CN109777808B - A polypeptide and its application in localized expression of foreign protein in nucleus and chloroplast - Google Patents

Abstract

The invention relates to a polypeptide and application thereof in positioning expression of exogenous protein in cell nucleus and chloroplast, belongs to the field of molecular genetics, and provides a DNA sequence for encoding the polypeptide, application thereof in positioning expression of exogenous protein in cell nucleus and chloroplast and a related method. In particular to a sequence from a moso bamboo genome, a polypeptide coded by the sequence can express exogenous protein in chloroplast and nucleus, can be used as a marker gene for co-localization of chloroplast and nucleus, and provides a reliable comparison method for subcellular localization. The invention firstly clones a sequence of a phyllostachys pubescens gene, and introduces yellow fluorescent protein connected with the C end into chloroplast and cell nucleus. The transit peptide is fused with the fluorescent protein, observation in living cells can be carried out, time and labor are saved, and the method is a basic method for researching the functions of the protein.

Description

A polypeptide and its application in localized expression of foreign protein in nucleus and chloroplast

technical field

The invention belongs to the field of molecular genetics, and provides a DNA sequence encoding a polypeptide and its application and related methods for localizing and expressing foreign proteins in cell nucleus and chloroplast.

Background technique

The chloroplast is a plastid in plant cells whose thylakoid is located in a water-containing matrix, is photosynthetically active, and is surrounded by a double membrane. In addition to the chloroplast stroma, it contains two other lumens: the intermembranous space between the double-membrane outer and inner envelopes, and the lumen surrounded by thylakoids. Chloroplasts present in plant and algal cells are derived from primitive cyanobacterial endosymbionts. During the development from a free-living cyanobacterial ancestor to a highly specialized organelle, the chloroplast lost its autonomy. A key factor in this adaptive process is the loss of genetic material caused by gene transfer to the nucleus. This is why the vast majority of proteins in chloroplasts today are encoded by the nucleus and require an N-terminal presequence (transit peptide) to target them to the chloroplast. Some chloroplast-located proteins are encoded by the chloroplast genome. However, most (about 95%) are encoded by the nuclear genome, processed in the cytoplasm, and then transferred to the chloroplast ^[1] . These proteins are transported to the matrix by two translocons, termed TOC and TIC, across the outer and inner envelopes, respectively, mainly aided by cleavable transit peptides ^[2] . However, some particular proteins may enter the chloroplast without the transit peptide. After entering the chloroplast, the proteins are further targeted to specific subcompartments. Thylakoid-targeting proteins are transferred from the matrix through one of four pathways: the secretory (Sec) pathway, the signal recognition particle (SRP) pathway, the double arginine translocation (Tat) pathway, or the spontaneous pathway. Proteins that cross the thylakoid membrane into the lumen use the Sec or Tat pathway, while the integrated thylakoid membrane proteins use the SRP or spontaneous pathway ^{[3], [4]} . All of these pathways require energy except the proteins of the spontaneous pathway.

There have been many studies on chloroplast transit peptides, N-terminal chloroplast transit peptides are of highly variable length, contain few negatively charged residues, and are highly enriched in hydroxylated amino acids. Many thylakoid proteins have complex targeting signals with a typical transit peptide followed by a thylakoid targeting signal. The latter are often very similar to the signal peptides found on secreted proteins, and the nuclear localization signal consists of either one (unimolecular) or a pair (bimolecular) short positively charged sequences in the protein chain. The single-molecule nuclear localization signal has the short conserved sequence K(K/R)X(K/R) and binds to a pocket on the surface of the importin alpha receptor. In the bipartite nuclear localization signal, the unimolecular motif is combined with a second small cluster of basic residues, the first being 10–12 residues N-terminal ^[5] .

Problems existing in the prior art: the localization of proteins is often related to the function of the protein, and the location of the expression of the protein cannot be completely determined based on the sequence of the protein. Predicted expression location. Therefore, some markers of subcellular localization are usually needed as a control for subcellular localization. This study reports a sequence from the Phyllostachys pubescens genome. The polypeptide encoded by it can express exogenous proteins in the chloroplast and nucleus, and can be used in chloroplast and Nuclear co-expressed marker genes.

[1]. Abdallah, F., F. Salamini and D. Leister, A prediction of the size and evolutionary origin of the proteome of chloroplasts of Arabidopsis. Trends inplant science, 2000.5(4):p.141-2.

[2].Aronsson,H.and R.Jarvis,The Chloroplast Protein Import Apparatus,Its Components,and Their Roles,in Plant Cell Monographs,A.S.Sandelius and H.Aronsson,A.S.Sandelius and H.Aronsson^Editors.2009.p .89-123.

[3]. Jarvis, P. and C. Robinson, Mechanisms of protein import and routing in chloroplasts. CURRENT BIOLOGY, 2004.14(24):p.R1064-R1077.

[4]. Robinson, C., S.J. Thompson and C. Woolhead, Multiple pathways used for the targeting of thylakoid proteins in chloroplasts. Traffic (Copenhagen, Denmark), 2001. 2(4): p.245-51.

[5]. Emanuelsson, O. and G. von Heijne, Prediction of organellar targeting signals. Biochimica et biophysica acta, 2001.1541(1-2):p.114-9.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the present invention provides a sequence from Phyllostachys edulis genome, a polypeptide encoded by it can express exogenous protein in the chloroplast and nucleus, and it is proved by the yellow fluorescence signal of the YFP fusion protein connected to the N-terminus. .

In order to achieve the above object, the present invention adopts the following technical scheme:

A signal peptide and its coding sequence, its sequence is as follows:

1. BBRG22 DNA sequence:

ATGGCCATCCACCACGGCCAGTACTACCCGCTCCCGGACCCCGCCCAC GTCTACCCCACCTTCGCTGACTACCCCCATCACCACCGCTTCGCCGCCGTG CCGCCGCCGCCGCCGCACTACCCCTCGTGGGCAGGCGCCAGGTACTACAA CGGGCCCGGGTCCATCTCGCAGCCGATCAACGGCAGCCCGGTGACGCCCG GGCTATGGCAGGTCCCTGCCGGCGTTGGCGTGGGGACGCCGCTGGCCGCG CGACGTCAAGAACCGCCGGCGCCCCCTCCGTCGTTGCTCAGAGGCGAGGA GCCAGTGGTGGTGGGAGGACCTGGCTCGACGTCGTTCTCGCCGTCGACCT CCTCCTCGTCATCGTCCGCTTCGCCGCATGAGCGCCGCGCCCGGCCGGAGC ATAAAGAGAATGTGAGTTTGGATCTGAGCTTGTAG

2. BBRG22 amino acid sequence

MAIHHGQYYPLPDPAHVYPTFADYPHHHRFAAVPPPPPHYPSWAGARYYNGPGSISQPINGSPVTPGLWQVPAGVGVGTPLAARRQEPPAPPPSLLRGEEPVVVGGPGSTSFSPSTSSSSSSASPHERRARPEHKENVSLDLSL

A method for localized expression of exogenous protein in nucleus and chloroplast, comprising the following steps:

1. Construction of the signal peptide C-terminal linked YFP yellow fluorescent vector BBRG22-YFP

Clone BBRG22: Search the BambooGDB database (http://www.bamboogdb.org/) for the PH01001993G0350 sequence and name it BBRG22. Primers BBRG22-F: 5'-ATGGCCATCCACCACGGCCA-3' and BBRG22-R: 5'-CAAGCTCAGATCCAAACTCA-3' were designed according to the open reading frame (ORF) of the BBRG22 sequence in Phyllostachys pubescens. Total RNA from Moso bamboo leaves was prepared using a total RNA extraction kit (TiangenBiotech Co., Ltd., Beijing, China) according to the manufacturer's protocol.

First-strand cDNA was synthesized from 1 μg of total RNA using the GoScript™ reverse transcription system (Promega Biotech, Co., Ltd., Beijing, China). Using the cDNA as a template for PCR, amplification was performed with primers BBRG22-F and BBRG22-R using PrimeSTAR GXL DNA polymerase (Takara Biotech, Co., Ltd., Dalian, China). The PCR fragment was then cloned into pEntry and subsequently confirmed by sequencing (BioSune Biotech, Co., Ltd., Shanghai, China).

Further, the plant total RNA was extracted, using a kit (Tiangen Biotech Co., Ltd., Beijing, China) to prepare total RNA from Moso bamboo leaves according to the manufacturer's protocol. The extraction steps are as follows: (1) Homogenization treatment: 50-100 mg of Phyllostachys edulis leaves are quickly ground into powder in liquid nitrogen, and 450 μl of RL is added (before operation, β-mercaptoethanol is added to RL to a final concentration of 1%, such as 10 μl of β-mercaptoethanol is added to 1 ml of RL. mercaptoethanol. This lysis solution is best prepared immediately.), vortex vigorously to mix. (2) Transfer all the solutions to the filter column CS (the filter column CS is placed in the collection tube), centrifuge at 12,000 rpm (~13,400 × g) for 2-5 min, and carefully aspirate the supernatant in the collection tube to RNase-Free centrifugation In the tube, try to avoid touching the cell debris pellet in the collection tube with the pipette tip. (3) Slowly add 0.5 times the volume of supernatant anhydrous ethanol (usually 225 μl), mix well (precipitation may occur at this time), transfer the obtained solution and the precipitate into the adsorption column CR3, 12,000 rpm (~13,400 ×g) Centrifuge for 30-60sec, discard the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube. (4) Add 350 μl of deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm (~13,400×g) for 30-60 sec, pour off the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube. (5) Preparation of DNaseI working solution: Take 10 μl of DNaseI stock solution into a new RNase-Free centrifuge tube, add 70 μl of RDD solution, and mix gently. (6) Add 80 μl of DNaseI working solution to the center of the adsorption column CR3, and place at room temperature for 15 minutes. (7) Add 350 μl of deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm (~13,400×g) for 30-60 sec, pour off the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube. (8) Add 500 μl of rinse solution RW to the adsorption column CR3 (please check whether ethanol has been added before use), stand at room temperature for 2 min, centrifuge at 12,000 rpm (~13,400 × g) for 30-60 sec, and pour out the Waste liquid, put the adsorption column CR3 back into the collection tube. (9) Repeat step 8. (10) Centrifuge at 12,000 rpm (~13,400×g) for 2 min, and discard the waste liquid. The adsorption column CR3 was left at room temperature for a few minutes to completely dry the residual rinse solution in the adsorption material. (11) Put the adsorption column CR3 into a new RNase-Free centrifuge tube, drop 30-100 μl RNase-Free ddH2O into the middle of the adsorption membrane, leave it at room temperature for 2 minutes, and centrifuge at 12,000 rpm (~13,400 × g) for 2 minutes. Get the RNA solution. Note: The volume of elution buffer should not be less than 30 μl, and the recovery efficiency will be affected if the volume is too small. Please store RNA samples at -70°C. Discard the waste liquid in the collection tube and put the adsorption column CR3 back into the collection tube.

Further, 1 μg of total RNA was used to synthesize first-strand cDNA using GoScript™ reverse transcription system (Promega Biotech, Co., Ltd., Beijing, China). The details are as follows: (1) Each component is mixed and centrifuged briefly before use. Mix the following:

(2) Heating at 70°C for 5 minutes. Immediately cool the ice water for at least 5 minutes. Centrifuge for 10 seconds in a microcentrifuge. Store on ice until addition of reverse transcription mix.

(3) Prepare a reverse transcription reaction mixture, 15 μl per cDNA reaction; add sequentially on ice.

(4) Mix 15 μl of reverse transcription mix with 5 μl of RNA and primer mix.

(5) Annealing in a heating block at 25°C for 5 minutes.

(6) Extension in a 42°C heating block for up to one hour.

(7) Reverse transcriptase in a heat block at 70°C for 15 minutes.

Further, using the synthesized cDNA as a template for PCR, amplification was performed with primers BBRG22-F and BBRG22-R using PrimeSTAR GXL DNA polymerase (Takara Biotech, Co., Ltd., Dalian, China).

(1) Preparation of PCR reaction solution

(2) PCR reaction conditions

Pre-denaturation: 98°C for 30sec; denaturation: 98°C for 10sec, annealing: 55°C for 15sec, extension: 68°C for 10sec, 30Cycles; final extension: 68°C for 10min.

Further, 1 μl of TaKaR r Taq enzyme was added to the PCR product for reaction A and treated at 72°C for 30 min. The PCR was ligated into pEntry (Gateway entry vector) vector and confirmed by sequencing, Sequencing Company Beaushang (Fuzhou, China).

Further, the pEntry-T vector using method is as follows:

(1) Digest pEntry-T plasmid with XcmI restriction enzyme, (2) recover by electrophoresis gel, (3) add the product after PCR with T4 DNA ligase and the product of step 2, (4) transform Dh5α competent cells , (5) single clones were picked after culturing in LB medium for 18 hours, (6) plasmids were extracted, (7) PCR identification and sequencing.

Further, the plasmid extraction adopts the steps of Tiangen Plasmid Small Extraction Kit, and the operation steps are as follows:

(1) Column equilibration step: add 500 μl of equilibration solution BL to the adsorption column CP3 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm (~13,400×g) for 1 min, pour out the waste liquid in the collection tube, and remove the adsorption The column is put back into the collection tube. (Please use the same day treated column).

(2) Take 1-5ml of overnight cultured bacterial liquid, add it to a centrifuge tube, use a conventional desktop centrifuge, 12,000 rpm (~13,400 × g) for 1 min, and remove the supernatant as much as possible (if the bacterial liquid is too large, it can be centrifuged several times) Collect the bacterial pellet into a centrifuge tube).

(3) Add 250 μl of solution P1 (please check whether RNaseA has been added) into the centrifuge tube with the bacterial pellet left, and use a pipette or a vortex shaker to thoroughly suspend the bacterial pellet. Note: If there is an incompletely mixed bacterial block, it will affect the lysis, resulting in low extraction yield and purity.

(4) Add 250 μl of solution P2 to the centrifuge tube, and gently turn up and down 6-8 times to fully lyse the cells. Note: Mix gently, do not shake vigorously, so as not to interrupt the genomic DNA, resulting in the mixture of genomic DNA fragments in the extracted plasmid. At this time, the bacterial liquid should become clear and viscous, and the time should not exceed 5 min to avoid the damage of the plasmid. If it does not become clear, it may be due to excessive bacterial cells and incomplete lysis, and the amount of bacterial cells should be reduced.

(5) Add 350 μl of solution P3 to the centrifuge tube, and immediately turn it up and down gently for 6-8 times, and mix thoroughly. At this time, a white flocculent precipitate will appear. Centrifuge at 12,000 rpm (~13,400 xg) for 10 min. Note: P3 should be mixed immediately after addition to avoid local precipitation. If there are still tiny white precipitates in the supernatant, centrifuge again and take the supernatant.

(6) Transfer the supernatant collected in the previous step to the adsorption column CP3 with a pipette (the adsorption column is placed in the collection tube), taking care not to aspirate the precipitate as much as possible. Centrifuge at 12,000rpm (~13,400×g) for 30-60sec, pour off the waste liquid in the collection tube, and put the adsorption column CP3 into the collection tube.

(7) Optional step: add 500 μl of deproteinized solution PD to the adsorption column CP3, centrifuge at 12,000 rpm (~13,400×g) for 30-60 sec, discard the waste liquid in the collection tube, and put the adsorption column CP3 back into the collection tube middle.

(8) Add 600μl of rinsing solution PW to the adsorption column CP3 (please check whether absolute ethanol has been added), centrifuge at 12,000rpm (~13,400×g) for 30-60sec, pour out the waste liquid in the collection tube, and put the adsorption column CP3 is placed in a collection tube.

(9) Repeat step 8.

(10) Put the adsorption column CP3 into the collection tube, and centrifuge at 12,000 rpm (~13,400 × g) for 2 min, in order to remove the residual rinsing solution in the adsorption column.

(11) Put the adsorption column CP3 in a clean centrifuge tube, drop 50-100 μl of elution buffer EB to the middle of the adsorption membrane, leave it at room temperature for 2 minutes, and centrifuge the plasmid solution at 12,000 rpm (~13,400 × g) for 2 minutes. Collect into centrifuge tubes.

2. Construction of expression vector

The PCR fragment was cloned into pEntry by TA reaction, and then recombined (Invitrogen) with the destination vector pEarleyGate101 vector (35S:C-YFP) (Earley et al., 2006) by LR reaction to obtain pEarleyGate101-BBRG22 plasmid (35S∷BBRG22-YFP). All constructs were transformed into Agrobacterium tumefaciens strain GV3101.

Further, 100 μl of Agrobacterium tumefaciens cells were used for transformation of Agrobacterium tumefaciens plus less than 1 μl of plasmid, mixed and added to the electric shock cup, placed on ice for 5 minutes, wiped off the water outside the electric shock cup, and put into the electric shock transforming instrument (BTX, Model ECM630).

Further, the electric shock conversion conditions are: mode: 2.5kV/RESISTANCE High Voltage(HV); electric shock cup type: BTX Disposable Cuvette P/N 610 (1mmgap); capacitance: 50μF; resistance: 125Ω; voltage: 1.4kV; rated field Intensity (for reference): 14.4kV/cm; Rated pulse length (for reference): 5.0msec; after transformation, add 1ml of anti-anti-LB to the transformed bacterial solution, incubate at 28°C for 2 hours, centrifuge at 40,000g for 2 minutes, and discard the After the clear mixture was mixed, it was spread on the medium containing kana resistance, and cultured at 28°C for 48h.

3. Transient Expression of Recombinant Plasmids in Tobacco

(1) Incubate with 5 ml of LB liquid medium at 28°C for about 12 hours. (2) Centrifuge at 5000g at room temperature for 10min, discard the supernatant, and suspend the pellet with 3ml of MgCl2 (10mM) solution. (3) Measure the concentration and use OD600. (4) Dilute the Agrobacterium suspension with 10 mM MgCL2 to a total volume of 10 ml, and the concentration of each bacteria is OD600=0.5. (5) Add 150 μM acetosyringone. (6) The syringe is injected to the back of the tobacco, and the fluorescence can be observed 24 hours later. (7) Observation and imaging were performed with an inverted Leica TCS SP8X DLS microscope. For excitation of DAPI, YFP and Chlorophyll autofluorescence, 364, 488 and 594 nm lasers were used, respectively. Detected emission wavelengths were 380-388 (DAPI), 520-580 (YFP), and 680-750 (Chlorophyll).

Description of drawings

Fig. 1 is the outline drawing of the epidermal cells of tobacco leaves of the present invention.

Figure 2 is a diagram showing the effect of BBRG22 localization in chloroplast and nucleus in tobacco leaf epidermal cells of the present invention. Among them, the brighter part in the figure is a yellow signal under the microscope.

Fig. 3 is the autofluorescence signal of the chloroplast of the present invention. Among them, the brighter part in the figure is the red signal under the microscope.

Figure 4 is the nuclear localization signal of DAPI staining of the present invention. Among them, the brighter part in the figure is the blue signal under the microscope.

FIG. 5 is a superimposed layer of the present invention. Among them, Fig. 5 is a superimposed view of Figs. 2-4.

Fig. 6 is the PCR amplification band of BBRG22 of the present invention.

Detailed ways

In order to make the objectives, technical solutions and advantages of the invention clearer, the specific embodiments of the invention are described in detail below with reference to the accompanying drawings. Examples of these preferred embodiments are illustrated in the accompanying drawings. The embodiments of the invention shown in the drawings and described with reference to the drawings are merely exemplary and the invention is not limited to these embodiments.

Here, it should also be noted that, in order to avoid obscuring the technical solutions of the present invention due to unnecessary details, only structures and/or processing steps closely related to the solutions according to the present invention are shown in the drawings, while Other details of little relevance are omitted.

Example 1

This embodiment provides a kind of signal peptide and its coding sequence, and its sequence is as follows:

1. BBRG22 DNA sequence:

ATGGCCATCCACCACGGCCAGTACTACCCGCTCCCGGACCCCGCCCAC GTCTACCCCACCTTCGCTGACTACCCCCATCACCACCGCTTCGCCGCCGTG CCGCCGCCGCCGCCGCACTACCCCTCGTGGGCAGGCGCCAGGTACTACAA CGGGCCCGGGTCCATCTCGCAGCCGATCAACGGCAGCCCGGTGACGCCCG GGCTATGGCAGGTCCCTGCCGGCGTTGGCGTGGGGACGCCGCTGGCCGCG CGACGTCAAGAACCGCCGGCGCCCCCTCCGTCGTTGCTCAGAGGCGAGGA GCCAGTGGTGGTGGGAGGACCTGGCTCGACGTCGTTCTCGCCGTCGACCT CCTCCTCGTCATCGTCCGCTTCGCCGCATGAGCGCCGCGCCCGGCCGGAGC ATAAAGAGAATGTGAGTTTGGATCTGAGCTTGTAG

2. BBRG22 amino acid sequence

MAIHHGQYYPLPDPAHVYPTFADYPHHHRFAAVPPPPPHYPSWAGARYYNGPGSISQPINGSPVTPGLWQVPAGVGVGTPLAARRQEPPAPPPSLLRGEEPVVVGGPGSTSFSPSTSSSSSSASPHERRARPEHKENVSLDLSL

Example 2

This embodiment provides a method for localized expression of an exogenous protein in the nucleus and chloroplast, comprising the following steps:

1. Construction of the signal peptide C-terminal linked YFP yellow fluorescent vector BBRG22-YFP

Clone BBRG22: Search the BambooGDB database (http://www.bamboogdb.org/) for the PH01001993G0350 sequence and name it BBRG22. Primers BBRG22-F: 5'-ATGGCCATCCACCACGGCCA-3' and BBRG22-R: 5'-CAAGCTCAGATCCAAACTCA-3' were designed according to the open reading frame (ORF) of the BBRG22 sequence in Phyllostachys pubescens. Total RNA from Moso bamboo leaves was prepared using a total RNA extraction kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's protocol.

First-strand cDNA was synthesized from 1 μg of total RNA using the GoScript™ reverse transcription system (Promega Biotech, Co., Ltd., Beijing, China). Using the cDNA as a template for PCR, amplification was performed with primers BBRG22-F and BBRG22-R using PrimeSTAR GXL DNA polymerase (Takara Biotech, Co., Ltd., Dalian, China). The PCR fragment was then cloned into pEntry and subsequently confirmed by sequencing (BioSune Biotech, Co., Ltd., Shanghai, China).

Further, the plant total RNA was extracted, using a kit (Tiangen Biotech Co., Ltd., Beijing, China) to prepare total RNA from Moso bamboo leaves according to the manufacturer's protocol. The extraction steps are as follows: (1) Homogenization treatment: 50-100 mg of Phyllostachys edulis leaves are quickly ground into powder in liquid nitrogen, and 450 μl of RL is added (before the operation, β-mercaptoethanol is added to the RL to a final concentration of 1%, such as 10 μl of β-mercaptoethanol is added to 1 ml of RL. -Mercaptoethanol. This lysis solution is best prepared immediately.), vortex vigorously to mix. (2) Transfer all the solutions to the filter column CS (the filter column CS is placed in the collection tube), centrifuge at 12,000 rpm (~13,400 × g) for 2-5 min, and carefully aspirate the supernatant in the collection tube to RNase-Free centrifugation In the tube, try to avoid touching the cell debris pellet in the collection tube with the pipette tip. (3) Slowly add 0.5 times the volume of supernatant anhydrous ethanol (usually 225 μl), mix well (precipitation may occur at this time), transfer the obtained solution and the precipitate into the adsorption column CR3, 12,000 rpm (~13,400 ×g) Centrifuge for 30-60sec, discard the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube. (4) Add 350 μl of deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm (~13,400×g) for 30-60 sec, pour off the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube. (5) Preparation of DNase I working solution: Take 10 μl DNase I stock solution into a new RNase-Free centrifuge tube, add 70 μl RDD solution, and mix gently. (6) Add 80 μl of DNaseI working solution to the center of the adsorption column CR3, and place at room temperature for 15 minutes. (7) Add 350 μl of deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm (~13,400×g) for 30-60 sec, pour off the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube. (8) Add 500 μl of rinse solution RW to the adsorption column CR3 (please check whether ethanol has been added before use), stand at room temperature for 2 min, centrifuge at 12,000 rpm (~13,400 × g) for 30-60 sec, and pour out the Waste liquid, put the adsorption column CR3 back into the collection tube. (9) Repeat step 8. (10) Centrifuge at 12,000 rpm (~13,400×g) for 2 min, and discard the waste liquid. The adsorption column CR3 was left at room temperature for a few minutes to completely dry the residual rinse solution in the adsorption material. (11) Put the adsorption column CR3 into a new RNase-Free centrifuge tube, drop 30-100 μl RNase-Free ddH ₂ O into the middle of the adsorption membrane, leave it at room temperature for 2 minutes, and centrifuge at 12,000rpm (~13,400×g). 2min to obtain RNA solution. Note: The volume of elution buffer should not be less than 30 μl, and the recovery efficiency will be affected if the volume is too small. Please store RNA samples at -70°C. Discard the waste liquid in the collection tube and put the adsorption column CR3 back into the collection tube.

Further, 1 μg of total RNA was used to synthesize first-strand cDNA using GoScript™ reverse transcription system (Promega Biotech, Co., Ltd., Beijing, China). The details are as follows: (1) Each component is mixed and centrifuged briefly before use. Mix the following:

(2) Heating at 70°C for 5 minutes. Immediately cool the ice water for at least 5 minutes. Centrifuge for 10 seconds in a microcentrifuge. Store on ice until addition of reverse transcription mix.

(3) Prepare a reverse transcription reaction mixture, 15 μl of each cDNA reaction. Add in order on ice.

(4) Mix 15 μl of reverse transcription mix with 5 μl of RNA and primer mix.

(5) Annealing in a heating block at 25°C for 5 minutes.

(6) Extension in a 42°C heating block for up to one hour.

(7) Reverse transcriptase in a heat block at 70°C for 15 minutes.

Further, using the synthesized cDNA as a template for PCR, amplification was performed with primers BBRG22-F and BBRG22-R using PrimeSTAR GXL DNA polymerase (Takara Biotech, Co., Ltd., Dalian, China).

(1) Preparation of PCR reaction solution

(2) PCR reaction conditions

Pre-denaturation: 98°C for 30sec; denaturation: 98°C for 10sec, annealing: 55°C for 15sec, extension: 68°C for 10sec, 30Cycles; final extension: 68°C for 10min.

Further, 1 μl of TaKaR r Taq enzyme was added to the PCR product for reaction A and treated at 72°C for 30 min. The PCR was ligated into pEntry (Gateway entry vector) vector and confirmed by sequencing, Sequencing Company Beaushang (Fuzhou, China).

Further, the method of using the pEntry-TccDB vector is as follows:

(1) Digest pEntry-T plasmid with XcmI restriction enzyme, (2) recover by electrophoresis gel, (3) use T4 DNA ligase to PCR add the product of A and the product of step 2, (4) transform Dh5α competent cells , (5) single clones were picked after culturing in LB medium for 18 hours, (6) plasmids were extracted, (7) PCR identification and sequencing.

Further, the plasmid extraction adopts the steps of Tiangen Plasmid Small Extraction Kit, and the operation steps are as follows:

(1) Column equilibration step: add 500 μl of equilibration solution BL to the adsorption column CP3 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm (~13,400×g) for 1 min, pour out the waste liquid in the collection tube, and remove the adsorption The column is put back into the collection tube. (Please use the same day treated column).

(2) Take 1-5ml of overnight cultured bacterial liquid, add it to a centrifuge tube, use a conventional desktop centrifuge, 12,000 rpm (~13,400 × g) for 1 min, and remove the supernatant as much as possible (if the bacterial liquid is too large, it can be centrifuged several times) Collect the bacterial pellet into a centrifuge tube).

(3) Add 250 μl of solution P1 (please check whether RNaseA has been added) into the centrifuge tube with the bacterial pellet left, and use a pipette or a vortex shaker to thoroughly suspend the bacterial pellet. Note: If there is an incompletely mixed bacterial block, it will affect the lysis, resulting in low extraction yield and purity.

(4) Add 250 μl of solution P2 to the centrifuge tube, and gently turn up and down 6-8 times to fully lyse the cells. Note: Mix gently, do not shake vigorously, so as not to interrupt the genomic DNA, resulting in the mixture of genomic DNA fragments in the extracted plasmid. At this time, the bacterial liquid should become clear and viscous, and the time should not exceed 5 min to avoid the damage of the plasmid. If it does not become clear, it may be due to excessive bacterial cells and incomplete lysis, and the amount of bacterial cells should be reduced.

(5) Add 350 μl of solution P3 to the centrifuge tube, and immediately turn it up and down gently for 6-8 times, and mix thoroughly. At this time, a white flocculent precipitate will appear. Centrifuge at 12,000 rpm (~13,400 xg) for 10 min. Note: P3 should be mixed immediately after addition to avoid local precipitation. If there are still tiny white precipitates in the supernatant, centrifuge again and take the supernatant.

(6) Transfer the supernatant collected in the previous step to the adsorption column CP3 with a pipette (the adsorption column is placed in the collection tube), taking care not to aspirate the precipitate as much as possible. Centrifuge at 12,000rpm (~13,400×g) for 30-60sec, pour off the waste liquid in the collection tube, and put the adsorption column CP3 into the collection tube.

(7) Optional step: add 500 μl of deproteinized solution PD to the adsorption column CP3, centrifuge at 12,000 rpm (~13,400×g) for 30-60 sec, discard the waste liquid in the collection tube, and put the adsorption column CP3 back into the collection tube middle.

(8) Add 600μl of rinsing solution PW to the adsorption column CP3 (please check whether absolute ethanol has been added), centrifuge at 12,000rpm (~13,400×g) for 30-60sec, pour out the waste liquid in the collection tube, and put the adsorption column CP3 is placed in a collection tube.

(9) Repeat step 8.

(10) Put the adsorption column CP3 into the collection tube, and centrifuge at 12,000 rpm (~13,400 × g) for 2 min, in order to remove the residual rinsing solution in the adsorption column.

(11) Put the adsorption column CP3 in a clean centrifuge tube, drop 50-100 μl of elution buffer EB to the middle part of the adsorption membrane, leave it at room temperature for 2 minutes, and centrifuge the plasmid solution at 12,000 rpm (~13,400 × g) for 2 minutes. Collect into centrifuge tubes.

2. Construction of expression vector

The PCR fragment was cloned into pEntry by TA reaction, and then recombined (Invitrogen) with the destination vector pEarleyGate101 vector (35S:C-YFP) (Earley et al., 2006) by LR reaction to obtain pEarleyGate101-BBRG22 plasmid (35S∷BBRG22-YFP). All constructs were transformed into Agrobacterium tumefaciens strain GV3101.

Further, 100 μl of Agrobacterium tumefaciens cells were used for transformation of Agrobacterium tumefaciens plus less than 1 μl of plasmid, mixed and added to the electric shock cup, placed on ice for 5 minutes, wiped off the water outside the electric shock cup, and put into the electric shock transforming instrument (BTX, Model ECM630).

Further, the electric shock conversion conditions are: mode: 2.5kV/RESISTANCE High Voltage(HV); electric shock cup type: BTX Disposable Cuvette P/N610(1mmgap); capacitance: 50μF; resistance: 125Ω; voltage: 1.4kV; rated field strength (For reference): 14.4kV/cm; Rated pulse length (for reference): 5.0msec; Add 1ml of anti-anti-LB to the transformed bacterial solution, incubate at 28°C for 2 hours, centrifuge at 40,000g for 2 minutes, discard part of the supernatant After mixing, it was spread on the medium containing kana resistance and cultured at 28°C for 48h.

3. Transient Expression of Recombinant Plasmids in Tobacco

(1) Incubate with 5 ml of LB liquid medium at 28°C for about 12 hours. (2) Centrifuge at 5000g at room temperature for 10min, discard the supernatant, and suspend the pellet with 3ml of MgCl2 (10mM) solution. (3) Measure the concentration and use OD600. (4) Dilute the Agrobacterium suspension with 10 mM MgCL2 to a total volume of 10 ml, and the concentration of each bacteria is OD600=0.5. (5) Add 150 μM acetosyringone. (6) The syringe is injected into the back of the tobacco, and the fluorescence can be observed 24 hours later. (7) Observation and imaging were performed with an inverted Leica TCS SP8X DLS microscope. For excitation of DAPI, YFP and Chlorophyll autofluorescence, 364, 488 and 594 nm lasers were used, respectively. The emission wavelengths were detected at 380-388 (DAPI), 520-580 (YFP), and 680-750 (Chlorophyll), and the results are shown in Figures 1-5.

Example 3

Application of the DNA sequence and amino acid sequence of the signal peptide BBRG22 in the localization and expression of foreign proteins in the nucleus and chloroplast.

Beneficial effects: The subcellular localization information of proteins predicted by amino acid sequences is not yet very accurate, and it is even more difficult to predict proteins co-localized with multiple cellular structures, and the final determination needs to be confirmed by experiments; and large-scale proteomes Due to the difficulty of separating low-abundance proteins, the chemical method cannot avoid contamination in the preparation of protein samples, and it also has certain limitations. In this study, a sequence of a Moso bamboo gene was cloned, constructed into an expression vector, and transformed into Agrobacterium for transient expression in tobacco leaf epidermal cells. The fluorescence signal of BBRG22 was observed by confocal microscopy to be correlated with chloroplast autofluorescence and nuclear staining signals, respectively. Coincidence, indicating that BBRG22 can import the yellow fluorescent protein linked at the C-terminus into the chloroplast and nucleus. Similarly, the fusion of transit peptide and fluorescein protein can be observed in living cells, saving time and effort, and is the most basic method to study protein function. At the same time, the BBRG22-YFP fusion protein presented in this study can be used as a marker gene for co-localization of chloroplast and nucleus, providing a reliable alignment method for subcellular localization.

The above are only specific embodiments of the present application. It should be pointed out that for those skilled in the art, without departing from the principles of the present application, several improvements and modifications can also be made. It should be regarded as the protection scope of this application.

<110> Fujian Agriculture and Forestry University

<120> A polypeptide and its application in localized expression of foreign protein in nucleus and chloroplast

<140>201811512218.X

<141>2018-12-12

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211>435

<212> DNA

<213> Phyllostachys edulis

<400> 1

ATGGCCATCCACCACGGCCAGTACTACCCGCTCCCGGACCCCGCCCACGTCTACCCCACCTTCGCTGACTACCCCCATCACCACCGCTTCGCCGCCGTGCCGCCGCCGCCGCCGCACTACCCCTCGTGGGCAGGCGCCAGGTACTACAACGGGCCCGGGTCCATCTCGCAGCCGATCAACGGCAGCCCGGTGACGCCCGGGCTATGGCAGGTCCCTGCCGGCGTTGGCGTGGGGACGCCGCTGGCCGCGCGACGTCAAGAACCGCCGGCGCCCCCTCCGTCGTTGCTCAGAGGCGAGGAGCCAGTGGTGGTGGGAGGACCTGGCTCGACGTCGTTCTCGCCGTCGACCTCCTCCTCGTCATCGTCCGCTTCGCCGCATGAGCGCCGCGCCCGGCCGGAGCATAAAGAGAATGTGAGTTTGGATCTGAGCTTGTAG

<210> 2

<211>144

<212> PRT

<213> Phyllostachys edulis

<400> 2

MAIHHGQYYPLPDPAHVYPTFADYPHHHRFAAVPPPPPHYPSWAGARYYNGPGSISQPINGSPVTPGLWQVPAGVGVGTPLAARRQEPPAPPPSLLRGEEPVVVGGPGSTSFSPSTSSSSSSASPHERRARPEHKENVSLDLSL

Claims (2)

1. A DNA encoding the signal peptide BBRG22, wherein the DNA sequence encoding the signal peptide BBRG22 is shown in SEQ ID NO: 1 in the sequence listing. 2 . The application of the DNA encoding the signal peptide BBRG22 according to claim 1 for localizing and expressing foreign proteins in cell nucleus and chloroplast. 3 .

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