CN113528450A - Establishment and application of rice protoplast high-efficiency biotin labeling system - Google Patents
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Abstract
The invention discloses a method for marking rice protoplast by biotin, which comprises the steps of transferring a recombinant vector containing TurboID-Flag coding gene into the rice protoplast to obtain the rice protoplast containing the recombinant vector; and mixing the rice protoplast containing the recombinant vector with biotin to obtain a reaction system for reaction, thus obtaining the rice protoplast marked by the biotin. The high-efficiency biotin labeling system based on TurboID is used for rice research for the first time, and is established and optimized in a rice protoplast transient expression system; under the condition of a certain number of seedlings (100 seedlings, and sampling 9-10 days after sowing), the prepared protoplast can complete the labeling reaction at a lower biotin concentration (400 mu M) in a short time (2-3 hours) without adding cytotoxic substances. The obtained sample can be used for subsequent interactive protein identification, subcellular localization research and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to establishment and application of a rice protoplast high-efficiency biotin labeling system.
Background
Rice is an important food crop in the world and is one of the model plants commonly used for research. Rice protoplasts have been widely used in physiological, biochemical and molecular mechanism studies as a transient expression system. The protoplast-based experimental technology mainly comprises subcellular localization, gene transient expression analysis, protein interaction screening and verification and the like. When the interaction factors are screened, the traditional co-immunoprecipitation method is difficult to identify instantaneous or weak interaction protein; the biotin proximity labeling technology developed in recent years better solves the problem. However, the existing biotin proximity labeling system still has the defects of high required reaction temperature, long labeling time, addition of cytotoxic substances and the like, and is not widely applied to rice research.
Disclosure of Invention
The invention aims to solve the problems that the reaction temperature is high, the labeling time is long and a cytotoxic substance needs to be added in the application process of the conventional biotin proximity labeling system.
In order to solve at least one of the above problems, the present invention provides a method for labeling rice protoplasts with biotin, comprising transferring a recombinant vector comprising a gene encoding TurboID-Flag into rice protoplasts to obtain rice protoplasts comprising the recombinant vector; and mixing the rice protoplast containing the recombinant vector with biotin to obtain a reaction system for reaction to obtain the rice protoplast marked by the biotin, wherein the TurboID-Flag is a protein with an amino acid sequence of sequence 2. Sequence 2 comprises 328 amino acids.
The coding sequence of the TurboID-Flag coding gene is the 817-th 1800 th nucleotide of the sequence 1. (the sequence 1, the 817-1800 th nucleotide is the coding sequence of the coding strand of the TurboID-Flag-encoding gene)
Wherein the recombinant vector is pAN580-TurboID-Flag, and the sequence of the pAN580-TurboID-Flag is shown as sequence 1. The corresponding rice protoplast transformed with the recombinant vector is the protoplast pAN 580-TurboID-Flag.
Wherein the concentration of the biotin after the rice protoplast transformed with the recombinant vector is mixed with the biotin is 50-600 mu M.
Preferably, the concentration of biotin after the rice protoplast transformed with the recombinant vector is mixed with biotin is 400. mu.M.
Wherein the reaction time of the rice protoplast transformed with the recombinant vector after being mixed with biotin is 0.25 to 4 hours.
Preferably, the reaction time after the rice protoplast transformed with the recombinant vector is mixed with biotin is 2 hours.
The reaction system does not contain toxic substances. The toxic substance is hydrogen peroxide and the like.
Said counterThe system comprises protoplast pAN580-TurboID-Flag, solvent WI solution and biotin. Wherein the WI solution contains 0.5M mannitol, 20mM KCl and 4mM MES, water as solvent, pH 5.7, and protoplast pAN580-TurboID-Flag content of 2x105Per ml; the concentration of biotin was 200-400. mu.M.
The reaction system consists of protoplast pAN580-TurboID-Flag, WI solution, biotin and phosphate buffer solution.
The invention also provides a system (product) for preparing the rice protoplast marked by the biotin, wherein the system comprises the rice protoplast containing the recombinant vector and the biotin. The recombinant vector is a vector containing a TurboID-Flag sequence, and the TurboID-Flag sequence is a sequence shown by 1-328 th nucleotides of a sequence 1. The recombinant vector may be a vector containing only a TurboID-Flag sequence, and may be a novel sequence containing a TurboID-Flag sequence for encoding a fusion protein of TurboID-Flag with a target protein.
Wherein the reaction system does not contain toxic substances. The toxic substance is hydrogen peroxide and the like.
Wherein the reaction system comprises protoplast pAN580-TurboID-Flag, solvent WI solution and biotin. Wherein the WI solution contains 0.5M mannitol, 20mM KCl and 4mM MES, water as solvent, pH 5.7, and protoplast pAN580-TurboID-Flag content of 2x105Per ml; the concentration of biotin was 200-400. mu.M.
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 the pAN580-TurboID-Flag is shown as a sequence 2. The corresponding rice protoplast transformed with the recombinant vector is the protoplast pAN 580-TurboID-Flag.
Wherein the concentration of the biotin after the rice protoplast transformed with the recombinant vector is mixed with the biotin is 50-600 mu M.
Preferably, the concentration of biotin after the rice protoplast transformed with the recombinant vector is mixed with biotin is 400. mu.M.
The rice protoplast containing the recombinant vector and the application of the biotin in the preparation of the rice protoplast marked by the biotin also belong to the protection scope of the invention.
The invention also claims a recombinant vector for marking rice protoplasts with biotin, wherein the recombinant vector is pAN580-TurboID-Flag, and the sequence of the pAN580-TurboID-Flag is shown as sequence 2.
The application of the biotin-labeled rice protoplast system or the recombinant vector in the biotin-labeled rice protoplast is also within the protection scope of the invention.
The method for marking the rice protoplast by the biotin, the application of the rice protoplast system marked by the biotin or the recombinant vector in interactive protein identification or subcellular localization research also belong to the protection scope of the invention.
The method applies a proximity labeling system based on TurboID to rice research, and establishes and optimizes a protoplast high-efficiency biotin labeling technical system. Compared with the prior art, the system of the invention has the following obvious advantages: 1) the catalytic activity of the catalyst is greatly improved, and the reaction can be carried out under the condition of lower biotin concentration; 2) the marking time is shortened; 3) room temperature 20 ℃ without the need for higher temperatures (e.g. 37 ℃)4) without the need for addition of cytotoxic substances (e.g. hydrogen peroxide, etc.). The establishment of the system provides a usable tool for the research of functional genes of rice, particularly lays a foundation for the system screening and verification of instantaneous or weak interaction protein, and is expected to form a rice protoplasm high-efficiency biotin labeling detection kit.
Drawings
FIG. 1 is a schematic diagram of the construction of the vector of the present invention.
FIG. 2 shows the detection of transient expression of a vector constructed in the present invention in rice protoplasts;
Streptavidin-HRP was used to detect biotinylated protein levels; alpha-Flag was used to detect the expression of TurboID protein.
FIG. 3 is an optimization based on TurboID biotin labeling system (optimization of exogenously added biotin concentration) where Streptavidin-HRP was used to detect biotinylated protein levels; alpha-Flag was used to detect the expression of TurboID protein.
FIG. 4 is an optimization based on TurboID biotin labeling system (optimization of labeling time) wherein Streptavidin-HRP was used to detect biotinylated protein levels; alpha-Flag was used to detect the expression of TurboID protein.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
1. Construction of vectors
1) Construction of pAN580-TurboID-Flag based on biotin-labeled vector
pCAMBIA1305-TurboID plasmid (purchased from Addgene) as a template, and primer 1 (SEQ ID NO: 3) (5' -GGACCGGTCCCGGG)GGATCCATGAAAGACAATACTGTG-3 ', in which the underlined sequence is a BamH I recognition site) and primer 2 (5' -TGCAGCCGGGCGGCCGCTTTA)AGATCTCTTGT CATCGTCGTCCTTGTAGTCCTTTTCGGCAGACCGCA-3', wherein the underlined sequence is a Bgl II recognition site, as shown in the sequence 4) as a primer, and the obtained amplified fragment has the length of 1031bp, and comprises the complete coding region of the TurboID gene and the Flag tag coding sequence, and is named as the TurboID-Flag fragment.
The PCR reaction system is as follows: 0.2. mu.l of DNA (100 ng/. mu.l), 0.1. mu.l of primer 1(100 pmol/. mu.l), 0.1. mu.l of primer 2(100 pmol/. mu.l), 25. mu.l of 2 XBuffer, 4. mu.l of dNTP (2.5mM), 0.5. mu.l of PrimerStar (5 u/. mu.l, Takara Co., Ltd.), ddH2O20.1. mu.l, total volume 50. mu.l. The amplification reaction was performed on a PTC-200(MJ Research Inc.) PCR instrument under the following conditions: 2min at 98 ℃; 10sec at 98 ℃, 5sec at 50 ℃, 1min at 72 ℃ for 2 cycles; 10sec at 98 ℃, 5sec at 65 ℃, 1min at 72 ℃ and 33 cycles; 5min at 72 ℃. The PCR product was purified and recovered (kit purchased from Tiangen, Beijing) and detected by electrophoresis on a 1% agarose gel.
By using
HD Cloning Kit (Takara Co., Ltd.) obtained by replacing The sequence between The BamH I recognition site and The BglII recognition site on pAN580 vector (OsALMT7 maize plasmid Size and Grain Yield in Rice by media plasmid transfer, The Plant Cell) with TurboID-Flag fragment while keeping The other sequences unchanged
pAN580-TurboID-Flag vector. The In-Fusion recombination reaction system (10. mu.l) used was: PCR product 10-200ng, pAN580 vector 50-200ng, 5 XIn-Fusion HD Enzyme Premix 2. mu.l with ddH2O make up to 10. mu.l. After the tip was blown and mixed, the mixed system was reacted at 50 ℃ for 15min, and then placed on ice, and 2. mu.l of the reaction system was used to transform E.coli DH 5. alpha. competent cells (Tiangen Co.) by heat shock. All the transformed cells were spread evenly on LB solid medium containing 100mg/L ampicillin, and cultured at 37 ℃ for 12-16 h. Positive clones were picked and sequenced to obtain the vector pAN 580-TurboID-Flag. The sequence of the vector pAN580-TurboID-Flag is shown as sequence 1.
2) Construction of vector pAN580-GFP
The vector pAN580-GFP is a recombinant vector obtained by replacing 817 th to 1800 th sites in the sequence 1 with a green fluorescent protein GFP sequence and keeping other sequences unchanged.
The structure schematic diagram of the vector pAN580-GFP and the vector pAN580-TurboID-Flag is shown in FIG. 1, and the structure schematic diagram of the vector pAN580-GFP is shown by pAN580-GFP in FIG. 1; the structure of the vector pAN580-TurboID-Flag is indicated by pAN 580-TurboID-Flag.
2. Preparation of Rice protoplasts
The method described in the reference article (Zhang et al plant Methods 2011,7:30) contained 300. mu.l of rice protoplasts per reaction system, and transformed the vector pAN580-TurboID-Flag into rice protoplasts to give protoplasts pAN 580-TurboID-Flag; transforming the vector pAN580-GFP into a rice protoplast to obtain a protoplast pAN 580-GFP; experiments were performed with protoplast pAN580-GFP as negative control and protoplast pAN580-TurboID-Flag as experimental group.
Example 2 determination of whether pAN580-TurboID-Flag can be expressed in Rice protoplasts
The test was carried out using the protoplast pAN580-GFP prepared in example 1 as a negative control and the protoplast pAN580-TurboID-Flag as a test group, adding biotin (Sigma Aldrich) at the corresponding concentration according to the different test conditions and starting the labeling test, all at room temperature 20 ℃.
To examine whether the TurboID-based biotin proximity labeling technique can be applied to the rice protoplast transient expression system, the construct of example 1 was performed using the protoplast pAN580-GFP as a negative control (denoted by pAN580-GFP in FIG. 2) and using the protoplast pAN580-TurboID-Flag (denoted by pAN580-TurboID-Flag in FIG. 2)
pAN 580-TurboID-Flag) as an experimental group, and a biotin-added group (marked as + and with a final concentration of 200. mu.M) with no biotin added (marked as-), and a biotin-added group (marked as +) in a control group and an experimental group respectively, as follows:
1. the protoplast pAN580-TurboID-Flag prepared in example 1 was mixed with biotin to form a reaction system, wherein the concentration of the protoplast pAN580-TurboID-Flag in the reaction system was 2X105A solvent of WI solution (0.5M mannitol, 20mM KCl and 4mM MES contained in the WI solution, the solvent is water, and the pH is 5.7); the concentration of biotin is 200 μ M; biotin is obtained by adding a biotin solution (which is a phosphate buffer solution prepared by dissolving biotin in PH 7.5), and therefore the reaction system also contains a small amount of phosphate buffer solution. After 2 hours of reaction, protein sample 1 was obtained.
2. And (2) replacing the protoplast pAN580-TurboID-Flag in the step 1 with the protoplast pAN580-GFP, obtaining a protein sample 2 under the same reaction conditions, and carrying out the same detection on the protein sample 2 to respectively obtain strips corresponding to pAN580-GFP in the Streptavidin-HRP part and the anti-Flag part in the picture 2.
As shown in FIG. 2, it can be seen from the results of anti-Flag portion in FIG. 2 that pAN580-TurboID-Flag vector, regardless of whether exogenous biotin was added, can be expressed well in rice protoplasts; as can be seen from the results of the Streptavidin-HRP moiety in FIG. 2, the biotin labeling band was detected within 2 hours at an applied biotin concentration of 200. mu.M. The results in fig. 2 show that the biotin labeling process can be completed in a short time (2 hours) by the TurboID-based system, and that the application of the system to transient expression of rice protoplasts is feasible and efficient.
Example 3. determination of optimal concentration of biotin required for labeling:
in order to further optimize a biotin proximity labeling system based on TurboID and obtain a short-time and efficient labeling effect, the labeling efficiency is tested under different biotin concentrations, and the optimization conditions required by TurboID to complete efficient labeling are preliminarily ascertained.
Taking protoplast pAN580-TurboID-Flag and biotin with different addition amounts to form 7 groups of reaction systems (respectively systems A1-A7), wherein the systems A1-A7 all comprise 300 mul/tube of protoplastPlastid pAN580-TurboID-Flag at a concentration of 2X105Each ml, solvent is WI solution (0.5M mannitol, 20mM KCl and 4mM MES included in the WI solution, solvent is water, pH 5.7). Wherein, the system 1 is a control without adding biotin, and different amounts of biotin solutions (obtained by dissolving biotin in phosphate buffer with PH 7.5) are respectively added into the systems 2 to 7 until the biotin concentrations in the reaction systems are respectively: 50 μ M, 100 μ M, 200 μ M, 300 μ M, 400 μ M and 600 μ M. (other components in the reaction system were the same as in example 2) after 2 hours of reaction, a protein sample was obtained, and the effect of labeling was detected by Western blot as in step 1 in example 2; protein samples A1 to A7 were obtained in systems A1 to A7, respectively, corresponding to biotin concentrations of 0, 50. mu.M, 100. mu.M, 200. mu.M, 300. mu.M, 400. mu.M and 600. mu.M in FIG. 3.
The results are shown in FIG. 3, where Streptavidin-HRP in FIG. 3 indicates that protein samples were tested according to the detection line: separating the obtained protein sample on SDS-PAGE gel, and transferring the protein sample to an NC membrane; after blocking with PBST solution containing 2.5% BSA for 1 hour, Streptavidin (dilution 1: 2000, cat # ABCam, AB7403) was added for hybridization for 1 hour; after washing 4 more times with PBST solution (15 minutes each), imaging with Biorad ChemiDoc Touch; anti-Flag indicates that protein samples are detected according to a detection system: separating the obtained protein sample on SDS-PAGE gel, and transferring the protein sample to an NC membrane; after blocking with PBST solution containing 5% skim milk powder for 1 hour, a Flag antibody (dilution 1: 2000, cat # MBL, M185-7) was added and hybridized for 1 hour; after washing 4 more times with PBST solution (15 minutes each), imaging was performed with Biorad ChemiDoc Touch.
As can be seen from the anti-Flag results in FIG. 3, the pAN580-TurboID-Flag vectors in the systems 1-7 can be well expressed in rice protoplasts, regardless of whether exogenous biotin is added or not and regardless of the biotin concentration; as can be seen from the results of the Streptavidin-HRP fractions in FIG. 3, the level of biotinylated protein labeled with TurboID was continuously increased at the concentrations of 0-300. mu.M of the applied biotin, and reached a steady state at 400. mu.M (in the case of the change in the band indicated by the arrow on the right). When the biotin concentration was further increased (600. mu.M), the biotinylation level of the protein did not change significantly (the gray value of the biotinylation band did not increase or decrease significantly). This indicates that in the existing experimental system, when the exogenously added biotin concentration reaches 400. mu.M, the biotinylation labeling of the adjacent protein can be completed within a short time by TurboID.
Example 4. determination of optimal treatment time for biotin required for labeling:
in order to further optimize a biotin proximity labeling system based on TurboID and obtain a short-time and efficient labeling effect, the labeling efficiency is tested under different processing times, and the optimization conditions required by TurboID to complete efficient labeling are preliminarily ascertained.
The protoplast pAN580-TurboID-Flag prepared in example 1 was mixed with biotin and dispensed into 8 tubes, each tube was designated as tube B1-B8, and 300. mu.l of the mixture was 2X105The protoplasts were transferred from pAN580-TurboID-Flag per ml in WI solution (0.5M mannitol, 20mM KCl and 4mM MES contained in the WI solution, water as a solvent, pH 5.7) at a concentration of biotin of 400. mu.M in each tube (biotin was dissolved in phosphate buffer solution at pH 7.5 in advance and then added to the tube at a concentration of 400. mu.M), wherein the protein sample B1 was obtained by directly sampling after mixing tube B1, and protein samples B2-B8 were obtained by mixing the remaining tubes B2-B8 after 0.25, 0.5, 1, 2, 3, 4 and 6 hours, respectively, and the labeling effect was detected according to Western blot in step 1 of example 2, and the results are shown in FIG. 4, and protein samples B1-B8 correspond to the reaction times of 0.25, 0.5, 1, 2, 3, 4 and 6 hours in FIG. 4, respectively. Wherein Streptavidin-HRP in fig. 4 indicates that protein samples B1-B8 were tested according to the detection line: separating the obtained protein sample B1-B8 on SDS-PAGE gel, and transferring the protein sample to an NC membrane; after blocking with PBST solution containing 2.5% BSA for 1 hour, Streptavidin (dilution 1: 2000, cat # ABCam, AB7403) was added for hybridization for 1 hour; after washing 4 more times with PBST solution (15 minutes each), imaging with Biorad ChemiDoc Touch; anti-Flag indicates that protein samples B1-B8 were tested according to the detection system: separating the obtained protein sample B1-B8 on SDS-PAGE gel, and transferring the protein sample to an NC membrane; after blocking with PBST solution containing 5% skim milk powder for 1 hour, a Flag antibody (dilution 1: 2000, cat # MBL, M185-7) was added and hybridized for 1 hour; washed 4 times with PBST solution (15 min each) and then with Biorad ChemiDoc Touch imaging.
As can be seen from the anti-Flag results in FIG. 4, the pAN580-TurboID-Flag vector in 8 tubes of samples can be well expressed in rice protoplasts; as can be seen from the results of the Streptavidin-HRP portion in FIG. 3, the TurboID system biotinylated the protein within 15 minutes (0.25 hours) (as exemplified by the band indicated by the red arrow). The level of TurboID-mediated protein biotinylation continued to increase over the 0-3 hour labeling period. The marking effect is not obviously changed after the marking time is continuously prolonged to 4-6 hours. This indicates that, in the conventional experimental system, when the biotin concentration was 400. mu.M, TurboID was able to achieve a good labeling effect in a short time (2 to 3 hours).
The specific method of sampling detection in the above embodiment is as follows: 1ml of W5 solution was added to each tube and centrifuged at 200g for 5 minutes. Add 50. mu.l of 1 Xloading buffer, boil the sample for 10 minutes, centrifuge at 13000rpm for 1 minute, and take the supernatant to obtain a protein sample.
The invention applies a high-efficiency biotin labeling system based on TurboID to rice research for the first time. In a rice protoplast transient expression system, a TurboID-based high-efficiency biotin labeling system is established and optimized. Under the condition of a certain number of seedlings (100 seedlings, and sampling 9-10 days after sowing), the prepared protoplast can complete the labeling reaction at a lower biotin concentration (400 mu M) in a short time (2-3 hours) without adding cytotoxic substances. The obtained sample can be used for subsequent interactive protein identification, subcellular localization research and the like.
Compared with the prior art, the system has the advantages of short required labeling time, high catalytic efficiency, wide required reaction temperature range and the like, and is expected to play an important role in identifying transient or weak interaction proteins. In future experiments, the potential application value of the technology is further explored in different tissues and organs of rice, so that a powerful research tool and a powerful research platform are provided for the field of rice, and a kit is expected to be formed for the research field to use.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> institute of crop science of Chinese academy of agricultural sciences
<120> establishment and application of rice protoplast high-efficiency biotin labeling system
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ctggaactgt tcgagcagga aggcctggct ccatatctgc cacggtggga gaagctggat 1620
aacttcatca atagacccgt gaagctgatc attggggaca aagagatttt cgggattagc 1680
cgggggattg ataaacaggg agccctgctg ctggaacagg acggagttat caaaccctgg 1740
atgggcggag aaatcagtct gcggtctgcc gaaaaggact acaaggacga cgatgacaag 1800
agatcttaaa gcggccgccc ggctgcagtt caaacatttg gcaataaagt ttcttaagat 1860
tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc 1920
atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag 1980
tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata 2040
aattatcgcg cgcggtgtca tctatgttac tagatcggga attcgatatc aagcttatcg 2100
ataccgtcga cctcgagggg gggcccggta cccagctttt gttcccttta gtgagggtta 2160
atttcgagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 2220
acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 2280
gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 2340
tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 2400
cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 2460
gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 2520
aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 2580
gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 2640
aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 2700
gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 2760
ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 2820
cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 2880
ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 2940
actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 3000
tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 3060
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 3120
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 3180
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 3240
ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 3300
tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 3360
agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 3420
gtcgtgtaga taactacgat acgggagggc 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 agggttattg 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 gaacaagagt ccactattaa 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 (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 (Artificial Sequence)
<400> 3
ggaccggtcc cgggggatcc atgaaagaca atactgtg 38
<210> 4
<211> 68
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tgcagccggg cggccgcttt aagatctctt gtcatcgtcg tccttgtagt ccttttcggc 60
agaccgca 68
Claims (10)
1. A method for labeling rice protoplasts with biotin comprises the steps of transferring a recombinant vector containing a TurboID-Flag coding gene into the rice protoplasts to obtain the rice protoplasts containing the recombinant vector; and mixing the rice protoplast containing the recombinant vector with biotin to obtain a reaction system for reaction to obtain the rice protoplast marked by the biotin, wherein the TurboID-Flag is a protein with an amino acid sequence of sequence 2.
2. The method as claimed in claim 1, wherein the coding sequence of the TurboID-Flag coding gene is nucleotides 1 to 328 of sequence 1.
3. The method according to claim 1 or 2, wherein the concentration of biotin in the reaction system is 50 to 600. mu.M.
4. The process according to any one of claims 1 to 3, wherein the reaction time is from 0.25 to 4 hours.
5. The method according to any one of claims 1 to 4, wherein the reaction system is free of toxic substances.
6. A system for preparing a biotin-labeled rice protoplast, comprising the rice protoplast containing the recombinant vector according to any one of claims 1 to 5 and biotin.
7. The system for preparing rice protoplasts labeled with biotin according to claim 6, wherein the reaction system comprises protoplasts, biotin, mannitol, potassium chloride, MES buffer solution and phosphate buffer.
8. Use of rice protoplasts comprising the recombinant vector according to any one of claims 1 to 5 and biotin for the preparation of biotin-labeled rice protoplasts.
9. A recombinant vector for biotin labeling of rice protoplasts is pAN580-TurboID-Flag, and the nucleotide sequence of the pAN580-TurboID-Flag is shown as a sequence 1.
10. Use of the method of claims 1-5, the system of biotinylated rice protoplasts of claims 5-7, or the recombinant vector of claim 9 for the identification of biotinylated rice protoplasts, interactive protein, or subcellular localization.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118496384A (en) * | 2024-07-22 | 2024-08-16 | 湘湖实验室(农业浙江省实验室) | Recombinant protein for marking plant ubiquitination modified protein and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103002879A (en) * | 2010-07-01 | 2013-03-27 | 阿昂梅迪克斯公司 | Microvesicles derived from cell protoplast, and use thereof |
| CN103146757A (en) * | 2007-12-21 | 2013-06-12 | 凯津公司 | Improved mutagenesis method introducing mutagenic nucleobase into plant protoplast by mediation of polyethylene glycol |
| JP2013247961A (en) * | 2013-08-21 | 2013-12-12 | Keygene Nv | Method for target alteration of double chain acceptor dna sequence in plant cell protoplast |
| WO2019143529A1 (en) * | 2018-01-17 | 2019-07-25 | The Board Of Trustees Of The Leland Stanford Junior University | Engineered promiscuous biotin ligases for efficient proximity labeling |
-
2020
- 2020-04-22 CN CN202010320853.9A patent/CN113528450B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146757A (en) * | 2007-12-21 | 2013-06-12 | 凯津公司 | Improved mutagenesis method introducing mutagenic nucleobase into plant protoplast by mediation of polyethylene glycol |
| CN103002879A (en) * | 2010-07-01 | 2013-03-27 | 阿昂梅迪克斯公司 | Microvesicles derived from cell protoplast, and use thereof |
| JP2013247961A (en) * | 2013-08-21 | 2013-12-12 | Keygene Nv | Method for target alteration of double chain acceptor dna sequence in plant cell protoplast |
| WO2019143529A1 (en) * | 2018-01-17 | 2019-07-25 | The Board Of Trustees Of The Leland Stanford Junior University | Engineered promiscuous biotin ligases for efficient proximity labeling |
Non-Patent Citations (2)
| Title |
|---|
| ANDREA MAIR等: "Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID", 《ELIFE》 * |
| 杜阳春等: "活细胞内亚细胞结构蛋白质组学研究新技术——几种邻近标记策略的应用及比较", 《生物化学与生物物理进展》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118496384A (en) * | 2024-07-22 | 2024-08-16 | 湘湖实验室(农业浙江省实验室) | Recombinant protein for marking plant ubiquitination modified protein and application thereof |
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