CN112608372B - Chloroplast-cell membrane double-localization gene, protein coded by same and application thereof - Google Patents

Chloroplast-cell membrane double-localization gene, protein coded by same and application thereof Download PDF

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CN112608372B
CN112608372B CN202011589395.5A CN202011589395A CN112608372B CN 112608372 B CN112608372 B CN 112608372B CN 202011589395 A CN202011589395 A CN 202011589395A CN 112608372 B CN112608372 B CN 112608372B
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金刚
陈涛
覃旭
刘明
黄显雅
彭欣怡
吴密
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Abstract

The invention belongs to the field of plant molecular biology, and relates to a gene for encoding a protein in chloroplast-cell membrane double localization, in particular to a chloroplast-cell membrane double localization gene, and an encoded protein and application thereof. The nucleotide sequence of the gene is shown as SEQ ID No. 2. The amino acid sequence of the gene coding protein NtODB is shown as SEQ ID No. 1. The fusion of the NtODB protein and the fluorescent reporter protein can be used as a double-localization marker of plant cell chloroplasts and cell membranes,NtODBthe gene can be used as a marker gene for plant chloroplast-cell membrane double expression. Will beNtODBThe fusion expression of the gene and the fluorescent reporter gene is used as a marker of chloroplast and cell membrane, and can provide accurate contrast during subcellular localization research of the target gene, thereby effectively overcoming the defect of predicting protein subcellular localization according to the amino acid sequence.

Description

Chloroplast-cell membrane double-localization gene, protein coded by same and application thereof
Technical Field
The invention belongs to the field of molecular biology, and relates to a tobacco chloroplast localization and cell membrane localization marker gene, in particular to a chloroplast-cell membrane double localization gene, and a coded protein and application thereof.
Background
Cells are the fundamental functional units of living organisms that perform vital activities. The eukaryotic cells of the plant comprise subcellular structures such as mitochondria, chloroplasts, peroxisomes, lysosomes, endoplasmic reticulum, cell nuclei, golgi apparatus, vacuoles, vesicles and the like. The study of subcellular localization of proteins in cells is important for analysis of protein function.
Subcellular localization refers to the presence of a protein or gene expression product at a specific site within a cell, such as the nucleus, cell membrane or a specific organelle.
Experimental localization and bioinformatics prediction are two means commonly used for subcellular localization. The biological informatics prediction method has the advantage of providing pre-reference information by using big data, thereby playing a good auxiliary role for the traditional experimental method and saving time cost. However, it is difficult to make accurate predictions, especially for proteins that are localized to multiple organelles. Common experimental methods for subcellular localization of plant proteins include fusion reporter gene localization, immunohistochemical localization, co-separation marker enzyme assisted localization, and proteomics localization techniques. Fusion reporter gene mapping is the most common method among them. The principle is that a target protein gene is fused with a reporter gene which is easy to detect, a fusion gene expression vector is constructed, fusion protein is expressed, and then the target protein is positioned by means of the characteristics of a reporter gene expression product. The target protein is usually fused with a reporter gene (such as green fluorescent protein gene GFP) for expression, and the expression site of fluorescence is observed under a confocal laser microscope so as to locate the target protein in cells. The living cell localization research is a method which is relatively common at present by utilizing the GFP fusion protein technology, the molecular weight of GFP protein is 27kD, and green fluorescence can be generated after laser irradiation of a laser scanning copolymerization microscope, so that the protein is precisely localized. Green Fluorescent Protein (GFP) gene and its 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, mcnana, mOrange) and the like are also commonly used subcellular localization reporter genes. Although the fusion reporter gene positioning method has long time consumption and high cost through experiments, instruments and equipment are expensive, and experimental results need artificial experience judgment, so that the fusion reporter gene positioning method has certain subjectivity. However, the result of the positioning method is relatively accurate, so that the defect of a bioinformatics prediction method can be effectively overcome, and the method has incomparable advantages. The method has the advantages of easy operation, strong applicability, short period and the like, can be applied to real-time positioning and dynamic research of living bodies, and has high sensitivity. However, the localization results of fusion reporter gene localization may differ from the distribution of the protein under in vivo conditions; too strong or weak expression of the fusion protein may cause unclear localization results, and in particular, the localization of membrane proteins such as plasma membranes may be greatly affected. And the development of the organelle fluorescent marker as a control can make up for the defect of a fusion reporter gene positioning method.
Disclosure of Invention
The invention discovers genes for locating tobacco chloroplast and tobacco cell membrane, provides a chloroplast-cell membrane double-locating gene, coded protein and application thereof, provides a new choice for tobacco chloroplast and cell membrane locating markers, and discovers that tobacco ODB1 protein has a function of locating cell membrane for the first time.
The technical scheme of the invention is realized as follows:
a positioning protein NtODB, wherein the amino acid sequence of the NtODB protein is shown as SEQ ID No. 1.
A gene for encoding NtODB protein, the nucleotide sequence of the gene is shown as SEQ ID No. 2.
The application of the gene in the double-localization expression of exogenous proteins in chloroplasts and cell membranes.
An expression vector containing the gene, wherein the expression vector is a subcellular localization vector.
The subcellular localization vector is applied to chloroplast-cell membrane double localization or chloroplast-cell membrane labeling.
The application comprises the following steps:
(1) According toNtODBDesigning primer pair for gene, amplifying by PCR to obtain amplified product, enzyme cutting, recovering, connecting with T carrier to obtain intermediate carrier, and amplifying with intermediate carrier as templateNtODBThe gene (the upstream and downstream of which is introduced with enzyme cutting sites) is connected with a blank expression vector pBI121-GFP after enzyme cutting recovery to obtain a pBI 121-NtODB/GFP subcellular localization vector;
(2) The pBI 121-NtODB/GFP subcellular localization vector was transiently expressed in tobacco.
The invention has the following beneficial effects:
1. the plant cell chloroplast and cell membrane localization marker enriches subcellular localization marker libraries, and has the effect of double localization of one marker gene in chloroplast and cell membrane.
2. The invention is characterized in thatNtODBThe plant expression vector fused by the gene and the fluorescent reporter gene GFP can stably express fusion protein in plants, green fluorescence can be overlapped with chlorophyll autored fluorescence, chloroplast can be specifically marked, a evidence is provided for chloroplast marking, and a fluorescent signal on a cell membrane is clear and stable.
3. The invention is characterized in thatNtODBThe gene is derived from tobacco, has constitutive expression characteristics, can be stably transformed into plants, and can stably mark plant cell chloroplasts and cell membranes for a long time. As a marker control gene for chloroplast-cell membrane double localization, the defect of predicting subcellular localization according to the gene sequence is also overcome.
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In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph of hydrophobicity/hydrophilicity of the tobacco NtODB protein.
FIG. 2 shows the analysis of the transmembrane region of the NtODB protein.
FIG. 3 is a map of the phosphorylation sites of the NtODB protein.
FIG. 4 is a diagram of the conserved regions of the NtODB protein of Nicotiana tabacum.
FIG. 5 shows the prediction of the secondary structure of the tobacco NtODB protein.
FIG. 6 is a three-level structure of the tobacco NtODB protein domain region.
FIG. 7 is a diagram ofNtODBSpatiotemporal expression pattern in tobacco.
FIG. 8 is the localization of pBI 121-NtODB/GFP in the epidermal cells of this cigarette.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without any inventive effort, are intended to be within the scope of the invention.
A positioning protein NtODB, wherein the amino acid sequence of the NtODB protein is shown as SEQ ID No. 1.
A gene for encoding NtODB protein, the nucleotide sequence of the gene is shown as SEQ ID No. 2.
The application of the protein and the gene for encoding the protein in chloroplast-cell membrane double-localization or mesochloroplast-cell membrane labeling.
An expression vector containing the gene, wherein the expression vector is a subcellular localization vector.
The subcellular localization vector is applied to chloroplast-cell membrane double localization or middle chloroplast-cell membrane labeling.
The application comprises the following steps:
(1) According toNtODBDesigning primer pair for gene, amplifying by PCR to obtain amplified product, enzyme cutting, recovering, connecting with T carrier to obtain intermediate carrier, and amplifying with intermediate carrier as templateNtODBThe gene (the upstream and downstream of which is introduced with enzyme cutting sites) is connected with a blank expression vector pBI121-GFP after enzyme cutting recovery to obtain a pBI 121-NtODB/GFP subcellular localization vector;
(2) The pBI 121-NtODB/GFP subcellular localization vector was transiently expressed in tobacco.
Specific:
the common tobacco and the raw tobacco used in the research are potted in the test base tobacco greenhouse (east longitude 108 DEG 20', north latitude 22 DEG 53') of the Guangxi subtropical crop institute. The common tobacco material is Guangxi sunning tobacco local variety Niu Li tobacco. After the plant tissue material is quickly frozen by liquid nitrogen, the plant tissue material is transported to an ultralow temperature refrigerator at the temperature of minus 80 ℃ for preservation by dry ice.
RNA extraction and reverse transcription were performed using routine experimental procedures: 1 g tobacco tissue was placed in a mortar, poured into liquid nitrogen, and ground to powder. And then extracting RNA by using a tenna prep Pure polysaccharide polyphenol plant total RNA extraction kit. The total RNA mass was detected by agarose gel electrophoresis, and then the total RNA mass and concentration were detected with a micro nucleic acid protein meter. Then utilizing Oligo (dT) to the total RNA of the tobacco 18 And the reverse transcription kit for Repran (HiScript 1st Strand cDNA Synthesis Kit).
Example 1: tobacco leafODB1Cloning and analysis of genes
The tobacco proteome database was searched by BLASTP homology alignment, and one sequence (GenBank accession number xm_ 016606610.1) having the highest similarity to the query sequence, coverage, and expected value of e was selected as a subject. PCR amplification of tobaccoNtODBThe reaction condition of the gene is 95 ℃ for 4 min;95 ℃ for 30 s,56 ℃ for 30 s,72 ℃ for 1 min,32 cycles; and at 72℃for 10 min. And (3) detecting the PCR product by 1.0% agarose gel electrophoresis, and then cutting the PCR product into gel to recover and purify the target band. Then connecting with pMD19-T vector, transforming E.coli DH5 alpha competent, screening positive transformant and transferring it to Jin Weizhi company to obtain the invented productSanger sequencing.
Extracting total RNA of stems, petals, leaves and sepals in the reproductive phase of common tobacco, and reversely transcribing the extracted RNA into cDNA. Designing and constructing a primer pBI121-ODB1-F/R (Table 1) of a NtODB subcellular localization vector, and amplifying by taking pMD19-T-NtODB as a templateNtODBA complete coding sequence. Amplification product is passed throughKpnI andXba i double enzyme cutting, then connected into the same quiltXbaI andKpn and I, double enzyme digestion and recovery of the purified blank expression vector pBI121-GFP to obtain a pBI 121-NtODB/GFP fusion expression vector.
The constructed subcellular localization vector is used for transforming competent cells of agrobacterium EHA105, and positive transformants are selected and cultured in a YEB liquid medium containing kanamycin in a shaking way for 12-16 h. Preparing agrobacterium suspension, and regulating each bacterial liquidOD 600 0.1-0.8, adopts a transient expression method,
the tobacco transient transformation steps are as follows:
1. amplifying and shaking the successfully detected agrobacterium tumefaciens bacteria liquid overnight at 28 ℃ and 200 rpm;
2. taking 1-1.5 ml bacterial liquid, and adding the bacterial liquid into a sterilized 1.5 ml centrifuge tube; at 8000rpm for 2 min, the thalli are precipitated (room temperature), the supernatant is removed, 1 ml permeate is added, and the thalli are suspended; repeating the centrifugation step to further remove a small amount of antibiotics;
3. diluting a small amount of suspension bacteria liquid by 10 times, measuring an OD600 value, and multiplying the OD600 value by 10 to obtain the OD600 value of the suspension bacteria liquid; and (3) injection: if the OD600 value of the suspension is between 1.5 and 2.0, it is indicated that a large number of dead bacteria cells exist, and the transformation efficiency is reduced;
4. determining the titer of the suspension to the permeate, calculating the dilution factor such that the final suspension (for infestation) is 0.5-5.0 ml, the OD600 is 0.4 (0.1-0.8, not more than 1 as required), and typically 0.5-1.0 ml is satisfactory;
5. preparing a final suspension bacterial liquid in a 1.5 ml centrifuge tube, standing for 1 to 3 hours at room temperature, and preparing for infection; before infection, putting tobacco under a white fluorescent lamp for 1 h to open air holes; selecting three or four leaves for infection (infection between two veins), selecting two leaves for one plant,infecting a bacterial liquid; with the syringe with the needle removed, the back of the blade to be turned (0.5 cm) 2 ) Or piercing with a small needle to remove the waxy layer;
6. marking the region to be transferred by a marker pen before infection; sucking the final suspension into a1 ml needle-removed syringe; the injector is opposite to the to-be-rotated area on the back of the blade, one hand presses the upper surface of the blade, the other hand lightly pushes the piston until the liquid is seen to spread, other parts are infected, and after the infection, the infected area is marked by a marker pen; spraying water on the leaves, sleeving a fresh-keeping bag, putting the infected tobacco back into a culture room, and standing overnight in darkness; opening the fresh-keeping bag on the 2 nd day, and ensuring the highest expression level 2-3 days after injection; and cutting an affected area, tearing the epidermis, and carrying out laser confocal microscope observation.
The leaf of Nicotiana benthamiana was injected with Agrobacterium that successfully transformed the plasmid of interest and was dark cultured 48 h. The affected area was excised, the epidermis was peeled off, and the distribution of GFP fluorescence in tobacco cells was observed using a laser confocal microscope. To be disconnected fromNtODBThe pBI121-GFP blank vector of the gene was used as a control.
Primer sequences and uses for Table 1
Figure 278912DEST_PATH_IMAGE002
The amino acid sequence of the Arabidopsis ODB1 protein is utilized to carry out Blastp homology alignment of common tobacco protein sequences in NCBI database, and a sequence with highest similarity, coverage and e expected value is XM_016606610.1 in GenBank accession number. The complete ORF of this cDNA is 75-653bp, the initiation and termination codons are ATG and TGA, respectively, the expressed protein contains 192 amino acids, the cDNA sequence is complete, the named geneNtODBThe expressed protein was NtODB. Analysis of physicochemical parameters of cDNA expressed protein using ProtParam tool, molecular formula, molecular weight and theoretical isoelectric point of the protein are C 943 H 1479 N 269 O 295 S 8 21.56 kDa and 8.38, are basic proteins; fat coefficient, number of negative charge residues (Asp+Glu) and positive chargeThe number of residues (Arg+Lys) was 67.50, 25.00 and 27.00, respectively; the instability coefficient and the average total hydrophilicity were 47.91 and-0.596, respectively, belonging to the unstable hydrophilic proteins. The NtODB protein is predicted to contain 20 amino acids, up to serine (Ser), 15.1%. ProtScale analysis was performed on the primary structure of the tobacco NtODB protein, and Lys at position 135 Tyr and 42 of the polypeptide chain had the highest (1.478) and lowest scores (-3.722), respectively, the former being the most hydrophobic and the latter being the most hydrophilic (FIG. 1).
Example 2: subcellular localization and transmembrane analysis
Analyzing the basic physicochemical indexes, hydrophilicity/hydrophobicity of the expressed protein by utilizing online software protparam and protscale respectively; analyzing the protein subcellular localization and transmembrane region using online software ProtComp v9.0 and TMHMM tools; analyzing the phosphorylation site and the signal peptide by using on-line software of SignalP4.1; the high-level structure of the domains was analyzed using SWISS-MODEL online software.
Using ProtComp v.9.0 on-line software to predict the subcellular localization of the NtODB protein (table 2), which has a higher overall score in the extracellular (2.22), chloroplast (2.19) and endoplasmic reticulum (2.11); according to the pentameric algorithm, the protein is higher in the mitochondria (3.62). From this, it was predicted that the NtODB protein might be distributed in the organelle. The NtODB protein transmembrane region was analyzed on-line using TMHMM v.2.0 software, and the protein did not have an obvious transmembrane region and did not belong to the membrane-bound protein.
TABLE 2 subcellular localization analysis of Nicotiana tabacum NtODB protein
Figure DEST_PATH_IMAGE003
Example 3: phosphorylation site and signal peptide analysis
Protein phosphorylation is at the end of the signal transmission chain, and the purpose of regulating gene activity can be achieved by phosphorylating transcription factors. The protein contained the number of serine, threonine and tyrosine phosphorylation sites using NetPhos 3.1 service for online analysis (fig. 3). Respectively 18, 4. Using the TargetP 2.0 assay, the probability of NtODB being a mitochondrial transporter was 93.92% with excision sites between amino acid residues 24-25. Implying the organelle localization properties of the NtODB protein.
Example 4: analysis of conserved domains and higher order structures
Analysis of the conserved domain of the NtODB protein using on-line software CD-Search (FIG. 4), the highly conserved RAD52 domain between amino acid residues 70-185 of the protein, indicated cloning by the present studyNtODBThe encoded protein of the gene belongs to a DNA repair protein RAD52 family member. Analysis of the secondary structure of the NtODB protein using SOPMA revealed that the polypeptide chain contained 3 amino acid sequences, 35.94% alpha helix, 16.67% extended chain, 5.73% beta-sheet and 41.67% random coil sequence (fig. 5). The three-level structure of the protein was predicted using SWISS-MODEL, and the spatial structure consisted mainly of alpha helices and random coils (FIG. 6).
Example 5:NtODBanalysis of Gene expression
Tobacco obtained according to clone sequencingNtODBGene design qRT-PCR primer for tobaccoActinIs the reference gene (Table 1). Relative expression levels of target genes were detected using a LightCycler 480 II fluorescent quantitative PCR apparatus from Roche, switzerland, 3 biological replicates per sample, 2 were used -ΔΔCt A method of manufacturing the same.
Extracting total RNA from organs such as stems, leaves, petals, sepals and the like of common tobacco in the full bloom stage. By common tobaccoNtActinqRT-PCR detection using gene as reference geneNtODBRelative expression levels of genes. The results show that the method has the advantages of,NtODBthe relative expression amount of the constitutive expression characteristics of different organs of the common tobacco in the full bloom stage is obviously different among the different organs. The most abundant expression was found in leaves, and the most abundant expression was found in stems and sepals, and the least abundant expression was found in petals (fig. 7).
In order to further study the subcellular localization of the NtODB protein, the distribution of the green fluorescent protein is observed under a laser confocal microscope by taking an empty carrier as a control, and as a result, the fluorescent signal of the transferred pBI121-GFP is found to be distributed in the whole cell; while the green fluorescent signal of the pBI121-NtODB-GFP fusion protein was localized to the chloroplasts and cell membranes (FIG. 8).
Discussion of analysis
This study showed that NtODB is constitutively expressed in different organs of tobacco. However, there is a difference in the relative expression levels in different organs, and the abundance of expression in the petals of the reproductive organ is lower than that in other organs. Samach et al found that the Arabidopsis ODB1 protein was localized in chloroplasts, nuclei and chloroplasts, but not in the ODB1 cell membrane, by subcellular localization experiments. The subcellular localization conclusion of other plant ODB1 homologous proteins is not reported. This study showed that tobacco NtODB is expressed only in chloroplasts and cell membranes. This is consistent with the software-predicted NtODB subcellular localization results. Can be effectively used as a chloroplast-cell membrane double-localization marker gene.
<110> Guangxi Zhuang autonomous region subtropical crop institute (Guangxi subtropical agricultural product processing institute)
<120> a chloroplast-cell membrane double-localization gene, protein encoded thereby and use thereof
<141> 2020-12-29
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Met Ala Phe Ser Ser Leu Arg Ser Thr Ser Ser Ser Val Leu Asn Arg
1 5 10 15
Val Arg Phe Cys Gln Cys Ser Phe Ser Ala Lys Pro Ser Ser Ser Ser
20 25 30
Ser His Arg Ala Val Lys Asp Lys Asp Lys Asp Lys Glu Lys Met Met
35 40 45
Ser Asp Ser Asp Arg Glu Thr Pro Glu Tyr Asp Val Pro Thr Ser Gly
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Ile Ser Arg Pro Leu Ser Glu Ile Leu Lys Glu Leu Asn Lys Lys Val
65 70 75 80
Pro Asp Ser Leu Ile Lys Phe Arg His Glu Pro Asn Gly Phe Ser Leu
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Lys Tyr Ile Pro Trp His Ile Val Asn Arg Ile Leu Asn Leu His Ala
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Pro Glu Trp Ser Gly Glu Val Arg Ser Ile Asn Tyr Ser Ser Asp Gly
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Lys Ser Val Ser Val Val Tyr Arg Val Thr Leu Tyr Gly Thr Asp Ala
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Glu Ile Tyr Arg Glu Ser Thr Gly Thr Ala Ser Val Asp Asp Pro Gly
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gacaaagaca aagagaaaat gatgtctgat tccgacaggg aaacgccgga gtacgatgtc 180
ccaacttcag gaatcagccg accactttct gagatactca aagagctgaa caagaaggtc 240
cccgattccc tcatcaagtt tcgccacgaa cctaatggct tctccctcaa atatatcccc 300
tggcatattg tgaataggat tctgaattta catgctccag aatggtctgg cgaagttcga 360
agcatcaatt actcatctga tggcaagtca gtttcagttg tttaccgtgt aacactatat 420
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tatggggatc ctgtgcagaa ggcagagggt atggcatttc gtcgagcttg tgctcgattt 540
gggctgggac ttcatcttta tcatgaggac atgtcatag 579

Claims (2)

1. An application of a localization protein NtODB gene in double localization expression of exogenous proteins in tobacco chloroplasts and cell membranes is characterized in that: the nucleotide sequence of the location protein NtODB gene is shown as SEQ ID No. 2.
2. The use according to claim 1, characterized by the steps of:
(1) According toNtODBDesigning primer pair for gene, amplifying by PCR to obtain amplified product, enzyme cutting, recovering, connecting with T carrier to obtain intermediate carrier, and amplifying with intermediate carrier as templateNtODBThe genes are connected with a blank expression vector pBI121-GFP after being recovered by enzyme digestion, and a pBI 121-NtODB/GFP subcellular localization vector is obtained;
(2) The pBI 121-NtODB/GFP subcellular localization vector was transiently expressed in tobacco.
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