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Method for ultrahigh-level and targeting expression of salmon calcitonin protein in oleosin of transgenic rape by using chromosome substitution technology

A salmon calcitonin and gene technology, which is applied in the field of specific expression technology with vegetable oil bodies, can solve the problems of low probability of homologous recombination and difficulty in transgenic plants

Inactive Publication Date: 2012-03-14
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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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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  • Method for ultrahigh-level and targeting expression of salmon calcitonin protein in oleosin of transgenic rape by using chromosome substitution technology
  • Method for ultrahigh-level and targeting expression of salmon calcitonin protein in oleosin of transgenic rape by using chromosome substitution technology
  • Method for ultrahigh-level and targeting expression of salmon calcitonin protein in oleosin of transgenic rape by using chromosome substitution technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, the cloning of Saccharomyces cerevisiae RAD54 gene

[0043] 1. Extraction of Saccharomyces cerevisiae genome

[0044] 1) Pick a single colony of yeast (purchased from China Industrial Microbiology Culture Collection Management Center, No. 31141), inoculate in 10ml YPD (1% yeast extract, 2% glucose, 1.5% agar powder, 2% peptone) liquid medium Incubate overnight in a shaker at 30°C;

[0045] 2) Take 1ml of the yeast liquid cultured overnight, centrifuge at 5000rpm for 5min, remove the supernatant, add 1ml of sterile water, resuspend the pellet, centrifuge at 5000rpm for 1min, remove the supernatant, add 200μl of bacteria-breaking buffer (1% SDS, 2 % TritionX-100, 100mM NaCl, 10mM Tris-Cl pH8.0, 1mM EDTA), shake vigorously.

[0046] 3) Add 200 μl phenol / chloroform / isoamyl alcohol (volume ratio 25:24:1), shake vigorously, centrifuge at 12,000 rpm for 5 minutes, take the supernatant, add an equal volume of chloroform, shake it upside down, centrifuge at 12,00...

Embodiment 2

[0055] Embodiment 2, the construction of RAD54 plant expression vector pRAD3301

[0056] 1. Construction of intermediate vector p3301

[0057] The expression cassette of gene pRAD3301 constructed in this example includes the following gene expression regulatory elements: 35S promoter, RAD54 gene and NOS terminator. First, pTΩ4A was double-digested with restriction endonucleases EcoRI and HindIII to recover a 999bp fragment, which was ligated with the same digested plant vector pCAMBIA3301 to obtain an intermediate vector named p3301. EcoRI and HindIII double enzyme digestion was used for verification, and the enzyme digestion identification results were as follows: image 3 As shown, the results prove that the pTΩ4A fragment has been linked to pCAMBIA3301.

[0058] 2. Construction of RAD54 plant expression vector pRAD3301

[0059] Plasmid RAD54 was digested with restriction endonuclease PstI, and a 2700bp fragment was recovered, which was ligated with vector p3301 cut with ...

Embodiment 3

[0060] Embodiment 3, the cloning of rapeseed oleosin gene 5' upstream sequence

[0061] Plant material: Rapeseed variety Zhongshuang No. 4, when seedlings are 4 weeks old.

[0062] 1. Design of chromosomal walking primers for the 5' upstream sequence of oleosin gene

[0063] 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.

[0064] 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

[0065] GSP1: 5'-TAATCTCCGCCGCGTCTCTCTCTAAAC-3'

[0066] GSP2: 5'-CC...

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Abstract

The invention provides a method for realizing targeting substitution by comprehensively using specific expression and gene targeting of plant oleosin and for ultrahigh-level expression of salmon calcitonin protein in transgenic rapeseeds. According to the invention, rape 5' UTR sequence is obtained by cloning 1900 bp with Race; an inherent endogenous sequence in the genome of rape is substituted by 5' UTR sequence of rape oleosin gene--sesame oleosin gene+CT fusion protein gene'--Pnos-NPTII-Tnos--3' UTR sequence of rape oleosin gene; a transgenic rape plant and a transgenic rape line are obtained by using the method of inplanta. According to detection results, the endogenous sequence in the genome of rape is substituted by a new sequence, which enables targeting expression of a target gene to be successfully realized and ultrahigh-level expression of salmon calcitonin protein in transgenic rapeseeds to be achieved. The method has the following remarkable advantages: the expression level of target protein is high, and the expressed protein is easy to store and purify.

Description

technical field [0001] The present invention relates to a method for chromosome replacement by comprehensively utilizing plant oil body specific expression technology and gene targeting technology, in particular to the use of Race technology to clone the 1900 bp rapeseed 5'UTR sequence and yeast chromosome reconstruction protein ScRAD54, named "Rapeseed oleosin gene 5' UTR sequence -'Sesame oleosin+CT fusion protein gene'-Pnos-NPTII-Tnos-rape oleosin gene 3'UTR sequence"replaces the inherent endogenous sequence in the rapeseed genome"rape oleosin gene 5'UTR sequence-rape oleosin gene-rape oleosin Gene 3'UTR sequence", a method for super-high expression of salmon calcitonin protein in transgenic rapeseed oil bodies. 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 se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/12C12N15/31C12N15/63C12N15/82C12N5/10C12N1/15C12N1/19C12N1/21C07K14/575A01H5/00
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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