CN113528450B - Establishment and application of rice protoplasm high-efficiency biotin marking system - Google Patents

Abstract

The invention discloses a method for labeling rice protoplasts with biotin. The method includes transferring a recombinant vector containing a TurboID-Flag encoding gene into a rice protoplast to obtain a rice protoplast containing a recombinant vector; The rice protoplasts are mixed with biotin to obtain a reaction system for reaction, and biotin-labeled rice protoplasts are obtained. The present invention applies the high-efficiency biotin labeling system based on TurboID to rice research for the first time. In the rice protoplast transient expression system, the high-efficiency biotin labeling system based on TurboID is established and optimized; under the condition that the number of seedlings used is certain (100 plants Seedlings, sampled 9-10 days after sowing), the prepared protoplasts can complete the labeling reaction at a lower biotin concentration (400 μM) and in a short time (2-3 hours) without adding cytotoxic substances. The obtained samples can be used for subsequent identification of interacting proteins, subcellular localization studies, etc.

Description

Establishment and application of an efficient biotin labeling system for rice protoplasts

Technical field

The invention relates to the field of biotechnology, and in particular to the establishment and application of a high-efficiency biotin labeling system for rice protoplasts.

Background technique

Rice is an important food crop in the world and one of the commonly used model plants in research. As a transient expression system, rice protoplasts have been widely used in physiological, biochemical and molecular mechanism research. Protoplast-based experimental technologies mainly include subcellular localization, gene transient expression analysis, protein interaction screening and verification, etc. When screening interacting factors, it is difficult to identify transient or weakly interacting proteins using traditional co-immunoprecipitation methods. However, the biotin proximity labeling technology developed in recent years has better solved this problem. However, the existing biotin proximity labeling system still has shortcomings such as high reaction temperature, long labeling time, and the need to add cytotoxic substances, and has not been widely used in rice research.

Contents of the invention

The problem to be solved by the present invention is that during the application process of the existing biotin proximity labeling system, the required reaction temperature is high, the labeling time is long, and cytotoxic substances need to be added.

In order to solve at least one of the above problems, the present invention provides a method for labeling rice protoplasts with biotin. The method includes transforming a recombinant vector containing a TurboID-Flag encoding gene into rice protoplasts to obtain rice containing the recombinant vector. In the protoplasts; the rice protoplasts containing the recombinant vector are mixed with biotin to obtain a reaction system for reaction to obtain biotin-labeled rice protoplasts, wherein the TurboID-Flag is a protein whose amino acid sequence is sequence 2. Sequence 2 contains 328 amino acids.

The coding sequence of the TurboID-Flag encoding gene is nucleotides 817-1800 of sequence 1. (Nucleotides 817-1800 of sequence 1 are the coding sequence of the coding chain of the TurboID-Flag coding gene)

Wherein, the recombinant vector is pAN580-TurboID-Flag, and the sequence of pAN580-TurboID-Flag is shown in Sequence 1. The corresponding rice protoplast transformed with the recombinant vector is the protoplast pAN580-TurboID-Flag.

Wherein, the concentration of biotin after mixing the rice protoplasts transformed with the recombinant vector and biotin is 50-600 μM.

Preferably, the concentration of biotin after mixing the rice protoplasts transformed with the recombinant vector and biotin is 400 μM.

Wherein, the reaction time after mixing the rice protoplasts transformed with the recombinant vector and biotin is 0.25-4 hours.

Preferably, the reaction time after mixing the rice protoplasts transformed with the recombinant vector and biotin is 2 hours.

The reaction system does not contain toxic substances. The toxic substances are hydrogen peroxide and the like.

The reaction system includes protoplast pAN580-TurboID-Flag, and the solvent is WI solution and biotin. Wherein, the WI solution contains 0.5M mannitol, 20mM KCl and 4mM MES, the solvent is water, pH=5.7, the content of protoplast pAN580-TurboID-Flag is ^2x105 /ml; the concentration of biotin is 200-400μM .

The reaction system consists of protoplast pAN580-TurboID-Flag, WI solution, biotin and phosphate buffer solution.

The present invention also provides a system (product) for preparing biotin-labeled rice protoplasts, which system includes any of the above-mentioned rice protoplasts containing recombinant vectors and biotin. That is, it includes rice protoplasts and biotin transformed with a recombinant vector. The recombinant vector is a vector containing a TurboID-Flag sequence. The TurboID-Flag sequence is the sequence shown in nucleotides 817-1800 of Sequence 1. The recombinant vector may be a vector containing only the TurboID-Flag sequence, or may contain a new sequence including the TurboID-Flag sequence for encoding a fusion protein formed by TurboID-Flag and the target protein.

Wherein, the reaction system does not contain toxic substances. The toxic substances are hydrogen peroxide and the like.

Wherein, the reaction system includes protoplast pAN580-TurboID-Flag, and the solvent is WI solution and biotin. Wherein, the WI solution contains 0.5M mannitol, 20mM KCl and 4mM MES, the solvent is water, pH=5.7, the content of protoplast pAN580-TurboID-Flag is ^2x105 /ml; the concentration of biotin is 200-400μM .

Wherein, the reaction system consists of protoplast pAN580-TurboID-Flag, WI solution, biotin and phosphate buffer solution.

Wherein, the vector containing the TurboID-Flag sequence is pAN580-TurboID-Flag, and the sequence of pAN580-TurboID-Flag is shown in Sequence 2. The corresponding rice protoplast transformed with the recombinant vector is the protoplast pAN580-TurboID-Flag.

Wherein, the concentration of biotin after mixing the rice protoplasts transformed with the recombinant vector and biotin is 50-600 μM.

Preferably, the concentration of biotin after mixing the rice protoplasts transformed with the recombinant vector and biotin is 400 μM.

The application of the rice protoplasts containing the recombinant vector and biotin in preparing biotin-labeled rice protoplasts should also be within the protection scope of the present invention.

The present invention also claims a recombinant vector for biotin-labeled rice protoplasts. The recombinant vector is pAN580-TurboID-Flag, and the sequence of pAN580-TurboID-Flag is shown in Sequence 2.

The application of the biotin-labeled rice protoplast system or the recombinant vector in biotin-labeled rice protoplasts should also be within the protection scope of the present invention.

The biotin-labeled rice protoplast method, the biotin-labeled rice protoplast system or the application of the recombinant vector in the identification of interacting proteins or subcellular localization research should also be within the protection scope of the present invention.

The present invention applies the proximity labeling system based on TurboID to rice research, and establishes and optimizes a high-efficiency biotin labeling technology system for protoplasts. Compared with the existing technology, the system of the present invention has the following obvious advantages: 1) its catalytic activity is greatly improved, and the reaction can be carried out at a lower biotin concentration; 2) the labeling time is shortened; 3) the room temperature is 20°C It can be carried out at low temperatures without the need for higher temperatures (such as 37°C). 4) There is no need to add cytotoxic substances (such as hydrogen peroxide, etc.). The establishment of this system will provide available tools for rice functional gene research, especially lay the foundation for the systematic screening and verification of transient or weakly interacting proteins, and is expected to form an efficient biotin labeling detection kit for rice protoplasm.

Description of the drawings

Figure 1 is a schematic diagram of the vector construction of the present invention.

Figure 2 shows the transient expression detection of the vector constructed in the present invention in rice protoplasts;

Streptavidin-HRP is used to detect biotinylated protein levels; α-Flag is used to detect TurboID protein expression.

Figure 3 shows the optimization of the TurboID biotin labeling system (optimization of the concentration of exogenously added biotin). Streptavidin-HRP is used to detect biotinylated protein levels; α-Flag is used to detect TurboID protein expression.

Figure 4 shows the optimization of the TurboID biotin labeling system (optimization of labeling time). Streptavidin-HRP is used to detect biotinylated protein levels; α-Flag is used to detect TurboID protein expression.

Detailed ways

The present invention will be described in further detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

The experimental methods in the following examples are all conventional methods unless otherwise specified. Materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

Example 1

1. Construction of carrier

1) Construction based on biotin-labeled vector pAN580-TurboID-Flag

Using pCAMBIA1305-TurboID plasmid (purchased from Addgene Company) as a template, primer 1 (sequence 3) (5'-GGACCGGTCCCGGG GGATCC ATGAAAGACAATACTGTG-3', the sequence shown underline is the BamHI recognition site) and primer 2 (5 '-TGCAGCCGGGCGGCCGCTTTA AGATCT CTTGT CATCGTCGTCCTTGTAGTCCTTTTCGGCAGACCGCA-3', where the underlined sequence is the Bgl II recognition site, as shown in Sequence 4) is a primer for PCR amplification, and the length of the amplified fragment obtained is 1031bp, which contains the TurboID gene The complete coding region and Flag tag coding sequence were named as TurboID-Flag fragment.

The PCR reaction system is: DNA (100ng/μl) 0.2μl, primer 1 (100pmol/μl) 0.1μl, primer 2 (100pmol/μl) 0.1μl, 2x Buffer 25μl, dNTP (2.5mM) 4μl, PrimerStar (5u /μl, Takara Company) 0.5μl, ddH ₂ O 20.1μl, the total volume is 50μl. The amplification reaction was performed on a PTC-200 (MJ Research Inc.) PCR instrument. The reaction conditions were: 98°C for 2 min; 98°C for 10 sec, 50°C for 5 sec, and 72°C for 1 min, 2 cycles; 98°C for 10 sec, 65°C for 5 sec. 72℃ 1min, 33 cycles; 72℃ 5min. The PCR product was purified and recovered (the kit was purchased from Beijing Tiangen Company) and detected by 1% agarose gel electrophoresis.

Use In- HD Cloning Kit recombination kit (Takara Company), using TurboID-Flag fragment to replace the BamHI recognition site and BglII recognition site on the pAN580 vector (OsALMT7 Maintains Panicle Size and Grain Yield in Rice by Mediating Malate Transport, The Plant Cell) between sequences and keeping other sequences unchanged, we get

pAN580-TurboID-Flag vector. The In-Fusion recombination reaction system (10 μl) used is: 10-200 ng of PCR product, 50-200 ng of pAN580 vector, 2 μl of 5×In-Fusion HD Enzyme Premix, and make up to 10 μl with ddH ₂ O. After pipetting and mixing with the pipette tip, the mixed system was reacted at 50°C for 15 minutes, then placed on ice. 2 μl of the reaction system was used to transform Escherichia coli DH5α competent cells (Tiangen Company) using the heat shock method. All transformed cells were evenly spread on LB solid medium containing 100 mg/L ampicillin and cultured at 37°C for 12-16 hours. Pick positive clones, sequence them, and obtain the vector pAN580-TurboID-Flag. The sequence of the vector pAN580-TurboID-Flag is shown in Sequence 1.

2) Construction of vector pAN580-GFP

The vector pAN580-GFP is a recombinant vector obtained by replacing positions 817-1800 in sequence 1 with the green fluorescent protein GFP sequence and keeping other sequences unchanged.

The schematic structural diagram of vector pAN580-GFP and vector pAN580-TurboID-Flag is shown in Figure 1. In Figure 1, pAN580-GFP is used to represent the structural schematic of vector pAN580-GFP; pAN580-TurboID-Flag is used to represent the structure of vector pAN580-TurboID-Flag. Structural representation.

2. Preparation of rice protoplasts

Referring to the method in the article (Zhang et al. Plant Methods 2011, 7:30), each reaction system contains 300 μl of rice protoplasts, and the vector pAN580-TurboID-Flag is transformed into the rice protoplasts to obtain the protoplast pAN580-TurboID-Flag. ; The vector pAN580-GFP was transformed into rice protoplasts to obtain protoplast pAN580-GFP; protoplast pAN580-GFP was used as a negative control, and protoplast pAN580-TurboID-Flag was used as the experimental group to conduct experiments.

Example 2 Determination of whether pAN580-TurboID-Flag can be expressed in rice protoplasts

The protoplast pAN580-GFP prepared in Example 1 was used as the negative control, and the protoplast pAN580-TurboID-Flag was used as the experimental group. According to different experimental conditions, corresponding concentrations of biotin (Sigma Aldrich) were added, and the labeling experiment was started. The experiments were all conducted at room temperature 20°C.

In order to test whether the biotin proximity labeling technology based on TurboID can be applied to the rice protoplast transient expression system, the protoplast pAN580-GFP constructed in Example 1 was used as a negative control (shown as pAN580-GFP in Figure 2). Body pAN580-TurboID-Flag (used in Figure 2

pAN580-TurboID-Flag) was used as the experimental group. In the control and experimental groups, the biotin-free (marked as -) and biotin-added groups (marked as +, final concentration is 200 μM) were set respectively. The specific steps are as follows:

1. Mix the protoplast pAN580-TurboID-Flag prepared in Example 1 and biotin to form a reaction system, wherein the concentration of protoplast pAN580-TurboID-Flag in the reaction system is 2x10 ⁵ /ml, and the solvent is WI solution (0.5M mannitol, 20mM KCl and 4mM MES included in the WI solution, the solvent is water, pH=5.7); the concentration of biotin is 200 μM; biotin is obtained by adding biotin solution (the biotin solution is Biotin is dissolved in phosphate buffer (PH=7.5), so the reaction system also contains a small amount of phosphate buffer. After 2 hours of reaction, protein sample 1 was obtained.

Protein sample 1 was used to detect the labeling effect by Western blot: After the obtained protein sample was separated on SDS-PAGE gel, it was transferred to NC membrane; after blocking with PBST solution containing 2.5% BSA for 1 hour, Streptavidin (dilution 1 : 2000, Catalog No.: ABCam, AB7403) hybridize for 1 hour; then wash 4 times with PBST solution (15 minutes each time), image with Biorad ChemiDoc Touch, and obtain the band corresponding to pAN580-TurboID-Flag in the Streptavidin-HRP part in Figure 2 ; Separate the obtained protein sample 1 on SDS-PAGE gel and transfer it to NC membrane; after blocking for 1 hour with PBST solution containing 5% skimmed milk powder, add Flag antibody (dilution 1:2000, product number: MBL, M185-7) hybridized for 1 hour; washed 4 times with PBST solution (15 minutes each time), imaged with Biorad ChemiDoc Touch, and obtained the band corresponding to pAN580-TurboID-Flag in the anti-Flag part of Figure 2.

2. Replace the protoplast pAN580-TurboID-Flag in step 1 with the protoplast pAN580-GFP. Under the same reaction conditions, protein sample 2 is obtained. The protein sample 2 is subjected to the same detection to obtain the Streptavidin-HRP part and figure in Figure 2. The band corresponding to pAN580-GFP in the 2 anti-Flag section.

The results are shown in Figure 2. From the results of the anti-Flag part in Figure 2, it can be seen that the pAN580-TurboID-Flag vector can be well expressed in rice protoplasts regardless of whether exogenous biotin is added or not. Biotin has nothing to do with it; it can be seen from the results of the Streptavidin-HRP part in Figure 2 that under the conditions of an added biotin concentration of 200 μM, the biotin-labeled band can be detected within 2 hours. The results in Figure 2 show that the TurboID-based system can complete the biotin labeling process in a short time (2 hours), and it is feasible and efficient to apply this system to transient expression of rice protoplasts.

Example 3. Determining the optimal concentration of biotin required for labeling:

In order to further optimize the biotin proximity labeling system based on TurboID and achieve short-term and efficient labeling effects, the labeling efficiency was tested under different biotin concentrations, and the optimization conditions required for TurboID to complete efficient labeling were initially explored.

Take protoplast pAN580-TurboID-Flag and different added amounts of biotin to form 7 groups of reaction systems (systems A1-A7 respectively). Systems A1-A7 all include 300 μl/tube of protoplast pAN580-TurboID-Flag, concentration 2x10 ⁵ /ml, the solvent is WI solution (the WI solution includes 0.5M mannitol, 20mM KCl and 4mM MES, the solvent is water, pH=5.7). System 1 is a control without adding biotin, and different amounts of biotin solutions (the solutions are obtained by dissolving biotin in phosphate buffer with pH = 7.5) are added to the reaction system in systems 2-7. The biotin concentrations are: 50μM, 100μM, 200μM, 300μM, 400μM and 600μM. (Other components in the reaction system are the same as in Example 2) After 2 hours of reaction, a protein sample was obtained, and the labeling effect was detected by Western blot according to step 1 in Example 2; protein samples A1-A7 were obtained in systems A1-A7, respectively. , corresponding to the biotin concentrations of 0, 50 μM, 100 μM, 200 μM, 300 μM, 400 μM and 600 μM in Figure 3.

The results are shown in Figure 3. Streptavidin-HRP in Figure 3 indicates that the protein samples were separated according to the detection system: after separation on SDS-PAGE gel, the obtained protein samples were transferred to NC membrane; PBST containing 2.5% BSA was used. After blocking the solution for 1 hour, add Streptavidin (dilution 1:2000, product number: ABCam, AB7403) for hybridization for 1 hour; then wash with PBST solution 4 times (15 minutes each time), and image with Biorad ChemiDoc Touch; anti-Flag indicates According to the detection system, the protein samples were separated as follows: after separation on SDS-PAGE gel, the obtained protein samples were transferred to NC membrane; after blocking with PBST solution containing 5% skim milk powder for 1 hour, Flag antibody (dilution 1: 2000, Catalog No.: MBL, M185-7) hybridized for 1 hour; then washed 4 times with PBST solution (15 minutes each time), and then imaged with Biorad ChemiDoc Touch.

It can be seen from the anti-Flag results in Figure 3 that the pAN580-TurboID-Flag vectors in systems 1-7 can be well expressed in rice protoplasts, regardless of whether exogenous biotin is added and the concentration of biotin; from It can be seen from the results of the Streptavidin-HRP part in Figure 3 that when the external biotin concentration is 0-300 μM, the level of biotinylated protein labeled by TurboID continues to increase and reaches a stable state at 400 μM (shown by the arrow on the right band changes as an example). When the biotin concentration continued to increase (600 μM), the protein biotinylation level did not change significantly (the gray value of the biotinylation band did not increase or decrease significantly). This shows that in the existing experimental system, when the concentration of exogenously added biotin reaches 400 μM, TurboID can complete the biotinylation labeling of adjacent proteins in a short time.

Example 4. Determining the optimal biotin treatment time required for labeling:

In order to further optimize the biotin proximity labeling system based on TurboID and achieve short-term and efficient labeling effects, the labeling efficiency was tested under different processing times, and the optimization conditions required for TurboID to complete efficient labeling were preliminarily explored.

The protoplast pAN580-TurboID-Flag prepared in Example 1 was mixed with biotin and divided into 8 tubes, respectively marked as tubes B1-B8. Each tube contained 300 μl of protoplast pAN580-TurboID-Flag at a concentration of 2x10 ⁵ /ml. The solvent is WI solution (the WI solution includes 0.5M mannitol, 20mM KCl and 4mM MES, the solvent is water, pH=5.7), and the biotin in each tube reaches a concentration of 400 μM (biotin is pre-dissolved in pH=5.7). 7.5 of the phosphate buffer, add the biotin solution into the test tube to a concentration of 400 μM). Tube B1 is mixed and directly sampled to obtain protein sample B1. The remaining tubes B2-B8 react with 0.25, 0.5, 1, 2, 3, and 4 respectively. After 6 hours, protein samples B2-B8 were obtained. The labeling effect was detected by Westernblot in step 1 of Example 2. The results are shown in Figure 4. Protein samples B1-B8 correspond to the reaction times of 0, 0.25, and 0 in Figure 4 respectively. 0.5, 1, 2, 3, 4 and 6 hours. Among them, Streptavidin-HRP in Figure 4 indicates that the protein samples B1-B8 are separated according to the detection system: after the obtained protein samples B1-B8 are separated on the SDS-PAGE gel, they are transferred to the NC membrane; PBST solution containing 2.5% BSA is used. After blocking for 1 hour, add Streptavidin (dilution 1:2000, product number: ABCam, AB7403) for hybridization for 1 hour; wash 4 times with PBST solution (15 minutes each time), and image with Biorad ChemiDoc Touch; anti-Flag indicates that the Protein samples B1-B8 follow the detection system as follows: separate the obtained protein samples B1-B8 on SDS-PAGE gel and transfer to NC membrane; block with PBST solution containing 5% skimmed milk powder for 1 hour, then add Flag antibody (Dilution 1:2000, Catalog No.: MBL, M185-7) hybridize for 1 hour; wash 4 times with PBST solution (15 minutes each time), and image with Biorad ChemiDocTouch.

It can be seen from the anti-Flag results in Figure 4 that the pAN580-TurboID-Flag vector in the 8 tube samples can be well expressed in rice protoplasts; it can be seen from the results of the Streptavidin-HRP part in Figure 3 that TurboID The system can biotinylate proteins within 15 minutes (0.25 hours) (take the band shown by the red arrow as an example). During the 0-3 hour mark time, TurboID-mediated protein biotinylation levels continued to increase. Continue to extend the marking time to 4-6 hours, and the marking effect does not change significantly. This shows that under the current experimental system, when the biotin concentration is 400 μM, TurboID can achieve good labeling effect in a short time (2-3 hours).

The specific method of sampling and testing in the above embodiments is as follows: add 1 ml of W5 solution to each tube of reaction system, and centrifuge at 200g for 5 minutes. Add 50 μl of 1x loading buffer, boil the sample for 10 minutes, centrifuge at 13,000 rpm for 1 minute, and take the supernatant to obtain a protein sample.

This invention uses a high-efficiency biotin labeling system based on TurboID for rice research for the first time. In the rice protoplast transient expression system, an efficient biotin labeling system based on TurboID was established and optimized. When a certain number of seedlings are used (100 seedlings, sampled 9-10 days after sowing), the prepared protoplasts can be labeled at a lower biotin concentration (400 μM) and in a short time (2-3 hours) reaction without adding cytotoxic substances. The obtained samples can be used for subsequent identification of interacting proteins, subcellular localization studies, etc.

Compared with previous methods, this system has many advantages such as short labeling time, high catalytic efficiency, and wide reaction temperature range, and is expected to play an important role in identifying transient or weakly interacting proteins. In future experiments, we will try to further explore the potential application value of this technology in different tissues and organs of rice. This will provide a powerful research tool and platform for the rice field, and is expected to form a kit for use by colleagues in the research field.

The present invention has been described in detail above. For those skilled in the art, the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. Although specific embodiments of the present invention have been shown, it should be understood that further modifications can be made to the invention. In short, based on the principles of the present invention, this application is intended to include any changes, uses, or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some essential features may be applied within the scope of the appended claims below.

sequence list

<110> Institute of Crop Science, Chinese Academy of Agricultural Sciences

<120> Establishment and application of an efficient biotin labeling system for rice protoplasts

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agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 3420

gtcgtgtaga taactacgat acggggagggc ttaccatctg gccccagtgc tgcaatgata 3480

ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 3540

gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 3600

cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 3660

acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 3720

cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 3780

cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 3840

ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 3900

tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 3960

atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 4020

tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 4080

actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 4140

aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 4200

ctcatactct tcctttttca atattattga agcatttatc agggttatattg tctcatgagc 4260

ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 4320

cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt 4380

gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa 4440

aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagtccactattaa 4500

agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac 4560

gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga 4620

accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa 4680

aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc 4740

tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc 4800

cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc 4860

agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc 4920

agtcacgacg ttgtaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat 4980

tg 4982

<210> 2

<211> 328

<212> PRT

<213> Artificial Sequence

<400> 2

Met Lys Asp Asn Thr Val Pro Leu Lys Leu Ile Ala Leu Leu Ala Asn

1 5 10 15

Gly Glu Phe His Ser Gly Glu Gln Leu Gly Glu Thr Leu Gly Met Ser

20 25 30

Arg Ala Ala Ile Asn Lys His Ile Gln Thr Leu Arg Asp Trp Gly Val

35 40 45

Asp Val Phe Thr Val Pro Gly Lys Gly Tyr Ser Leu Pro Glu Pro Ile

50 55 60

Pro Leu Leu Asn Ala Lys Gln Ile Leu Gly Gln Leu Asp Gly Gly Ser

65 70 75 80

Val Ala Val Leu Pro Val Val Asp Ser Thr Asn Gln Tyr Leu Leu Asp

85 90 95

Arg Ile Gly Glu Leu Lys Ser Gly Asp Ala Cys Ile Ala Glu Tyr Gln

100 105 110

Gln Ala Gly Arg Gly Ser Arg Gly Arg Lys Trp Phe Ser Pro Phe Gly

115 120 125

Ala Asn Leu Tyr Leu Ser Met Phe Trp Arg Leu Lys Arg Gly Pro Ala

130 135 140

Ala Ile Gly Leu Gly Pro Val Ile Gly Ile Val Met Ala Glu Ala Leu

145 150 155 160

Arg Lys Leu Gly Ala Asp Lys Val Arg Val Lys Trp Pro Asn Asp Leu

165 170 175

Tyr Leu Gln Asp Arg Lys Leu Ala Gly Ile Leu Val Glu Leu Ala Gly

180 185 190

Ile Thr Gly Asp Ala Ala Gln Ile Val Ile Gly Ala Gly Ile Asn Val

195 200 205

Ala Met Arg Arg Val Glu Glu Ser Val Val Asn Gln Gly Trp Ile Thr

210 215 220

Leu Gln Glu Ala Gly Ile Asn Leu Asp Arg Asn Thr Leu Ala Ala Thr

225 230 235 240

Leu Ile Arg Glu Leu Arg Ala Ala Leu Glu Leu Phe Glu Gln Glu Gly

245 250 255

Leu Ala Pro Tyr Leu Pro Arg Trp Glu Lys Leu Asp Asn Phe Ile Asn

260 265 270

Arg Pro Val Lys Leu Ile Ile Gly Asp Lys Glu Ile Phe Gly Ile Ser

275 280 285

Arg Gly Ile Asp Lys Gln Gly Ala Leu Leu Leu Glu Gln Asp Gly Val

290 295 300

Ile Lys Pro Trp Met Gly Gly Glu Ile Ser Leu Arg Ser Ala Glu Lys

305 310 315 320

Asp Tyr Lys Asp Asp Asp Asp Lys

325

<210> 3

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 3

ggaccggtcc cgggggatcc atgaaagaca atactgtg 38

<210> 4

<211> 68

<212> DNA

<213> Artificial Sequence

<400> 4

tgcagccggg cggccgcttt aagatctctt gtcatcgtcg tccttgtagt ccttttcggc 60

agaccgca 68

Claims (10)

1. A method for labeling rice protoplasts with biotin, characterized in that the method includes transferring the recombinant vector containing the TurboID-Flag encoding gene into the rice protoplasts to obtain the rice protoplasts containing the recombinant vector; The rice protoplasts of the recombinant vector are mixed with biotin to obtain a reaction system for reaction, and biotin-labeled rice protoplasts are obtained, wherein the amino acid sequence of the TurboID-Flag is shown in SEQ ID NO: 2. 2. The method according to claim 1, characterized in that the coding sequence of the TurboID-Flag encoding gene is shown in positions 817-1800 of SEQ ID NO: 1. 3. The method according to claim 1 or 2, characterized in that the concentration of biotin in the reaction system is 50-600 μM. 4. The method according to claim 1 or 2, characterized in that the reaction time is 0.25-4 hours. 5. The method according to claim 1 or 2, characterized in that the reaction system does not contain toxic substances. 6. A system for preparing biotin-labeled rice protoplasts, characterized in that the system includes the rice protoplasts containing the recombinant vector according to any one of claims 1-5 and biotin. 7. The system for preparing biotin-labeled rice protoplasts according to claim 6, wherein the reaction system is composed of protoplasts, biotin, mannitol, potassium chloride, MES buffer solution and phosphate buffer. 8. Use of the rice protoplasts containing the recombinant vector and biotin according to any one of claims 1 to 5 in the preparation of biotin-labeled rice protoplasts. 9. A recombinant vector for biotin-labeled rice protoplasts, the recombinant vector is pAN580-TurboID-Flag, and the nucleotide sequence of pAN580-TurboID-Flag is shown in SEQ ID NO: 1. 10. The method of claims 1-5, the biotin-labeled rice protoplast system of claims 6-7 or the recombinant vector of claim 9 in biotin-labeled rice protoplasts, identification of interacting proteins or subcellular localization applications in.