Cloning of rape Oleosin5'UTR sequence and application of Oleosin5'UTR sequence in elaioplast targeting expression

A technology of rapeseed in the sequence list, applied in the direction of application, DNA/RNA fragments, using vectors to introduce foreign genetic material, etc., can solve the problems of difficult transgenic plants and low probability of homologous recombination

Active Publication Date: 2012-03-14
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the principle and technical route of gene targeting are not complicated, it is still very difficult to obtain site-specific integration of transgenic plants due to the very low probability of homologous recombination between exogenous DNA and target cell DNA sequences in higher eukaryotic cells. Work

Method used

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  • Cloning of rape Oleosin5'UTR sequence and application of Oleosin5'UTR sequence in elaioplast targeting expression
  • Cloning of rape Oleosin5'UTR sequence and application of Oleosin5'UTR sequence in elaioplast targeting expression
  • Cloning of rape Oleosin5'UTR sequence and application of Oleosin5'UTR sequence in elaioplast targeting expression

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Embodiment 1

[0032] Embodiment 1, the cloning of rapeseed oleosin gene 5' upstream sequence

[0033] Plant material: Rapeseed variety Zhongshuang No. 4, when seedlings are 4 weeks old.

[0034] 1. Design of chromosomal walking primers for the 5' upstream sequence of oleosin gene

[0035] The size of the homologous sequence is an important factor affecting the efficiency of gene targeting. When constructing a gene targeting vector, it is generally required to have a DNA homologous sequence of about 2 kb, but the 5' upstream sequence of the oleosin gene (sequence number M63985) registered on Genbank The size is only 944bp, for which, chromosome walking is needed to further clone the upstream sequence of oleosin.

[0036] According to the 5'UTR sequence of oleosin gene registered on Genbank and GenomeWalker TM According to the requirements of Universal Kit, design 2 specific primers, the primer sequences are as follows

[0037] GSP1: 5'-TAATCTCCGCCGCGTCTCTCTCTAAAC-3'

[0038] GSP2: 5'-CCAA...

Embodiment 2

[0062] Example 2, Cloning of Gene Targeting Vector Elements

[0063] Experimental materials: Rapeseed Zhongshuang 4, Escherichia coli JM109.

[0064] 1. Cloning of the whole gene of rapeseed oil body protein Oleosin

[0065] In the present invention, the full length of oleosin gene (including exon1+intron1+exon2+intron2+polyA) is used as another homologous recombination sequence for gene targeting. Primers were designed according to the oleosin gene registered on Genbank (sequence number M63985), and the oleosin gene was amplified by PCR from the rapeseed genome. Specific steps are as follows:

[0066] 1) Rapeseed Genomic DNA Extraction

[0067] Take 25-100 mg of fresh rapeseed leaves, add liquid nitrogen to grind into powder, and transfer to a 1.5 mL centrifuge tube. Add 450μL extraction buffer (100mmol·L -1 Tris-Cl, pH 8.0, 500mmol·L -1 NaCl, 10mmol L -1 β-mercaptoethanol, 50mmol·L -1 EDTA, pH 8.0) and 100 μL 10% SDS, shake vigorously. 65℃ water bath for 30min, add ...

Embodiment 3

[0114] Embodiment 3, the acquisition of transgenic rapeseed

[0115] Plant material: Rapeseed Zhongshuang No. 4

[0116] 1. Plant expression vectors pRAD3301 and pGTO

[0117] The constructed Agrobacterium tumefaciens LBA4404 was transformed by heat shock method. The specific method is as follows:

[0118] 1) Using Promega's WizardTM plus minipreps DNA purification system, extract plasmid DNA and add it to 100 μ LBA4404 competent cells, mix well, and ice-bath for 5 minutes.

[0119] 2) Freeze the centrifuge tube in liquid nitrogen for 5 minutes, and quickly transfer to a 37°C water bath for 5 minutes.

[0120] 3) Add 1 mL of YEB liquid medium, and recover on a shaker at 28° C. at 250 rpm for 4-5 hours.

[0121]4) Take an appropriate amount of bacterial liquid and spread it on the YEB solid medium containing 50 mg / L rifampicin and 100 mg / L Kan (kanamycin), and culture it at 28°C for 24-48 hours.

[0122] Pick a single colony and extract plasmid DNA by alkali lysis method t...

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Abstract

The invention discloses a 5'UTR sequence of a rape oleosin gene and an application of the 5'UTR sequence in the gene targeting expression. The 5'UTR sequence of the oleosin gene is one of the following nucleotide sequences: 1, a DNA sequence represented by SEQ ID NO:1 in a sequence table; and 2, a nucleotide sequence which can be hybridized with the DNA sequence limited by the SEQ ID NO:1 in the sequence table under highly strict conditions. The sequence represented by SEQ ID NO:1 and other elements construct a plant expression vector pGOT which comprises the following four complete expression boxes: a P35S-codA-Tnos expression box, a 1900bp rape oleosin gene 5'UTR-"sesame oleosin + CT fusion protein gene"-Pnos-NPTII-Tnos expression box, a rape oleosin gene 3'DNA sequence expression box, and a P35S-codA-Tnos expression box. Transgenic rape plants and lines are obtained through an in planta method. According to the invention, a gene targeting technology is utilized to insert an exogenous target gene into the 3' terminus of the oleosin gene, so the expression of the exogenous gene in plants is greatly improved, and a new platform is established for the massive production of a medicinal protein with a bioreactor.

Description

technical field [0001] The present invention relates to the comprehensive utilization of plant oil body specific expression technology and gene targeting technology to carry out homologous chromosome-directed recombination, especially relates to the cloning of UTR sequence at the 5' end of Oleosin gene derived from Brassica napus oil body protein and its application in gene targeting technology . Background technique [0002] Gene targeting was produced in the late 1970s and early 1980s. It refers to the introduction of exogenous target genes carrying selectable markers into recipient cells by certain experimental methods, and then through exogenous DNA sequences and receptor cell chromosomes. Recombination occurs between homologous DNA sequences, and finally the exogenous DNA is integrated into a predetermined site in the genome of the recipient cell, changing the genetic characteristics of the cell, or a site-directed mutation is performed on a predetermined receptor site,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63C12N15/82C12N5/10C12N1/15C12N1/19C12N1/21A01H5/00
Inventor 刘昱辉李珊珊李梅贾士荣
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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