CN105823890A - Subcellular localization kit constructed through sorghum mosaic virus P3N-PIPO - Google Patents
Subcellular localization kit constructed through sorghum mosaic virus P3N-PIPO Download PDFInfo
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Abstract
The invention relates to a plant subcellular localization kit constructed through sorghum mosaic virus P3N-PIPO. The kit comprises four plasmodesma localization vectors SrMV-P3N-PIPO-GFP, pSrMV-P3N-PIPO-RFP, pSrMV-P3N-PIPO-YFP and pSrMV-P3N-PIPO-CFP which are marked with green, red, yellow and cyan fluorescent proteins respectively, four contrast vectors pSAT6-EGFP-C1, pSAT6-ERFP-C1, pSAT6-EYFP-C1 and pSAT6-ECFP-C1, without specific subcellular localization, restriction endonucleases Xho I, Hind III, water without RNase and infection liquid. Through the adoption of the kit, the fact that whether a target gene expression product has the feature of plasmodesma localization can be rapidly ascertained.
Description
Present disclosure relates to a kind of test kit, is specifically related to a kind of Asia utilizing sorghum mosaic virus P3N-PIPO to build
Cell location reagent box.The present invention utilize be respectively provided with green fluorescent protein (green fluorescent protein, GFP) labelling,
Red fluorescent protein (red fluorescent protein, RFP), yellow fluorescence protein (yellow fluorescent protein, YFP)
Determine with the special plasmodesmata (Plasmodesma, PD) that has of cyan fluorescent protein (cyan fluorescent protein, CFP)
Protein S rMV-P3N-PIPO of bit function, indicates whether gene expression albumen to be verified has the characteristic of plasmodesmata location, belongs to
In biological technical field.
The background technology development along with the development of biotechnology, especially genomics and the progress of sequencing technologies, more and more
New gene is found, and has defined mass data.Specify the function of unknown gene, inquire into its potential using value, be base
Because group learns the ultimate aim of research.Protein is the expression product of gene, the Subcellular Localization of protein and the structure of protein and
Functional relationship is close, and protein necessarily be in suitable position its function of competence exertion.For a new gene, specify it and express
The product i.e. Subcellular Localization of protein, can be that the function studying this gene provides important clue.At present, determine that protein is sub-
The method of cell location mainly has three kinds: Cell Fractionation method, electron-microscopic analysis, laser confocal, wherein laser copolymerization
Burnt method is most widely used.Laser confocal utilize mark fluorescent albumen be stimulated produce fluorescence signal, can see intuitively
Observe the subcellular location at target protein place.When the Subcellular Localization of target protein is studied, need positive control,
I.e. there is the albumen with detectable label of special subcellular fraction status, the location of evidence agnoprotein.
Plasmodesmata (Plasmodesma, PD) is interior Cytoplasm and the endoplasmic reticulum connecting flanking cell through cell wall of plant
Desmotubule, is the important channel of intercellular substance transport and information transmission.The structure of PD is extremely complex, the completeest
All clear Chu, its structural model is constantly in and supplements more new state.The permeability of PD is regulated by many factors, and small-molecule substance is permissible
By plasmodesmata free diffusing, but the macromole such as protein, nucleic acid or macromolecule complex such as virion, ribosome
The intercellular of protein complexes etc. moves and is regulated and controled by PD.For the conduction of plant intracellular signal and the research of the aspect such as matter transportation
Speech, although the encoding proteins Subcellular Localization of the bioinformatics means gene to being separated to can be utilized to be predicted, but must
This gene coded protein must be done Subcellular Localization and carry out biological experiment, specify it and can be positioned PD, and then judge this albumen
Whether participate in intracellular signal transduction and matter transportation, provide experimental evidence intuitively for studying its function.
2008, Chung etc. utilized bioinformatics method, to include corn mosaic virus (Sugarcane mosaic virus,
SCMV), sorghum mosaic virus (Sorghum mosaic virus, SrMV) and Caulis Sacchari sinensis stripe mosaic virus (Sugarcane streak
Mosaic virus, SCSMV) etc. show at the virus analysis of 48 interior marmor upsilon sections, exist at P3 gene internal
One conserved structure G1-2A6-7, ribosome passes through+2 frameshit in this conserved structure can translate the albumen of an about 6-7kDa
(PIPO), this albumen is combined with the N end of P3 albumen, forms the fusion protein of an about 25kDa, i.e. P3N-PIPO.
Chung etc. have cloned the P3N-PIPO coded sequence of Brassica 2 et 4 (Turnip mosaic virus, TuMV), and pass through
Experiment confirms that TuMV-P3N-PIPO is positioned plasmodesmata.Research shows, P3N-PIPO is likely to marmor upsilon
Floating preteins, this albumen by with host factor interaction, make virus realize intercellular by plasmodesmata and move, and then to host
Set up systematicness infect (Choi etc., 2005；Chung etc., 2008；Wen etc., 2010；Wei etc., 2010；Vijayapalani etc.,
2012).But the Subcellular Localization of relevant SCMV-P3N-PIPO, SrMV-P3N-PIPO and SCSMV-P3N-PIPO is ground
Study carefully and have not been reported.
Summary of the invention it is an object of the invention to provide a kind of Subcellular Localization utilizing sorghum mosaic virus P3N-PIPO to build
Test kit, whether being positioned plant plasmodesmata for testing goal gene expression product provides instruction.
For realizing the purpose of the present invention, technical scheme is as follows.
Utilize fusion DNA vaccine technology, with the P3 gene coded sequence of SrMV as template, clone the code sequence of P3N-PIPO
Row, are building up to carry on different fluorescent marker protein gene plant expression vector, convert Agrobacterium, inject Ben Shi Tobacco Leaves,
Use corresponding laser excitation under laser confocal microscope and gather transmitting optical signal, can be at the intercellular of Ben Shi cigarette epithelial cell
Even observe clear bright scattergram picture on silk position, it was demonstrated that P3N-PIPO is positioned plasmodesmata.Accordingly, Wo Menli
Plant sub-cellular location reagent box is constructed with the P3N-PIPO of SrMV.
A kind of Subcellular Localization test kit utilizing sorghum mosaic virus P3N-PIPO to build of the present invention, it is characterised in that this examination
Agent box is made up of following reagent:
(1) the PD positioning carrier pSrMV-P3N-PIPO-GFP of Green Fluorescent Protein, as positive control, 1 pipe,
100ng/ μ L, 50 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(2) the PD positioning carrier pSrMV-P3N-PIPO-RFP of red fluorescent protein marker, as positive control, 1 pipe,
100ng/ μ L, 50 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(3) the PD positioning carrier pSrMV-P3N-PIPO-YFP of yellow fluorescence protein labelling, as positive control, 1 pipe,
100ng/ μ L, 50 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(4) the PD positioning carrier pSrMV-P3N-PIPO-CFP of cyan fluorescent protein labelling, as positive control, 1 pipe,
100ng/ μ L, 50 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(5) carrier pSAT6-EGFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(6) carrier pSAT6-ERFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(7) carrier pSAT6-EYFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(8) carrier pSAT6-ECFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(9) restricted enzyme Xho I:1 pipe, 500units, 100 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(10) restricted enzyme Hind III:1 pipe, 500units, 100 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(11) without RNase water: 2 bottles, 100mL/ bottle, 4 DEG C of stored refrigerated are standby；
(12) infecting liquid: 1 pipe, 50mL/ manages, and-20 DEG C of freezen protective are standby；Described infect liquid, including D-Glucose, 250
mg；MES, 5mL；Na3PO4·12H2O, 5mL；Acetosyringone, 5 μ L；Add ddH2O to cumulative volume 50mL.
The construction method of the PD positioning carrier pSrMV-P3N-PIPO-GFP of described Green Fluorescent Protein: use Xho I
With Hind III, carrier pSAT6-EGFP-C1 (GenBank:AY818374) is carried out double digestion, then with T4DNA even
Connect enzyme to be connected with Insert Fragment S by digestion products, and convert to competence escherichia coli (Escherichia coli) DH5 α,
Picking positive colony is verified；
The construction method of the PD positioning carrier pSrMV-P3N-PIPO-RFP of described red fluorescent protein marker: use Xho I
With Hind III, carrier pSAT6-ERFP-C1 (GenBank:DQ005474) is carried out double digestion, then connect with T4DNA
Digestion products is connected by enzyme with Insert Fragment S, and converts to competence escherichia coli (Escherichia coli) DH5 α, chooses
Take positive colony checking；
The construction method of the PD positioning carrier pSrMV-P3N-PIPO-YFP of described yellow fluorescence protein labelling: use Xho I
With Hind III, carrier pSAT6-EYFP-C1 (GenBank:AY818380) is carried out double digestion, then with T4DNA even
Connect enzyme to be connected with Insert Fragment S by digestion products, and convert to competence escherichia coli (Escherichia coli) DH5 α,
Picking positive colony is verified；
The construction method of the PD positioning carrier pSrMV-P3N-PIPO-CFP of described cyan fluorescent protein labelling: use Xho I
With Hind III, carrier pSAT6-ECFP-C1 (GenBank:AY818374) is carried out double digestion, then with T4DNA even
Connect enzyme to be connected with Insert Fragment S by digestion products, and convert to competence escherichia coli (Escherichia coli) DH5 α,
Picking positive colony is verified.
The nucleotides sequence of described Insert Fragment S is classified as the nucleotide sequence shown in SEQ ID NO:11 in sequence table.
Advantages of the present invention and beneficial effect: use the test kit of the present invention quickly can be building up to genes of interest with fluorescence mark
In the carrier of note, specify whether its expression product has the characteristic of plasmodesmata location.This test kit provides 4 kinds of fluorescent labelinies
Carrier, provides more choices for user, meets research different genes expression product and is positioned plasmodesmata the most altogether
Demand.Use this test kit can be rapidly completed the Subcellular Localization research work of destination gene expression product.
Accompanying drawing illustrates:
Fig. 1 is the plasmid map of the carrier pSrMV-P3N-PIPO-GFP of Green Fluorescent Protein；
Fig. 2 is the plasmid map of the carrier pSrMV-P3N-PIPO-RFP of red fluorescent protein marker；
Fig. 3 is the plasmid map of the carrier pSrMV-P3N-PIPO-YFP of yellow fluorescence protein labelling；
Fig. 4 is the plasmid map of the carrier pSrMV-P3N-PIPO-CFP of cyan fluorescent protein labelling；
Fig. 5 is the plasmid map of the arabidopsis AtREM1.3 Subcellular Localization carrier pAtREM1.3-RFP of red fluorescence labelling；
Fig. 6 is the composing picture of pAtREM1.3-RFP and pSrMV-P3N-PIPO-GFP Subcellular Localization figure；
Fig. 7 is the Subcellular Localization of GFP carrier pSAT6-EGFP-C1；
Fig. 8 is the Subcellular Localization of RFP carrier pSAT6-ERFP-C1.
Detailed description of the invention, in order to be further elucidated with the present invention rather than limit the present invention, is illustrated below in conjunction with embodiment.Under
State experimental technique described in embodiment, if no special instructions, be conventional method.Described reagent and biomaterial are as without special theory
Bright the most commercially obtain.
The clone of embodiment one: SrMV-P3N-PIPO coded sequence
The present invention utilizes round pcr, has cloned the P3 albumen of sorghum mosaic virus (Sorghum mosaic virus, SrMV)
Coded sequence；With P3 gene as template, utilize fusion DNA vaccine technology, clone the coded sequence of the P3N-PIPO of SrMV,
And this sequence is connected in the expression vector with different fluorescent protein labelings, it is thus achieved that 4 special can be positioned intercellular connection
The subcellular fraction expression vector of silk, i.e. the PD positioning carrier pSrMV-P3NPIPO-GFP of Green Fluorescent Protein, red fluorescence
The PD positioning carrier pSrMV-P3NPIPO-RFP of protein labeling, the PD positioning carrier of yellow fluorescence protein labelling
The PD positioning carrier pSrMV-P3NPIPO-CFP of pSrMV-P3NPIPO-YFP and cyan fluorescent protein labelling.Vector construction
Method is as follows:
(1) acquisition of SrMV-P3 coded sequence: the coded sequence for SrMV-P3 designs specific PCR primers SrMV-P3-F
(forward primer) and SrMV-P3-R (downstream primer), with the cDNA of SrMV as template, carry out PCR amplification, by PCR
Product is separated and recovered from by agarose gel electrophoresis, it is thus achieved that the coded sequence of SrMV-P3.Described forward primer
The nucleotides sequence of SrMV-P3-F is classified as the nucleotide sequence shown in SEQ ID NO:1 in sequence table；Downstream primer SrMV-P3-R
Nucleotides sequence be classified as the nucleotide sequence shown in SEQ ID NO:2 in sequence table；The nucleotides sequence of SrMV-P3 coded sequence
It is classified as the nucleotide sequence shown in SEQ ID NO:3 in sequence table；
(2) acquisition of SrMV-P3N coded sequence: design specific PCR primers, SrMV-P3N-F (forward primer) and
SrMV-P3N-R (downstream primer), introduces restriction enzyme site Xho I in forward primer SrMV-P3N-F.Compile with SrMV-P3
Code sequence is template, uses primer SrMV-P3N-F and SrMV-P3N-R to carry out PCR amplification, and reaction system is 25 μ L:
10 × PCR Buffer 2.5 μ L, dNTPs 2.0 μ L, forward primer SrMV-P3N-F (10 μm ol/L) 1.0 μ L, downstream primer
SrMV-P3N-R (10 μm ol/L) 1.0 μ L, Taq enzyme (5U/ μ L) 0.125 μ L, ddH2O 17.375 μ L, template (cDNA)
1.0 μ L, cumulative volume 25 μ L.PCR response procedures: 94 DEG C of denaturations 4min；Then 35 circulations of operation, 94 DEG C of 30s,
50 DEG C of 30s, 72 DEG C of 1min；Last 72 DEG C of 10min.After PCR reaction terminates, by PCR primer by agarose gel electricity
Swimming separates, and reclaims and be concentrated into 400ng/ μ L, it is thus achieved that the coded sequence of SrMV-P3N.Described forward primer SrMV-P3N-F
Nucleotides sequence be classified as the nucleotide sequence shown in SEQ ID NO:4 in sequence table；The nucleoside of downstream primer SrMV-P3N-R
Acid sequence is the nucleotide sequence shown in SEQ ID NO:5 in sequence table；The nucleotides sequence of SrMV-P3N coded sequence is classified as
Nucleotide sequence shown in SEQ ID NO:6 in sequence table；
(3) acquisition of SrMV-PIPO coded sequence: design specific PCR primers, SrMV-PIPO-F (forward primer) and
SrMV-PIPO-R (downstream primer), introduces restriction enzyme site Hind III in downstream primer SrMV-P3N-R.With SrMV-P3
Coded sequence is template, uses primer SrMV-PIPO-F and SrMV-PIPO-R to carry out PCR amplification, and reaction system is 25 μ L:
10 × PCR Buffer 2.5 μ L, dNTPs 2.0 μ L, forward primer SrMV-PIPO-F (10 μm ol/L) 1.0 μ L, downstream is drawn
Thing SrMV-PIPO-R (10 μm ol/L) 1.0 μ L, Taq enzyme (5U/ μ L) 0.125 μ L, ddH2O 17.375 μ L, template (cDNA)
1.0 μ L, cumulative volume 25 μ L.PCR response procedures: 94 DEG C of denaturations 4min；Then 35 circulations of operation, 94 DEG C of 30s,
50 DEG C of 30s, 72 DEG C of 1min；Last 72 DEG C of 10min.After PCR reaction terminates, by PCR primer by agarose gel electricity
Swimming separates, and reclaims and be concentrated into 400ng/ μ L, it is thus achieved that the coded sequence of SrMV-PIPO.Described forward primer
The nucleotides sequence of SrMV-PIPO-F is classified as the nucleotide sequence shown in SEQ ID NO:7 in sequence table；Downstream primer
The nucleotides sequence of SrMV-PIPO-R is classified as the nucleotide sequence shown in SEQ ID NO:8 in sequence table；SrMV-PIPO encodes
The nucleotides sequence of sequence is classified as the nucleotide sequence shown in SEQ ID NO:9 in sequence table；
(4) acquisition of SrMV-P3N-PIPO coded sequence: concentration is the PCR of P3N and PIPO of 400ng/ μ L
Product as template according to 1:1 mixing, uses primer SrMV-P3N-F and SrMV-PIPO-R, utilizes fusion DNA vaccine method
Clone's SrMV-P3NPIPO coded sequence.PCR reaction system is 25 μ L:10 × PCR Buffer 2.5 μ L, dNTPs 2.0 μ L,
Forward primer SrMV-P3N-F (10 μm ol/L) 1.0 μ L, downstream primer SrMV-PIPO-R (10 μm ol/L) 1.0 μ L, Taq
Enzyme (5U/ μ L) 0.125 μ L, ddH2O 12.375 μ L, template (cDNA) 6.0 μ L, cumulative volume 25 μ L.PCR reaction interval
Sequence: 94 DEG C of denaturations 4min；Then 35 circulations, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min are run；Last 72 DEG C 10
min.After PCR reaction terminates, PCR primer is separated and recovered from by agarose gel electrophoresis, it is thus achieved that
The coded sequence of SrMV-P3N-PIPO.The nucleotides sequence of described SrMV-P3N-PIPO coded sequence is classified as SEQ in sequence table
Nucleotide sequence shown in ID NO:10；
(5) acquisition of Insert Fragment S: use Xho I and Hind III that SrMV-P3N-PIPO coded sequence is carried out double digestion,
Digestion products is separated and recovered from by agarose gel electrophoresis, it is thus achieved that Insert Fragment S；The nucleoside of described Insert Fragment S
Acid sequence is the nucleotide sequence shown in SEQ ID NO:11 in sequence table；
(6) structure of the PD positioning carrier pSrMV-P3N-PIPO-GFP of Green Fluorescent Protein: use Xho I and Hind
III carries out double digestion to carrier pSAT6-EGFP-C1 (GenBank:AY818374), and digestion products is passed through agarose gel
Electrophoresis is separated and recovered from.Then with T4DNA ligase, digestion products is connected with Insert Fragment S, and product will be connected
Converting to competence escherichia coli (Escherichia coli) DH5 α, picking positive colony is verified, obtains green fluorescent protein
The PD positioning carrier pSrMV-P3N-PIPO-GFP of labelling, its plasmid map is as shown in Figure 1；
(7) structure of the PD positioning carrier pSrMV-P3N-PIPO-RFP of red fluorescent protein marker: use Xho I and Hind
III carries out double digestion to carrier pSAT6-ERFP-C1 (GenBank:DQ005474), and digestion products is passed through agarose gel
Electrophoresis is separated and recovered from.Then with T4DNA ligase, digestion products is connected with Insert Fragment S, and product will be connected
Converting to competence escherichia coli (Escherichia coli) DH5 α, picking positive colony is verified, obtains red fluorescent protein
The PD positioning carrier pSrMV-P3N-PIPO-RFP of labelling, its plasmid map is as shown in Figure 2；
(8) structure of the PD positioning carrier pSrMV-P3N-PIPO-YFP of yellow fluorescence protein labelling: use Xho I and Hind
III carries out double digestion to carrier pSAT6-EYFP-C1 (GenBank:AY818380), and digestion products is passed through agarose gel
Electrophoresis is separated and recovered from.Then with T4DNA ligase, digestion products is connected with Insert Fragment S, and product will be connected
Converting to by state escherichia coli (Escherichia coli) DH5 α, picking positive colony is verified, obtains yellow fluorescence protein mark
The PD positioning carrier pSrMV-P3N-PIPO-YFP of note, its plasmid map is as shown in Figure 3；
(9) structure of the PD positioning carrier pSrMV-P3N-PIPO-CFP of cyan fluorescent protein labelling: use Xho I and Hind
III carries out double digestion to carrier pSAT6-ECFP-C1 (GenBank:AY818374), and digestion products is passed through agarose gel
Electrophoresis is separated and recovered from.Then with T4DNA ligase, digestion products is connected with Insert Fragment S, and product will be connected
Converting to competence escherichia coli (Escherichia coli) DH5 α, picking positive colony is verified, obtains cyan fluorescent protein
The PD positioning carrier pSrMV-P3N-PIPO-CFP of labelling, its plasmid map is as shown in Figure 4.
Embodiment two: the Subcellular Localization of arabidopsis AtREM1.3
1, the clone of arabidopsis AtREM1.3 gene
From arabidopsis, clone a Remorin gene, be cloned in cloning vehicle pMD19-T.Phylogenetic analysis table
This gene bright belongs to the 3rd type of the 1st subgroup of Remorin gene family, named AtREM1.3.Document shows Remorin
Can be positioned plasma membrane and plasmodesmata, but bioinformatic analysis shows, Remorin does not has membrane spaning domain and signal peptide.
It is to be positioned plasmodesmata for checking AtREM1.3, uses this test kit that it is carried out Subcellular Localization research.Described
The nucleotides sequence of AtREM1.3 coded sequence is classified as the nucleotide sequence shown in SEQ ID NO:12 in sequence table；
2, the structure of Subcellular Localization carrier pAtREM1.3-RFP
(1) design specific PCR primers, AtREM1.3-F (forward primer) and AtREM1.3-R (downstream primer), upper
Trip primer AtREM1.3-F introduces restriction enzyme site Xho I, downstream primer AtREM1.3-R introduces restriction enzyme site Hind III.
Use this to primer, with the cloning vehicle of AtREM1.3 gene as template, carry out PCR amplification.PCR reaction system is 25 μ L:
10 × PCR Buffer 2.5 μ L, dNTPs 2.0 μ L, forward primer AtREM1.3-F (10 μm ol/L) 1.0 μ L, downstream primer
AtREM1.3-R (10 μm ol/L) 1.0 μ L, Taq enzyme (5U/ μ L) 0.125 μ L, ddH2O 17.375 μ L, template (cDNA)
1.0 μ L, cumulative volume 25 μ L.PCR response procedures: 94 DEG C of denaturations 4min；Then 35 circulations of operation, 94 DEG C of 30s,
50 DEG C of 30s, 72 DEG C of 1min；Last 72 DEG C of 10min.After PCR reaction terminates, use Xho I and Hind III that PCR is produced
Thing carries out double digestion, is separated and recovered from by agarose gel electrophoresis by digestion products, it is thus achieved that Insert Fragment InsTarget；
The nucleotides sequence of described forward primer AtREM1.3-F is classified as the nucleotide sequence shown in SEQ ID NO:13 in sequence table；Under
The nucleotides sequence of trip primer AtREM1.3-R is classified as the nucleotide sequence shown in SEQ ID NO:14 in sequence table；Insert Fragment
The nucleotides sequence of InsTarget is classified as the nucleotide sequence shown in SEQ ID NO:15 in sequence table；
(2) take RFP control vector, use Xho I and Hind III carrier pSAT6-ERFP-C1 to be carried out double digestion, by enzyme
Cut product to be separated and recovered from by agarose gel electrophoresis.Then with T4DNA ligase by digestion products and Insert Fragment
InsTarget connects, and is converted to competence escherichia coli (Escherichia coli) DH5 α by connection product, and picking is positive
Clone's checking, obtains the PD positioning carrier pAtREM1.3-RFP of red fluorescent protein marker, and its plasmid map is as shown in Figure 5；
3, Subcellular Localization experiment
(1) use frozen-thawed method, respectively the PD of Subcellular Localization carrier pAtREM1.3-RFP and GFP labelling is positioned
Carrier pSrMV-P3N-PIPO-GFP proceeds in competent Agrobacterium EHA105 bacterial strain；(contain in 5mL LB culture medium
Rif, 34 μ g/mL；Kan, 50 μ g/mL) the middle Agrobacterium cultivating conversion, 28 DEG C, 200rpm overnight incubation；
(2) under room temperature, 5,000rpm, centrifugal 5min, abandon supernatant, add 1mL and infect the resuspended bacterium solution of liquid；
(3) repeat step (2), wash away the antibiotic contained in culture medium；
(4) add 1mL and infect liquid, measure bacterium solution OD600, and regulate OD600Value is to 0.1；
(5) 750 μ L bacterium solution are taken in 2mL sterile centrifugation tube, mixing, place 3～5h；Will be containing Subcellular Localization carrier
The bacterium solution of pAtREM1.3-GFP and the bacterium solution geometric ratio mixing containing YFP labeled vector, carry out infecting experiment；
(6) take healthy Ben Shi cigarette plant (before infecting, photo-irradiation treatment Nicotiana tabacum L. 1h), select the blade that two panels is big, at tobacco leaf
The sheet back side (between two veins) injection bacterium solution also carries out labelling；
(7) Nicotiana tabacum L. infected is put cultivate under normal operation.Take after 2 days and infect blade 1-2cm2, the back side upward, is used and is swashed
Light confocal microscopy.When gathering fluorescence signal, same cell is gathered respectively different fluorescence signal.To be detected
PAtREM1.3-RFP labelling is red fluorescent protein, when gathering red fluorescent protein signal, there will be in position shown in arrow
Bright spot, shows that AtREM1.3 expresses on cell membrane, but cannot determine that it is positioned PD；This test kit provides PD location
Carrier pSrMV-P3N-PIPO-GFP labelling is green fluorescent protein, when gathering green florescent signal, also there will be bright spot,
Show that bright spot position is plasmodesmata；When two pictures are merged, aobvious after bright red spot and the green accurate superposition of bright spot
It is shown as the bright spot of yellow, shows that AtREM1.3 is positioned plasmodesmata, as shown in Figure 6.
(8) according to said procedure and method, by GFP carrier pSAT6-EGFP-C1 and RFP carrier pSAT6-ERFP-C1
Ben Shi Tobacco Leaves epidermis cell is expressed, under laser confocal microscope, detects fluorescence signal.Green florescent signal is for spreading shape
State (Fig. 7), red fluorescent is spread state (Fig. 8), shows that green fluorescent protein and red fluorescent protein do not have special
Subcellular Localization, the Subcellular Localization of target protein is not green or red fluorescent protein causes, thus, it was demonstrated that AtREM1.3
Plasmodesmata can be positioned.
Described agarose gel electrophoresis, with reference to agarose gel in " Molecular Cloning: A Laboratory guide " (second edition) chapter 6 first segment
The method of electrophoresis；The described product that will connect converts to competence bacillus coli DH 5 alpha, and method for transformation is with reference to " molecular cloning is real
Test guide " (second edition) chapter 1 Section five prepares method colibacillary with transformed competence colibacillus with calcium chloride；Described containing sun
The picking of the bacterium colony of sex clone, with reference to containing the thin of recombiant plasmid in " Molecular Cloning: A Laboratory guide " (second edition) chapter 1 Section six
The authentication method of bacterium bacterium colony；The method of described extraction plasmid DNA, with reference to plasmid extraction kit description；Described enzyme action side
Method, with reference to the description of restricted enzyme；Described recovery method, reclaims test kit description with reference to glue；Described T4-DNA
Ligase is attached method, with reference to T4-DNA ligase operating instruction.
Claims (5)
1. the Subcellular Localization test kit utilizing sorghum mosaic virus P3N-PIPO to build, it is characterised in that this test kit by
Following reagent forms:
(1) the PD positioning carrier pSrMV-P3N-PIPO-GFP of Green Fluorescent Protein, as positive control, 1 pipe,
100ng/ μ L, 50 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(2) the PD positioning carrier pSrMV-P3N-PIPO-RFP of red fluorescent protein marker, as positive control, 1 pipe,
100ng/ μ L, 50 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(3) the PD positioning carrier pSrMV-P3N-PIPO-YFP of yellow fluorescence protein labelling, as positive control, 1 pipe,
100ng/ μ L, 50 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(4) the PD positioning carrier pSrMV-P3N-PIPO-CFP of cyan fluorescent protein labelling, as positive control, 1 pipe,
100ng/ μ L, 50 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(5) carrier pSAT6-EGFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(6) carrier pSAT6-ERFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(7) carrier pSAT6-EYFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(8) carrier pSAT6-ECFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes ,-20 DEG C of freezen protective are standby；
(9) restricted enzyme Xho I:1 pipe, 500units, 100 μ L/ pipes；
(10) restricted enzyme Hind III:1 pipe, 500units, 100 μ L/ pipes；
(11) without RNase water: 2 bottles, 100mL/ bottle；
(12) infecting liquid: 1 pipe, 50mL/ manages, and-20 DEG C of freezen protective are standby.
A kind of Subcellular Localization test kit utilizing sorghum mosaic virus P3N-PIPO to build the most according to claim 1,
It is characterized in that the construction method of the PD positioning carrier pSrMV-P3N-PIPO-GFP of described Green Fluorescent Protein: use
Xho I and Hind III carries out double digestion to carrier pSAT6-EGFP-C1, is carried out point by agarose gel electrophoresis by digestion products
From and reclaim；Then with T4DNA ligase, digestion products is connected with Insert Fragment S, and product conversion will be connected to impression
In state escherichia coli (Escherichia coli) DH5 α, picking positive colony is verified, obtains the PD of Green Fluorescent Protein
Positioning carrier pSrMV-P3N-PIPO-GFP；The nucleotides sequence of described Insert Fragment S is classified as SEQ ID NO:11 in sequence table
Shown nucleotide sequence.
A kind of Subcellular Localization test kit utilizing sorghum mosaic virus P3N-PIPO to build the most according to claim 1,
It is characterized in that the construction method of the PD positioning carrier pSrMV-P3N-PIPO-RFP of described red fluorescent protein marker: use
Xho I and Hind III carries out double digestion to carrier pSAT6-ERFP-C1, is carried out point by agarose gel electrophoresis by digestion products
From and reclaim；Then with T4DNA ligase, digestion products is connected with Insert Fragment S, and product conversion will be connected to impression
In state bacillus coli DH 5 alpha, picking positive colony is verified, obtains the PD positioning carrier of red fluorescent protein marker
pSrMV-P3N-PIPO-RFP；The nucleotides sequence of described Insert Fragment S is classified as the core shown in SEQ ID NO:11 in sequence table
Nucleotide sequence.
A kind of Subcellular Localization test kit utilizing sorghum mosaic virus P3N-PIPO to build the most according to claim 1,
It is characterized in that the construction method of the PD positioning carrier pSrMV-P3N-PIPO-YFP of described yellow fluorescence protein labelling: use
Xho I and Hind III carries out double digestion to carrier pSAT6-EYFP-C1, is carried out point by agarose gel electrophoresis by digestion products
From and reclaim；Then with T4DNA ligase, digestion products is connected with Insert Fragment S, and product conversion will be connected to impression
In state bacillus coli DH 5 alpha, picking positive colony is verified, obtains the PD positioning carrier of yellow fluorescence protein labelling
pSrMV-P3N-PIPO-YFP；The nucleotides sequence of described Insert Fragment S is classified as the core shown in SEQ ID NO:11 in sequence table
Nucleotide sequence.
A kind of Subcellular Localization test kit utilizing sorghum mosaic virus P3N-PIPO to build the most according to claim 1,
It is characterized in that the construction method of the PD positioning carrier pSrMV-P3N-PIPO-CFP of described cyan fluorescent protein labelling: use
Xho I and Hind III carries out double digestion to carrier pSAT6-ECFP-C1, is carried out point by agarose gel electrophoresis by digestion products
From and reclaim；Then with T4DNA ligase, digestion products is connected with Insert Fragment S, and product conversion will be connected to impression
In state bacillus coli DH 5 alpha, picking positive colony is verified, obtains the PD positioning carrier of cyan fluorescent protein labelling
pSrMV-P3N-PIPO-CFP；The nucleotides sequence of described Insert Fragment S is classified as the core shown in SEQ ID NO:11 in sequence table
Nucleotide sequence.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017818A (en) * | 2014-06-24 | 2014-09-03 | 中山大学 | Inflammasome activity reporting system for sub-cellular localization and application thereof |
| CN104212816A (en) * | 2013-05-31 | 2014-12-17 | 河北农业大学 | Zea mays zinc iron-regulated transporter ZmZIPs genes and applications thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104212816A (en) * | 2013-05-31 | 2014-12-17 | 河北农业大学 | Zea mays zinc iron-regulated transporter ZmZIPs genes and applications thereof |
| CN104017818A (en) * | 2014-06-24 | 2014-09-03 | 中山大学 | Inflammasome activity reporting system for sub-cellular localization and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| BETTY Y.-W. CHUNG ET AL: "An overlapping essential gene in the Potyviridae", 《PNAS》 * |
| TAIYUN WEI ET AL: "Formation of Complexes at Plasmodesmata for Potyvirus Intercellular Movement Is Mediated by the Viral Protein P3N-PIPO", 《PLOS PATHOGENS》 * |
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