CN107058373A - One carry NtRBSC1 genes transient expression vector - Google Patents
One carry NtRBSC1 genes transient expression vector Download PDFInfo
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Abstract
The invention belongs to biological technical field, and in particular to one carries tobacco ribulose 1, the restructuring transient expression vector of the small ylidene gene of 5 diphosphonic acid carboxylases.The present invention utilizes the gene constructed transient expression vector NtRBSC1 pFF19 GFP of tobacco NtRBSC1, after the carrier transformation of tobacco cell, fluorescent marker gene GFP and NtRBSC1 fusion protein can be enable in tobacco protoplast, and then Subcellular Localization is carried out to NtRBSC1 albumen by the position of laser confocal microscope detection fusion albumen, and the position of chloroplaset is shown.In general, this mode is method that is a kind of easy and accurately being positioned in living cells, it can be that research tobacco cell chloroplaset and related gene provide definite reference frame, be easy to the further research of tobacco NtRBSC1 genes and its deeply developing for function.
Description
Technical field
The invention belongs to biological technical field, and in particular to one carries a ribulose-1,5-bisphosphate, 5- diphosphonic acid carboxylases
Small ylidene gene（NtRBSC1）The restructuring transient expression vector of gene.
Background technology
During chloroplaset is plant cell, surrounded by duplicature, containing chlorophyll, photosynthetic organelle can be carried out.Leaf
The thylakoid being made up of membrane vesicle is suspended with green body matrix, chloroplast DNA is included, is a kind of plastid, there is circular, oval or disk
3 kinds of forms of shape.The chlorophyll a, b that chloroplaset contains absorbs green glow at least, and green glow is reflected, therefore blade is in green.Chloroplaset is flat
Spherical, thick about 2.5 microns, about 5 microns of diameter has duplicature, inside there is the enzyme and lamella contained in interstitial, interstitial in dissolved state.
Lamella is formed by the thylakoid pile heap of the hollow plate-like closed, and thylakoid is to form energy-rich compound atriphos (ATP) institute
It is required, it is plant " nutriment manufacturing shop " and " energy switching station ".
Ribulose-1,5-bisphosphate, 5- diphosphonic acid carboxylases（Ribulose bisphosphate carboxylase/oxygenase）,
RuBisCO is generally abbreviated as, is a kind of enzyme (EC 4.1.1.39), molecular weight is about 53kD, by 8 large subunits and 8 small subunits
Composition, is the key enzyme of decision carbon assimilation speed in photosynthesis, is also the marker enzyme of plant chloroplast.It is photosynthetic
First main carbon fixation reaction is catalyzed in Calvin cycle, is to be stored up in organism by the carbon dioxide conversion dissociated in air
Can molecule, such as sucrose molecule.For the enzyme, clone, the analysis of the enzyme coding gene are still focused primarily upon in the prior art
Deng basic work, and research situations such as to specific positioning of the enzyme in cell and its mechanism of action still lacks relatively
It is weary.
The content of the invention
The present invention mainly uses tobacco ribulose-1,5-bisphosphate, 5- diphosphonic acid carboxylase small ylidene genes（Ntab0062040,
NtRBSC1）Transient expression vector is constructed, using the expression vector, tobacco NtRBSC1 genes can be preferably studied in tobacco
The mechanism of action and the marker gene as tobacco chloroplast, so as to carry out chloroplaset common location research.
Details are as follows for the technical scheme that the application is taken.
One carry NtRBSC1 genes transient expression vector, the carrier carries NtRBSC1（Ntab0062040）
With fluorescent marker protein GFP gene（Reporter gene is used as using fluorescent marker protein GFP genes）, its preparation method includes
Following steps：
（1）PCR is expanded, and obtains tobacco ribulose-1,5-bisphosphate, and 5- diphosphonic acid carboxylase small ylidene gene NtRBSC1 are specially：
Using the big gold dollar tobacco bred of safflower as material, its total serum IgE is extracted, and further reverse transcription obtains cDNA, as template, if
NtRBSC1-F/NtRBSC1-R primer pairs are counted, enters performing PCR amplification, obtains NtRBSC1 amplified production, and amplified production is entered
Row is reclaimed；
The NtRBSC1-F/NtRBSC1-R primer pairs, particular sequence is：
NtRBSC1-F:CGGGGTACCATGTCTGTCTCAAGTT, wherein GGTACC partial sequences are KpnI restriction enzyme sites,
NtRBSC1-R:CGCGGATCCAGATGCTACAGG, wherein GGATCC partial sequences are BamHI restriction enzyme sites；
The NtRBSC1 genes, its base sequence is as shown in SEQ ID NO.1；
（2）Digestion, and connect, it is specially：
To step（1）The middle amplified production for reclaiming obtained NtRBSC1, double digestion processing is carried out using KpnI and BamHI, and
Reclaim digestion products；
It is same that double digestion processing is carried out using KpnI and BamHI simultaneously to pFF19-GFP carriers, and reclaim digestion products；
50 μ L double digestions System Designs are with reference to as follows：
Buffer K, 2.5 μ L；
BamHI, 2.5 μ L；
SphI, 2.5 μ L；
Step（1）In NtRBSC1 amplified production（Or pFF19-GFP carriers）, 20 μ L；
Aqua sterilisa adds to 50 μ L；
Using T4 DNA ligases by the double digestion of the cohesive end of above-mentioned NtRBSC1 digestion products and pFF19-GFP carriers
The cohesive end of product is attached, and 10 μ L linked systems are as follows with reference to setting：
NtRBSC1 digestion products, 1 μ L；
PFF19-GFP carrier digestion products, 1 μ L；
T4 DNA ligases, 1 μ L；
ddH2O adds to 10 μ L；
16 DEG C of connections are stayed overnight；
（3）Conversion, is screened, and is specially：
By step（2）In connection product conversion conversion bacillus coli DH 5 alpha competent cell, carry out resistance screening, and picking sun
Property bacterium colony carry out sequence verification, for verifying that correct bacterial strain is enlarged culture, and it is standby to extract plasmid；
This plasmid is to contain tobacco ribulose-1,5-bisphosphate, 5- diphosphonic acid carboxylase small ylidene gene NtRBSC1 and fluorescent marker protein
The transient expression vector of GFP genes, NtRBSC1-pFF19-GFP is named as by this plasmid vector.
Application of the transient expression vector for carrying NtRBSC1 genes in tobacco, by NtRBSC1-pFF19-GFP
Plasmid vector passes through Biolistic mediated transformation method transformation of tobacco cell suspending liquid（As BY-2 tobacco cells suspend）, or pass through
The protoplast transformation transformation of tobacco protoplast of PEG mediations, the h of room temperature light culture 8 ~ 16, passes through laser co-focusing after conversion
Micro- sem observation suspension cell or protoplast, after gene expression, the fusion protein of the gene and green fluorescent protein is due to plant
The chloroplaset of thing, the carrier can as tobacco chloroplast common location expression vector；In other words, by fusion protein in cell
In fluorescence display position can position tobacco NtRBSC1, consequently facilitating the positioning of tobacco chloroplast and its mechanism of action are ground
Study carefully.
On the basis of previous work, inventor is cloned into a ribulose-1,5-bisphosphate, the small ylidene gene of 5- diphosphonic acid carboxylases
（Internal number Ntab0062040）, it is named as NtRBCS1.Further to study the function of the gene, inventor utilizes this
After the gene constructed transient expression vector NtRBSC1-pFF19-GFP of NtRBSC1, the carrier transformation of tobacco cell, it can make
Fluorescent marker protein GFP can be co-expressed with NtRBSC1 albumen in suspension cell（That is, fluorescence mark has been carried out to NtRBSC1
Note）, and then Subcellular Localization is carried out to NtRBSC1 albumen by the position of laser confocal microscope detection fusion albumen.
In general, this mode is method that is a kind of easy and accurately being positioned in living cells, can be thin for research tobacco
Born of the same parents' chloroplaset and related gene provide definite reference frame, is easy to further research and its function of tobacco NtRBSC1 genes
Deeply develop.
Brief description of the drawings
Fig. 1 is constructed transient expression vector NtRBSC1-pFF19-GFP digestion verification figure, and wherein M is DL 2000
DNA marker, 1 is NtRBSC1-pFF19-GFP plasmid enzyme restriction figures；
Fig. 2 is laser co-focusing micrograph after constructed transient expression vector NtRBSC1-pFF19-GFP transformation of tobacco cells, from
Fluorescent places can be positioned to tobacco NtRBSC1 in figure；" Tag " is fluorescence labels passage in figure, and " Chi " is logical for chlorophyll
Road, " DIC " is light field passage, and " Merge " is superimposed for passage.
Embodiment
Explanation is further explained to the application with reference to embodiment, before specific embodiment is introduced, with regard to following realities
Apply and be related to part biological material, experiment reagent and experimental facilities in example, briefly introduce and be described as follows.
Biomaterial：
The big gold dollar of tobacco safflower, purchased from the southern center of Yunnan Academy of Tobacco Agricultural Science's Chinese tobacco breeding research；It is real
During testing, cultivate in Zhengzhou Tobacco Research Institute of CNTC gene center illumination cultivation, prepare RNA sample
When, take the tobacco young leaflet tablet of grand cross phase to carry out associative operation；
Primer is synthesized and sequencing company has raw work bioengineering（Shanghai）Limited company completes；
PFF19-GFP carriers, are existing biomaterial or are prepared by prior art；
Experiment reagent：
PCR reaction kits, Beijing Quanshijin Biotechnology Co., Ltd's product；
Reverse transcription reagent box, SMART MMLV reverse transcriptase, KpnI, BamHI enzyme, T4 DNA ligases（Solution I connect
Connect enzyme）Deng being TaKaRa Bioisystech Co., Ltd product；
Experimental facilities：
Laser confocal microscope, German Leica SP5 STED CW.
Embodiment 1
The transient expression vector for carrying NtRBSC1 genes provided herein, the carrier carries NtRBSC1
（Ntab0062040））With fluorescent marker protein GFP gene（Reporter gene is used as using fluorescent marker protein GFP genes）,
Details are as follows for its preparation method.
（1）PCR is expanded, and obtains NtRBSC1 genes, is specially：
Using the big gold dollar tobacco bred of safflower as material, its total serum IgE is extracted（RIN≥7.5）, and further reverse transcription obtains cDNA, with
This is template, designs NtRBSC1-F/NtRBSC1-R primer pairs, enters performing PCR amplification, obtains the amplification production of NtRBSC1 genes
Thing, and reclaimed.
The NtRBSC1-F/NtRBSC1-R primer pairs, using DNAMAN6.0, with reference to the design of NtRBSC1 gene orders
Into the NtRBSC1 genes, its base sequence such as SEQ ID NO：Shown in 1；Designed primer particular sequence is：
NtRBSC1-F:CGGGGTACCATGTCTGTCTCAAGTT, wherein GGTACC partial sequences are KpnI restriction enzyme sites,
NtRBSC1-R:CGCGGATCCAGATGCTACAGG, wherein GGATCC partial sequences are BamHI restriction enzyme sites.
Artificial synthesized primer is used into Tris-EDTA respectively（pH=8.0）Solution is diluted to 10 μM of working concentration, carries out
PCR is expanded, when PCR is expanded, and the design of 50 μ L reaction systems is as follows：
10 × II buffer, 5 μ L；
CDNA, 2 μ L；
NtRBSC1-F, 2.5 μ L；
NtRBSC1-R, 2.5 μ L；
Trans Taq HiFi archaeal dna polymerases, 0.3 μ L；
DNTP, 4 μ L；
Aqua sterilisa adds to 50 μ L；
Pcr amplification reaction condition specifically may be configured as：94 DEG C of pre-degeneration 5min；94 DEG C, 30s, 62 DEG C, 30s, 72 DEG C, 40s, 35
Individual circulation；72 DEG C again, 10min, 16 DEG C, 10min；
Pcr amplification product is subjected to electrophoresis detection, and reclaimed, 4 DEG C of institute's recovery product saves backup or directly carried out subsequent operation
Experiment.
（2）Digestion, and connect, it is specially：
To step（1）Middle reclaimed NtRBSC1 genes, carry out double digestion processing, and reclaim digestion production using KpnI and BamHI
Thing；
It is same that double digestion processing is carried out using KpnI and BamHI simultaneously to pFF19-GFP carriers, and reclaim digestion products.
When digestion is handled, 50 μ L double digestions System Designs are with reference to as follows：
BufferK, 2.5 μ L；
BamHI, 2.5 μ L；
SphI, 2.5 μ L；
Step（1）In NtRBSC1 genes amplified production（Or pFF19-GFP carriers）, 20 μ L；
Aqua sterilisa, adds to 50 μ L.
Using T4 DNA ligases by the cohesive end of said gene NtRBSC1 digestion products and pFF19-GFP carriers
The cohesive end of double digestion product be attached, 10 μ L linked systems are as follows with reference to setting：
NtRBSC1 digestion products, 1 μ L；
PFF19-GFP carrier digestion products, 1 μ L；
T4 DNA ligases, 1 μ L；
ddH2O adds to 10 μ L；
16 DEG C of connections are stayed overnight（More than 8 hours）.
（3）Conversion, is screened, and is specially：
By step（2）In connection product, with heat shock method conversion conversion bacillus coli DH 5 alpha competent cell, containing Amp (ammonia
Parasiticin, 50 μ g/mL) LB culture mediums on carry out resistance screening, and the positive bacterium colony of picking extracts plasmid and carries out digestion verification,
Digestion verification result is as shown in figure 1, represent that plasmid restructuring is correct；Further sequencing is carried out to the correct recombinant plasmid of digestion verification to test
Card, it is ensured that construction of recombinant plasmid is errorless.
For verifying that correct bacterial strain is enlarged culture, and it is standby to extract plasmid；This plasmid be NtRBSC1 genes and
The transient expression vector of fluorescent marker protein GFP genes, NtRBSC1-pFF19-GFP is named as by this plasmid vector.
Embodiment 2
By the tobacco protoplast conversion method that prepared NtRBSC1-pFF19-GFP plasmid vectors are mediated by PEG in embodiment 1
Transformation of tobacco protoplast, further passes through confocal laser scanning microscope, you can more intuitively position tobacco
NtRBCS1, so as to establish Research foundation and application foundation for related gene research, related experiment is briefly discussed below.
（One）Tobacco protoplast is prepared, concrete operations are：
1st, 0.2g Cellulase R-10 are weighed（Solarbio）、0.08g Macerozyme R-10（Solarbio）, add
Into 20mL enzyme liquid storages, 55 DEG C of water-bath 10min add 200uL 1M CaCl after cooling2（10mM）, 0.02g BSA
（0.1%）, 0.45 μm of micro porous filtration is made enzymolysis liquid, moves into standby in 100mL small beakers；
2nd, the tobacco leaf of growth 4 weeks is taken（The big gold dollar of safflower, the good plump blade of health, upgrowth situation）, each conversion is about
10, disleaf leading edge and petiole are removed, the wide strips of 1mm are cut into blade, are placed in the enzymolysis liquid of step 1, masking foil parcel lucifuge,
23 DEG C, 40 ~ 50 rpm enzymolysis, 2.5 ~ 3h；
3rd, the enzymolysis liquid of step 2 is filtered with 100 ~ 200 mesh sieve, filtrate is placed in 50mL centrifuge tubes, 4 DEG C, 60g, brake
=4 centrifugation 15min；
4th, supernatant is abandoned, precipitation gently dispels washing with the W5 solution of 4mL ice baths.4 DEG C, the centrifugation of 100g, brake=4 3min；
5th, supernatant is abandoned, precipitation is resuspended with the W5 solution of 4mL ice baths again, and 30min is placed on ice；
6th, 23 DEG C, the centrifugation 1min of 100g, brake=4, abandon supernatant, precipitation is resuspended with mmg solution, each conversion plus 100 μ L
mmg。
（Two）The tobacco protoplast of PEG mediations is converted, and concrete operations are：
7th, 10 ~ 20 μ g plasmids are taken（PFF19-NtTIP1.1-GFP plasmids prepared by embodiment 1）In 1.5mL EP pipes, add
Protoplast in 100 μ L steps 6 is soft to mix；
8th, 110 μ L PEG-Ca solution is added, it is soft to mix, place 20 ~ 30min；
9th, 440 μ L W5 solution is added, soft to mix, 23 DEG C, 100g, brake=4 centrifugation 1min；
10th, abandon supernatant, add 500 μ L W5 solution, soft to mix, immigration is cleaned up and with six holes of W5 solution rinses
In plate, 1mL is supplied with W5 solution, is somewhat mixed；
23 DEG C, 6 ~ 18h of masking foil parcel lucifuge culture；Then confocal laser scanning microscope is carried out.
（Three）Confocal laser scanning microscope, specific operation process is：
Draw appropriate Protoplast suspension first to drip on slide, gently covered；Finally by laser co-focusing
Microscope is observed；
During observation, slide is inverted and is put on objective table, is focused with bright-field, found and sent out with dark field after getting a clear view
Go out the cell of bright green fluorescence；The z-axis of setting laser co-focusing is taken pictures to cell.
As a result it is as shown in Figure 2.Fluorescent places distribution of specific, shape in the cell as can be seen that in Fig. 2 are analyzed Fig. 2
The autofluorescence of state size structure and plant chloroplast overlaps, therefore, and gene that can be by the use of this carrier as chloroplaset is fixed altogether
Position carrier.
It is to be understood that during aforesaid operations, it is as follows that portion of reagent prepares briefing：
200mM MES（3.904g/100mL）, KOH adjustment pH=5.7；
1M mannitol（18.218g/100mL）；
1M CaCl2（11.1g/100mL）；
Enzyme liquid storage（200mL）, including：
A, 0.4M mannitol（80mL, 1M mannitol）；
B, 20mM MES（20mL、200mM MES）；
C, 20mM KCl（0.2982g KCl）；
mmg（10mL）, including：
A, 0.4M mannitol（4mL 1M mannitol）；
B, 4mM MES（200uL 200mM MES）；
C, 15mM MgCl2（The anhydrous MgCl of 0.03049g2Or 0.065g MgCl2·6H2O）；
W5（100mL）, including：
154mM NaCl（0.8999g NaCl）；
125mM CaCl2（12.5mL 1M CaCl2）；
5mM KCl（0.037275g KCl）；
2mM MES（1mL 200mM MES）；
PEG(40%)-Ca（1mL）, including：
PEG4000 (Sigma), 0.4g；
H2O, 0.3mL；
1M mannitol, 0.2mL；
1MCaCl2, 0.1mL.
When the tobacco suspension cell mediated using particle gun carries out dependent conversion operation experiments（With U.S. Bio-rad's
Exemplified by the desk-top particle guns of PDS1000）, specific steps refer to as follows：
（One）Tobacco suspension cell is prepared, and evoked callus is standby；Detailed process is as follows：
Take the callus of culture growth in advance（Such as the BY-2 tobacco cells cultivated in BY-2 culture mediums）, with the spoon pressure of sterilizing
After broken, in access suspension cell fluid nutrient medium, shaking table culture a couple of days is to when being appropriate for via Particle Bombardment Transformation（It is thin with BY-2
Exemplified by born of the same parents, when suspension cell is glassy yellow, you can carry out Biolistic mediated transformation operation）；
（Two）By Biolistic mediated transformation method, prepared NtRBSC1-pFF19-GFP plasmids conversion in embodiment 1 is entered into cigarette
In careless suspension cell, detailed process is described as follows.
First, bronze suspension is prepared, detailed process is：
First, 60 mg bronzes are taken to be added in 1.5mL centrifuge tubes；Then 1mL 70% second newly prepared is added into centrifuge tube
Alcoholic solution, be vortexed 3 ~ 5min, then stands 15min, and then 3000rpm centrifuges 5s, finally carefully removes supernatant；This step 3 time；
Then, 1mL sterilized waters are added, 1min is vibrated, then stand 1min, last 3000rpm centrifuges 2s, removes supernatant；
Finally, 50% glycerine 1mL is added（That is, the μ g/ μ L of bronze concentration 60）, -20 DEG C save backup.
Secondly, micro- bullet is prepared（By taking 6 secondary amounts as an example）, detailed process is：
The bronze solution prepared in step 1 is taken, vortex 5min moves into 50 μ L solution after 1.5mL sterilizings in centrifuge tube；
Under vorticity, the NtRBSC1-pFF19-GFP plasmids prepared in embodiment 1 are added in centrifuge tube successively（Final concentration 1
μg/μL）、50 μL CaCl2（2.5 M, autoclaving）, 20 μ L spermidines（0.1 M, filtration sterilization）, then fully vibration 3
Min, then 1min is stood, to ensure that plasmid is fully settled on bronze surface again；
3000 rpm centrifuge 2 s, as fully as possible supernatant discarding, add 140 μ L 70% ethanol washing bronze, but to the greatest extent
Amount should not touch bronze；
3000 rpm centrifuge 2 s, as fully as possible supernatant discarding, add 140 μ L absolute ethyl alcohols washing bronze；
3000 rpm centrifuge 2 s, and supernatant discarding, adds 48 μ L absolute ethyl alcohols, then flick tube wall, make as fully as possible
Bronze is mixed, and now, micro- bullet in each centrifuge tube is available for 6 rifles to use.
Via Particle Bombardment Transformation, detailed process is：
Particle gun is placed on superclean bench to carry out sterile working, detailed process has：First by 70% ethanol to vacuum
Room carries out disinfection；Disk can be split again, stops net and micro- bullet film 70% ethanol, 30 min of sterilizing, be subsequently placed on aseptic filter paper
Dry standby；
The middle section that the uniform points of the even μ L of genophore-bronze mixed liquor 6 are hanged in step 2 in sterile micro- bullet carrier film is taken, so
It is standby after being dried up on superclean bench；The fixed lid of disk loading can be split to screw；Micro- missile-borne body and the resistance of micro- bullet are will be equipped with again
Backstop loads micro- bullet emitter；
Voltage-stabilized power supply, vavuum pump and nitrogen threshold switch are opened, by acceptor material（Step（One）In prepared suspension cell）It is put into
Sample room, sets the cm of range 9, acceptor material is bombarded with particle gun；After bombardment, exhaust takes out sample, uses sealed membrane
Seal up；The 28 DEG C of avoid light places of suspension cell bombarded are stayed overnight in plant incubator, laser co-focusing then can be carried out
Micro- sem observation.
SEQUENCE LISTING
<110>Zhengzhou Tobacco Research Institute of CNTC
<120>One carry NtRBSC1 genes transient expression vector
<130> none
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 510
<212> DNA
<213> Nicotiana tabacum
<400> 1
atgtctgtct caagttttgt tgcagctcca attgctggat caacctatct tggtttgaaa 60
tccaacaaca ccaacctttt tcctgctaag gatacaatca cttggaatcg aaaaactatc 120
tccaatggct ctaaaactta ctgcatgaag acttggaatc cgatcgacaa caagaaattc 180
gagacactct catatctgcc acctctatca gaagattcga ttgcaaagga ggttgattac 240
atgatccaaa agggctggat tccttgcctt gaatttgatc aggtgggata tgtacgcagg 300
gaaaatagca gcatgccagg ctactatgat ggaaggtatt ggacactgtg gaagcttccc 360
atgtttggct gcaatgactc atcccaagtt ctgaatgaaa tccaagattg caagaaagcc 420
tacccaaatg ctttcattcg ttgtttggca tttgacaatg ttaaacaggt tcagtgcatg 480
gcctttctca ttcagaaacc tgtagcatag 510
Claims (4)
1. a transient expression vector for carrying NtRBSC1 genes, it is characterised in that the carrier carry tobacco ribulose-
The small ylidene gene NtRBSC1 of 1,5- diphosphonic acid carboxylase and a fluorescent marker protein GFP gene, make as follows
It is standby to form：
（1）PCR is expanded, and obtains tobacco ribulose-1,5-bisphosphate, and the small ylidene gene NtRBSC1 of 5- diphosphonic acid carboxylases is specially：
Tobacco-containing material total serum IgE is extracted, reverse transcription obtains cDNA, as template, designs NtRBSC1-F/NtRBSC1-R primer pairs,
Enter performing PCR to expand, acquisition tobacco ribulose-1,5-bisphosphate, the small ylidene gene NtRBSC1 of 5- diphosphonic acid carboxylases amplified production, and
Amplified production is reclaimed；
The NtRBSC1-F/NtRBSC1-R primer pairs, particular sequence is：
NtRBSC1-F:CGGGGTACCATGTCTGTCTCAAGTT,
NtRBSC1-R: CGCGGATCCAGATGCTACAGG；
The NtRBSC1 genes, its base sequence is as shown in SEQ ID NO.1；
（2）Digestion, and connect, it is specially：
To step（1）The middle amplified production for reclaiming obtained gene NtRBSC1, is carried out at double digestion using KpnI and BamHI
Reason, and reclaim digestion products；
It is same that double digestion processing is carried out using KpnI and BamHI simultaneously to pFF19-GFP carriers, and reclaim digestion products；
Using T4 DNA ligases by above-mentioned tobacco ribulose-1,5-bisphosphate, the small ylidene gene NtRBSC1 of 5- diphosphonic acid carboxylases enzyme
The cohesive end for cutting the cohesive end of product and the double digestion product of pFF19-GFP carriers is attached；
（3）Conversion, is screened, and is specially：
By step（2）In connection product conversion conversion bacillus coli DH 5 alpha competent cell, carry out resistance screening, and picking sun
Property bacterium colony checking, for verifying that correct bacterial strain is enlarged culture, and extract plasmid, this plasmid vector is transient expression vector
NtRBSC1-pFF19-GFP。
2. the transient expression vector of NtRBSC1 genes is carried as claimed in claim 1, it is characterised in that step（1）In, institute
Tobacco-containing material is stated for the big gold dollar of safflower.
3. application of the transient expression vector of NtRBSC1 genes in tobacco is carried described in claim 1, it is characterised in that
NtRBSC1-pFF19-GFP plasmid vectors are mediated by Biolistic mediated transformation method transformation of tobacco cell suspending liquid or PEG former
Raw plastid transformation method transformation of tobacco protoplast, the h of room temperature light culture 8 ~ 16 is suspended thin by confocal laser scanning microscope
Born of the same parents or tobacco plasm, tobacco chloroplast is positioned by fluorescence display position of the fusion protein in cell.
4. carrying application of the transient expression vector of NtRBSC1 genes in tobacco as claimed in claim 3, its feature exists
In the tobacco cell suspension is BY-2 tobacco cell suspension, and the tobacco protoplast is the tobacco of the big gold dollar of safflower
Protoplast.
Priority Applications (1)
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| CN109609543A (en) * | 2018-12-29 | 2019-04-12 | 青岛袁策集团有限公司 | It is a kind of for identifying rice to the transient expression vector of bakanae disease resistance |
| CN110199708A (en) * | 2019-06-27 | 2019-09-06 | 杭州师范大学 | A method of detection rice leaf Chloroplast distribution situation after light processing |
| CN110199708B (en) * | 2019-06-27 | 2021-09-28 | 杭州师范大学 | Method for detecting distribution condition of chloroplasts in rice leaves after light treatment |
| CN115044611A (en) * | 2022-06-29 | 2022-09-13 | 河北农业大学 | Tobacco instantaneous transformation method convenient to operate |
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