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A kind of long oyster tyrosinase1 promoter and its recombinant expression vector and application

A technology of expression vector and promoter, applied in the field of molecular biology

Active Publication Date: 2020-12-08
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] In early mollusc development studies, no promoters are currently available

Method used

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  • A kind of long oyster tyrosinase1 promoter and its recombinant expression vector and application
  • A kind of long oyster tyrosinase1 promoter and its recombinant expression vector and application
  • A kind of long oyster tyrosinase1 promoter and its recombinant expression vector and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Promoter amplification:

[0020] Genomic DNA of the long oyster (Crassostrea gigas) was extracted using a marine animal tissue genomic DNA extraction kit (TIANGEN, catalog number: DP324), according to the long oyster genome database ( http: / / www.oysterdb.com ) to design long oyster gene promoter-specific primers.

[0021] (Upstream primer Cgtyr1PmF, add 16 bases of vector pEGFP-1 homologous end 1, downstream primer Cgtyr1PmR, add 16 bases of vector pEGFP-1 homologous end 2)

[0022] Using the gDNA of the long oyster extracted above as a template, use Premix Taq TM (LA Taq TM Version 2.0) (Takara, catalog number: RR900Q) polymerase for PCR amplification. As shown in Table 1.

[0023] Table 1 PCR system for gene promoter amplification

[0024] composition volume Premix Taq(LA TaqVersion 2.0) 25μl DNA template 2μl Upstream primer Cgtyr1PmF (10μM) 2μl Downstream primer Cgtyr1PmR (10μM) 2μl Ultra-pure water 19μl

[0...

Embodiment 2

[0029] cgtyr1::GFP recombinant vector construction:

[0030] The pEGFP-1 plasmid (provided by Zhang Xiaojun, Institute of Oceanology, Chinese Academy of Sciences, can also be ordered from Youbio (http: / / www.youbio.cn / ), article number: VT1106, its base sequence is shown in sequence 2) with BamHI Enzyme digestion and linearization, the PCR recovery product cgtyr1 obtained as above was activated and linearized pEGFP-1 was In-Fusion ligated (In-Fusion HD Cloning Kit, TaKaRa, catalog number: 638909), transformed into Escherichia coli, and picked positive Transformant sequencing (shown as the cgtyr1 promoter sequence) proved to be accurate, specifically:

[0031] In-Fusion connection system:

[0032] composition volume PCR recovery product (cgtyr1 promoter) 1.5μl Ultra-pure water 3.5μl Linearized pEGFP-1 3μl Infusion Mix 2μl total capacity 10μl

[0033] The two fragments described above were mixed according to the connection syst...

Embodiment 3

[0036] Expression of GFP in early mollusk larvae

[0037] The extracted recombinant vector cgtyr1::GFP was formulated into injection solution: recombinant vector 50ng / uL, phenol red 0.05%.

[0038] Put the sexually mature scallops into 70ml fresh seawater culture cups (one per cup), and collect sperm and egg cells after spawning and ejaculation.

[0039] Transfer the collected ovum cells into a petri dish filled with 5ml of fresh sea water, and place them under a dissecting microscope. The input air pressure of the microinjector (WARNER, catalog number: PLI-100A) was maintained at 200 Pa. Use a Microloader (eppendorf, catalog number: 5242956003) to draw 0.5ul injection solution into the injection needle Femtotip II (eppendorf, catalog number: 5242957000), and connect the injection needle to the syringe. Set injection parameters: input air pressure 200pa, balance air pressure 3pa, injection pressure 15pa, injection time 0.08s. The injection volume is indicated by the phenol ...

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Abstract

The invention relates to the technology of molecular biology, in particular to a crassostrea gigas tyrosinase 1 promoter and a recombinant expression vector and application thereof. The promoter is abase sequence as shown in SEQ ID No.1, and the base sequence has 80% or more homology and has the function of the promoter. Through injection of the recombinant expression vector, expression of a target gene is started, and the target gene can be expressed in shellfish larvae.

Description

technical field [0001] The invention relates to molecular biology technology, in particular to a long oyster tyrosinase 1 promoter and its recombinant expression vector and application. Background technique [0002] In the study of early mollusk development, no promoters are currently available. Tyrosinase is a copper-containing oxidase that is involved in numerous biochemical processes, including shell formation, pigment synthesis, and immune responses. The tyrosinase 1 promoter can drive the expression of a reporter gene in shellfish larvae, which facilitates the developmental study of molluscs. Contents of the invention [0003] The object of the present invention is to provide a long oyster tyrosinase 1 promoter and its recombinant expression vector and application. [0004] In order to achieve the above object, the technical solution adopted by the present invention is: [0005] A long oyster tyrosinase 1 promoter, the promoter is the base sequence shown in SEQ ID ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85
CPCA01K2267/01C07K14/43504C12N15/8509
Inventor 郇聘王倩刘保忠谭素建王鸿霞
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI