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Stentor coeruleus RNA interference expression vector as well as construction method and application thereof

A technology of expression vectors and hornworms, applied in the direction of DNA / RNA fragments, applications, recombinant DNA technology, etc., can solve the problems of regulating the expression of foreign genes

Active Publication Date: 2020-07-31
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there is no research on the regulation of exogenous gene expression based on the expression system of Synechocystis sp. PCC6803. It is used in the application of RNAi expression vectors in trumpet worms and inhibits the expression of target genes in trumpet worms.

Method used

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  • Stentor coeruleus RNA interference expression vector as well as construction method and application thereof
  • Stentor coeruleus RNA interference expression vector as well as construction method and application thereof
  • Stentor coeruleus RNA interference expression vector as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] Example 1: Construction of plasmid pSCT3C

[0117] 1. Extraction of Synechocystis PCC6803 DNA by CTAB method

[0118] (1) Add 1mL OD 580 =1 Synechocystis PCC6803 was transferred to an appropriate amount of BG11 medium, placed at a temperature of 30°C, shaking at a speed of 100rpm, and 40μmol photons m -2 the s -1 Cultured under continuous light conditions for 3 days;

[0119] (2) Take 2 mL of Synechocystis sp. PCC6803 in a centrifuge tube, centrifuge at 12,000 rpm in an Eppendorf centrifuge for 2 min (at room temperature), collect the algae precipitate, and discard the supernatant with a 1 mL pipette (product of Eppendorf);

[0120] (3) Prepare CTAB extraction buffer: 2% CTAB + 100mM Tris-HCl + 20mM EDTA (pH 8.0) + 1.4M NaCl + 2% β-mercaptoethanol (ready to use);

[0121] (4) Add 567 μL of TE buffer solution to the algae pellet, pipette repeatedly to resuspend it, add 30 μL of 10% SDS and 15 μL of proteinase K, mix well, and incubate at 37°C for 1 hour;

[0122] (5...

Embodiment 2

[0174] Example 2: Construction of plasmids pSCT3C-Mob1-633, pSCT3C-Mob1-317 and pSCT3C-Mob1-347

[0175] 1. Kit method for extracting the DNA of Trumpeter cerevisiae

[0176] (1) The blue trumpet worm is cultured in a petri dish (90mm in diameter) containing 30mL of MSM medium, at a temperature of 20°C, in a dark environment, and under the condition of supplementing Clostridium longum as food, and grows for 2-3 days;

[0177] (2) The petri dish was placed under a dissecting microscope, and 10 T. cerevisiae cells were sucked into the PCR tube with a mouth pipette for use, and the excess liquid was sucked away;

[0178] (3) For the process of extracting the DNA of Trumpetinus cerevisiae, refer to the instructions of the Shanghai Beiman REDExtract-Amp Tissue PCR Kit kit, dissolve the DNA in sterile Milli-Q water, and freeze it at -20°C.

[0179] 2. PCR amplification of different segments of the Mob1 gene of Trumpeter cerevisiae

[0180] (1) according to the whole genome sequenc...

Embodiment 3

[0212] Example 3: Transfection of plasmids pSCT3C-Mob1-633, pSCT3C-Mob1-317 and pSCT3C-Mob1-347 into Synechocystis sp. PCC6803

[0213] 1. Cell culture

[0214] (1) Culture Synechocystis sp. PCC6803 in 10mL BG11 medium, temperature 30°C, shaking at 100rpm and 40μmol photons m -2 the s -1 Under continuous light conditions, until the concentration of algal liquid grows to OD 580 = 1;

[0215] (2) Escherichia coli DH10B containing plasmids pSCT3C-Mob1-633, pSCT3C-Mob1-317 and pSCT3C-Mob1-347 were cultured in 10 mL LB medium (containing final concentrations of 50 μg / mL Car, 25 μg / mL Cm and 50 μg / mL mL Km), the temperature is 37°C, the rotation speed is 200rpm, and there is no light until the concentration of the bacterial solution grows to OD 600 =1.

[0216] 2. Joint transfer

[0217] (1) Collect 2 mL of Synechocystis PCC6803 and Escherichia coli DH10B in the logarithmic growth phase respectively in a centrifuge tube, centrifuge at 13,000 rpm for 2 min (room temperature), col...

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Abstract

The invention discloses a Stentor coeruleus RNA interference expression vector as well as a construction method and application thereof. The method comprises the following steps of 1) obtaining a pSCTGA plasmid skeleton by using a known sequence of plasmid pSCTGA; (2) amplifying a promoter of a C-phycocyanin beta subunit through PCR (Polymerase Chain Reaction) by using synechocystis PCC6803 genomeDNA (Deoxyribose Nucleic Acid); 3) amplifying multiple cloning sites of the plasmid through PCR (Polymerase Chain Reaction) by using the plasmid L4440; and 4) finally constructing a RNAi expression vector pSCT3C by using a DNA fragment obtained by PCR amplification through homologous recombinant fusion PCR and a one-step cloning method. The method comprises the following steps of transfecting a RNAi expression vector containing a target gene into an Escherichia coli DH10B strain by using a calcium chloride method, and transfecting the Escherichia coli DH10B strain into synechocystis PCC6803 by using a conjugational transfer method to successfully obtain a positive monoclonal algae strain, and then interfering the expression of a target gene in the Stentor coendeus in a feeding mode. In conclusion, the method is easy to implement and convenient to operate, and has the feasibility of being applied to gene interference of other ciliates.

Description

【Technical field】 [0001] The invention belongs to the field of biotechnology, and relates to a construction method and application of a hornworm RNAi expression vector. 【Background technique】 [0002] The blue trumpet beetle is a relatively common ciliate in my country. It belongs to the ciliated phylum Polymembrane Heterochaetes in protozoa, and is widely distributed in freshwater environments all over the world. The cell shape is conical or trumpet-shaped; there is a chain bead-shaped large nucleus and many small nuclei adjacent to the large nucleus; the front of the cell is wide and has mouthparts; the appendages are located in the narrow posterior. When the worm stretches, the body length can reach 1-2mm. Rows of blue-green pigment particles are distributed between the moving body cords on the surface and along the longitudinal axis, which is an important part of plankton in fresh water. Different from the model organism Tetrahymena, the blue trumpet worm cannot use gen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/65A01N57/16A01P7/04
CPCC12N15/113C12N15/8218C12N15/65A01N57/16C12N2310/14
Inventor 缪炜韦薇柴小翠张巨源张承才
Owner INST OF AQUATIC LIFE ACAD SINICA
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