CN103926226A - Method for detecting plant protein sub-cellular localization - Google Patents
Method for detecting plant protein sub-cellular localization Download PDFInfo
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Abstract
The invention discloses a method for detecting the plant protein sub-cellular localization. The method comprises the following steps: (1) cloning fusion genes (a to-be-detected plant protein gene and a reporter gene) in a plant expression vector so as to obtain a recombinant expression vector; (2) converting the recombinant expression vector into agrobacterium tumefaciens so as to obtain recombinant agrobacterium tumefaciens; (3) adding an agrobacterium tumefaciens infection buffer solution into the recombinant agrobacterium tumefaciens, adjusting the OD600 to be 0.1-0.3, and standing for 1-2h at the temperature being 20-25 DEG C so as to obtain an infection bacterium liquid; (4) pricking holes in plant leaves by a needle in a manner of keeping away from veins, and injecting the infection bacterium liquid in the plant leaves from the holes by an injection syringe without a needle; and (5) cultivating plants for 2 days, fetching the leaves each of which the area is 1-2cm<2> from the injection parts, and detecting the sub-cellular localization of to-be-detected plant protein in the plants according to the reporter gene. Tests prove that the method has the advantages of being easy to operate, fast, good in marking effect and good in repeatability; the method can be used for adjusting and controlling of gene expression, the functional study on intrones, and the like; by using the method, a technological base for the study on new gene functions and the adjusting and the controlling of the gene expression is established.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method that detects phytoprotein Subcellular Localization.
Background technology
Subcellular Localization refers to that certain albumen or expression product are at intracellular concrete Present site.For example in core, in kytoplasm or exist on cell membrane.
The Subcellular Localization of protein and its Function are closely related.Protein is the direct agent of biological function, and the protein distributing is in order the prerequisite that ensures living individual normal growth, growth, is exactly in order to understand better life process so understand the proteins subcellular location of these processes.Growing of plant, the variation in Adaptable growth stage, and can resist poor environment, and the molecular biology of these processes and biochemical basis are exactly protein.The Subcellular Localization that detects phytoprotein has vital role for degeneration-resistant, the High-yielding Mechanism of understanding important crops.
Derive from green fluorescent protein (the green fluorescent protein of jellyfish, GFP), its endogenous fluorophor be subject to ultraviolet light or when blue-light excited (395nm and 479nm) can efficiently launch apparent green fluorescence, be the desirable probe of gene expression and protein positioning in monitoring living cells and tissue.Be widely used as reporter gene, carried out the cytologic experiment research of live body.
About phytoprotein in the Position Research in cell, what great majority adopted was via Particle Bombardment Transformation method in the past.But gene gun conversion method is higher to operator's technical requirement, and consumptive material price in particle gun operation is very high, Preparatory work of experiment complexity, and success ratio is not high especially.
Therefore, be badly in need of a kind of method that can quick and precisely detect phytoprotein Subcellular Localization.
Summary of the invention
The object of this invention is to provide a kind of method that detects phytoprotein Subcellular Localization.
The method of detection phytoprotein Subcellular Localization provided by the present invention, specifically can comprise the steps:
(1) encoding gene by phytoprotein to be measured and reporter gene are merged to the fusion gene cloning forming in plant expression vector, obtain expressing the recombinant expression carrier of described fusion;
Wherein, reporter gene is protein that a kind of coding can be detected or the gene of enzyme.Described reporter gene both can be blended in 5 ' end of the encoding gene of described phytoprotein to be measured, also can be blended in 3 ' end of the encoding gene of described phytoprotein to be measured.
(2) described recombinant expression carrier is converted in Agrobacterium competent cell, obtains the Agrobacterium of recombinating;
(3) in described restructuring Agrobacterium, add Agrobacterium to infect damping fluid, regulate OD
600be that 0.1～0.3(is as 0.3), 20-25 DEG C (as 25 DEG C) leave standstill 1-2h(as 1h), obtain infecting bacterium liquid;
(4) on plant leaf blade, avoid vein with the syringe needle of syringe and prick hole, described in then inciting somebody to action with the described syringe of removing described syringe needle, infect bacterium liquid and be injected in described plant leaf blade from Zha Kongchu;
(5) cultivate described plant 30h-48h(as 48h) after, cut 1-2cm from injection site
2blade as detected materials; Detect in described detected materials by the described fusion Subcellular Localization of the protein obtaining of encoding according to described reporter gene, thereby determine the Subcellular Localization of described phytoprotein to be measured in described plant.
In the step (4) of described method, on described plant leaf blade, prick hole (as 2 distances hole that is 3cm) with the syringe needle of described syringe, and with remove described syringe needle described syringe will described in infect bacterium liquid and be injected in described plant leaf blade from Zha Kongchu, be all to operate from the back side of described plant leaf blade.
In the step (4) of described method, when infecting bacterium liquid described in injection in described plant leaf blade, injection volume is every cm
2described in blade injection 0.2mL, infect bacterium liquid.
In the step (1) of described method, the encoding gene that described reporter gene is fluorescin; Described fluorescin is specially enhanced green fluorescence protein (referred to as EGFP, amino acid sequence is sequence 1 in sequence table).Accordingly, in step (5), detect in described detected materials by the encode Subcellular Localization of the protein obtaining of described fusion according to described reporter gene, for: will after described detected materials compressing tablet, adopt fluorescent microscope or laser confocal microscope, by imaging analysis, the protein obtaining of being encoded by described fusion is carried out to Subcellular Localization.Wherein, described detected materials (is cut to 1-2cm from injection site
2blade) carry out compressing tablet and be: vacuum side of blade is placed on microslide upward, and on described microslide, drips a 2-3 and drip distilled water, blade is soaked completely, covered.In addition, while adopting fluorescent microscope or laser confocal microscope to carry out imaging analysis, need to corresponding microscope operation parameter be set according to the excitation wavelength of described fluorescin.
In the step (1) of described method, described plant expression vector can be any carrier for expression of plants, as pBI121 etc.In the present invention, in described plant expression vector, starting the promoter that described fusion transcribes is CaMV35S promoter; Stopping the terminator that described fusion transcribes is NOS terminator.More concrete, described plant expression vector is pCAMBIA2300 carrier.
Accordingly, in the step (1) of described method, the final described recombinant expression carrier obtaining that builds is as lower arbitrary:
(a1) recombinant vector (called after pCAMBIA2300-EGFP-BiP2) of DNA molecular shown in sequence 4 in insetion sequence table between the restriction enzyme site SmaI of pCAMBIA2300 carrier and PstI;
(a2) recombinant vector (the called after pCAMBIA2300-EGFP-PIP2 of DNA molecular shown in sequence 3 in insetion sequence table between the restriction enzyme site SmaI of pCAMBIA2300 carrier and PstI; 1).
(a3) recombinant vector (called after pCAMBIA2300-EGFP-HDEL) of DNA molecular shown in sequence 5 in insetion sequence table between the restriction enzyme site SalI of pCAMBIA2300 carrier and PstI.
In the step (2) of described method, the method that described recombinant expression carrier is converted in described Agrobacterium competent cell is heat shock method or electric shocking method.In one embodiment of the invention, the method adopting is heat shock method.
Described Agrobacterium can be Agrobacterium GV3101, Agrobacterium LBA4404, Agrobacterium EHA105 or Agrobacterium AGL1.In one embodiment of the invention, described Agrobacterium is specially Agrobacterium GV3101.
In the step (3) of described method, adding described Agrobacterium to infect before damping fluid in described restructuring Agrobacterium, also comprise the steps: the described restructuring Agrobacterium of step (2) gained to be inoculated in fluid nutrient medium and to be cultured to exponential phase, centrifugal (5min as centrifugal in 6000rmp) collects thalline;
Wherein, described exponential phase is the period of the specific growth rate maximum of described restructuring Agrobacterium; The biomass that the thalline that described specific growth rate is unit mass per hour increases.
The solvent of described fluid nutrient medium is water, and solute and concentration thereof are as follows: yeast extract 10g/L, peptone 10g/L, NaCl5g/L, pH7.4.
In the step (3) of described method, the pH that described Agrobacterium is infected damping fluid is 5.4, and solvent is water, and solute and concentration thereof are as follows: D-Glucose 5g/L, MES 50mM, Na
3pO
412H
2o2mM, acetosyringone 0.1mM.Described Agrobacterium is infected damping fluid can not autoclaving.
In the step (4) of described method, described syringe is 1mL syringe.In one embodiment of the invention, described syringe is specifically 1mL plastic injector for temporary use.
In the step (5) of described method, described " cultivate described plant 30h-48h(as 48h) " is: by as described in plant under cellar culture condition the normal 30h-48h(that cultivates as 48h).
In described method, described plant is dicotyledon; Described dicotyledon can be tobacco or arabidopsis.Described tobacco specifically can be this life cigarette (Nicotiana benthamiana) or common tobacco (Nicotiana tobacum), and described arabidopsis can be columbia (Col-0) or Landsberg erecta (Ler).
In one embodiment of the invention, described plant is this life cigarette (Nicotiana benthamiana), is specially 2 months this life cigarettes (Nicotiana benthamiana) of growth.Accordingly, in the step (5) of described method, described " cultivate described plant 30h-48h(as 48h) " is: by as described in this life cigarette (Nicotiana benthamiana) 16 hours illumination/8 hour dark, the condition of 22 DEG C of constant temperature is cultivated 30h-48h(as 48h).
Another object of the present invention is to provide a kind of product for detection of phytoprotein Subcellular Localization.
Product for detection of phytoprotein Subcellular Localization provided by the present invention, can comprise independent packaging as lower all or part of:
(a) above-described plant expression vector;
(b) above-described Agrobacterium;
(c) above-described Agrobacterium is infected damping fluid;
(d) above-described fluid nutrient medium;
(e) above-described syringe.
The invention provides a kind of to phytoprotein Subcellular Localization carry out simply, the method for express-analysis.This method has the following advantages: 1) simple to operate, quick: do not need complicated instrument and equipment and reagent, and low to operator's technical requirement; 2) mark is effective: Agrobacterium is infected method can obtain a large amount of fluorescence labeled cells, can obtain the particle gun method high-level efficiency that is beyond one's reach; 3) reproducible: this method can reach 100% success ratio.This method is equally applicable to the analysis of other phytoprotein Subcellular Localization.
The present invention not only can apply to the Subcellular Localization research of protein, but also can be for fields such as gene expression regulation and introne functional studies, thereby function and expression regulation to further other new gene of research established technical foundation.
Brief description of the drawings
Fig. 1 is the schematic flow sheet that adopts agriculture bacillus mediated instant expression method phytoprotein to be carried out to Subcellular Localization detection.Wherein, E-1, E-2 and E-3 are three different vegetable protein HDEL, BiP2 and the PIP2 that is followed successively by fluoroscopic examination; The Subcellular Localization image of 1 albumen.
Embodiment
The experimental technique using in following embodiment if no special instructions, is conventional method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
PCAMBIA2300 carrier: be recorded in " Gong Yuanyong; Feng Yongkun; Ni Wanchao etc. the structure of plant expression vector pCAMBIA2300-35S-GUS-CaMVterm and checking. Chinese biological engineering magazine, 03 phase in 2013 " literary composition, the public can obtain from Institute of Botany, Chinese Academy of Sciences.
This life cigarette (Nicotiana benthamiana): be recorded in " Cui Haitao; Li Chunxia; Wang Hongyan etc. the foundation of the cultivation of this life cigarette tissue and genetic conversion system. Shandong science, 01 phase in 2006 " literary composition, the public can obtain from Institute of Botany, Chinese Academy of Sciences.
Agrobacterium GV3101: be recorded in " Xiao Weimin, Zhao Mingchen, Zou Min etc. arabidopsis is injured and inoculate Agrobacterium tumefaciems GV3101 to the impact of transcribing. Journal of Agricultural Biotechnology, 05 phase in 2013 " literary composition, the public can obtain from Institute of Botany, Chinese Academy of Sciences.
Embodiment 1, detection phytoprotein Subcellular Localization
In the present embodiment, adopt agriculture bacillus mediated instant expression method to carry out Subcellular Localization detection to phytoprotein, its schematic flow sheet as shown in Figure 1.
1, the structure of recombinant expression carrier and qualification
With arabidopsis HDEL albumen, BiP2 albumen and PIP2; 1 albumen is respectively as phytoprotein to be measured, the encoding gene (nucleotide sequence of enhanced green fluorescence protein is as shown in sequence 2) of the encoding gene of albumen and enhanced green fluorescence protein (amino acid sequence is as shown in sequence 1) is merged to the fusion gene cloning forming in plant expression vector pCAMBIA2300, obtain expressing the recombinant expression carrier of described fusion.Concrete operations are as follows:
(1) structure of pCAMBIA2300-EGFP carrier
Taking the encoding gene (sequence 2) of enhanced green fluorescence protein as template, adopt primer EGFP-F and EGFP-R to carry out pcr amplification.
EGFP-F:5 '-GTCGACATGGGCAAAGGAGAAGAACT-3 ' (underscore part is the recognition sequence of Sal I, and sequence is thereafter the 1-20 position of sequence 2);
EGFP-R:5 '-
cTGCAGtTATTTGTATAGTTCATCCATGC-3 ' (underscore part is the recognition sequence of Pst I, and sequence is thereafter the reverse complementary sequence of the 695-717 position of sequence 2).
Gained PCR product is carried out to double digestion with restriction enzyme Sal I and Pst I, reclaim enzyme and cut product, be connected with the pCAMBIA2300 carrier framework large fragment through same double digestion, obtain recombinant plasmid.The recombinant plasmid called after pCAMBIA2300-EGFP of DNA fragmentation shown in sequence 2 will be shown to have inserted between the restriction enzyme site Sal I of pCAMBIA2300 carrier and Pst I through order-checking.
(2) structure of recombinant expression carrier pCAMBIA2300-EGFP-BiP2
Taking the encoding gene (" 1-2007+TAA of sequence 4 ") of arabidopsis BiP2 albumen as template, adopt primer BiP2-F and BiP2-R to carry out pcr amplification.
BiP2-F:5 '-
cCCGGGaTGGCTCGCTCGTTTGGAGC-3 ' (underscore part is the recognition sequence of Sma I, and sequence is thereafter the 1-20 position of sequence 4);
BiP2-R:5 '-
tCTAGAgAGCTCATCGTGAGACTCAT-3 ' (underscore part is the recognition sequence of Xba I, and sequence is thereafter the reverse complementary sequence of the 1987-2007 position of sequence 4).
Gained PCR product is carried out to double digestion with restriction endonuclease sma I and Xba I, reclaim enzyme and cut product, be connected with the pCAMBIA2300-EGFP carrier framework large fragment through same double digestion, obtain recombinant plasmid.The recombinant plasmid called after pCAMBIA2300-EGFP-BiP2 of DNA fragmentation shown in the 1-2007 position of sequence 4 will be shown to have inserted between the restriction enzyme site Sma I of pCAMBIA2300-EGFP carrier and Xba I through order-checking.
In recombinant expression carrier pCAMBIA2300-EGFP-BiP2, there is the encoding gene (in sequence table shown in sequence 4) of BiP2-EGFP fusion, the promoter that the encoding gene of startup BiP2-EGFP fusion is transcribed is CaMV35S promoter, and terminator is NOS terminator.
(3) structure of recombinant expression carrier pCAMBIA2300-EGFP-BiP2
With arabidopsis PIP2; The encoding gene (" 1-861+TAA of sequence 3 ") of 1 albumen is template, adopts primer PIP2; 1-F and PIP2; 1-R carries out pcr amplification.
PIP2; 1-F:5 '-
cCCGGGaTGGCAAAGGATGTGGAAGCCGTTC-3 ' (underscore part is the recognition sequence of Sma I, and sequence is thereafter the 1-25 position of sequence 3);
PIP2; 1-R:5 '-
tCTAGAgACGTTGGCAGCACTTCTGAAT-3 ' (underscore part is the recognition sequence of Xba I, and sequence is thereafter the reverse complementary sequence of the 840-861 position of sequence 3).
Gained PCR product is carried out to double digestion with restriction endonuclease sma I and Xba I, reclaim enzyme and cut product, be connected with the pCAMBIA2300-EGFP carrier framework large fragment through same double digestion, obtain recombinant plasmid.The recombinant plasmid called after pCAMBIA2300-EGFP-PIP2 of DNA fragmentation shown in the 1-861 position of sequence 3 will be shown to have inserted between the restriction enzyme site Sma I of pCAMBIA2300-EGFP carrier and Xba I through order-checking; 1.
At recombinant expression carrier pCAMBIA2300-EGFP-PIP2; In 1, there is PIP2; The encoding gene of 1-EGFP fusion (in sequence table shown in sequence 3), starts PIP2; The promoter that the encoding gene of 1-EGFP fusion is transcribed is CaMV35S promoter, and terminator is NOS terminator.
(4) structure of recombinant expression carrier pCAMBIA2300-EGFP-HDEL
Taking the encoding gene (sequence 2) of enhanced green fluorescence protein as template, adopt primer EGFP-F and EGFP-HDEL-R to carry out pcr amplification.
EGFP-F:5 '-
gTCGACaTGGGCAAAGGAGAAGAACT-3 ' (underscore part is the recognition sequence of Sal I, and sequence is thereafter the 1-20 position of sequence 5);
EGFP-HDEL-R：
5 '-CTGCAGCTAGAGCTCATCGTGTTTGTATAGTTCATCCATGC-3 ' (underscore part is the recognition sequence of Pst I, and sequence is thereafter the reverse complementary sequence of the 695-729 position of sequence 5).
Gained PCR product is carried out to double digestion with restriction enzyme Sal I and Pst I, reclaim enzyme and cut product, be connected with the pCAMBIA2300 carrier framework large fragment through same double digestion, obtain recombinant plasmid.The recombinant plasmid called after pCAMBIA2300-EGFP-HDEL of DNA fragmentation shown in sequence 5 will be shown to have inserted between the restriction enzyme site Sal I of pCAMBIA2300 carrier and Pst I through order-checking.
In recombinant expression carrier pCAMBIA2300-EGFP-HDEL, there is the encoding gene (in sequence table shown in sequence 5) of EGFP-HDEL fusion, the promoter that the encoding gene of startup EGFP-HDEL fusion is transcribed is CaMV35S promoter, and terminator is NOS terminator.
2, acquisition and the qualification of restructuring Agrobacterium
The recombinant expression carrier pCAMBIA2300-EGFP-HDEL that step 1 is obtained, pCAMBIA2300-EGFP-BiP2, pCAMBIA2300-EGFP-PIP2; 1 is converted in the competent cell of Agrobacterium GV3101 by heat shock method respectively, obtains the Agrobacterium of recombinating.It is as follows that heat shock method transforms concrete operations: the recombinant expression carrier pCAMBIA2300-EGFP-HDEL that 1 μ L step 1 is obtained, pCAMBIA2300-EGFP-BiP2 or pCAMBIA2300-EGFP-PIP2; 1 joins in the 1.5mL centrifuge tube that contains 30 μ L Agrobacterium GV3101 competent cells, and is placed in and hatches 30 minutes on ice; Hatch after end, centrifuge tube is placed in to liquid nitrogen freezing 5 minutes, in 37 DEG C of water-baths, hatch 5 minutes subsequently.Add 800 μ L YEP fluid nutrient mediums (formula: solvent is water, and solute and concentration thereof are as follows: yeast extract 10g/L, peptone 10g/L, NaCl5g/L, pH7.4), 28 DEG C of shaking table 180rpm recoveries 2 hours.After recovery finishes, draw 100 μ L bacterium liquid and coat (interpolation 50 μ M Kan and 50 μ M Rif) on YEP solid medium, in 28 DEG C of incubators, cultivate 2 days.
Gained restructuring Agrobacterium is carried out to Molecular Identification: employing primer EGFP-F and EGFP-R(sequence are the same) restructuring Agrobacterium is carried out to pcr amplification.By show to contain size through qualification, to be about the restructuring Agrobacterium of 700bp object fragment positive.According to the difference of entrained recombinant expression carrier, by three kinds of positive Agrobacteriums called after GV3101/pCAMBIA2300-EGFP-HDEL respectively, GV3101/pCAMBIA2300-EGFP-BiP2 and GV3101/pCAMBIA2300-EGFP-PIP2; 1.
Arrange to the contrast that proceeds to pCAMBIA2300-EGFP empty carrier in Agrobacterium simultaneously.The restructuring Agrobacterium called after GV3101/pCAMBIA2300-EGFP of pCAMBIA2300-EGFP empty carrier will be proceeded to.
3, infect the preparation of bacterium liquid
Positive restructuring Agrobacterium GV3101/pCAMBIA2300-EGFP-HDEL, GV3101/pCAMBIA2300-EGFP-BiP2, GV3101/pCAMBIA2300-EGFP-PIP2 will be identified in step 2; 1 or the monoclonal bacterium colony of GV3101/pCAMBIA2300-EGFP in YEP fluid nutrient medium (filling a prescription the same), be cultured to exponential phase.The centrifugal 5min of 6000rpm collects thalline.In gained thalline, add Agrobacterium to infect damping fluid, resuspended thalline is to OD
600value is 0.3, and room temperature (25 DEG C) leaves standstill after 1h, obtains infecting bacterium liquid, for infecting.
4, injection tobacco leaf
For trying plant: this uncured tobacco (Nicotiana benthamiana) of growing 2 months.
With 1mL plastic injector for temporary use (Beijing Suo Laibao Science and Technology Ltd. product, its catalog number is ZSQ1mL) syringe needle prick two apertures apart from 3cm at this life cigarette (Nicotiana benthamiana) vacuum side of blade, and avoid vein (being beneficial to infect the infiltration of bacterium liquid).Remove syringe needle, described in inciting somebody to action with the needle tubing of needle-less, infect bacterium liquid and be injected in described plant leaf blade from Zha Kongchu, slowly bacterium liquid is injected between the vein of blade.Injection volume is every cm
2blade area injection volume is 0.2mL.
5, Subcellular Localization detects
Tobacco leaf after the injection that step 4 is obtained, in 16 hours illumination/8 hour dark, is normally cultivated 2 days under 22 DEG C of constant temperatures.Then, cut 1-2cm from injection site
2blade, vacuum side of blade is placed on microslide upward, and on described microslide drip a 2-3 drip distilled water, blade is soaked completely, covered.Compressing tablet finishes rear directly in the lower observation of laser confocal microscope (ZEISS LSM510META), Ar/Kr laser, and excitation wavelength is 488nm, receiving spectrum parameter is BP500-550IR.Each sample is all got the imaging of axis optical section.
Experiment in triplicate.
Result shows: in the visual field of choosing at random, observed a large amount of fluorescence labeled cell (more than 95% cell is labeled fluorescence, as long as substantially infect the wet blade position of immersion, nearly all can be luminous).EGFP protein signal is positioned whole cell, all has green fluorescence in tenuigenin and on cell membrane.The green fluorescence signal framing of EGFP-HDEL fusion is in endoplasmic reticulum (as shown in the E1 in E in Fig. 1), and the green fluorescence signal framing of BiP2-EGFP fusion is in plasmodesmus (as shown in the E2 in E in Fig. 1), PIP2; The green fluorescence of 1-EGFP fusion is positioned cell membrane (as shown in the E3 in E in Fig. 1).More than adopt Subcellular Localization and HDEL, BiP2 and the PIP2 of three albumen that the inventive method obtains, the relevant report of the Subcellular Localization of 1 albumen unanimously (refers to " Yokota E, et al. (2008) An isoform of myosin XI is responsible for the translocation of endoplasmic reticulum in tobacco cultured BY-2cells.J.Exp.Bot.:ern280. ", " Schirawski J, et al. (2005) Endoplasmic Reticulum Glucosidase II Is Required for Pathogenicity of Ustilago maydis.Plant Cell17 (12): 3532-3543. " and " Li X, et al. (2011) Single-molecule analysis of PIP2, 1dynamics and partitioning reveals multiple modes of Arabidopsis plasma membrane aquaporin regulation.Plant Cell23 (10): 3780-3797. ").Visible, in method provided by the present invention, the existence of EGFP albumen does not affect the Subcellular Localization of testing protein, and the method success ratio is high, reproducible, repeats experiment for three times and all obtains identical experimental result.
Claims (10)
1. a method that detects phytoprotein Subcellular Localization, comprises the steps:
(1) encoding gene by phytoprotein to be measured and reporter gene are merged to the fusion gene cloning forming in plant expression vector, obtain expressing the recombinant expression carrier of described fusion;
(2) described recombinant expression carrier is converted in Agrobacterium competent cell, obtains the Agrobacterium of recombinating;
(3) in described restructuring Agrobacterium, add Agrobacterium to infect damping fluid, regulate OD
600be 0.1～0.3,20-25 DEG C of standing 1-2h, obtain infecting bacterium liquid;
(4) on plant leaf blade, avoid vein with the syringe needle of syringe and prick hole, described in then inciting somebody to action with the described syringe of removing described syringe needle, infect bacterium liquid and be injected in described plant leaf blade from Zha Kongchu;
(5) cultivate after described plant 30-48h, cut 1-2cm from injection site
2blade as detected materials; Detect in described detected materials by the described fusion Subcellular Localization of the protein obtaining of encoding according to described reporter gene, thereby determine the Subcellular Localization of described phytoprotein to be measured in described plant.
2. method according to claim 1, it is characterized in that: in step (4), on described plant leaf blade, prick hole with the syringe needle of described syringe, and with remove described syringe needle described syringe will described in infect bacterium liquid and be injected in described plant leaf blade from Zha Kongchu, be all to operate from the back side of described plant leaf blade.
3. method according to claim 1 and 2, is characterized in that: in step (1), and the encoding gene that described reporter gene is fluorescin; Described fluorescin is specially enhanced green fluorescence protein;
In step (5), detect in described detected materials by the encode Subcellular Localization of the protein obtaining of described fusion according to described reporter gene, for: will after described detected materials compressing tablet, adopt fluorescent microscope or laser confocal microscope, by imaging analysis, the protein obtaining of being encoded by described fusion is carried out to Subcellular Localization.
4. according to arbitrary described method in claim 1-3, it is characterized in that: in step (1), in described plant expression vector, starting the promoter that described fusion transcribes is CaMV35S promoter; In described plant expression vector, stopping the promoter that described fusion transcribes is NOS terminator;
Described plant expression vector is specially pCAMBIA2300 carrier.
5. according to arbitrary described method in claim 1-4, it is characterized in that: in step (2), the method that described recombinant expression carrier is converted in described Agrobacterium competent cell is heat shock method or electric shocking method; And/or
Described Agrobacterium is Agrobacterium GV3101, Agrobacterium LBA4404, Agrobacterium EHA105 or Agrobacterium AGL1.
6. according to arbitrary described method in claim 1-5, it is characterized in that: in step (3), adding described Agrobacterium to infect before damping fluid in described restructuring Agrobacterium, also comprise the steps: the described restructuring Agrobacterium of step (2) gained to be inoculated in and in fluid nutrient medium, to be cultured to exponential phase, centrifugal collection thalline;
The solvent of described fluid nutrient medium is water, and solute and concentration thereof are as follows: yeast extract 10g/L; Peptone 10g/L; NaCl5g/L.
7. according to arbitrary described method in claim 1-6, it is characterized in that: in step (3), the pH that described Agrobacterium is infected damping fluid is 5.4, and solvent is water, and solute and concentration thereof are as follows: D-Glucose 5g/L, MES 50mM, Na
3pO
412H
2o2mM, acetosyringone 0.1mM.
8. according to arbitrary described method in claim 1-7, it is characterized in that: in step (4), described syringe is 1mL syringe.
9. according to arbitrary described method in claim 1-8, it is characterized in that: described plant is dicotyledon;
Described dicotyledon is specially tobacco or arabidopsis.
10. for detection of the product of phytoprotein Subcellular Localization, comprise independent packaging as lower all or part of:
(a) plant expression vector of claim 1-9 described in arbitrary;
(b) Agrobacterium of claim 1-9 described in arbitrary;
(c) Agrobacterium of claim 1-9 described in arbitrary infected damping fluid;
(d) fluid nutrient medium of claim 1-9 described in arbitrary;
(e) syringe of claim 1-9 described in arbitrary.
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| CN108642127A (en) * | 2018-03-29 | 2018-10-12 | 福建农林大学 | Application of the rice E3 ubiquitin ligases in detecting plant nucleolus subcellular localization |
| CN109266669A (en) * | 2018-10-10 | 2019-01-25 | 北京林业大学 | A kind of method of quick positioning protein matter position in plant leaf blade guard cell |
| CN114223426A (en) * | 2021-12-27 | 2022-03-25 | 中国热带农业科学院热带生物技术研究所 | Monocotyledon leaf liquid injection method |
| CN114921489A (en) * | 2022-04-29 | 2022-08-19 | 中国农业科学院蔬菜花卉研究所 | Method for detecting subcellular localization by using lily leaves |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007096192A2 (en) * | 2006-02-23 | 2007-08-30 | Era Biotech, S.A. | Production of biologically active proteins |
| CN101240290A (en) * | 2008-02-29 | 2008-08-13 | 杭州师范大学 | Plant transgene expression vector containing green fluorescence protein gene and its construction method and application |
-
2014
- 2014-04-17 CN CN201410155769.0A patent/CN103926226A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007096192A2 (en) * | 2006-02-23 | 2007-08-30 | Era Biotech, S.A. | Production of biologically active proteins |
| CN101240290A (en) * | 2008-02-29 | 2008-08-13 | 杭州师范大学 | Plant transgene expression vector containing green fluorescence protein gene and its construction method and application |
Non-Patent Citations (2)
| Title |
|---|
| 王雪: "一个新的烟草糖基转移酶基因sm-Ngt1在转基因烟草中的功能研究", 《中国优秀硕士学位论文全文数据库（电子期刊） 农业科技辑》 * |
| 程广伟: "NtNAC-R1介导烟株打顶诱导烟碱生物合成过程", 《中国优秀硕士学位论文全文数据库（电子期刊） 农业科技辑》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106771243A (en) * | 2016-12-23 | 2017-05-31 | 福建农林大学 | The kit and detection method of indicator plant kernel and Ke Hao body Subcellular Localizations |
| CN107794292A (en) * | 2017-11-02 | 2018-03-13 | 南京农业大学 | A kind of Subcellular Localization kit for infecting tobacco and its application |
| CN107881192A (en) * | 2017-11-15 | 2018-04-06 | 北京林业大学 | A kind of method of albumen homodimer distribution situation on observation of plant cell membrane |
| CN108642127A (en) * | 2018-03-29 | 2018-10-12 | 福建农林大学 | Application of the rice E3 ubiquitin ligases in detecting plant nucleolus subcellular localization |
| CN109266669A (en) * | 2018-10-10 | 2019-01-25 | 北京林业大学 | A kind of method of quick positioning protein matter position in plant leaf blade guard cell |
| CN114223426A (en) * | 2021-12-27 | 2022-03-25 | 中国热带农业科学院热带生物技术研究所 | Monocotyledon leaf liquid injection method |
| CN114921489A (en) * | 2022-04-29 | 2022-08-19 | 中国农业科学院蔬菜花卉研究所 | Method for detecting subcellular localization by using lily leaves |
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