CN105823890B - A kind of Subcellular Localization kit built using sorghum mosaic virus P3N PIPO - Google Patents
A kind of Subcellular Localization kit built using sorghum mosaic virus P3N PIPO Download PDFInfo
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Abstract
The present invention relates to a kind of plant sub-cellular location reagent box built using sorghum mosaic virus P3N PIPO, including 4 plasmodesmus positioning carriers for marking green, red, yellow and cyan fluorescent protein respectively：SrMV P3N PIPO GFP, pSrMV P3N PIPO RFP, pSrMV P3N PIPO YFP and pSrMV P3N PIPO CFP；4 control vectors without special Subcellular Localization：PSAT6 EGFP C1, pSAT6 ERFP C1, pSAT6 EYFP C1 and pSAT6 ECFP C1；Restriction enzyme Xho I, Hind III, without RNase water and infect liquid.Whether there can be the characteristic of plasmodesmus positioning with fast explicit destination gene expression product using this kit.
Description
The present invention relates to a kind of kit for technical field, and in particular to one kind utilizes sorghum mosaic virus P3N-PIPO structures
The Subcellular Localization kit built.The present invention utilizes and is respectively provided with green fluorescent protein (green fluorescent
Protein, GFP) mark, red fluorescent protein (red fluorescent protein, RFP), yellow fluorescence protein
(yellow fluorescent protein, YFP) and cyan fluorescent protein (cyan fluorescent protein, CFP)
The Protein S rMV-P3N-PIPO with special plasmodesmus (Plasmodesma, PD) positioning function, indicate gene to be verified
Whether expressing protein has the characteristic of plasmodesmus positioning, belongs to biological technical field.
Background technology is got over the development of the development of biotechnology, especially genomics and the progress of sequencing technologies
It is found come more new genes, has formd mass data.The function of unknown gene is specified, it is inquired into and potentially applies valency
Value, is the ultimate aim of genomics research.Protein is the expression product of gene, the Subcellular Localization and protein of protein
26S Proteasome Structure and Function it is in close relations, protein necessarily be in suitable its function of position competence exertion.It is bright for a new gene
The Subcellular Localization of its true expression product, that is, protein, can provide important clue to study the function of the gene.At present, determine
The method of proteins subcellular location mainly has three kinds：Cell Fractionation method, electron-microscopic analysis, laser confocal, wherein
Laser confocal is most widely used.Laser confocal is stimulated the fluorescence signal of generation using mark fluorescent albumen, can
Intuitively to observe the subcellular location where target protein.When the Subcellular Localization to target protein is studied, need
Positive control, the i.e. albumen with detectable label with special subcellular fraction status are wanted, proves the positioning of agnoprotein.
Plasmodesmus (Plasmodesma, PD) is through the cytoplasm of cell membrane connection flanking cell and interior in plant
The desmotubule of matter net, is the important channel of intercellular substance transport and information transmission.The structure of PD is extremely complex, so far still
It is not fully apparent from, its structural model is constantly in supplement more new state.The permeability of PD is adjusted by many factors, small molecule thing
Matter can by plasmodesmus free diffusing, but the macromolecular such as protein, nucleic acid or macromolecule complex such as virion,
The intercellular movement of ribosomal protein complex etc. is regulated and controled by PD.For the conduction of plant intracellular signal and matter transportation etc.
For research, although can be carried out using the encoding proteins Subcellular Localization of gene of the bioinformatics means to being separated to pre-
Survey, but Subcellular Localization must be done to the gene coded protein and carries out biological experiment, can specify it be positioned at PD, and then
Judge whether the albumen participates in intracellular signal transduction and matter transportation, intuitively experimental evidence is provided to study its function.
2008, Chung etc. utilized bioinformatics method, to including corn mosaic virus (Sugarcane mosaic
Virus, SCMV), sorghum mosaic virus (Sorghum mosaic virus, SrMV) and sugarcane stripe mosaic virus
The virus analysis of 48 marmor upsilon sections including (Sugarcane streak mosaic virus, SCSMV) etc. shows,
In P3 gene internals, there are a conserved structure G1-2A6-7, ribosomes can translate one in the conserved structure by+2 frameshit
The albumen (PIPO) of about 6-7kDa, the albumen are combined with the N-terminal of P3 albumen, form the fusion protein of an about 25kDa, i.e.,
P3N-PIPO.Chung etc. has cloned the P3N-PIPO code sequences of Brassica 2 et 4 (Turnip mosaic virus, TuMV)
Row, and experiments prove that TuMV-P3N-PIPO is positioned at plasmodesmus.Research shows that P3N-PIPO is likely to Ma Ling
The floating preteins of potato Y virus, the albumen by with host factor interaction, make virus by plasmodesmus realize intercellular move, into
And host is established systematicness infect (Choi etc., 2005；Chung etc., 2008；Wen etc., 2010；Wei etc., 2010；
Vijayapalani etc., 2012).But the Asia of related SCMV-P3N-PIPO, SrMV-P3N-PIPO and SCSMV-P3N-PIPO
Cellular localization research has not been reported.
The object of the present invention is to provide a kind of subcellular fraction built using sorghum mosaic virus P3N-PIPO for the content of the invention
Location reagent box, instruction is provided for whether testing goal gene expression product is positioned at plant plasmodesmus.
To achieve the object of the present invention, technical scheme is as follows.
Using fusion DNA vaccine technology, using the P3 gene coded sequences of SrMV as template, the code sequence of P3N-PIPO has been cloned
Row, are building up to and carry on different fluorescent marker protein gene plant expression vectors, convert Agrobacterium, inject Ben's Tobacco Leaves,
Corresponding laser excitation is used under laser confocal microscope and gathers transmitting optical signal, you can in the born of the same parents of Ben Shi cigarette epithelial cell
Observe clearly bright scattergram picture on plasmodesmata position, it was demonstrated that P3N-PIPO is positioned at plasmodesmus.Accordingly, Wo Menli
Plant sub-cellular location reagent box is constructed with the P3N-PIPO of SrMV.
A kind of Subcellular Localization kit built using sorghum mosaic virus P3N-PIPO of the present invention, it is characterised in that
The kit is made of following reagent：
(1) the PD positioning carrier pSrMV-P3N-PIPO-GFP of Green Fluorescent Protein, are managed as positive control, 1,
100ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；
(2) the PD positioning carrier pSrMV-P3N-PIPO-RFP of red fluorescent protein marker, are managed as positive control, 1,
100ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；
(3) the PD positioning carrier pSrMV-P3N-PIPO-YFP of yellow fluorescence protein mark, are managed as positive control, 1,
100ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；
(4) the PD positioning carrier pSrMV-P3N-PIPO-CFP of cyan fluorescent protein mark, are managed as positive control, 1,
100ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；
(5) carrier pSAT6-EGFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；
(6) carrier pSAT6-ERFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；
(7) carrier pSAT6-EYFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；
(8) carrier pSAT6-ECFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；
(9) restriction enzyme Xho I：1 pipe, 500units, 100 μ L/ pipe, -20 DEG C of freezen protectives are spare；
(10) restriction enzyme Hind III：1 pipe, 500units, 100 μ L/ pipe, -20 DEG C of freezen protectives are spare；
(11) without RNase water：2 bottles, 100mL/ bottles, 4 DEG C stored refrigerated spare；
(12) liquid is infected：1 pipe, 50mL/ pipes, -20 DEG C of freezen protectives are spare；It is described to infect liquid, including D-Glucose,
250mg；MES, 5mL；Na3PO4·12H2O, 5mL；Acetosyringone, 5 μ L；Add ddH2O to cumulative volume 50mL.
The construction method of the PD positioning carriers pSrMV-P3N-PIPO-GFP of the Green Fluorescent Protein：Use Xho
I and Hind III are to carrier pSAT6-EGFP-C1 (GenBank:AY818374 double digestion) is carried out, then with T4DNA ligases
Digestion products are connected with Insert Fragment S, and are converted into competence Escherichia coli (Escherichia coli) DH5 α, picking
Positive colony is verified；
The construction method of the PD positioning carriers pSrMV-P3N-PIPO-RFP of the red fluorescent protein marker：Use Xho
I and Hind III are to carrier pSAT6-ERFP-C1 (GenBank:DQ005474 double digestion) is carried out, then with T4DNA ligases
Digestion products are connected with Insert Fragment S, and are converted into competence Escherichia coli (Escherichia coli) DH5 α, picking
Positive colony is verified；
The construction method of the PD positioning carriers pSrMV-P3N-PIPO-YFP of the yellow fluorescence protein mark：Use Xho
I and Hind III are to carrier pSAT6-EYFP-C1 (GenBank：AY818380 double digestion) is carried out, then with T4DNA ligases
Digestion products are connected with Insert Fragment S, and are converted into competence Escherichia coli (Escherichia coli) DH5 α, picking
Positive colony is verified；
The construction method of the PD positioning carriers pSrMV-P3N-PIPO-CFP of the cyan fluorescent protein mark：Use Xho
I and Hind III are to carrier pSAT6-ECFP-C1 (GenBank：AY818374 double digestion) is carried out, then with T4DNA ligases
Digestion products are connected with Insert Fragment S, and are converted into competence Escherichia coli (Escherichia coli) DH5 α, picking
Positive colony is verified.
The nucleotides sequence of the Insert Fragment S is classified as SEQ ID NO in sequence table：Nucleotide sequence shown in 11.
The advantages of the present invention：Quickly target gene can be building up to and carried using the kit of the present invention
In the carrier of fluorescent marker, specify whether its expression product has the characteristic of plasmodesmus positioning.This kit provide 4 kinds it is glimmering
Signal carrier, provides more choices for user, and meeting research different genes expression product, whether common location is in born of the same parents
The demand of plasmodesmata.The Subcellular Localization research work of destination gene expression product can be rapidly completed using this kit.
Brief description of the drawings：
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 mark；
Fig. 4 is the plasmid map of the carrier pSrMV-P3N-PIPO-CFP of cyan fluorescent protein mark；
Fig. 5 is the plasmid figure of the arabidopsis AtREM1.3 Subcellular Localization carriers pAtREM1.3-RFP of red fluorescence mark
Spectrum；
Fig. 6 is the composing picture of pAtREM1.3-RFP and pSrMV-P3N-PIPO-GFP Subcellular Localization figures；
Fig. 7 is the Subcellular Localization of GFP carriers pSAT6-EGFP-C1；
Fig. 8 is the Subcellular Localization of RFP carriers pSAT6-ERFP-C1.
Embodiment is subject to reference to embodiments in order to which the present invention is furture elucidated rather than the limitation present invention
Explanation.Experimental method described in following embodiments, is conventional method unless otherwise specified.The reagent and biomaterial are such as
No specified otherwise commercially obtains.
Embodiment one：The clone of SrMV-P3N-PIPO coded sequences
The present invention utilizes round pcr, has cloned the P3 eggs of sorghum mosaic virus (Sorghum mosaic virus, SrMV)
White coded sequence；Using P3 genes as template, using fusion DNA vaccine technology, the coded sequence of the P3N-PIPO of SrMV has been cloned, and will
The sequence is connected in the expression vector with different fluorescent protein labelings, and plasmodesmata can be specifically positioned at by obtaining 4
Subcellular fraction expression vector, i.e. Green Fluorescent Protein PD positioning carrier pSrMV-P3NPIPO-GFP, red fluorescent protein
The PD positioning carrier pSrMV-P3NPIPO-RFP of mark, the PD positioning carriers pSrMV-P3NPIPO- of yellow fluorescence protein mark
The PD positioning carriers pSrMV-P3NPIPO-CFP of YFP and cyan fluorescent protein mark.Carrier construction method is as follows：
(1) acquisition of SrMV-P3 coded sequences：For the coded sequence design specific PCR primers SrMV-P3- of SrMV-P3
F (sense primer) and SrMV-P3-R (anti-sense primer), using the cDNA of SrMV as template, carries out PCR amplification, PCR product is passed through
Agarose gel electrophoresis is separated and recovered from, and obtains the coded sequence of SrMV-P3.The core of the sense primer SrMV-P3-F
Nucleotide sequence is SEQ ID NO in sequence table：Nucleotide sequence shown in 1；The nucleotides sequence of anti-sense primer SrMV-P3-R is classified as
SEQ ID NO in sequence table：Nucleotide sequence shown in 2；The nucleotides sequence of SrMV-P3 coded sequences is classified as SEQ in sequence table
ID NO：Nucleotide sequence shown in 3；
(2) acquisition of SrMV-P3N coded sequences：Design specific PCR primers, SrMV-P3N-F (sense primer) and SrMV-
P3N-R (anti-sense primer), introduces restriction enzyme site Xho I in sense primer SrMV-P3N-F.Using SrMV-P3 coded sequences as mould
Plate, carries out PCR amplification, reaction system is 25 μ L using primer SrMV-P3N-F and SrMV-P3N-R：10×PCR Buffer
2.5 μ L, dNTPs 2.0 μ L, sense primer SrMV-P3N-F (10 μm of ol/L) 1.0 μ L, anti-sense primer SrMV-P3N-R (10 μ
Mol/L) 1.0 μ L, Taq enzyme (5U/ μ L) 0.125 μ L, ddH217.375 μ L of O, 1.0 μ L of template (cDNA), 25 μ L of cumulative volume.PCR
Response procedures：94 DEG C of pre-degeneration 4min；Then 35 circulations, 94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min are run；Last 72 DEG C
10min.PCR is separated by agarose gel electrophoresis after reaction, by PCR product, is recycled and is concentrated into 400ng/ μ L,
Obtain the coded sequence of SrMV-P3N.The nucleotides sequence of the sense primer SrMV-P3N-F is classified as SEQ ID NO in sequence table：
Nucleotide sequence shown in 4；The nucleotides sequence of anti-sense primer SrMV-P3N-R is classified as SEQ ID NO in sequence table：Core shown in 5
Nucleotide sequence；The nucleotides sequence of SrMV-P3N coded sequences is classified as SEQ ID NO in sequence table：Nucleotide sequence shown in 6；
(3) acquisition of SrMV-PIPO coded sequences：Design specific PCR primers, SrMV-PIPO-F (sense primer) and
SrMV-PIPO-R (anti-sense primer), introduces restriction enzyme site Hind III in anti-sense primer SrMV-P3N-R.Compiled with SrMV-P3
Code sequence is template, carries out PCR amplification using primer SrMV-PIPO-F and SrMV-PIPO-R, reaction system is 25 μ L：10×
PCR Buffer 2.5 μ L, dNTPs 2.0 μ L, sense primer SrMV-PIPO-F (10 μm of ol/L) 1.0 μ L, anti-sense primer SrMV-
PIPO-R (10 μm of ol/L) 1.0 μ L, Taq enzyme (5U/ μ L) 0.125 μ L, ddH217.375 μ L of O, 1.0 μ L of template (cDNA), it is overall
25 μ L of product.PCR response procedures：94 DEG C of pre-degeneration 4min；Then 35 circulations, 94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 1min are run；
Last 72 DEG C of 10min.PCR is separated by agarose gel electrophoresis after reaction, by PCR product, is recycled and is concentrated into
400ng/ μ L, obtain the coded sequence of SrMV-PIPO.The nucleotides sequence of the sense primer SrMV-PIPO-F is classified as sequence table
Middle SEQ ID NO：Nucleotide sequence shown in 7；The nucleotides sequence of anti-sense primer SrMV-PIPO-R is classified as SEQ ID in sequence table
NO：Nucleotide sequence shown in 8；The nucleotides sequence of SrMV-PIPO coded sequences is classified as SEQ ID NO in sequence table：Shown in 9
Nucleotide sequence；
(4) acquisition of SrMV-P3N-PIPO coded sequences：It is the PCR product of the P3N and PIPO of 400ng/ μ L by concentration
According to 1：1 mixing is used as template, and using primer SrMV-P3N-F and SrMV-PIPO-R, SrMV- is cloned using fusion DNA vaccine method
P3NPIPO coded sequences.PCR reaction systems are 25 μ L：10 × PCR Buffer, 2.5 2.0 μ L of μ L, dNTPs, sense primer
SrMV-P3N-F (10 μm of ol/L) 1.0 μ L, anti-sense primer SrMV-PIPO-R (10 μm of ol/L) 1.0 μ L, Taq enzyme (5U/ μ L)
0.125 μ L, ddH212.375 μ L of O, 6.0 μ L of template (cDNA), 25 μ L of cumulative volume.PCR response procedures：94 DEG C of pre-degeneration 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 of 10min.After reaction, PCR is produced by PCR
Thing is separated and recovered from by agarose gel electrophoresis, obtains the coded sequence of SrMV-P3N-PIPO.The SrMV-P3N-
The nucleotides sequence of PIPO coded sequences is classified as SEQ ID NO in sequence table：Nucleotide sequence shown in 10；
(5) acquisition of Insert Fragment S：Double enzymes are carried out to SrMV-P3N-PIPO coded sequences using Xho I and Hind III
Cut, digestion products are separated and recovered from by agarose gel electrophoresis, obtain Insert Fragment S；The core of the Insert Fragment S
Nucleotide sequence is SEQ ID NO in sequence table：Nucleotide sequence shown in 11；
(6) structure of the PD positioning carriers pSrMV-P3N-PIPO-GFP of Green Fluorescent Protein：Using Xho I and
Hind III are to carrier pSAT6-EGFP-C1 (GenBank:AY818374 double digestion) is carried out, digestion products are passed through into agarose
Gel electrophoresis is separated and recovered from.Then digestion products are connected with Insert Fragment S with T4DNA ligases, and connection is produced
Thing is converted into competence Escherichia coli (Escherichia coli) DH5 α, and the verification of picking positive colony, obtains green fluorescence
The PD positioning carrier pSrMV-P3N-PIPO-GFP of protein labeling, its plasmid map are as shown in Figure 1；
(7) structure of the PD positioning carriers pSrMV-P3N-PIPO-RFP of red fluorescent protein marker：Using Xho I and
Hind III are to carrier pSAT6-ERFP-C1 (GenBank:DQ005474 double digestion) is carried out, digestion products are passed through into agarose
Gel electrophoresis is separated and recovered from.Then digestion products are connected with Insert Fragment S with T4DNA ligases, and connection is produced
Thing is converted into competence Escherichia coli (Escherichia coli) DH5 α, and the verification of picking positive colony, obtains red fluorescence
The PD positioning carrier pSrMV-P3N-PIPO-RFP of protein labeling, its plasmid map are as shown in Figure 2；
(8) structure of the PD positioning carriers pSrMV-P3N-PIPO-YFP of yellow fluorescence protein mark：Using Xho I and
Hind III are to carrier pSAT6-EYFP-C1 (GenBank：AY818380 double digestion) is carried out, digestion products are passed through into agarose
Gel electrophoresis is separated and recovered from.Then digestion products are connected with Insert Fragment S with T4DNA ligases, and connection is produced
Thing is converted in by state Escherichia coli (Escherichia coli) DH5 α, and the verification of picking positive colony, obtains yellow fluorescence egg
The PD positioning carrier pSrMV-P3N-PIPO-YFP of white marker, its plasmid map are as shown in Figure 3；
(9) structure of the PD positioning carriers pSrMV-P3N-PIPO-CFP of cyan fluorescent protein mark：Using Xho I and
Hind III are to carrier pSAT6-ECFP-C1 (GenBank：AY818374 double digestion) is carried out, digestion products are passed through into agarose
Gel electrophoresis is separated and recovered from.Then digestion products are connected with Insert Fragment S with T4DNA ligases, and connection is produced
Thing is converted into competence Escherichia coli (Escherichia coli) DH5 α, and the verification of picking positive colony, obtains hanced cyan fluorescent
The PD positioning carrier pSrMV-P3N-PIPO-CFP of protein labeling, its plasmid map are as shown in Figure 4.
Embodiment two：The Subcellular Localization of arabidopsis AtREM1.3
1st, the clone of arabidopsis AtREM1.3 genes
A Remorin gene has been cloned from arabidopsis, has been cloned into cloning vector pMD19-T.Chadogram point
Analysis shows that the gene belongs to the 3rd type of the 1st subgroup of Remorin gene families, is named as AtREM1.3.Document shows
Remorin can be positioned at plasma membrane and plasmodesmus, but bioinformatic analysis shows, Remorin does not have transmembrane domain
And signal peptide.It is that can be positioned at plasmodesmus for verification AtREM1.3, carrying out Subcellular Localization to it using this kit grinds
Study carefully.The nucleotides sequence of the AtREM1.3 coded sequences is classified as SEQ ID NO in sequence table：Nucleotide sequence shown in 12；
2nd, the structure of Subcellular Localization carrier pAtREM1.3-RFP
(1) specific PCR primers, AtREM1.3-F (sense primer) and AtREM1.3-R (anti-sense primer) are designed, in upstream
Restriction enzyme site Xho I are introduced in primer AtREM1.3-F, restriction enzyme site Hind III are introduced in anti-sense primer AtREM1.3-R.
Using this to primer, using the cloning vector of AtREM1.3 genes as template, PCR amplification is carried out.PCR reaction systems are 25 μ L：10
× PCR Buffer 2.5 μ L, dNTPs 2.0 μ L, sense primer AtREM1.3-F (10 μm of ol/L) 1.0 μ L, anti-sense primer
AtREM1.3-R (10 μm of ol/L) 1.0 μ L, Taq enzyme (5U/ μ L) 0.125 μ L, ddH217.375 μ L of O, 1.0 μ L of template (cDNA),
25 μ L of cumulative volume.PCR response procedures：94 DEG C of pre-degeneration 4min；Then run 35 to circulate, 94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C
1min；Last 72 DEG C of 10min.PCR carries out double digestion to PCR product after reaction, using Xho I and Hind III, by enzyme
Cut product to be separated and recovered from by agarose gel electrophoresis, obtain Insert Fragment InsTarget；The sense primer
The nucleotides sequence of AtREM1.3-F is classified as SEQ ID NO in sequence table：Nucleotide sequence shown in 13；Anti-sense primer
The nucleotides sequence of AtREM1.3-R is classified as SEQ ID NO in sequence table：Nucleotide sequence shown in 14；Insert Fragment
The nucleotides sequence of InsTarget is classified as SEQ ID NO in sequence table：Nucleotide sequence shown in 15；
(2) RFP control vectors are taken, double digestion is carried out to carrier pSAT6-ERFP-C1 using Xho I and Hind III, will
Digestion products are separated and recovered from by agarose gel electrophoresis.Then by digestion products and it is inserted into piece with T4DNA ligases
Section InsTarget connections, and connection product is converted into competence Escherichia coli (Escherichia coli) DH5 α, picking
Positive colony is verified, obtains the PD positioning carrier pAtREM1.3-RFP of red fluorescent protein marker, its plasmid map such as Fig. 5 institutes
Show；
3rd, Subcellular Localization is tested
(1) frozen-thawed method is used, respectively positions the PD of Subcellular Localization carrier pAtREM1.3-RFP and GFP mark
Carrier pSrMV-P3N-PIPO-GFP is transferred in the Agrobacterium EHA105 bacterial strains of competence；(contain Rif, 34 μ in 5mL LB culture mediums
g/mL；Kan, 50 μ g/mL) in culture conversion Agrobacterium, 28 DEG C, 200rpm overnight incubations；
(2) under room temperature, 5,000rpm, 5min is centrifuged, abandons supernatant, 1mL is added and infects liquid resuspended bacterium solution；
(3) repeat step (2), wash away the antibiotic contained in culture medium；
(4) add 1mL and infect liquid, measure bacterium solution OD600, and adjust OD600It is worth to 0.1；
(5) 750 μ L bacterium solutions are taken in 2mL sterile centrifugation tubes, are mixed, place 3~5h；Subcellular Localization carrier will be contained
The bacterium solution of pAtREM1.3-GFP and the bacterium solution grade ratio mixing containing YFP labeled vectors, carry out infecting experiment；
(6) healthy Ben Shi cigarette plant (before infecting, photo-irradiation treatment tobacco 1h) is taken, the big blade of two panels is selected, in tobacco
Vacuum side of blade (between two veins) injects bacterium solution and carries out mark；
(7) tobacco infected is put and cultivated under normal operation.Taken after 2 days and infect blade 1-2cm2, the back side upward, is used
Confocal laser scanning microscope.When gathering fluorescence signal, different fluorescence signals are gathered respectively to same cell.It is to be detected
PAtREM1.3-RFP mark be red fluorescent protein, gather red fluorescent protein signal when, can go out in position shown in arrow
Existing bright spot, shows that AtREM1.3 is expressed on cell membrane, but can not determine that it is positioned at PD；This kit provides PD positioning and carries
Body pSrMV-P3N-PIPO-GFP marks are green fluorescent proteins, when gathering green florescent signal, bright spot also occur, shows
Bright spot position is plasmodesmus；When two pictures are merged, shown after bright red spot and the accurate superposition of green bright spot
For the bright spot of yellow, show that AtREM1.3 is positioned at plasmodesmus, as shown in Figure 6.
(8) according to above procedure and method, GFP carrier pSAT6-EGFP-C1 and RFP carriers pSAT6-ERFP-C1 is existed
Expressed in Ben Shi cigarette epidermal leaf cells, fluorescence signal is detected under laser confocal microscope.Green florescent signal is distribution shape
State (Fig. 7), red fluorescent are spread state (Fig. 8), show green fluorescent protein and red fluorescent protein do not have it is special
Subcellular Localization, caused by green or red fluorescent protein, thus, it was demonstrated that AtREM1.3 the Subcellular Localization of target protein is not
Plasmodesmus can be positioned at.
The agarose gel electrophoresis, reference《Molecular Cloning:A Laboratory guide》Agarose in (second edition) chapter 6 first segment
The method of gel electrophoresis；It is described to convert connection product into competence bacillus coli DH 5 alpha, method for transformation reference《Molecular cloning
Experiment guide》In Section five of (second edition) chapter 1 the method with transformed competence colibacillus Escherichia coli is prepared with calcium chloride；It is described to contain
There are the picking of the bacterium colony of positive colony, reference《Molecular Cloning:A Laboratory guide》Contain recombinant plasmid in Section six of (second edition) chapter 1
Bacterial clump identification method；The method of the extraction Plasmid DNA, with reference to plasmid extraction kit specification；The digestion
Method, with reference to the specification of restriction enzyme；The recovery method, with reference to plastic recovery kit specification；It is described to use T4-
DNA ligase is attached method, with reference to T4-DNA ligase operational manuals.
Claims (2)
- A kind of 1. preparation method of the PD positioning carriers of fluorescent protein labeling, it is characterised in that：(1) for coded sequence design the specific PCR primers SrMV-P3-F and SrMV-P3-R of SrMV-P3, with the cDNA of SrMV For template, PCR amplification is carried out, PCR product is separated and recovered from by agarose gel electrophoresis, obtains the volume of SrMV-P3 Code sequence；The nucleotides sequence of the SrMV-P3-F is classified as SEQ ID NO in sequence table：Nucleotide sequence shown in 1；SrMV- The nucleotides sequence of P3-R is classified as SEQ ID NO in sequence table：Nucleotide sequence shown in 2；The nucleotide of SrMV-P3 coded sequences Sequence is SEQ ID NO in sequence table：Nucleotide sequence shown in 3；(2) specific PCR primers SrMV-P3N-F and SrMV-P3N-R are designed, restriction enzyme site Xho I are introduced in SrMV-P3N-F； Using SrMV-P3 coded sequences as template, PCR amplification is carried out using primer SrMV-P3N-F and SrMV-P3N-R, PCR reactions terminate Afterwards, PCR product is separated by agarose gel electrophoresis, recycles and be concentrated into 400ng/ μ L, obtain the volume of SrMV-P3N Code sequence；The nucleotides sequence of the SrMV-P3N-F is classified as SEQ ID NO in sequence table：Nucleotide sequence shown in 4；SrMV- The nucleotides sequence of P3N-R is classified as SEQ ID NO in sequence table：Nucleotide sequence shown in 5；The nucleosides of SrMV-P3N coded sequences Acid sequence is SEQ ID NO in sequence table：Nucleotide sequence shown in 6；(3) acquisition of SrMV-PIPO coded sequences：Specific PCR primers SrMV-PIPO-F and SrMV-PIPO-R are designed, Restriction enzyme site Hind III are introduced in SrMV-P3N-R；Using SrMV-P3 coded sequences as template, primer SrMV-PIPO-F is used PCR amplification is carried out with SrMV-PIPO-R, PCR is separated by agarose gel electrophoresis after reaction, by PCR product, is returned Receive and be concentrated into 400ng/ μ L, obtain the coded sequence of SrMV-PIPO；The nucleotides sequence of the SrMV-PIPO-F is classified as sequence SEQ ID NO in table：Nucleotide sequence shown in 7；The nucleotides sequence of SrMV-PIPO-R is classified as SEQ ID NO in sequence table：8 Shown nucleotide sequence；The nucleotides sequence of SrMV-PIPO coded sequences is classified as SEQ ID NO in sequence table：Nucleosides shown in 9 Acid sequence；(4) acquisition of SrMV-P3N-PIPO coded sequences：Be by concentration the P3N and PIPO of 400ng/ μ L PCR product according to 1 ︰ 1 mixing is used as template, and using primer SrMV-P3N-F and SrMV-PIPO-R, SrMV- is cloned using fusion DNA vaccine method P3NPIPO coded sequences；PCR is separated and recovered from by agarose gel electrophoresis after reaction, by PCR product, is obtained The coded sequence of SrMV-P3N-PIPO；The nucleotides sequence of the SrMV-P3N-PIPO coded sequences is classified as SEQ ID in sequence table NO：Nucleotide sequence shown in 10；(5) acquisition of Insert Fragment S：Double digestion is carried out to SrMV-P3N-PIPO coded sequences using Xho I and Hind III, Digestion products are separated and recovered from by agarose gel electrophoresis, obtain Insert Fragment S；The nucleosides of the Insert Fragment S Acid sequence is SEQ ID NO in sequence table：Nucleotide sequence shown in 11；(6) acquisition of the PD positioning carriers of fluorescent protein labeling：Using Xho I and Hind III respectively to carrier pSAT6- EGFP-C1, pSAT6-ERFP-C1, pSAT6-EYFP-C1 and pSAT6-ECFP-C1 carry out double digestion, and digestion products are passed through fine jade Sepharose electrophoresis is separated and recovered from；Then digestion products are connected with Insert Fragment S with T4DNA ligases, and will be even Thing of practicing midwifery is converted into competence bacillus coli DH 5 alpha, and picking positive colony verification, the fluorescent protein labeling PD obtained respectively determines Position carrier pSrMV-P3N-PIPO-GFP, pSrMV-P3N-PIPO-RFP, pSrMV-P3N-PIPO-YFP, pSrMV-P3N- PIPO-CFP；The nucleotides sequence of the Insert Fragment S is classified as SEQ ID NO in sequence table：Nucleotide sequence shown in 11.
- A kind of 2. subcellular fraction of the PD positioning carriers comprising the fluorescent protein labeling being prepared using claim 1 the method Location reagent box, it is characterised in that the kit is made 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 protectives are spare；(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 protectives are spare；(3) the PD positioning carrier pSrMV-P3N-PIPO-YFP of yellow fluorescence protein mark, as positive control, 1 pipe, 100ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；(4) the PD positioning carrier pSrMV-P3N-PIPO-CFP of cyan fluorescent protein mark, as positive control, 1 pipe, 100ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；(5) carrier pSAT6-EGFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；(6) carrier pSAT6-ERFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；(7) carrier pSAT6-EYFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；(8) carrier pSAT6-ECFP-C1,1 pipe, 500ng/ μ L, 50 μ L/ pipes, -20 DEG C of freezen protectives are spare；(9) restriction enzyme Xho I：1 pipe, 500units, 100 μ L/ pipe；(10) restriction enzyme Hind III：1 pipe, 500units, 100 μ L/ pipe；(11) without RNase water：2 bottles, 100mL/ bottles；(12) liquid is infected：1 pipe, 50mL/ pipes, -20 DEG C of freezen protectives are spare.
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| 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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| 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 |
|---|
| An overlapping essential gene in the Potyviridae;Betty Y.-W. Chung et al;《PNAS》;20080415;第15卷(第15期);第5897-5902页 * |
| Formation of Complexes at Plasmodesmata for Potyvirus Intercellular Movement Is Mediated by the Viral Protein P3N-PIPO;Taiyun Wei et al;《PLoS Pathogens》;20100624;第6卷(第6期);第1-12页 * |
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