation carrier used in the present invention is pJD, such as figure 2 As shown, it is generated by adding the luciferase reporter gene to the genetic transformation plasmid pH124 of Chlamydomonas reinhardtii preserved in our laboratory. It not only contains the HSP70A-RBCS2 promoter sequence and RBCS2 terminator sequence, but also carries HindIII, Nhel, Apol , EcoRI, PmacI, Pstl, KpnI and other multiple cloning sites, which can be inserted into exogenous target genes and expressed in Chlamydomonas reinhardtii. Moreover, the plasmid PJD contains the expression cassette RBCS2::ble::RBCS2, which can make the transformed host cells acquire resistance to bleomycin, and is used as a selection marker for Chlamydomonas transformation in the present invention. The plasmid pJD124 is published in the specification of the application number 201410521...

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Abstract

The invention discloses a recombinant expression vector of a Chlamydomonas reinhardtii Dof (DNA binding with one finger) gene as well as a construction method and an application of the recombinant expression vector. The recombinant expression vector suitable for overexpressing the Dof gene in Chlamydomonas reinhardtii is constructed and is transferred into a host, namely, a Chlamydomonas reinhardtii cell, and genetically modified Chlamydomonas reinhardtii is obtained. The expression quantity of CrDof in the Chlamydomonas reinhardtii is remarkably increased; the overexpressed CrDof regulates the expression quantity of lipid metabolism related genes, and the lipid content of genetically modified Chlamydomonas reinhardtii cells is remarkably increased. The recombinant expression vector of the Chlamydomonas reinhardtii Dof gene as well as the construction method and the application of the recombinant expression vector has great significance in enhancement of lipid metabolism gene regulation and increase of microalgae lipid content, and has greater application value and prospect in the technical field of genetic engineering.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a recombinant expression vector of Chlamydomonas reinhardtii Dof gene and its construction method and application. Background technique [0002] With the massive consumption of fossil energy, the development of bio-energy has been paid more and more attention. Compared with fossil fuels, biodiesel has the advantages of being renewable and not polluting the environment. The raw material for biodiesel production has changed from higher land plants to microalgae in recent years. Because microalgae has the outstanding characteristics of not occupying cultivated land and high oil production rate among many raw materials. Triacylglycerol (TAG) and other compounds in microalgae can be used to produce biodiesel. Some oil-producing microalgae have very high oil content. The oil content in Botryococcus braunii is 57%-64%, and the oil content in Neochlorisoleoabundans is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N15/66C12N1/13C12R1/89
CPCC12N15/79C07K14/415C12N15/66C12N2800/60C12N2830/34C12N2830/36
Inventor 黄瑛谢鑫凤贾彬胡章立
Owner SHENZHEN UNIV
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