A cell nucleus localization gene and its encoded protein and application

Abstract

The present invention relates to the field of molecular biology technology, and in particular to a cell nucleus localization gene, a protein encoded by the gene, and an application thereof. The nucleotide sequence of the gene is shown in SEQ ID No.2. The amino acid sequence of the gene-encoded protein NtODB2 is shown in SEQ ID No.1. In the present invention, the fusion of NtODB2 protein and fluorescent reporter protein can be used as a localization marker for the cell nucleus of plant cells, and the NtODB2 gene can be used as a marker gene specifically expressed in the cell nucleus of a plant cell. The fusion expression of the NtODB2 gene and the fluorescent reporter gene as a marker for the cell nucleus can provide an accurate control when studying the subcellular localization of the target gene, which effectively makes up for the deficiency of predicting the subcellular localization of proteins based on amino acid sequences.

Description

[Technical field]

[0001] The invention relates to the technical field of molecular biology, and in particular to a cell nucleus localization gene and a protein encoded by the gene and application thereof. [Background technology]

[0002] Cells are the basic functional units of organisms for life activities. In plant eukaryotic cells, subcellular structures include mitochondria, chloroplasts, peroxisomes, lysosomes, endoplasmic reticulum, nuclei, Golgi bodies, vacuoles, vesicles, etc. Studying the subcellular localization of proteins in cells is very important for analyzing protein functions.

[0003] Subcellular localization refers to the specific location of a protein or a gene expression product in a cell, such as the nucleus, cell membrane or a specific organelle.

[0004] Experimental localization and bioinformatics prediction are two commonly used methods for subcellular localization. Bioinformatics prediction methods have the advantage of using big data to provide advance reference information, so they play a good auxiliary role in traditional experimental methods and save time costs. However, it is difficult to make accurate predictions, especially for proteins localized in multiple organelles. Common experimental methods for subcellular localization of plant proteins include fusion reporter gene localization, immunohistochemical localization, co-isolation marker enzyme-assisted localization, and proteomics localization technology. Fusion reporter gene localization is the most commonly used method. The principle is to fuse the target protein gene with an easily detectable reporter gene, construct a fusion gene expression vector, express the fusion protein, and then locate the target protein with the help of the characteristics of the reporter gene expression product. Usually, the target protein is fused with a reporter gene (such as the green fluorescent protein gene GFP) and the expression site of fluorescence is observed under a laser confocal microscope to localize the target protein in the cell. Using GFP fusion protein technology to conduct live cell localization research is currently a more common method, and the molecular weight of GFP protein is 27kD. After being irradiated by laser scanning confocal microscope, it can produce a green fluorescence, thereby accurately locating the protein. Green fluorescent protein (GFP) gene and fluorescent mutants (yellow fluorescent protein (YFP) gene, blue fluorescent protein (BFP) gene, cyan fluorescent protein (CFP)) and red fluorescent protein (RFP) gene and its mutants (mCherry, mStrawberry, mTangerine, mBanana, mOrange) are also commonly used subcellular localization reporter genes. Although the fusion reporter gene localization method is time-consuming and costly through experiments, the instruments and equipment are expensive, and the experimental results require human experience judgment, which has a certain degree of subjectivity. However, the results of this positioning method are relatively accurate, which can effectively make up for the shortcomings of bioinformatics prediction methods and have incomparable advantages of prediction methods. It has the advantages of easy operation, strong applicability, and short cycle, and can be applied to real-time positioning and dynamic research of living bodies with high sensitivity. However, the localization results of the fusion reporter gene localization method may be different from the distribution of proteins under in vivo conditions; too strong or too weak expression of the fusion protein may make the localization results unclear, especially for membrane proteins such as the plasma membrane. The development of organelle fluorescent markers as controls can make up for the shortcomings of the fusion reporter gene localization method. [Summary of the invention]

[0005] In view of the above, the present invention discovered a gene for locating the tobacco cell nucleus, proposed a cell nucleus localization gene and its encoded protein and application, and provided a new option for tobacco cell nucleus localization markers.

[0006] The present invention provides a localization protein NtODB2, and the amino acid sequence of the NtODB2 protein is shown as SEQ ID No.1.

[0007] Specifically:

[0008] MAFSSLHSTSSSVLNRVRFCQRSFSAKPSSNRAVKDKDRKKMMSDCDRETPEYDVPTSGISRPLSEILKELNKKVPDSLIKFRHEPNGFSLKYIPWHIVNRILNLHAPEWSGEVRSITYSSDGKSVSVVYRVTLYGTDAEIYRESTGTASVDDPGYGDPVQKAEGMAFRRACARFGLGLHLYHEDMS.

[0009] Another object of the present invention is to provide a gene encoding NtODB2 protein, the nucleotide sequence of the gene is shown in SEQ ID No.2.

[0010] Specifically:

[0011] .

[0012] The present invention also provides the application of the above-mentioned gene expression exogenous protein in the cell nucleus.

[0013] The present invention also provides an expression vector containing the above gene, wherein the expression vector is a subcellular localization vector, and the subcellular localization vector is used in cell nucleus labeling.

[0014] In the present invention, further, the application comprises the following steps:

[0015] (1) Designing primer pairs based on the NtODB2 gene, amplifying the product by PCR, recovering the product by restriction digestion and connecting it to the T vector to obtain an intermediate vector, then amplifying the NtODB2 gene using the intermediate vector as a template (introducing restriction sites upstream and downstream), recovering the product by restriction digestion and connecting it to the blank expression vector pBI121-GFP to obtain the NtODB2-GFP subcellular localization vector;

[0016] (3) Transient expression of NtODB2-GFP subcellular localization vector in tobacco.

[0017] The present invention has the following beneficial effects:

[0018] 1. The plant cell nuclear localization marker of the present invention enriches the subcellular localization marker library and has the effect of localizing a marker gene in the cell nucleus.

[0019] 2. The NtODB2 gene of the present invention is derived from tobacco, has constitutive expression characteristics, can be stably transformed into plants, and can stably mark plant cell nuclei for a long time. As a marker control gene for nuclear localization, it also makes up for the deficiency of predicting its subcellular localization based on gene sequence.

Brief Description of the Drawings

[0020] Figure 1 This is the hydrophobicity / hydrophilicity diagram of tobacco NtODB2 protein;

[0021] Figure 2 Analysis of the transmembrane region of NtODB2 protein;

[0022] Figure 3 This is the predicted phosphorylation site map of NtODB2 protein;

[0023] Figure 4 This is the predicted tertiary structure of the tobacco NtODB2 protein domain region;

[0024] Figure 5 is the spatiotemporal expression pattern of NtODB2 in tobacco;

[0025] Figure 6This is the localization map of NtODB2-GFP in epidermal cells of Nicotiana benthamiana (scale bar = 10 μm). [Specific implementation method]

[0026] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0027] Embodiment 1:

[0028] This embodiment provides a localization protein NtODB2, and the amino acid sequence of the NtODB2 protein is shown in SEQ ID No.1.

[0029] Specifically:

[0030] MAFSSLHSTSSSVLNRVRFCQRSFSAKPSSNRAVKDKDRKKMMSDCDRETPEYDVPTSGISRPLSEILKELNKKVPDSLIKFRHEPNGFSLKYIPWHIVNRILNLHAPEWSGEVRSITYSSDGKSVSVVYRVTLYGTDAEIYRESTGTASVDDPGYGDPVQKAEGMAFRRACARFGLGLHLYHEDMS.

[0031] This embodiment also provides a gene encoding NtODB2 protein, and the nucleotide sequence of the gene is shown in SEQ ID No.2.

[0032] Specifically:

[0033] .

[0034] This embodiment also provides an expression vector containing the above gene, wherein the expression vector is a subcellular localization vector, which is used in cell nucleus labeling.

[0035] This embodiment also provides an application of the above gene to localize and express foreign protein in the cell nucleus, and the application comprises the following steps:

[0036] (1) Primers were designed based on the NtODB2 gene, and the amplified product was obtained by PCR amplification. The amplified product was recovered by restriction digestion and connected to the T vector to obtain an intermediate vector. The NtODB2 gene was amplified using the intermediate vector as a template (with restriction digestion sites introduced upstream and downstream). After restriction digestion and recovery, the product was connected to the blank expression vector pBI121-GFP to obtain the NtODB2-GFP subcellular localization vector; (2) The NtODB2-GFP subcellular localization vector was transiently expressed in tobacco.

[0037] Specifically, the common tobacco and the native tobacco used in this embodiment were potted in the tobacco greenhouse of the Guangxi Subtropical Crops Research Institute Experimental Base (108°20′ east longitude, 22°53′ north latitude). The common tobacco material was the Guangxi sun-dried tobacco local variety "Niuliyan". The plant tissue material was quickly frozen with liquid nitrogen and then transported to a -80°C ultra-low temperature refrigerator with dry ice for storage.

[0038] RNA extraction and reverse transcription were performed using conventional experimental procedures: 1 g of tobacco tissue was placed in a mortar and ground into powder using liquid nitrogen. RNA was then extracted using the Tiangen RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit. The quality of total RNA was tested by agarose gel electrophoresis, and the quality and concentration of total RNA were tested using a micro-nucleic acid protein analyzer. The tobacco total RNA was then reverse transcribed using Oligo(dT)18 and the HiScript 1st Strand cDNA Synthesis Kit.

[0039] Embodiment 2:

[0040] cDNA cloning of NtODB2 gene:

[0041] The tobacco proteome database was searched by BLASTP homology comparison, and a sequence with lower similarity, coverage, and expected value than the query sequence (GenBank accession number XM_016658280.1) was selected as the research object. The reaction conditions for PCR amplification of tobacco NtODB2 gene were 95℃4min; 95℃30s, 56℃30s, 72℃1min, 32 cycles; 72℃10min. The PCR product was detected by 1.0% agarose gel electrophoresis and the gel was cut to recover the purified target band. It was then connected with the pMD19-T vector, transformed into Escherichia coli DH5α competent state, and the positive transformants were screened and sent to Jinweizhi Company for Sanger sequencing.

[0042] Total RNA from stems, petals, leaves, and sepals of common tobacco during the reproductive growth period was extracted and reverse transcribed into cDNA. Primers pBI121-ODB2-F / R (Table 1) were designed to construct the NtODB2 subcellular localization vector, and pMD19-T-NtODB2 was used as a template to amplify the complete coding sequence of NtODB2. The amplified product was double-digested with KpnⅠ and XbaI, and then connected to the blank expression vector pBI121-GFP, which was also double-digested with XbaI and KpnⅠ and then recovered and purified, to obtain the NtODB2-GFP fusion expression vector.

[0043] The constructed subcellular localization vector was transformed into Agrobacterium EHA105 competent cells, and the positive transformants were selected and cultured in YEB liquid medium containing kanamycin for 12-16 hours.600 is 0.1-0.8, using the instantaneous expression method;

[0044] The steps for transient transformation of tobacco are:

[0045] (1) Shake the successfully detected Agrobacterium culture overnight at 28°C and 200 rpm;

[0046] (2) Take 1-1.5 ml of bacterial solution and add it to a sterilized 1.5 ml centrifuge tube; centrifuge at 8000 rpm for 2 min to precipitate the bacteria (at room temperature), remove the supernatant, add 1 ml of permeate, and suspend the bacteria; repeat the centrifugation step to further remove a small amount of antibiotics;

[0047] (3) Take a small amount of the suspended bacterial solution and dilute it 10 times, and measure the OD 600 The value was multiplied by 10 and used as the OD of the bacterial suspension. 600 Note: If the OD of the bacterial suspension is 600 A value between 1.5 and 2.0 indicates a large number of dead bacterial cells, which will reduce the efficiency of transformation;

[0048] (4) Determine the titer of the bacterial suspension to the infiltration solution and calculate the dilution factor so that the final bacterial suspension (for infection) is 0.5-5.0 ml, OD 600 The concentration of the final suspension is 0.4 (0.1-0.8 as needed, not more than 1), usually 0.5-1.0ml of the final suspension is sufficient for infection;

[0049] (5) Prepare the final suspension of bacterial solution in a 1.5 ml centrifuge tube and let it stand at room temperature for 1-3 hours to prepare for infection. Before infection, place the tobacco under a white fluorescent light for 1 hour to open its stomata. Select the third and fourth leaves for infection (infection between two leaf veins). Select two leaves from one plant and infect one bacterial solution. Use a syringe with the needle removed to gently rub the back of the leaf to be transformed (0.5 cm 2 ), or pierce it with a small needle to remove its waxy layer;

[0050] (6) Before infection, mark the area to be transferred with a marker pen; draw the final bacterial suspension into a 1 ml syringe without a needle; point the syringe at the area to be transferred on the back of the leaf, press the leaf with one hand, and gently push the piston with the other hand until the liquid spreads, then infect other parts. After infection, circle the infected area with a marker pen; then spray the leaves with water, put them in a fresh-keeping bag, put the infected tobacco back into the culture room, and leave it in the dark overnight; open the fresh-keeping bag on the second day, and the expression level is highest 2-3 days after injection; cut the infected area, tear off the epidermis to prepare slides, and observe under a laser confocal microscope.

[0051] The Agrobacterium successfully transformed with the target plasmid was injected into the leaves of Nicotiana benthamiana and cultured in the dark for 48 hours. The infected area was cut, the epidermis was torn off and sliced, and the distribution of GFP fluorescence in tobacco cells was observed using a laser confocal microscope. The pBI121-GFP blank vector without the NtODB2 gene was used as a control.

[0052] Table 1 Primer sequences and uses

[0053]

[0054] In Table 1, the underline indicates the introduced restriction site.

[0055] Sequencing analysis of the amplified product of the NtODB2 gene showed that the complete ORF frame of the amplified cDNA was 56-619bp, the start and stop codons were ATG and TAG, respectively, and the expressed protein contained 187 amino acids. The sequence has a high affinity with the DNA repair RAD52-like protein of Ningxia wolfberry and sweet potato, with sequence identities of 84.04% and 70.90%, respectively, and can be aligned to the beginning and end of the homologous protein, that is, the cDNA sequence is complete, the gene is named NtODB2, and the expressed protein is NtODB2.

[0056] Embodiment 3:

[0057] Analysis of physicochemical properties of NtODB2 protein:

[0058] The ProtParam tool was used to analyze the physicochemical parameters of the cDNA expressed protein. The molecular formula, molecular weight and theoretical isoelectric point of the protein were C 926 H 1454 N 266 O 283 S8, 21.10kDa and 8.94, which is an alkaline protein; the fat coefficient, the number of negatively charged residues (Asp+Glu) and the number of positively charged residues (Arg+Lys) are 69.30, 23.00 and 27.00 respectively; the instability coefficient and the average total hydrophilicity are 45.71 and -0.565 respectively, which is an unstable hydrophilic protein. It is predicted that the NtODB2 protein contains 20 amino acids, the most abundant of which is serine (Ser), accounting for 13.4%. The primary structure of tobacco NtODB2 protein was analyzed by ProtScale. The 129th Val and the 38th Asp of the polypeptide chain have the highest (1.478) and the lowest score (-2.589), respectively. The former is the most hydrophobic and the latter is the most hydrophilic ( Figure 1 ).

[0059] Embodiment 4:

[0060] Subcellular localization and transmembrane region analysis:

[0061] The subcellular localization of NtODB2 protein was predicted using ProtComp v.9.0 online software (Table 2). NtODB2 protein had a high overall score in the extracellular space (2.25), chloroplasts (2.18), and endoplasmic reticulum (1.92); and according to the pentamer algorithm, the protein had a high score in mitochondria (3.24). Based on this, it was predicted that NtODB2 protein might be a secretory protein distributed in organelles. The transmembrane region of NtODB2 protein was analyzed online using TMHMM v.2.0 software. The protein had no obvious transmembrane region and was not a membrane-bound protein ( Figure 2 ).

[0062] Table 2 Subcellular localization analysis of NtODB2 protein in common tobacco

[0063]

[0064] Embodiment 5:

[0065] Phosphorylation site and signal peptide analysis:

[0066] Protein phosphorylation is at the end of the signal transduction chain and can regulate gene activity by phosphorylating transcription factors. Figure 3 ), the number of serine, threonine and tyrosine phosphorylation sites contained in this protein are 19, 4 and 4 respectively. Using TargetP 2.0 analysis, the possibility that NtODB2 is a mitochondrial transporter is 55.92%, and the excision site is between the 24th and 25th amino acid residues, suggesting the organelle localization of NtODB2 protein.

[0067] Embodiment 6:

[0068] Analysis of conserved domains and higher-order structures:

[0069] The tertiary structure of the protein was predicted using SWISS-MODEL, and the spatial structure was mainly composed of α-helix and random coil ( Figure 4 ).

[0070] Embodiment 7:

[0071] Expression analysis of NtODB2 gene:

[0072] Total RNA was extracted from stems, leaves, petals and sepals of tobacco at full flowering stage. qRT-PCR was used to detect the relative expression level of NtODB2 gene using NtActin gene of tobacco as internal reference gene. The results showed that although NtODB2 showed constitutive expression characteristics in different organs of tobacco at full flowering stage, its relative expression level was significantly different among different organs. The highest expression abundance was in leaves, followed by sepals and stems, and the lowest expression abundance was in petals ( Figure 5 ).

[0073] In order to further study the subcellular localization of NtODB2 protein, the empty vector was used as a control and the distribution of green fluorescent protein was observed under a laser confocal microscope. The results showed that the fluorescence signal of pBI121-GFP was distributed throughout the cell; while the green fluorescence signal of NtODB2-GFP fusion protein was localized in the cell nucleus ( Figure 6 ).

[0074] Analysis and discussion:

[0075] The study of this application shows that NtODB2 is constitutively expressed in different organs of tobacco. However, there are certain differences in the relative expression levels in different organs, and the expression abundance in the petals of reproductive organs is lower than that in other organs. This study shows that tobacco NtODB2 is only expressed in the nucleus. This is consistent with the results of NtODB2 subcellular localization based on software prediction. It can be effectively used as a nuclear localization marker gene.

[0076] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A localization protein NtODB2, characterized in that: The amino acid sequence of the NtODB2 protein is shown in SEQ ID No.

1.

2. A gene encoding NtODB2 protein, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID No.

2.

3. Use of the gene-expressed exogenous protein for localization in the cell nucleus according to claim 2.

4. The use according to claim 3, characterized in that: The steps include: (1) Designing primer pairs based on the NtODB2 gene, obtaining an amplification product by PCR amplification, recovering the amplification product by enzyme digestion and connecting it to a T vector to obtain an intermediate vector, then amplifying the NtODB2 gene using the intermediate vector as a template, recovering the amplification product by enzyme digestion and connecting it to a blank expression vector pBI121-GFP to obtain an NtODB2-GFP subcellular localization vector; (2) The NtODB2-GFP subcellular localization vector was transiently expressed in tobacco.

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