CN112980842B - A non-coding nucleotide sequence and its application in improving the expression level of foreign genes - Google Patents

Abstract

The invention discloses a non-coding nucleotide sequence and its application in improving the expression level of exogenous genes, which belongs to the field of biotechnology. The nucleotide sequence is as SEQ ID NO.1. Compared with the prior art, the Nucleotide sequences can significantly increase the expression level of exogenous genes at the translation level, provide a new way for the overexpression of exogenous genes, significantly promote the expression of proteins encoded by exogenous genes, and have strong application value.

Description

A non-coding nucleotide sequence and its role in increasing the expression level of foreign genes application

technical field

The present invention relates to the field of biotechnology, in particular to a nucleotide sequence, in particular to a non-coding nucleotide sequence and its application in improving the expression level of exogenous genes.

Background technique

With the development of protein engineering, improving the expression level of foreign genes can improve the production efficiency of proteins, which has become the focus of people's research. At present, improving the expression level of foreign genes mainly includes: increasing the strength of promoters, shortening promoters and cloning genes The distance between them, the development of efficient translation initiation sequences, the development of efficient transcription termination regions, the improvement of plasmid copy number and stability, the improvement of protein translation levels, the reduction of cellular metabolic load, and the improvement of expression protein stability (to prevent degradation), etc., these methods It has the characteristics of high protein expression level, and is currently the mainstream method for improving the expression level of exogenous genes.

In vitro expression of fusion proteins refers to the process of recombining two genes through DNA technology, then expressing, separating and purifying them in vitro to obtain the target protein, and the transient expression system of plants is one of the most common technical methods for expressing fusion proteins in vitro. The plant transient expression system is a technology that transfers the target gene into the target cell in a relatively short period of time, and establishes a temporary and efficient expression system in the cell to obtain a short-term high-level expression of the target gene. However, as far as the current technology is concerned, the use of plant transient expression systems to express fusion proteins in vitro has low protein expression levels and has been widely criticized. Therefore, it is not one of the main methods to increase the expression level of exogenous genes.

Contents of the invention

The purpose of the present invention is to provide a non-coding nucleotide sequence and its application in improving the expression level of foreign genes, so as to solve the technical problem that the protein expression level is not high when the plant transient expression system expresses the fusion protein in vitro.

The present invention is achieved through the following technical solutions:

The present invention finds that the subgenome 5' non-coding sequence of Maize yellow mosaic virus (Maize yellow mosaic virus) plays an important role in gene regulation, and the subgenome 5' non-coding sequence can affect the gene by regulating the translation initiation process. Since the 5' non-coding sequence has the characteristics of regulating gene expression, its characteristics can be used to improve the expression level of foreign genes. Based on this, the present invention provides a non-coding nucleotide sequence as shown in SEQ ID NO.1 derived from maize yellow mosaic virus, and the full length of the nucleotide sequence is 75bp.

The present invention also provides the application of the above-mentioned non-coding nucleotide sequence in improving the expression level of foreign genes in plants.

As a further optimization scheme of the present invention, the method for increasing the expression level of foreign genes in plants is as follows: connecting the above-mentioned non-coding nucleotide sequence to the upstream of the start codon of the coding region of the foreign gene to construct a fusion fragment, Then the fusion fragment is connected to the plant expression vector, and the plant expression vector is introduced into the plant body to realize the expression of the exogenous gene in the plant body.

As a further optimization scheme of the present invention, the plant is tobacco.

Compared with the prior art, the present invention has the following advantages: the present invention provides a non-coding nucleotide sequence and its application in improving the expression level of foreign genes, and the nucleotide sequence can significantly increase the level of translation of foreign genes. The expression level of the gene provides a new way for the overexpression of the exogenous gene, and can significantly promote the expression of the protein encoded by the exogenous gene, which has strong application value.

Description of drawings

Fig. 1 is a comparison chart of the results of increasing the expression level of GFP in tobacco leaves after the non-coding nucleotide sequence of the present invention is linked to the upstream of the initiation codon of GFP.

Detailed ways

Example 1

1. Materials

The methods used in this example are conventional methods known to those skilled in the art unless otherwise specified, and the reagents and other materials used are all commercially available products unless otherwise specified.

2. Method

2.1 Obtaining non-coding nucleotide sequences

(1) Trizol method was used to extract the total RNA of maize infected with maize yellow mosaic virus and reverse transcribed it into cDNA.

(2) The obtained cDNA is used as a template, and the first primer pair shown in SEQ ID NO.2-3 is used for PCR reaction;

SEQ ID NO. 2: F1: ATACAGAAGCTTCGAAGATA

SEQ ID NO. 3: R1: GGCTGCTTATGTGTGAGACT

The amplification system was: 5 μl of 10×EX Taq buffer, 5 μl of 2.5mM dNTPs, 2 μl of 10 μM P1 and P2, 1 μl of EXTaq enzyme (5U/μl) (purchased from Dalian Takara Company), and 50 μl of sterile water.

The amplification reaction conditions were: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 10 min.

The target product was obtained with a size of 75bp.

(3) After the PCR product was purified by Qiagen gel purification kit (Qiagen Company, Germany), it was purified by Promega -T EASY Vector (Promega, USA) for TA cloning of PCR fragments, the conditions are as follows: 2×Promegaligase buffer 5 μl, /> -T EASY Vector 1 μl, PCR recovered fragments 2 μl, Promega T4-ligase 1 μl (Promega, USA), sterile water 1 μl, mixed and reacted at 4°C for 16 hours.

(4) After the reaction is complete, take 10 μl of the reaction solution and add it to a centrifuge tube containing 100 μl of Escherichia coli competent cells, mix gently and place on ice for 30 minutes, heat shock at 42°C for 90 seconds, immediately place on ice for 2-3 minutes, and pour Add 890 μl of LB liquid medium to the centrifuge tube. Place the centrifuge tube on a shaker at 37°C for 1 hour, then spread 300 μl of culture solution on solid LB medium containing 100 mmol/L ampicillin, 1 mmol/L IPTG and 10 mg/L, and culture at 37°C for 14-16 hours. Then select the white plaque and send it to Qingke Company for sequencing. Clones containing the inserted sequence were designated T-UTR.

2.2 Construction of plant expression vectors

Using molecular biology experimental techniques, the obtained sequence was connected to the multiple cloning site of the entry vector pDonr221 (Prutin Biotechnology (Beijing) Co., Ltd.), and the conventional plant expression vector construction was carried out. The specific steps included:

(1) Using T-UTR as a template, use the second primer pair to carry out PCR amplification, and use restriction endonucleases EcolRI and XbaI to double-enzyme-cut the PCR amplification product and the entry vector pDonr221 respectively, connect them, and construct and obtain the entry vector pDonor221UTR; The second primer pair is:

SEQ ID NO. 4: F2: cccaagcttATGGTGAGCAAGGGCGAGGAGCT

SEQ ID NO. 5: R2: gctctagaGGCTGCTTATGTGTGAGACT.

(2) The entry vector pDonor221UTR was connected to the plant expression vector pGWB451 (Protin Biotechnology (Beijing) Co., Ltd.) by LR reaction to obtain pGWB451UTR; the empty vector pGWB451 was also subjected to LR reaction for comparison.

2.3 Expression of exogenous genes

Transiently transform tobacco leaves with pGWB451 obtained from the above pGWB451UTR and LR reactions, and use a fluorescence microscope to detect the expression of GFP in tobacco leaves after 4 days. The specific steps include:

The two plasmids were transformed into Agrobacterium GV3101, and when the concentration of the bacterial solution was OD ₆₀₀ =1, centrifuged at 5000 rpm for 5 minutes at 4°C, and the supernatant was removed to retain the bacterial cells. The bacteria were mixed in the infection solution (10mM MES, 200uM acetosyringone, 10mM magnesium chloride), and stood at room temperature for 3h before use. Inject the back of Nicotiana benthamiana leaves at the 6-leaf stage with a syringe without a needle, and inject the bacterial solution between the leaf veins by pressure. After the injection, place the tobacco at 22° C. for 4 days. The green fluorescence intensity of GFP was observed by fluorescent confocal microscope.

The results are shown in Figure 1, where it can be seen that in the tobacco leaves transformed with pGWB451UTR, the clarity of green fluorescence is significantly higher than that of the control pGWB451, indicating that the insertion of the nucleotide of the present invention directly improves the expression level of the exogenous GFP gene .

The above is a detailed implementation mode and specific operation process of the present invention, which is implemented on the premise of the technical solution of the present invention, but the protection scope of the present invention is not limited to the above-mentioned embodiments.

sequence listing

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Claims (3)

1. The application of a non-coding nucleotide sequence in improving the expression level of foreign genes in plants, characterized in that the nucleotide sequence is as shown in SEQ ID NO.1. 2. The application of a non-coding nucleotide sequence according to claim 1 in improving the expression level of exogenous genes in plants, characterized in that, the method for improving the expression levels of exogenous genes in plants is: Link the above-mentioned non-coding nucleotide sequence to the upstream of the start codon of the coding region of the foreign gene, construct the fusion fragment, then connect the fusion fragment to the plant expression vector, and introduce the plant expression vector into the plant to realize the expression of the foreign gene in the plant. In vivo expression. 3. The application of a non-coding nucleotide sequence according to claim 1 in increasing the expression level of exogenous genes in plants, characterized in that the plants are tobacco.