CN109777808B

CN109777808B - Polypeptide and application thereof in positioning expression of foreign protein in cell nucleus and chloroplast

Info

Publication number: CN109777808B
Application number: CN201811512218.XA
Authority: CN
Inventors: 王文斐; 郭泽军; 杨雪莲; 殷魁星
Original assignee: Fujian Agriculture and Forestry University
Current assignee: Fujian Agriculture and Forestry University
Priority date: 2018-12-11
Filing date: 2018-12-11
Publication date: 2020-09-15
Anticipated expiration: 2038-12-11
Also published as: CN109777808A

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

Polypeptide and application thereof in positioning expression of foreign protein in cell nucleus and chloroplast

Technical Field

The invention belongs to the field of molecular genetics, and provides a DNA sequence for encoding polypeptide, application of the DNA sequence in positioning expression of foreign protein in cell nucleus and chloroplast and a related method.

Background

Chloroplasts are plastids in plant cells, with thylakoids located in a water-containing matrix, having photosynthetic activity, surrounded by a bilayer membrane. In addition to the chloroplast stroma, two additional lumens are included: the space between the two-film outer and inner envelope, and the cavity surrounded by the thylakoid body. Chloroplasts present in plant and algal cells are derived from the original cyanobacterial endosymbiont. Chloroplasts lose autonomy in the process of developing from a free-living cyanobacterial ancestor into a highly specialized organelle. One key element of this adaptation process is the loss of genetic material resulting from gene transfer into the nucleus. This is why today most proteins in chloroplasts are encoded by the nucleus and some chloroplast-localized proteins are encoded by the chloroplast genome that require an N-terminal pro-sequence (transit peptide) to target them to the chloroplast. However, most (about 95%) are encoded by the nuclear genome, processed in the cytoplasm, and then transferred to chloroplasts^[1]. These proteins pass through two translocons (called TOC and TI)C) Transport to the matrix across the outer and inner envelopes, respectively, is mainly assisted by a cleavable transport peptide^[2]. However, some particular proteins may enter the chloroplast without a transit peptide. Upon entry into the chloroplast, the protein is further targeted to specific sub-compartments. Thylakoid targeted proteins are transferred from the stroma by one of four pathways: a secretory (Sec) pathway, a Signal Recognition Particle (SRP) pathway, a twin arginine translocation (Tat) pathway, or a spontaneous pathway. Proteins that cross the thylakoid membrane into the lumen use the Sec or Tat pathway, while integrated thylakoid membrane proteins use the SRP or spontaneous pathway^[3]，[4]. All of these pathways are energy demanding, except for proteins of the spontaneous pathway.

There have been many studies on chloroplast transit peptides, the N-terminal chloroplast transit peptide being of highly variable length, containing few negatively charged residues, and being highly enriched in hydroxylated amino acids many thylakoid proteins have a complex targeting signal, with a typical transit peptide, followed by a thylakoid targeting signal, the latter being generally very similar to the signal peptide found on secreted proteins, the nuclear localization signal consisting of one (single molecule) or a pair (bi-molecule) of short positively charged sequences in the protein chain^[5]。

The problems existing in the prior art are as follows: the localization of proteins is often related to the functions of proteins, so that the expression positions of proteins cannot be completely determined according to the sequences of the proteins, and the expression positions of proteins which are simultaneously localized in a plurality of subcellular organelle structures are more difficult to accurately predict. Therefore, when subcellular localization is carried out, a plurality of subcellular localization markers are usually needed for comparison, and the research reports a sequence from a phyllostachys pubescens genome, wherein a polypeptide coded by the sequence can express foreign proteins in chloroplasts and cell nuclei, and can be used for marker genes co-expressed by the chloroplasts and the cell nuclei.

[1].Abdallah,F.,F.Salamini and D.Leister,A prediction of the size andevolutionary 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 routingin chloroplasts.CURRENT BIOLOGY,2004.14(24):p.R1064-R1077.

[4].Robinson,C.,S.J.Thompson and C.Woolhead,Multiple pathways usedfor 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 targetingsignals. Biochimica et biophysica acta,2001.1541(1-2):p.114-9.

Disclosure of Invention

In view of the defects of the prior art, the invention provides a sequence from a moso bamboo genome, and the sequence encodes a polypeptide which can express foreign proteins in chloroplasts and cell nucleuses and is proved by a yellow fluorescent signal of YFP fusion protein connected to the N end.

In order to achieve the purpose, the invention adopts the following technical scheme:

a signal peptide and a coding sequence thereof, wherein the sequence is as follows:

BBRG22 DNA sequence:

ATGGCCATCCACCACGGCCAGTACTACCCGCTCCCGGACCCCGCCCAC GTCTACCCCACCTTCGCTGACTACCCCCATCACCACCGCTTCGCCGCCGTG CCGCCGCCGCCGCCGCACTACCCCTCGTGGGCAGGCGCCAGGTACTACAA CGGGCCCGGGTCCATCTCGCAGCCGATCAACGGCAGCCCGGTGACGCCCG GGCTATGGCAGGTCCCTGCCGGCGTTGGCGTGGGGACGCCGCTGGCCGCG CGACGTCAAGAACCGCCGGCGCCCCCTCCGTCGTTGCTCAGAGGCGAGGA GCCAGTGGTGGTGGGAGGACCTGGCTCGACGTCGTTCTCGCCGTCGACCT CCTCCTCGTCATCGTCCGCTTCGCCGCATGAGCGCCGCGCCCGGCCGGAGC ATAAAGAGAATGTGAGTTTGGATCTGAGCTTGTAG

BBRG22 amino acid sequence

MAIHHGQYYPLPDPAHVYPTFADYPHHHRFAAVPPPPPHYPSWAGARYY NGPGSISQPINGSPVTPGLWQVPAGVGVGTPLAARRQEPPAPPPSLLRGEEPVV VGGPGSTSFSPSTSSSSSSASPHERRARPEHKENVSLDLSL

A method for the localized expression of foreign proteins in cell nucleus and chloroplast comprises the following steps: the method comprises the following steps:

1. construction of YFP yellow fluorescent vector connected with signal peptide C-terminal BBRG22-YFP

Clone BBRG 22: the PH01001993G0350 sequence was searched in the BamHoGDB database (http:// www.bamboogdb.org /), and named BBRG 22. Designing and designing a primer BBRG22-F according to an Open Reading Frame (ORF) of a BBRG22 sequence in the phyllostachys pubescens: 5'-ATGGCCATCCACCACGGCCA-3' and BBRG 22-R: 5'-CAAGCTCAGATCCAAACTCA-3' are provided. 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 using 1. mu.g total RNA using the GoScript TM reverse transcription system (Promega Biotech, Co., Ltd., Beijing, China). The cDNA was used as template for PCR and amplified using PrimeSTAR GXL DNA polymerase (TakaraBiotech, Co., Ltd., Dalian, China) with primers BBRG22-F and BBRG 22-R. The PCR fragment was then cloned into pEntry and subsequently confirmed by sequencing (biosone Biotech, co., ltd., Shanghai, China).

Further, the plant total RNA was extracted, and total RNA from Moso bamboo leaves was prepared using a kit (Tiangen Biotech co., ltd., Beijing, China) according to the manufacturer's protocol. The extraction steps are as follows: (1) homogenizing, namely rapidly grinding 50-100mg of moso bamboo leaves into powder in liquid nitrogen, adding 450 mu lRL (adding beta-mercaptoethanol into RL to the final concentration of 1% before operation, such as adding 10 mu l of beta-mercaptoethanol into 1ml of RL, preferably preparing the lysate in situ), and vortexing, vigorously and uniformly mixing. (2) All solutions were transferred to the filter column CS (filter column CS placed in the collection tube), centrifuged at 12,000rpm (-13,400 Xg) for 2-5min, the supernatant from the collection tube carefully pipetted into the RNase-Free centrifuge tube, and the tip was kept from touching the cell debris pellet in the collection tube as much as possible. (3) 0.5 volume of the supernatant of absolute ethanol (usually 225. mu.l) was slowly added and mixed (in which case precipitation may occur), the resulting solution and the precipitate were transferred to an adsorption column CR3, centrifuged at 12,000rpm (13,400 Xg) for 30-60sec, the waste liquid in the collection tube was discarded, and the adsorption column CR3 was returned to the collection tube. (4) 350 μ l of deproteinizing solution RW1 was added to the adsorption column CR3, centrifuged at 12,000rpm (. about.13,400 Xg) for 30-60sec, the waste liquid in the collection tube was discarded, and the adsorption column CR3 was returned to the collection tube. (5) Preparing DNaseI working solution: add 10. mu.l DNaseI stock into a new RNase-Free centrifuge tube, add 70. mu.l RDD solution, mix gently. (6) 80. mu.l of DNaseI working solution was added to the center of the adsorption column CR3 and the mixture was left at room temperature for 15 min. (7) 350 μ l of deproteinizing solution RW1 was added to the adsorption column CR3, centrifuged at 12,000rpm (. about.13,400 Xg) for 30-60sec, the waste liquid in the collection tube was discarded, and the adsorption column CR3 was returned to the collection tube. (8) Add 500. mu.l of rinsing solution RW (previously checked for ethanol addition) to the adsorption column CR3, let stand at room temperature for 2min, centrifuge at 12,000rpm (13,400 Xg) for 30-60sec, discard the waste from the collection tube, and replace the adsorption column CR3 back into the collection tube. (9) And repeating the step 8. (10) Centrifuge at 12,000rpm (. about.13,400 Xg) for 2min and discard the waste. The adsorption column CR3 was left at room temperature for several minutes to thoroughly dry the residual rinse solution from the adsorption material. (11) The adsorption column CR3 was placed in a new RNase-Free centrifuge tube, 30-100. mu.l RNase-Free ddH2O was added dropwise to the middle of the adsorption membrane, left at room temperature for 2min, and centrifuged at 12,000rpm (13,400 Xg) for 2min to obtain an RNA solution. Note that: the volume of elution buffer should not be less than 30. mu.l, and too small a volume affects the recovery efficiency. The RNA samples were stored at-70 ℃. The trap was drained and the adsorption column CR3 was returned to the trap.

Further, first strand cDNA was synthesized using 1. mu.g of total RNA using a GoScript TM reverse transcription system (Promega Biotech, Co., Ltd., Beijing, China). The method comprises the following specific steps: (1) each component was mixed and briefly centrifuged prior to use. The following were mixed:

(2) heating at 70 deg.C for 5 min. The ice water was immediately cooled for at least 5 minutes. Centrifuge in a microcentrifuge for 10 seconds. Store on ice until addition of reverse transcription mixture.

(3) Preparing reverse transcription reaction mixture, reacting 15 μ l of each cDNA; the additions were in order on ice.

(4) Mu.l of the reverse transcription mix was mixed with 5. mu.l of the RNA and primer mix.

(5) Anneal in 25 ℃ heat block for 5 minutes.

(6) Extended in a heating block at 42 ℃ for up to one hour.

(7) Reverse transcriptase was done in a 70 ℃ heat block for 15 minutes.

Further, the synthesized cDNA was used as a template for PCR, and amplification was performed using PrimeSTAR GXL DNA polymerase (Takara Biotech, Co., Ltd., Dalian, China) with primers BBRG22-F and BBRG 22-R.

(1) Preparation of PCR reaction solution

(2) PCR reaction conditions

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

Further, A reaction was added to the PCR product, and 1. mu.l of TaKaRa r Taq enzyme was added thereto and treated at 72 ℃ for 30 min. PCR was ligated into the pentry (gateway entry vector) vector and subsequently confirmed by sequencing, inc.

Further, the pEntry-T vector is used as follows:

(1) digesting pEntry-T plasmid with XcmI restriction enzyme, (2) recovering electrophoresis gel, (3) adding A product and step 2 product by using T4DNA ligase PCR, (4) transforming Dh5 alpha competent cell, (5) picking single clone after 18 hours of LB culture medium culture, (6) extracting plasmid, (7) identifying PCR and sequencing.

Further, the plasmid extraction adopts the steps of a small Tiangen plasmid extraction kit, and the operation steps are as follows:

(1) column equilibration step: 500. mu.l of the equilibration solution BL was added to the adsorption column CP3 (the adsorption column was placed in the collection tube), centrifuged at 12,000rpm (. about.13,400 Xg) for 1min, the waste solution in the collection tube was decanted, and the adsorption column was replaced in the collection tube. (please use the column treated the day).

(2) 1-5ml of overnight-cultured bacterial liquid is taken and added into a centrifuge tube, and is centrifuged for 1min at 12,000rpm (13,400 Xg) by using a conventional desktop centrifuge, and the supernatant is removed as much as possible (when the bacterial liquid is more, the bacterial precipitate can be collected into one centrifuge tube by centrifuging for many times).

(3) To the tube containing the pellet was added 250. mu.l of solution P1 (please check if RNaseA had been added first), and the pellet was suspended thoroughly using a pipette or vortex shaker. Note that: if the bacteria block is not thoroughly mixed, the lysis is affected, and the extraction amount and the purity are low.

(4) 250. mu.l of the solution P2 was added to the tube and gently turned upside down 6 to 8 times to lyse the cells sufficiently. Note that: gently mix without vigorous shaking to avoid disrupting the genomic DNA and resulting in mixing of genomic DNA fragments with the extracted plasmid. At this time, the bacterial liquid should be clear and viscous, and the time for using the bacterial liquid should not exceed 5min so as to prevent the plasmid from being damaged. If the cells are not clear, the cells may be too much and the lysis is incomplete, so that the cell mass should be reduced.

(5) Add 350. mu.l of solution P3 to the tube, turn gently up and down 6-8 times immediately, mix well, at which time white flocculent precipitate will appear. Centrifuge at 12,000rpm (. about.13,400 Xg) for 10 min. Note that: the P3 should be mixed immediately after addition to avoid local precipitation. If there is a small white precipitate in the supernatant, the supernatant can be centrifuged again.

(6) The supernatant collected in the previous step was pipetted into adsorption column CP3 (the adsorption column was put into the collection tube), taking care not to aspirate the pellet as much as possible. Centrifuge at 12,000rpm (13,400 Xg) for 30-60sec, remove waste liquid from the collection tube, and place adsorption column CP3 in the collection tube.

(7) Optional steps are as follows: add 500. mu.l deproteinized liquid PD to the adsorption column CP3, centrifuge at 12,000rpm (. about.13,400 Xg) for 30-60sec, discard the waste liquid from the collection tube, and replace adsorption column CP3 back into the collection tube.

(8) Add 600. mu.l of rinsing solution PW (please check if absolute ethanol has been added) to the adsorption column CP3, centrifuge at 12,000rpm (. about.13,400 Xg) for 30-60sec, dump the waste liquid from the collection tube, and place the adsorption column CP3 into the collection tube.

(9) Operation 8 is repeated.

(10) The adsorption column CP3 was placed in a collection tube and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min in order to remove the residual rinse from the adsorption column.

(11) The adsorption column CP3 was placed in a clean centrifuge tube, 50-100. mu.l of elution buffer EB was added dropwise to the middle of the adsorption membrane, and the mixture was left at room temperature for 2min and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min to collect the plasmid solution in the centrifuge tube.

2. Construction of expression vectors

The PCR fragment was cloned into pEntry by TA reaction and then recombined (Invitrogen) with the pEarley Gate101 vector (35S: C-YFP) (Earley et al, 2006) of the target vector by LR reaction to obtain the pEarley Gate101-BBRG22 plasmid (35S: BBRG 22-YFP). All constructs were transformed into Agrobacterium tumefaciens (Agrobacterium tumefaciens) strain GV 3101.

Further, 100. mu.l of Agrobacterium tumefaciens cells were used for transformation, less than 1. mu.l of plasmid was added to the cells, mixed, added to an electric cuvette, left on ice for 5 minutes, wiped to dry the water outside the electric cuvette, and placed in an electric shock transformation apparatus (BTX, Model ECM 630).

Further, the shock conversion conditions are: mode (2): 2.5kV/RESISTANCE High Voltage (HV); the type of the electric shock cup: BTX dispersible Cuvette P/N610(1 mmgap); capacitance: 50 muF; resistance: 125 omega; voltage: 1.4 kV; nominal field strength (for reference): 14.4 kV/cm; nominal pulse length (for reference): 5.0 msec; adding 1ml of non-antibiotic LB into the transformed bacterial liquid, culturing for 2 hours at 28 ℃ in a shaking way, centrifuging for 2 minutes at 40000g, discarding part of supernatant, mixing uniformly, coating on a kana resistant culture medium, and culturing for 48 hours at 28 ℃.

3. Tobacco transient expression of recombinant plasmids

(1) 5ml of LB liquid medium was used to culture the cells at 28 ℃ for about 12 hours. (2) Centrifuge at 5000g for 10min at room temperature, discard the supernatant and suspend the pellet with 3ml MgCL2(10 mM). (3) The concentration was measured using OD 600. (4) The agrobacterium suspension was diluted with 10mM MgCL2 to a total volume of 10ml, with an OD600 of 0.5 per bacterium. (5) Add 150. mu.M acetosyringone. (6) The syringe is injected to the back of the tobacco, and fluorescence can be observed after 24. (7) Observations and imaging were performed with an inverted Leica TCS SP8X DLS microscope. For excitation of DAPI, YFP and Chlorophyll (chlorophyl) autofluorescence, 364, 488 and 594nm lasers were used, respectively. The emission wavelengths were detected at 380-.

Drawings

FIG. 1 is a contour map of epidermal cells of tobacco leaves according to the present invention.

FIG. 2 is a graph showing the effect of BBRG22 in locating in chloroplasts and nuclei in epidermal cells of tobacco leaves of the present invention. Wherein the lighter parts in the figure are yellow signals under a microscope.

FIG. 3 shows the chloroplast autofluorescence signal of the present invention. Wherein the brighter part in the figure is a red signal under a microscope.

FIG. 4 shows the nuclear localization signals of DAPI staining of the present invention. Wherein the brighter parts in the figure are blue signals under the microscope.

Fig. 5 is a superimposed layer according to the invention. Wherein fig. 5 is an overlay of fig. 2-4.

FIG. 6 shows the PCR amplified band of BBRG22 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present 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 in accordance with the drawings are exemplary only, and the invention is not limited to these embodiments.

It should be noted that, in order to avoid obscuring the technical solution of the present invention with unnecessary details, only the structures and/or processing steps closely related to the solution according to the present invention are shown in the drawings, and other details that are not relevant are omitted.

Example 1

This example provides a signal peptide and its coding sequence, the sequence of which is as follows:

BBRG22 DNA sequence:

BBRG22 amino acid sequence

Example 2

This example provides a method for localized expression of a foreign protein in the nucleus and chloroplast: the method comprises the following steps:

Further, the total RNA of the plant is extracted by using a kit (Tiangen Biotech Co., Ltd., Beijing, China), the total RNA from Moso bamboo leaves is prepared according to the manufacturer's protocol (1) homogenization treatment in which 50-100mg of Moso bamboo leaves are quickly ground into powder in liquid nitrogen, 450. mu. lRL is added (β -mercaptoethanol is added to RL to a final concentration of 1% before operation, e.g., 10. mu.l of β -mercaptoethanol is added to 1ml RL), vortex shaking and mixing (2) all the solutions are transferred to a filtration column CS (where the filtration column CS is placed in a collection tube), 12,000rpm (13,400 × g) is centrifuged for 2-5min, supernatant in a collection tube is sucked into a centrifuge tube of RNN-Free, the supernatant in the centrifuge tube, the pipette tip is transferred to a centrifuge tube (3) which is slowly added to a 0.5 times the supernatant volume of the supernatant (3. mu.5 rpm) of the adsorption column, the supernatant is placed into a centrifuge tube 35-60. mu.5 rpm), the centrifuge tube (where the supernatant is placed back into a centrifuge tube 35-60 g of the centrifuge column), the centrifuge tube (where the centrifuge column RS 12) is placed into a centrifuge tube (where the centrifuge tube 19-60 g of the centrifuge column RS 35. mu.5 rpm), the supernatant is placed into a centrifuge tube (35-60) and the centrifuge tube (35. mu.5. mu.g of the centrifuge tube), the centrifuge tube (35-60) is placed into a centrifuge tube), the centrifuge tube (35-60.60) and the centrifuge tube (where the centrifuge tube), the centrifuge tube (35-60) is placed into a centrifuge tube), the centrifuge tube (where the supernatant), the centrifuge tube (where the centrifuge tube), the centrifuge tube (where the supernatant is placed into a centrifuge tube), the centrifuge tube (35-60 g of the centrifuge tube), the centrifuge tube (35-60 g of the centrifuge tube), the supernatant is placed into the supernatant), the centrifuge tube (35-60) is placed into the centrifuge tube (where the centrifuge tube), the pipette tube (where the pipette tube (13) is placed into the pipette tube (where the pipette tube), the pipette tube (where the pipette tube) is placed into the pipette tube), the pipette tube (13) is placed into the pipette tube (where the pipette tube), the pipette tube (where the pipette tube) is placed, the pipette tube) is placed into the pipette tube (where the pipette tube) is placed into the pipette tubeThe adsorption column CR3 is put into a new RNase-Free centrifuge tube, and 30-100 μ l RNase-Free ddH is suspended and dripped into the middle part of the adsorption membrane₂O, standing at room temperature for 2min, centrifuging at 12,000rpm (-13,400 × g) for 2min to obtain RNA solution, taking care that the volume of the elution buffer solution is not less than 30 mu l, the volume is too small to influence the recovery efficiency, storing the RNA sample at-70 ℃, removing waste liquid in the collecting tube, and putting the adsorption column CR3 back into the collecting tube.

(3) Reverse transcription reaction mixtures were prepared, 15. mu.l each cDNA was reacted. The additions were in order on ice.

(5) Anneal in 25 ℃ heat block for 5 minutes.

(6) Extended in a heating block at 42 ℃ for up to one hour.

(7) Reverse transcriptase was done in a 70 ℃ heat block for 15 minutes.

(1) Preparation of PCR reaction solution

(2) PCR reaction conditions

Further, the pEntry-TccDB vector was used as follows:

(9) Operation 8 is repeated.

2. Construction of expression vectors

3. Tobacco transient expression of recombinant plasmids

(1) 5ml of LB liquid medium was used to culture the cells at 28 ℃ for about 12 hours. (2) Centrifuge at 5000g for 10min at room temperature, discard the supernatant and suspend the pellet with 3ml MgCL2(10 mM). (3) The concentration was measured using OD 600. (4) The agrobacterium suspension was diluted with 10mM MgCL2 to a total volume of 10ml, with an OD600 of 0.5 per bacterium. (5) Add 150. mu.M acetosyringone. (6) The syringe is injected to the back of the tobacco, and fluorescence can be observed after 24. (7) Observations and imaging were performed with an inverted Leica TCS SP8X DLS microscope. For excitation of DAPI, YFP and Chlorophyll (chlorophyl) autofluorescence, 364, 488 and 594nm lasers were used, respectively. The emission wavelengths were measured at 380-.

Example 3

The application of the DNA sequence and the amino acid sequence of the signal peptide BBRG22 in the positioning expression of foreign protein in nucleus and chloroplast.

Has the advantages that: the subcellular localization information of the protein predicted by the amino acid sequence has not high accuracy at present, is difficult to predict the protein co-localized by a plurality of cell structures, and the final determination needs to be verified by experiments; the large-scale proteomics method cannot avoid pollution in the preparation of protein samples due to the difficulty in separating low-abundance proteins, and has certain limitations. The research clones a sequence of a phyllostachys pubescens gene, constructs the sequence on an expression vector, converts agrobacterium tumefaciens, and then transiently expresses the phyllostachys pubescens gene in epidermal cells of tobacco leaves, and uses a confocal laser microscope to observe that BBRG22 fluorescent signals are respectively superposed with chloroplast autofluorescence and cell nucleus staining signals, which shows that BBRG22 can guide yellow fluorescent protein connected to a C end into chloroplasts and cell nuclei. The similar fusion of the transit peptide and the fluorescein protein can be used for observation in living cells, saves time and labor and is the most basic method for researching the protein function. Meanwhile, the BBRG22-YFP fusion protein provided by the research can be used as a co-localization marker gene of chloroplast and nucleus, and a reliable comparison method is provided for subcellular localization.

The foregoing is directed to embodiments of the present application and it is noted that numerous modifications and adaptations may be made by those skilled in the art without departing from the principles of the present application and are intended to be within the scope of the present application.

<110> Fujian agriculture and forestry university

<120> polypeptide and application thereof in positioning expression of foreign protein in cell 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

1. A DNA coding for a signal peptide BBRG22, wherein the DNA sequence of the signal peptide BBRG22 is shown in a sequence table SEQ ID NO: 1 is shown.

2. Use of the DNA encoding the signal peptide BBRG22 according to claim 1 for the targeted expression of foreign proteins in the nucleus and chloroplast.