CN111607600B - Specific marker for plant cell chloroplast and application thereof - Google Patents

Abstract

The invention discloses a specific marker of plant cell chloroplast, which is RxLR132^aa21‑119A gene; also discloses the protein coded by the specific marker and the application of the specific marker in the fluorescence detection of plant chlorophyll. Rxlr132 of the invention^aa21‑119The fluorescence of the fusion protein expressed after the fusion of the gene and the fluorescence reporter gene can be superposed with the autofluorescence of chlorophyll, so that the chloroplast can be specifically marked, and the specific positioning of the chloroplast is not influenced by the fluorescence reporter gene. The plant cell chloroplast specific marker enriches a chloroplast marker library, can provide a plurality of candidate marker selections for chloroplast localization research of different plant proteins, and has important significance.

Description

Specific marker for plant cell chloroplast and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a specific marker for chloroplast of plant cells and application thereof.

Background

Cells are the basic structural and functional units of an organism. In eukaryotic cells, biofilms divide eukaryotic cells into different subcellular structures including mitochondria, chloroplasts, peroxisomes, lysosomes, endoplasmic reticulum, nuclei, golgi apparatus, vacuoles, vesicles, and the like, enabling multiple chemical reactions to be carried out simultaneously within the cell without interfering with each other. The various proteins function in each compartment of the cell in their functional order. The research on the subcellular localization of proteins is crucial to the exploration and analysis of protein functions and is an essential link for systematically understanding plant growth and development, morphogenesis, resistance to various abiotic stresses and the like.

The fusion reporter gene localization method is a common means for subcellular localization, and comprises the steps of fusing a target protein gene with a reporter gene easy to detect, constructing a fusion gene expression vector, expressing the fusion protein, and then localizing the target protein by means of the characteristics of a reporter gene expression product. As for the expression product of the reporter gene, fluorescent protein and beta-Glucuronidase (GUS) are mainly included, and the fluorescent reporter gene method is most widely applied.

The fluorescence reporter gene method is to fuse a target gene with a fluorescence reporter gene which is easy to detect, then introduce the fusion gene into an expression vector, transform proper cells by methods such as callus transformation, gene gun, agrobacterium-mediated transient transformation and the like, express fusion protein, and detect the fluorescence of the fluorescence protein under a fluorescence microscope or a laser confocal microscope so as to intuitively determine the accurate positioning of the target protein in the cells. Fluorescent reporter genes typically include the 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, mbana, mororange), and the like. The fluorescence reporter gene method has the advantages of high sensitivity, strong applicability, easy operation, relatively short test period, capability of keeping the natural characteristics of the protein, no toxicity to living cells, capability of observing the dynamic change of the protein in a living body and the like, and avoids the complex methods of purifying the protein, marking fluorescent dyes such as fluorescein isothiocyanate and the like, and introducing the fluorescent dyes into the cells through microinjection or other modes, thereby leading the accurate positioning of the living cells for researching the protein to be simple and easy, and being the method which is most widely applied in the current protein subcellular positioning research.

The fluorescence reporter gene method for marking the organelles has important significance for researching the dynamic change and the function of the organelles. Firstly, the fluorescent protein gene and the organelle positioning sequence are fused and expressed to position the fluorescent protein in a specific organelle and a membrane system, so that the morphological characteristics, the number, the distribution rule, the dynamic change, the function, the endomembrane system and the like of the organelle can be visually observed in vivo. If GFP and the endoplasmic reticulum localization signal KDEL are constructed as a fusion protein, a membrane-coat-like network structure can be observed in the endoplasmic reticulum of the transformed cells, and the endoplasmic reticulum marker facilitates the study of the structure, function and vesicle trafficking of the endomembrane system. GFP is fused with actin, tubulin and other cytoskeletal acting proteins, and can be used for researching the interaction between components of plant cytoskeleton and cytoskeletal mechanics. Secondly, as the types of organelles in the cell are more, and the sizes of partial organelles are similar, the organelles are difficult to distinguish, when the fluorescence of the unknown protein is observed to be possibly positioned on a certain specific organelle, the co-positioning analysis of the fluorescence of the unknown protein and the fluorescence of the organelle markers is required to be carried out, and the positioning of the unknown protein can be finally determined.

Chloroplasts are the most important, most prevalent plastids within plant cells, which are organelles that undergo photosynthesis. Chloroplast utilizes chlorophyll to convert light energy into chemical energy and CO₂And water is converted into sugar, which is the biological factory with the lowest cost and the most material resources in the world. AT present, fusion of FZL, APG2, AT5C48790 and other gene-encoded proteins and fluorescent proteins has been successfully used for labeling chloroplasts, but the number is still not large. The discovery of new chloroplast markers can enrich marker libraries, can provide multiple candidate marker selections for chloroplast localization research of different plant proteins, and has important significance.

Disclosure of Invention

The invention provides a specific marker of plant cell chloroplast and application thereof, which can enrich a marker library and provide a plurality of candidate marker selections for positioning research of different plant protein chloroplasts.

Plant cell chloroplast specific marker, which is RxLR132^aa21-119The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

Further, the above plant cellThe chloroplast specific marker is obtained by the following method: cloning of RxLR132 from Plasmopara viticola cDNA^aa21-119Coding region of the gene, transforming into Escherichia coli, screening positive clone, and performing DNA sequencing.

Further, cloning of the RxLR132^aa21-119The base sequence of the primer pair of the gene is shown as SEQ ID NO. 3 and SEQ ID NO. 4.

The invention also discloses a protein coded by the plant cell chloroplast specific marker, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

The invention also discloses application of the plant cell chloroplast specific marker in fluorescence detection of plant chlorophyll.

Further, the specific method of the application is as follows: subjecting said RxLR132 to^aa21-119Fusing the gene with a fluorescent reporter gene to obtain a fused gene; introducing the fusion gene into an expression vector to obtain a recombinant vector; transforming the recombinant vector into a host to obtain a transformant; the transformant is transformed into a target plant and used for marking chlorophyll.

Further, the fluorescent reporter gene includes GFP, YFP, BFP, CFP, RFP, mCherry, mStrawberry, mTangerine, mBanana, or mOrange.

Further, the expression vector comprises pBI 121.

Further, the host includes Escherichia coli or Agrobacterium.

Compared with the prior art, the invention has the following beneficial effects:

1. the plant cell chloroplast specific marker enriches a chloroplast marker library, can provide a plurality of candidate marker selections for chloroplast localization research of different plant proteins, and has important significance.

2. Rxlr132 of the invention^aa21-119The green fluorescence of the fusion protein expressed after the fusion of the gene and the fluorescence reporter gene can be superposed with the chlorophyll autofluorescence, the chloroplast can be specifically marked, and the specific positioning of the chloroplast is not influenced by the fluorescence reporter gene.

3. Rxlr132 of the invention^aa21-119The gene can be stably transformed into the plant, stably marks the chloroplast of plant cells for a long time, and does not influence the normal growth of the plant.

Drawings

FIG. 1 is a schematic representation of the pathology of grape downy mildew.

FIG. 2 is an electrophoretogram of RNA.

FIG. 3 is a PCR electrophoretogram.

FIG. 4 is a confocal laser microscopy image of protein.

FIG. 5 is a Western blotting analysis chart of protein.

FIG. 6 shows RxLR132^aa21-119Co-localization assay of GFP and chloroplast autofluorescence.

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, it should be noted that the examples are only for the purpose of further illustration and should not be construed as limiting the scope of the present invention.

Example 1

Obtaining of specific chloroplast marker of plant cell

1. Grapevine mildew sample collection

The grape downy mildew generally starts to attack in 6-7 months, and the most advanced stage of the attack is 8-9 months, and the best time for sampling is the most advanced stage. As shown in FIG. 1, grape downy mildew mainly damages grape leaves, producing yellow to brown polygonal lesions and a white mildew layer on the back of the leaf spots. The disease sample is stored in a self-sealing bag after being collected.

2. Extraction of downy grape fungus RNA

(1) A diseased tissue containing Plasmopara viticola (about 100mg) was taken out into a 1.5mL centrifuge tube, frozen with liquid nitrogen, and ground into powder.

(2) Add 600. mu.L of 65 ℃ pre-heated RNA extract (2% CTAB, 2% PVP-40, 2M NaCl, 20mM EDTA, 0.1M Tris-HCl, 1% beta-mercaptoethanol, 0.5g/L Spermidine, pH 8.0), vortex, shake and mix well, water bath 30min at 65 ℃ with M in mol/L, and reverse twice.

(3) 600 μ L of a water-saturated phenol/chloroform/isoamyl alcohol (25:24:1) solution was added, shaken vigorously for 10s and then centrifuged (room temperature, 13000rpm, 10 min).

(4) The supernatant was taken, added with an equal volume of chloroform, shaken vigorously for 10s, and then centrifuged (room temperature, 13000rpm, 2 min).

(5) The supernatant was taken, and an equal volume of 5M LiCl was added and precipitated overnight at 4 ℃.

(6) After centrifugation (4 ℃, 13000rpm, 20min), the supernatant was removed, and the pellet was washed once with 70% ethanol and once with absolute ethanol, and after air-drying, 500. mu.L of SSTE (1M NaCl, 0.5% SDS, 10mM Tris-HCl, 1mM EDTA, pH 8.0) was added to dissolve the pellet.

(7) Add 500. mu.L chloroform, shake vigorously for 10s and centrifuge (room temperature, 13000rpm, 2 min).

(8) The supernatant was taken and an equal volume of isopropanol was added to precipitate RNA1h at-20 ℃.

(9) After centrifugation (4 ℃, 13000rpm, 10min), the supernatant was removed. Washing the precipitate with 70% ethanol once, air drying, adding a proper amount of DEPC water to dissolve RNA, and performing electrophoresis detection, wherein 28S and 18S bands are complete as shown in figure 2, which shows that the RNA has high quality and meets the requirements of subsequent experiments.

All the consumables are RNase-free materials or are subjected to DEPC water treatment in advance and then are sterilized at high temperature.

3. Reverse transcription of RNA into cDNA

Reference is made to the TransScript One-Step gDNA Removal and cDNAsyntheses Super Mix kit from the company Beijing allkunjin (TransGen Biotech).

(1) A1.5 mL RNase free centrifuge tube was taken and the following ingredients were added:

(2) after incubation of the samples at 25 ℃ for 10min, the product was used for qPCR: incubating at 42 deg.C for 15 min; the product was used for PCR: incubate at 42 ℃ for 30 min.

(3) The sample was heated at 85 ℃ for 5 seconds to inactivate the TransScript RT/RI and the gDNARemover, and the sample was stored at-80 ℃ to obtain cDNA.

4. Cloning of RxLR132 Gene fragment RxLR132^aa21-119(300bp)

The full length of the RxLR132 gene was amplified by PCR using the cDNA as a template and the full length primers for the RxLR132 gene (see P1 and P2 in Table 1).

TABLE 1RxLR132 Gene primer information

Note: "CTAGA" in P3 and "GGTACC" in P4 are added cleavage sites.

The PCR reaction system is as follows:

the reaction procedure is as follows:

separating the PCR product by 2% agarose gel electrophoresis, distinguishing a target band under an ultraviolet lamp, cutting off a corresponding adhesive tape, and purifying and recovering the adhesive tape by a small fragment adhesive recovery kit, wherein the specific steps refer to the specification. Then, the RxLR132 gene fragment RxLR132 was amplified by PCR using the purified and recovered RxLR132 as a template and primers (P3 and P4) with XbaI and KpnI cleavage sites^aa21-119The PCR electrophoresis chart (FIG. 3) shows that the PCR product is about 300bp and accords with RxLR132^aa21-119Size. And then, recovering the PCR product by using a purification recovery kit to obtain a purified and recovered gene fragment.

Second, constructing a vector and sequencing verification

1. Construction of vectors

(1) The subcellular localization vectors pBI121-GFP and pBI121-mCherry and the purified and recovered gene fragment were digested simultaneously with XbaI and KpnI, respectively. The enzyme cutting system is as follows:

the enzyme digestion components are mixed evenly and cut for 2h at 37 ℃.

(2) The pBI121 carrier enzyme digestion product is purified and recovered by a PCR product recovery kit, and RxLR132^aa21-119The enzyme digestion product is purified and recovered by a small fragment purification and recovery kit.

(3) Connecting the vector and the target gene fragment, wherein the connecting body is:

the ligation components were mixed well and ligated overnight at 16 ℃.

2. Transformation of Escherichia coli

Each ligation system was added with 50. mu.L of E.coli DH 5. alpha. competent cells, mixed well and then left on ice for 30 min. Then, the mixture is heated at 42 ℃ for 90s and kept stand on ice for 2-3 min. Add 500. mu.L SOC liquid medium (2% Tryptone, 0.5% Yeast extract, 0.05% NaCl,2.5mM KCl,10mM MgCl)₂20mM glucose), resuscitative culture (37 ℃, 200rpm, 30 min). 200. mu.L of the bacterial suspension was applied to a plate of LB medium (1% Tryptone, 0.5% Yeast extract, 1% NaCl, 1.5% Agar) containing 50. mu.g/mL of kanamycin, and the plate was incubated at 37 ℃ for 36 hours in an incubator.

3. Positive transformant identification and preservation

And detecting whether the recombinant vectors pBI121-gene-GFP and pBI121-gene-mCherry are introduced into Escherichia coli by using a PCR technology. And (3) picking a small amount of thallus of a single colony from the LB plate by using a toothpick as a template of PCR reaction, and carrying out colony PCR verification by using corresponding primers. The colony PCR system is as follows:

the reaction procedure is as follows:

and (3) detecting the PCR product through electrophoresis, and obtaining the clone of which the amplified fragment is consistent with the target fragment as a positive clone according to the electrophoresis result. Positive clones were inoculated in LB liquid medium containing 50. mu.g/mL kanamycin for expanded culture. The correct plasmid was verified by sequencing and stored at-80 ℃ for use.

RxLR132^aa21-119The gene fragment is verified by sequencing, and the specific sequence information is as follows:

(1) the nucleotide sequence is shown as SEQ ID NO. 1;

(2) the amino acid sequence is shown as SEQ ID NO. 2.

Thirdly, transformation of recombinant plasmid

1. Preparation and transformation of agrobacterium electric shock competence

(1) The preserved Agrobacterium strain GV3101 was streaked on LB medium plate containing 50. mu.g/mL rifampicin (2 days at 30 ℃ C.).

(2) Single colonies were picked, inoculated in LB liquid medium containing 50. mu.g/mL rifampicin (30 ℃, 250rpm), and shaken to OD₆₀₀＝0.5。

(3) The bacterial solution was ice-cooled for 15min, and then dispensed into a sterile 50mL centrifuge tube, followed by centrifugation to collect the cells (4 ℃, 4000rpm, 15 min).

(4) The supernatant was discarded, and 30mL of ultrapure water (precooled at 4 ℃ C.) was added to resuspend and wash the cells, followed by centrifugation (15 min at 4 ℃ C., 4000 rpm).

(5) Repeat (4) twice.

(6) Discarding the supernatant, adding 5mL of 10% glycerol (precooling at 4 ℃) to resuspend the thalli, subpackaging 80 mu L of the thalli into 1.5mL of centrifuge tubes to obtain agrobacterium tumefaciens competence, quickly freezing by liquid nitrogen, and storing at-80 ℃ for later use.

2. Recombinant plasmid transformed agrobacterium tumefaciens

(1) The Agrobacterium was taken out and stored in a freezer at-80 ℃ for the competent state, and thawed on ice.

(2) 1 mu.L of plasmid to be transformed (pBI121 recombinant vector) is added into each tube of competence respectively, the mixture is mixed evenly, and the mixture is slowly added into an electric shock cup to prevent air bubbles from generating.

(3) The cuvette was placed in an electroporator and the conversion of the shock was performed at 2500V (mode P2).

(4) After the electric shock is finished, 1mL of pre-cooled LB liquid medium is added into the electric shock cup, 100 μ L of bacterial liquid is sucked and coated on an LB medium plate containing 50 μ g/mL kanamycin and 50 μ g/mL rifampicin.

(5) After the plate was cultured in an incubator at 30 ℃ for 2 days, a single colony was picked and subjected to colony PCR identification (refer to the PCR system and reaction procedure in "identification and preservation of Positive transformant" described above), and after amplification culture of positive clones, glycerol of 50% equivalent volume was added and the colonies were preserved at-80 ℃.

Four, subcellular localization and co-localization analysis

pBI121-RxLR132^aa21-119GFP and pBI121-RxLR132^aa21-119The mCherry adopts an agrobacterium-mediated nicotiana benthamiana transient expression system for expression, and the specific steps are as follows:

(1) agrobacterium (containing pBI121 recombinant vector) at-80 ℃ was taken and plate-activated in LB medium containing 50. mu.g/mL kanamycin and 50. mu.g/mL rifampicin (2 d culture at 30 ℃).

(2) Single colonies were picked up in 10mL LB liquid medium containing 50. mu.g/mL kanamycin and 50. mu.g/mL rifampicin, cultured at 30 ℃ and 250rpm to OD₆₀₀＝0.5～1.0。

(3) The cells were collected by centrifugation (24 ℃, 4000rpm, 5 min).

(4) The supernatant was discarded and 30mL of resuspension (10mM MgCl) was added₂) The cells were washed by resuspension and centrifuged (5 min at 24 ℃ C. and 4000 rpm).

(5) The supernatant was discarded and a resuspension (10mM MgCl) was added₂) Resuspending the cells and adjusting to OD₆₀₀(Co-expression, concentration of Agrobacterium containing different plasmids was adjusted to OD)₆₀₀Mixed again in equal volume ═ 0.8), and left to stand at 30 ℃ for 3 h.

(6) Taking tobacco potted seedlings growing for 5 weeks, and injecting the agrobacterium tumefaciens heavy suspension into tobacco leaves by using a syringe with a needle removed. The planting method of the tobacco comprises the following steps:

mixing the nutrient soil and vermiculite according to a ratio of 1:2, subpackaging in plastic flowerpots with the caliber of 10cm, sowing tobacco seeds in pots, and transplanting tobacco seedlings to new pots after 10 days, wherein 1 plant in each pot. Culturing in plant room with photoperiod of 12 h/dark of 12h and temperature of 22 deg.C, and periodically watering and fertilizing.

(7) And taking off the tobacco leaves after 2 days, and observing the fluorescence distribution condition under a laser confocal microscope. The excitation wavelength of GFP is 488nm, and the emission wavelength is 525 nm; the excitation wavelength of the mCherry is 580nm, and the emission wavelength is 610 nm; the excitation wavelength of chlorophyll autofluorescence is 488nm, and the emission wavelength is 628 nm.

Subcellular localization of the proteins was observed under confocal laser microscopy, and as shown in FIG. 4, GFP proteins localized to the nucleus and cytoplasm of tobacco cells. RxLR132^aa21-119The green fluorescence of GFP is distributed in the cytosol as an oval organelle, whose morphology is similar to that of chloroplasts. RxLR132 when the fluorescent reporter was replaced with mCheery^aa21-119The red fluorescence distribution and the green fluorescence distribution of the fusion protein expressed after the fusion of the gene and the fluorescence reporter gene are basically consistent, which indicates that the RxLR132 has the same color^aa21-119The specific localization of (a) is not affected by the fluorescent protein.

Fifthly, determining the size of the transient expression protein by protein immunoblotting (Western blot)

Western blot was performed to check whether the size of the transiently expressed protein was as expected.

(1) Collecting plant tissue, quick freezing with liquid nitrogen, and grinding into powder.

(2) Adding appropriate amount of protein extract (50mM Tris-HCl [ pH 7.5],150mM NaCl,1mM EDTA, 0.5% NP-40,1 Xcocktail, 1mM PMSF, 5. mu.M MG132) containing protease inhibitor into 2mL centrifuge tube, shaking, mixing, and ice-water bath for 10 min.

(3) Centrifugation was carried out at 13000rpm for 10min at 4 ℃ and the supernatant was transferred to a new 2mL centrifuge tube and subjected to Western blotting analysis after quantification by Bradford.

(4) SDS-PAGE electrophoresis gel preparation: according to the molecular cloning, separating gel with corresponding concentration is prepared according to the molecular weight of protein, the separating gel is flattened by isopropanol, after the separating gel is solidified, the isopropanol is poured out, 5% concentrated gel is poured on the separating gel, and the separating gel is used after being solidified.

(5) Protein sample treatment: taking a proper amount of protein sample to be detected, adding 5 xSDS-PAGE Loading Buffer, and carrying out boiling water bath for 10 min.

(6) Electrophoresis: and (3) taking a proper amount of protein sample for electrophoresis. The electrophoresis parameters are set as low voltage 60V running for 30min to run through the concentrated gel, and then the electrophoresis tank is placed in ice water to run for 60-90min at high voltage 180V.

(7) Film transfer: soaking the membrane transferring filter paper, the cellulose acetate membrane and the gel in a membrane transferring buffer solution, and placing the membrane transferring filter paper, the cellulose nitrate membrane (NC), the gel and the membrane transferring filter paper (cathode) in a semi-dry membrane transferring instrument according to the sequence of the (anode) membrane transferring filter paper, the (NC), the gel and the membrane transferring filter paper (cathode), wherein the membrane transferring parameters are as follows: 23V for 30 min.

(8) And (3) sealing: preparing 5% skimmed milk powder solution with TBST, placing NC membrane in appropriate amount of 5% milk powder, and sealing at room temperature for 1 hr.

(9) Primary antibody incubation: the blocked membranes were transferred to TBST solution (1: 10000 dilution) supplemented with primary anti-GFP, incubated at room temperature for 2h, and washed 3 times with TBST solution for 5min each time.

(10) And (3) secondary antibody incubation: the membrane was transferred to TBST solution (1: 10000 dilution) supplemented with goat anti-mouse secondary antibody and incubated for 1 h. Wash 3 times with TBST solution for 5min each time.

(11) And (3) developing: placing the membrane in X-ray cassette, dripping ECL color development solution on the membrane, covering with transparent membrane, and standing in dark for 2 min. The excess reaction solution was wiped off with filter paper, and the membrane was fixed with scotch tape. In a dark room, place the X-ray film right above the film and expose for 3-5 min. The film is then placed in a development apparatus for development.

(12) The NC film was taken out and placed in ponceau red dye solution to be dyed for 10s to confirm the transfer effect.

The expression of the proteins was analyzed by western blotting, and as shown in FIG. 5, a band of the corresponding size was detected for all the proteins (GFP 27 kD; RxLR 132)^aa21-119GFP 39kD), indicating that all proteins are normally expressed in tobacco.

Sixthly, co-localization analysis and verification of protein expression position

To determine RxLR132^aa21-119The exact localization of GFP in the cells, the above fusion proteins and the chloroplast autofluorescence were subjected to a co-localization analysis. The results are shown in FIG. 6, RxLR132^aa21-119Superposition of green fluorescence of GFP with chlorophyll autofluorescence, indicating RxLR132^aa21-119The gene is specifically positioned in chloroplast, namely, RxLR132 provided by the invention^aa21-119The gene can be used as a specific marker of plant cell chloroplast.

The present invention is described in detail with reference to the examples, but the embodiments of the present invention are not limited by the examples, and any other changes, substitutions, combinations, and simplifications made under the teaching of the patent core of the present invention are included in the protection scope of the present invention.

Sequence listing

<110> Shanghai university of transportation

<120> specific marker for plant cell chloroplast and application thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 300

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgctaataa gcgtgaccaa cgctgcagtg ggtgccaagc cggggcctca tgcaggtcgg 60

gacttagacg gaagcaccac atcaatgagc gtcaacgtgg acgacgaaga aagagggctt 120

tcagatatgc taaaaaggtt gcgttcaatg cttttcgatg cgaactctgc caccaaaggc 180

caggcgttga aaaaggacgc caaatccacc aaaagtgtta aagctgctgg tgctgcttca 240

aacgccaaga aggttggtca gctccgacac atgtggaatc agatcaaaaa tgttgagtac 300

<210> 2

<211> 100

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Leu Ile Ser Val Thr Asn Ala Ala Val Gly Ala Lys Pro Gly Pro

1 5 10 15

His Ala Gly Arg Asp Leu Asp Gly Ser Thr Thr Ser Met Ser Val Asn

20 25 30

Val Asp Asp Glu Glu Arg Gly Leu Ser Asp Met Leu Lys Arg Leu Arg

35 40 45

Ser Met Leu Phe Asp Ala Asn Ser Ala Thr Lys Gly Gln Ala Leu Lys

50 55 60

Lys Asp Ala Lys Ser Thr Lys Ser Val Lys Ala Ala Gly Ala Ala Ser

65 70 75 80

Asn Ala Lys Lys Val Gly Gln Leu Arg His Met Trp Asn Gln Ile Lys

85 90 95

Asn Val Glu Tyr

100

<210> 3

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gctctagaat gctaataagc gtgaccaacg 30

<210> 4

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggggtacccc gtactcaaca tttttgatct g 31

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgcgtcaaa ttcctcttgt cg 22

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ctcgtgaata tactgattcg 20

Claims (9)

1. A specific marker for plant cell chloroplasts, which is characterized in that: the specific marker of chloroplast of the plant cell is RxLR132^aa21-119The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. The plant cell chloroplast specific marker of claim 1, which is obtained by: cloning of RxLR132 from Plasmopara viticola cDNA^aa21-119Coding region of the gene, transforming into Escherichia coli, screening positive clone, and performing DNA sequencing.

3. The plant cell chloroplast specific marker of claim 2, wherein cloning of the RxLR132^{aa21 -119}The base sequence of the primer pair of the gene is shown as SEQ ID NO. 3 and SEQ ID NO. 4.

4. A plant cell chloroplast-specific marker according to any one of claims 1 to 3, wherein: the amino acid sequence of the protein is shown as SEQ ID NO. 2.

5. Use of a plant cell chloroplast specific marker according to any one of claims 1 to 3 in the detection of fluorescence from plant chlorophyll.

6. The application according to claim 5, wherein the specific application is: the RxLR132 is added^aa21-119Fusing the gene with a fluorescent reporter gene to obtain a fused gene; introducing the fusion gene into an expression vector to obtain a recombinant vector; transforming the recombinant vector into a host to obtain a transformant; the transformant is transformed into a target plant and used for marking chlorophyll.

7. Use according to claim 6, characterized in that: the fluorescent reporter gene comprises GFP, YFP, BFP, CFP, RFP, mCherry, mStrawberry, mTangerine, mBanana or mOrange.

8. Use according to claim 6, characterized in that: the expression vector comprises pBI 121.

9. Use according to claim 6, characterized in that: the host comprises Escherichia coli or Agrobacterium.

Images