CN109485708A - For detecting specific gene PgWOX4 and its detection method and the application of ginseng bundle trunk cell - Google Patents

For detecting specific gene PgWOX4 and its detection method and the application of ginseng bundle trunk cell Download PDF

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CN109485708A
CN109485708A CN201811329306.6A CN201811329306A CN109485708A CN 109485708 A CN109485708 A CN 109485708A CN 201811329306 A CN201811329306 A CN 201811329306A CN 109485708 A CN109485708 A CN 109485708A
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CN109485708B (en
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黄璐琦
刘娟
袁媛
蒋超
陈同
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Institute of Materia Medica of CAMS
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Abstract

The invention discloses for detecting ginseng bundle trunk cell specific gene PgWOX4 and its detection method and application.Specific gene PgWOX4 disclosed by the invention for detecting ginseng bundle trunk cell can be used for marking ginseng bundle trunk cell.The invention also discloses a kind of detection ginseng bundle trunk cell area or the methods of position.The present invention has filled up the blank of non-mode plant stem cell detection field, provides a set of feasible detection scheme for the positioning of non-mode plant stem cell, has extraordinary research potential and application prospect.

Description

For detecting specific gene PgWOX4 and its detection side of ginseng bundle trunk cell Method and application
Technical field
The present invention relates to field of biotechnology, and in particular to for detecting the specific gene of ginseng bundle trunk cell PgWOX4 and its detection method and application.
Background technique
Ginseng (Panax ginseng C.A.Mey.) is Araliaceae perennial herb plant, has 5000 in China The medication history of many years has effects that reinforce vital energy, veins takes off admittedly, reinforce the spleen to benefit the lung, production of sperm blood-nourishing, tranquilize the mind and promote the intelligence, is listed in " top grade " of Shennong's Herbal, enjoys the good reputation of " King of Herbs ".Under natural conditions, wild ginseng has extremely strong reparation energy Power can resist the erosion of pathogen and herbivore.It is extremely strong extensive that ginseng " Ding change " phenomenon shows that ginseng stem cell has Reactivation power and regeneration potential.Wherein, ginseng bundle trunk cell is to the development of ginseng leaf, the growth of stem, the overstriking of root and damage It repairs significant, however how to detect ginseng bundle trunk cell there is not been reported.
RNA hybridization in situ technique is a kind of using the known RNA of specific markers as phase in probe, with cell or tissue slice The genetic fragment answered combines (hybridization), and the hybrid (hybrids) of formation is through chromogenic reaction, to carry out essence to specific gene Determine the process of amount positioning.Whole mount in situ hybridization is that vegetable material progress in the case where integrally keeping relatively intact is in situ miscellaneous It hands over, it can be observed that the overall condition at gene expression position.Plant is observed by situ hybridization or whole mount in situ hybridization method The expression of object stem cell marker genes is a kind of method of common detection plant stem cell.But use is not yet seen The report of the in-situ hybridization method detection ginseng stem cell of stem cell marker genes.
Summary of the invention
It is an object of the present invention to provide a kind of protein.
Protein provided by the invention is following protein a) or b) or c) or d):
A) amino acid sequence is protein shown in sequence 2 or sequence 4；
B) fused protein that the N-terminal of the protein shown in sequence 2 or sequence 4 and/or C-terminal connection label obtain；
C) amino acid sequence shown in sequence 2 or sequence 4 by the substitution of one or several amino acid residues and/or is lacked Lose and/or add obtained protein with the same function；
D) with amino acid sequence shown in sequence 2 or sequence 4 with 75% or 75% or more homology and have it is identical The protein of function.
In order to make protein in a) convenient for purifying, can in sequence table protein shown in sequence 2 or sequence 4 amino End or carboxyl terminal connect upper label as shown in Table 1.
The sequence of table 1, label
It is above-mentioned c) in protein, the substitutions of one or several amino acid residues and/or deletion and/or addition is not More than the substitution and/or deletion and/or addition of 10 amino acid residues.
It is above-mentioned c) in protein can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression and obtain.
It is above-mentioned c) in protein encoding gene can by will in DNA sequence dna shown in sequence 1 or sequence 3 lack one Or the codon of several amino acid residues, and/or carry out the missense mutation of one or several base-pairs, and/or its 5 ' end and/ Or 3 ' end connect the coded sequence of label shown in table 1 and obtain.
It is above-mentioned d) in, " homology " include with amino acid sequence shown in sequence of the invention 2 or sequence 4 have 75% or It is higher or 80% or higher or 85% or higher or 90% or higher or 95% or more high homology amino acid sequence.
It is a further object to provide biomaterials relevant to above-mentioned protein.
Biomaterial relevant to above-mentioned protein provided by the invention is following A 1) any one of to A12):
A1 the nucleic acid molecules of above-mentioned protein) are encoded；
A2) contain A1) expression cassettes of the nucleic acid molecules；
A3) contain A1) recombinant vectors of the nucleic acid molecules；
A4) contain A2) recombinant vector of the expression cassette；
A5) contain A1) recombinant microorganisms of the nucleic acid molecules；
A6) contain A2) recombinant microorganism of the expression cassette；
A7) contain A3) recombinant microorganism of the recombinant vector；
A8) contain A4) recombinant microorganism of the recombinant vector；
A9) contain A1) the transgenic plant cells systems of the nucleic acid molecules；
A10) contain A2) the transgenic plant cells system of the expression cassette；
A11) contain A3) the transgenic plant cells system of the recombinant vector；
A12) contain A4) the transgenic plant cells system of the recombinant vector.
In above-mentioned biomaterial, A1) nucleic acid molecules be it is following 1) or 2) or 3) shown in gene:
1) its coded sequence is cDNA molecule or genomic DNA molecule shown in sequence 1 or sequence 3；
2) there is 75% or 75% or more identity with the nucleotide sequence 1) limited, and encodes the cDNA of above-mentioned protein Molecule or genomic DNA molecule；
1) or 2) 3) and the cDNA molecule of above-mentioned protein is encoded with the nucleotide sequence hybridization that limits under strict conditions Or genomic DNA molecule.
Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also To be RNA, such as mRNA or hnRNA.
Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation Method is mutated the nucleotide sequence of the above-mentioned protein of coding of the invention.Those are by manually modified, on coding The nucleotide sequence 75% of protein or the nucleotide of higher identity are stated, as long as encoding above-mentioned protein and there is identical function Can, it is derived from nucleotide sequence of the invention and to be equal to sequence of the invention.
Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair Amino acid sequence shown in bright coded sequence 2 or sequence 4 composition protein nucleotide sequence have 75% or higher, or The nucleotide sequence of 85% or higher or 90% or higher or 95% or higher identity.Identity can with the naked eye or calculate Machine software is evaluated.Using computer software, the identity between two or more sequences can be indicated with percentage (%), It can be used to evaluate the identity between correlated series.
Above-mentioned 75% or 75% or more identity can be 80%, 85%, 90% or 95% or more identity.
In above-mentioned biomaterial, the stringent condition is hybridized simultaneously at 68 DEG C in 2 × SSC, the solution of 0.1%SDS It washes film 2 times, each 5min, and in 0.5 × SSC, the solution of 0.1%SDS, hybridize at 68 DEG C and washes film 2 times, every time 15min；Or, hybridizing under the conditions of 65 DEG C in the solution of 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS and washing film.
In above-mentioned biomaterial, A2) described in the expression cassette containing the nucleic acid molecules for encoding above-mentioned protein be to refer to The DNA of above-mentioned protein is expressed in host cell, which may include not only the promoter for starting target gene transcription, may be used also Terminator including terminating target gene transcription.Further, the expression cassette may also include enhancer sequence.
Can be used for constructing A3) described in the expression vector of recombinant vector include double base agrobacterium vector and to can be used for plant micro- The carrier etc. of bullet bombardment.As pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, PCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA company) etc..The plant expression Carrier also may include 3 ' end untranslated regions of foreign gene, i.e., processes comprising polyadenylation signals and any other participation mRNA Or the DNA fragmentation of gene expression.When using gene constructed plant expression vector of the invention, enhancer also can be used, including turn over Enhancer or transcriptional enhancer are translated, these enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., But must be identical as the reading frame of coded sequence, to guarantee the correct translation of entire sequence.The translation control signal and starting The source of codon be it is extensive, can be natural, be also possible to synthesis.Translation initiation region can come from transcription initiation Region or structural gene.
In above-mentioned biomaterial, the carrier can be plasmid, sticking grain, bacteriophage or viral vectors.
In above-mentioned biomaterial, the microorganism can be yeast, bacterium, algae or fungi, such as Agrobacterium.
In above-mentioned biomaterial, the transgenic plant cells system, Transgenic plant tissue and genetically modified plants organ are equal It does not include propagation material.
The new application that the present invention has a purpose to be to provide above-mentioned protein or above-mentioned biomaterial again.
The present invention provides above-mentioned protein or above-mentioned biomaterial in detection ginseng bundle trunk cell area or position In application.
The present invention also provides the application of above-mentioned protein or above-mentioned biomaterial in positioning ginseng bundle trunk cell.
The present invention also provides above-mentioned protein or its encoding gene in as ginseng vascular bundle stem cell markers Using.
In above-mentioned application, the bundle trunk cell is the bundle trunk cell of blade and/or the bundle trunk of the aerial stem of plant The bundle trunk cell of cell and/or root.
It is a still further object of the present invention to provide a kind of detection ginseng bundle trunk cell area or the methods of position.
The method of detection ginseng bundle trunk cell area provided by the invention or position is by detecting ginseng to be measured sample The expressive site of above-mentioned protein or its encoding gene in product or position are realized.The protein or its encoding gene Expressive site or position are ginseng bundle trunk cell area or position.
In the above method, the bundle trunk cell is the bundle trunk cell of blade and/or the bundle trunk of the aerial stem of plant The bundle trunk cell of cell and/or root.
In the above method, the above-mentioned protein or its coding in ginseng to be measured sample are detected by whole mount in situ hybridization method The expressive site of gene or position.
Further, rna probe single strand RNA molecule as shown in sequence 7 used in the whole mount in situ hybridization method It is formed with single strand RNA molecule shown in sequence 8.
Final object of the present invention is to provide a kind of rna probe.
Single strand RNA molecule group shown in rna probe provided by the invention single strand RNA molecule as shown in sequence 7 and sequence 8 At.
Application of the above-mentioned rna probe in detection ginseng bundle trunk cell area or position also belongs to protection of the invention Range.
Application of the above-mentioned rna probe in positioning ginseng bundle trunk cell also belongs to protection scope of the present invention.
The present invention provides the specific gene PgWOX4 for detecting the maintenance of ginseng bundle trunk cell.The present invention provides The specific gene PgWOX4 for detecting ginseng bundle trunk cell can be used for marking ginseng bundle trunk cell.The present invention It additionally provides a kind of for detecting the detection method of ginseng bundle trunk cell area or position.The present invention has filled up non-mode plant The blank of object stem cell detection field provides a set of feasible detection scheme for the positioning of non-mode plant stem cell, has non- Often good research potential and application prospect.
Detailed description of the invention
Fig. 1 is PgWOX4 gene order.Wherein, WOX4-A and WOX4-B is respectively the most important two kinds of bases of ginseng PgWOX4 Because of type.
Fig. 2 is to detect ginseng bundle trunk cell using whole mount in situ hybridization method.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even Mean value.
The preparation method of 4%PFA solution in following embodiments is as follows: 1 × PBS (pH7.4), 65 DEG C of placement 2-3 are small When, after restoring room temperature, 10%DMSO, 0.1%Tween20,3%NP-40 is added, it is ready-to-use.
The formula of prehybridization solution 1 in following embodiments is as follows: 50% formamide, 5 × SSC, 3%SDS, 0.1%DTT, 0.1%Tween20.
The formula of prehybridization solution 2 in following embodiments is as follows: 50% formamide, 5 × SSC, 0.1%Tween20, 0.1mg/mL heparin.
The acquisition of the albumen of embodiment 1, PgWOX4 gene and its coding
1, the extraction of RNA
Take the sample of ginseng to be crushed with liquid nitrogen grinding, using TRIzolPlantRNAKit according to TRIzolPlantRNAKit specification extracts total serum IgE, and determines RNA integrality, nucleic acid/albumen with 1% agarose gel electrophoresis Quantitative instrument measures rna content.
2, the synthesis of cDNA
M-MLVRTasecDNASynthesisKit is pressed using M-MLVRTasecDNASynthesisKit (Takara) (Takara) specification synthesizes cDNA.
3, PCR amplification
The cDNA obtained using step 2 carries out PCR amplification as template, using WOX4-F and WOX4-R primer, obtains PCR production Object.Primer sequence is as follows:
WOX4-F:5 '-CTAATAGCAACACCGTCACCAT-3 '；
WOX4-R:5 '-CGAGATAGTCAGGGATACAACAAA-3 '.
4, cloning and sequencing
Glue recycling rear clone sequencing is carried out to PCR product, sequencing result shows: big containing one in the sequence of PCR amplification The small complete ORF frame for 711bp, is confirmed as the WOX4 gene of ginseng after comparing using smartBLAST.Since ginseng is four Times body, PgWOX4 gene is there are some sites SNPs, and different clones has found different sequences after cloning and sequencing, and common two Kind nucleotide sequence is respectively WOX4-A and WOX4-B (Fig. 1), by gene order shown in FIG. 1 and similarity > 95% or more Gene order Uniform Name is PgWOX4.Wherein, the nucleotide sequence of WOX4-A is as shown in sequence 1, the WOX4- of WOX4-A coding The amino acid sequence of A albumen is as shown in sequence 2, and the nucleotide sequence of WOX4-B is as shown in sequence 3, the WOX4-B of WOX4-B coding The amino acid sequence of albumen is as shown in sequence 4.
The application of embodiment 2, PgWOX4 gene in ginseng bundle trunk cell detection
Ginseng bundle trunk cell is detected using whole mount in situ hybridization method, the specific steps are as follows:
One, the preparation of PgWOX4 probe
1, the preparation of probe template
The genetic fragment in PgWOX4 gene open reading frame is carried out as template using the ginseng cDNA obtained in embodiment 1 PCR amplification, (PCR product is the mixture of genetic fragment shown in sequence 1 and sequence 3,1 He of sequence to the PCR product of acquisition herein The ratio of genetic fragment shown in sequence 3 is about 1:1) it is the template DNA that probe synthesizes.The primer of PCR amplification is as follows:
Forward primer: 5 '-CTAATAGCAACACCGTCACCAT-3 '；
Reverse primer: 5 '-CGAGATAGTCAGGGATACAACAAA-3 '.
2, the synthesis of single-stranded probe
The template DNA for taking 1 μ g step 1 to obtain adds water polishing to 11 μ L, and 65 DEG C are denaturalized 5 minutes, are immediately placed on ice.Match The system of single-stranded probe synthesis processed: 5 × transbuffer 4 μ L, DIGLabellingNTPmix (are purchased from Roche company, article No. 11277073910) 2 μ L, RNaseinhibitor (being purchased from TAKARA company, article No. 2313A) 1 μ L, 1 μ L of probe primer (control The positive probe primer that group uses, the reversed probe primer that experimental group uses), (control group uses just 2 μ L of single stranded RNA synthase The RNA synthase synthesized to probe is SP6RNApolymerase, and the RNA synthase for the reversed probe synthesis that experimental group uses is T7RNApolymerase).37 DEG C of heat preservation 2h.The DNaseI of 2 μ L RNase-free, 37 DEG C of incubation 15min is added.Incubation terminates Afterwards, add 20 μ L deionized formamides.The single-stranded probe of 1-2 μ L is taken, electrophoresis detection determines that probe band is single band.
Positive probe primer: 5 '-ATTTAGGTGACACTATAGAATAGGAACCCAACGCAAGAACAAATAG-3 '.
Reversed probe primer: 5 '-AATTAATACGACTCACTATAGGGGGGTGTAATGGGAAGAGTTCTAGGG-3 '.
Single strand RNA molecule shown in positive rna probe single strand RNA molecule shown in sequence 5 and sequence 6 forms；
Single strand RNA molecule shown in reversed rna probe single strand RNA molecule shown in sequence 7 and sequence 8 forms.
Two, whole mount in situ hybridization
Fresh leaves of panax ginseng, the aerial stem of plant, root (taking the transverse section at the lower 1.0-1.2cm of reed head) are acquired as sample material Material detects ginseng bundle trunk cell with the rna probe of the PgWOX4 genetic fragment of step 1 preparation.Specific steps It is as follows:
1, specimen material is put into fixer (4%PFA solution) and is fixed, vacuum suction is extremely in fixer for material Until bubble-free generates.After the completion of pumping, material is sunken to bottom, and the 4%PFA/PBST solution renewed is (containing 0.1% Tween20), after vacuumizing, 4 DEG C overnight.
2, after completing step 1, using PBST solution and 4%PFA solution replacement, so that material is suspended in PBST solution. Then (20% ethyl alcohol/PBST, 20min are replaced with ethyl alcohol and PBST solution gradient；40% ethyl alcohol/PBST, 20min；60% second Alcohol/PBST, 20min；80% ethyl alcohol/PBST, 20min；100% ethyl alcohol, 20min, 2 times).It is set again with ethyl alcohol to dimethylbenzene gradient Change (ethyl alcohol: dimethylbenzene=2:1,30min；Ethyl alcohol: dimethylbenzene=1:1,30min；Ethyl alcohol: dimethylbenzene=1:2,30min；Pure two Toluene, 1h).Finally (dimethylbenzene: ethyl alcohol=2:1,30min is replaced with dimethylbenzene to ethanol gradient again；Dimethylbenzene: ethyl alcohol=1: 1,30min；Dimethylbenzene: ethyl alcohol=1:2,30min；Straight alcohol, 30min, 3 times).
3, complete step 2 after, material is put in prehybridization solution 1,55 DEG C incubations 5-10 hours, then use Proteinase K exist 30min is digested at 37 DEG C, then is reacted with 5% glycine/PBST processing 5min with terminating.Material is finally put in prehybridization solution In 2,55 DEG C hybridization 2-4 hours.
4, after completing step 3, the tRNA that 1mg/mL is added into prehybridization solution 2 (is purchased from Roche, article No. is 1093274910) and the PgWOX4 denatured probe of 1 μ g (the positive probe and reversed probe that prepare in step 1), hybridization is for 24 hours.
5, after completing step 4, unbonded probe is washed off, is closed at room temperature using 5%BSA/PBST 1 hour.Then In the ratio of 1:2000, DIG antibody (being purchased from Roche, article No. 1093274910) is added in confining liquid, room temperature reaction 16 is small When.Chromogenic reaction is carried out with NBT/BCIP again, is protected from light 2-6 hours, is terminated and is reacted using 4%PFA.Finally use PBST solution Ginseng bundle trunk cell is detected using body formula mirror and microscope after washing.
As a result as shown in Fig. 2, as can be seen from the figure: the position of blue markings is leaves of panax ginseng bundle trunk cell (i.e. blade vascular bundle forming layer), cauline bundle stem cell (i.e. ground cauline bundle forming layer), root bundle trunk cell (i.e. root Vascular bundle forming layer and phellogen), the reversed rna probe of PgWOX4 gene is tieed up in leaves of panax ginseng bundle trunk cell, stem Hybridize in tube bank stem cell and root bundle trunk cell with the mRNA of PgWOX4 gene, illustrates PgWOX4 gene mainly in folium panacis japonici cum caule It is expressed in piece bundle trunk cell, cauline bundle stem cell and root bundle trunk cell, and the positive rna probe of PgWOX4 gene It cannot hybridize.The above results show: PgWOX4 gene can be used as the marker gene of ginseng bundle trunk cell, and based on this The whole mount in situ hybridization method of the reversed rna probe of the PgWOX4 gene of invention can effectively detect ginseng bundle trunk cell Position, and can intuitively, clearly observe ginseng bundle trunk cell.
Sequence table
<110>Institute Of Chinese Materia Medica Of China Academy of Chinese Medical Sciences
<120>for detecting specific gene PgWOX4 and its detection method and the application of ginseng bundle trunk cell
<160>8
<170>PatentIn version 3.5
<210>1
<211>711
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>1
atgggaggaa gcatgaaggt gcatcagttc gcacgtggat taatcagctg ggatcagcag 60
cacgaaccct ccctcacgct cggcctctgc aagcgtttac gccctcttat tcccaagaac 120
cccaccacca acagcgatac taatagcaac accgtcacca tcaccaacac caccacaacc 180
accaccgcta tccctttcga tcttaagagc tttattaaac ctgattgtgg ccccagaaaa 240
cccgatgttt catcctctga cgacaagaga gatcaattaa ctcaggtgga gacacatccg 300
ggagggacac ggtggaaccc aacgcaagaa caaataggga tattggagat gctgtatagg 360
gggggaatgc gcaccccaac cgcgcaacaa atagaacaaa ttaccaccca gctcggcaat 420
tatggcaaga tagaaggcaa gaacgtgttt tactggttcc aaaatcacaa agcacgcgaa 480
aggcaaaagc agaagcgtaa taacctcggc ctcggccata gtccacggag ctcctccact 540
acatttagca ccataacatt aagcacaagg ggtgatcaag aattggaaga gagtccaata 600
ttcaagaaaa aatgtaggac atggtcattt gaaagcatgg aagaggagga acataagaat 660
aagaccctag aactcttccc attacaccca gaattaagga agatgaatta a 711
<210>2
<211>236
<212>PRT
<213>artificial sequence (Artificial Sequence)
<400>2
Met Gly Gly Ser Met Lys Val His Gln Phe Ala Arg Gly Leu Ile Ser
1 5 10 15
Trp Asp Gln Gln His Glu Pro Ser Leu Thr Leu Gly Leu Cys Lys Arg
20 25 30
Leu Arg Pro Leu Ile Pro Lys Asn Pro Thr Thr Asn Ser Asp Thr Asn
35 40 45
Ser Asn Thr Val Thr Ile Thr Asn Thr Thr Thr Thr Thr Thr Ala Ile
50 55 60
Pro Phe Asp Leu Lys Ser Phe Ile Lys Pro Asp Cys Gly Pro Arg Lys
65 70 75 80
Pro Asp Val Ser Ser Ser Asp Asp Lys Arg Asp Gln Leu Thr Gln Val
85 90 95
Glu Thr His Pro Gly Gly Thr Arg Trp Asn Pro Thr Gln Glu Gln Ile
100 105 110
Gly Ile Leu Glu Met Leu Tyr Arg Gly Gly Met Arg Thr Pro Thr Ala
115 120 125
Gln Gln Ile Glu Gln Ile Thr Thr Gln Leu Gly Asn Tyr Gly Lys Ile
130 135 140
Glu Gly Lys Asn Val Phe Tyr Trp Phe Gln Asn His Lys Ala Arg Glu
145 150 155 160
Arg Gln Lys Gln Lys Arg Asn Asn Leu Gly Leu Gly His Ser Pro Arg
165 170 175
Ser Ser Ser Thr Thr Phe Ser Thr Ile Thr Leu Ser Thr Arg Gly Asp
180 185 190
Gln Glu Leu Glu Glu Ser Pro Ile Phe Lys Lys Lys Cys Arg Thr Trp
195 200 205
Ser Phe Glu Ser Met Glu Glu Glu Glu His Lys Asn Lys Thr Leu Glu
210 215 220
Leu Phe Pro Leu His Pro Glu Leu Arg Lys Met Asn
225 230 235
<210>3
<211>711
<212>DNA
<213>artificial sequence (Artificial Sequence)
<400>3
atgggaggaa gcatgaaggt gcatcagttc gcacgtggat taatcagctg ggatcagcag 60
cacgaaccct ccctcacgct cggcctctgc aagcgtttac gccctcttat tcccaagaac 120
cccaccacca acagcgatac taatagcaac accgtcacca tcaccaacac caccacaacc 180
accaccgcta tccctttcga tcttaagagc tttattaaac ctgattgtgg ccccagaaaa 240
cccgatgttt catcctctga cgacaagaga gatcaattaa ctcaggtgga gacacatccg 300
ggagggacac ggtggaaccc aacgcaagaa caaataggga tattggagat gctgtatagg 360
gggggaatgc gcaccccaac cgcgcaacaa atagaacaaa ttaccaccca gctcggcaat 420
tatggcaaga tagaaggcaa gaacgtgttt tactggttcc aaaatcacaa agcacgcgaa 480
aggcaaaagc agaagcgtaa taacctcggc ctcggccata gtctacggat ctcctccact 540
acatttagca ccataacatt aagcacaagg ggtgatcaag aattggaaaa gagtccaata 600
ttcaagaaaa aatgtaggac atggtcattc gaaagcatgg aagaggagga acataagaat 660
aagaccctaa aactcttccc attacaccca gaattaagga ggatgaatta a 711
<210>4
<211>236
<212>PRT
<213>artificial sequence (Artificial Sequence)
<400>4
Met Gly Gly Ser Met Lys Val His Gln Phe Ala Arg Gly Leu Ile Ser
1 5 10 15
Trp Asp Gln Gln His Glu Pro Ser Leu Thr Leu Gly Leu Cys Lys Arg
20 25 30
Leu Arg Pro Leu Ile Pro Lys Asn Pro Thr Thr Asn Ser Asp Thr Asn
35 40 45
Ser Asn Thr Val Thr Ile Thr Asn Thr Thr Thr Thr Thr Thr Ala Ile
50 55 60
Pro Phe Asp Leu Lys Ser Phe Ile Lys Pro Asp Cys Gly Pro Arg Lys
65 70 75 80
Pro Asp Val Ser Ser Ser Asp Asp Lys Arg Asp Gln Leu Thr Gln Val
85 90 95
Glu Thr His Pro Gly Gly Thr Arg Trp Asn Pro Thr Gln Glu Gln Ile
100 105 110
Gly Ile Leu Glu Met Leu Tyr Arg Gly Gly Met Arg Thr Pro Thr Ala
115 120 125
Gln Gln Ile Glu Gln Ile Thr Thr Gln Leu Gly Asn Tyr Gly Lys Ile
130 135 140
Glu Gly Lys Asn Val Phe Tyr Trp Phe Gln Asn His Lys Ala Arg Glu
145 150 155 160
Arg Gln Lys Gln Lys Arg Asn Asn Leu Gly Leu Gly His Ser Leu Arg
165 170 175
Ile Ser Ser Thr Thr Phe Ser Thr Ile Thr Leu Ser Thr Arg Gly Asp
180 185 190
Gln Glu Leu Glu Lys Ser Pro Ile Phe Lys Lys Lys Cys Arg Thr Trp
195 200 205
Ser Phe Glu Ser Met Glu Glu Glu Glu His Lys Asn Lys Thr Leu Lys
210 215 220
Leu Phe Pro Leu His Pro Glu Leu Arg Arg Met Asn
225 230 235
<210>5
<211>376
<212>RNA
<213>artificial sequence (Artificial Sequence)
<400>5
ggaacccaac gcaagaacaa auagggauau uggagaugcu guauaggggg ggaaugcgca 60
ccccaaccgc gcaacaaaua gaacaaauua ccacccagcu cggcaauuau ggcaagauag 120
aaggcaagaa cguguuuuac ugguuccaaa aucacaaagc acgcgaaagg caaaagcaga 180
agcguaauaa ccucggccuc ggccauaguc cacggagcuc cuccacuaca uuuagcacca 240
uaacauuaag cacaaggggu gaucaagaau uggaagagag uccaauauuc aagaaaaaau 300
guaggacaug gucauuugaa agcauggaag aggaggaaca uaagaauaag acccuagaac 360
ucuucccauu acaccc 376
<210>6
<211>376
<212>RNA
<213>artificial sequence (Artificial Sequence)
<400>6
ggaacccaac gcaagaacaa auagggauau uggagaugcu guauaggggg ggaaugcgca 60
ccccaaccgc gcaacaaaua gaacaaauua ccacccagcu cggcaauuau ggcaagauag 120
aaggcaagaa cguguuuuac ugguuccaaa aucacaaagc acgcgaaagg caaaagcaga 180
agcguaauaa ccucggccuc ggccauaguc uacggaucuc cuccacuaca uuuagcacca 240
uaacauuaag cacaaggggu gaucaagaau uggaaaagag uccaauauuc aagaaaaaau 300
guaggacaug gucauucgaa agcauggaag aggaggaaca uaagaauaag acccuaaaac 360
ucuucccauu acaccc 376
<210>7
<211>376
<212>RNA
<213>artificial sequence (Artificial Sequence)
<400>7
ggguguaaug ggaagaguuc uagggucuua uucuuauguu ccuccucuuc caugcuuuca 60
aaugaccaug uccuacauuu uuucuugaau auuggacucu cuuccaauuc uugaucaccc 120
cuugugcuua auguuauggu gcuaaaugua guggaggagc uccguggacu auggccgagg 180
ccgagguuau uacgcuucug cuuuugccuu ucgcgugcuu ugugauuuug gaaccaguaa 240
aacacguucu ugccuucuau cuugccauaa uugccgagcu gggugguaau uuguucuauu 300
uguugcgcgg uuggggugcg cauucccccc cuauacagca ucuccaauau cccuauuugu 360
ucuugcguug gguucc 376
<210>8
<211>376
<212>RNA
<213>artificial sequence (Artificial Sequence)
<400>8
ggguguaaug ggaagaguuu uagggucuua uucuuauguu ccuccucuuc caugcuuucg 60
aaugaccaug uccuacauuu uuucuugaau auuggacucu uuuccaauuc uugaucaccc 120
cuugugcuua auguuauggu gcuaaaugua guggaggaga uccguagacu auggccgagg 180
ccgagguuau uacgcuucug cuuuugccuu ucgcgugcuu ugugauuuug gaaccaguaa 240
aacacguucu ugccuucuau cuugccauaa uugccgagcu gggugguaau uuguucuauu 300
uguugcgcgg uuggggugcg cauucccccc cuauacagca ucuccaauau cccuauuugu 360
ucuugcguug gguucc 376

Claims (10)

1. protein is following protein a) or b) or c) or d):
A) amino acid sequence is protein shown in sequence 2 or sequence 4；
B) fused protein that the N-terminal of the protein shown in sequence 2 or sequence 4 and/or C-terminal connection label obtain；
C) amino acid sequence shown in sequence 2 or sequence 4 is passed through to the substitution and/or missing of one or several amino acid residues And/or the protein with the same function that addition obtains；
D) with amino acid sequence shown in sequence 2 or sequence 4 with 75% or 75% or more homology and with identical function Protein.
2. it is following A 1 biomaterial relevant to protein described in claim 1) any one of to A12):
A1 the nucleic acid molecules of protein described in claim 1) are encoded；
A2) contain A1) expression cassettes of the nucleic acid molecules；
A3) contain A1) recombinant vectors of the nucleic acid molecules；
A4) contain A2) recombinant vector of the expression cassette；
A5) contain A1) recombinant microorganisms of the nucleic acid molecules；
A6) contain A2) recombinant microorganism of the expression cassette；
A7) contain A3) recombinant microorganism of the recombinant vector；
A8) contain A4) recombinant microorganism of the recombinant vector；
A9) contain A1) the transgenic plant cells systems of the nucleic acid molecules；
A10) contain A2) the transgenic plant cells system of the expression cassette；
A11) contain A3) the transgenic plant cells system of the recombinant vector；
A12) contain A4) the transgenic plant cells system of the recombinant vector.
3. relevant biological material according to claim 2, it is characterised in that: A1) nucleic acid molecules be it is following 1) or 2) Or 3) shown in gene:
1) its coded sequence is cDNA molecule or genomic DNA molecule shown in sequence 1 or sequence 3；
2) there is 75% or 75% or more identity with the nucleotide sequence 1) limited, and encodes albumen described in claim 1 The cDNA molecule or genomic DNA molecule of matter；
1) or 2) 3) and protein described in claim 1 is encoded with the nucleotide sequence hybridization that limits under strict conditions CDNA molecule or genomic DNA molecule.
4. protein described in claim 1 or relevant biological material described in claim 2 or 3 are in detection ginseng bundle trunk Application in cell area or position；
Or, protein described in claim 1 or relevant biological material described in claim 2 or 3 are in positioning ginseng vascular bundle Application in stem cell；
Or, protein described in claim 1 or its encoding gene are as the application in ginseng vascular bundle stem cell markers.
5. application according to claim 4, it is characterised in that: the bundle trunk cell is the bundle trunk cell of blade And/or the aerial stem of plant bundle trunk cell and/or root bundle trunk cell.
6. a kind of method of detection ginseng bundle trunk cell area or position is by the right in detection ginseng to be measured sample It is required that the expressive site or position of protein described in 1 or its encoding gene are realized.
7. according to the method described in claim 6, it is characterized by: the bundle trunk cell is the bundle trunk cell of blade And/or the aerial stem of plant bundle trunk cell and/or root bundle trunk cell；
Or, detecting the protein described in claim 1 or its coding in ginseng to be measured sample by whole mount in situ hybridization method The expressive site of gene or position.
8. according to the method described in claim 7, it is characterized by: rna probe used in the whole mount in situ hybridization method Single strand RNA molecule shown in the single strand RNA molecule shown in sequence 7 and sequence 8 forms.
9. rna probe described in claim 8.
10. application of the rna probe as claimed in claim 9 in detection ginseng bundle trunk cell area or position；
Or, application of the rna probe as claimed in claim 9 in positioning ginseng bundle trunk cell.
CN201811329306.6A 2018-11-09 2018-11-09 Specific gene PgWOX4 for detecting ginseng vascular bundle stem cells, and detection method and application thereof Active CN109485708B (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104232679A (en) * 2009-01-28 2014-12-24 巴斯夫植物科学有限公司 Plants having enhanced yield-related traits and a method for making the same
CN104531723A (en) * 2015-01-31 2015-04-22 中南民族大学 Plant vascular bundle development gene sm-Nvas and application thereof
CN105164262A (en) * 2013-11-22 2015-12-16 孟加拉朱特研究所 Nucleotide sequence encoding WUSCHL-related homeobox4 (WOX4) protein from corchorus olitorius and corchorus capsularis and methods of use for same

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104232679A (en) * 2009-01-28 2014-12-24 巴斯夫植物科学有限公司 Plants having enhanced yield-related traits and a method for making the same
CN105164262A (en) * 2013-11-22 2015-12-16 孟加拉朱特研究所 Nucleotide sequence encoding WUSCHL-related homeobox4 (WOX4) protein from corchorus olitorius and corchorus capsularis and methods of use for same
CN104531723A (en) * 2015-01-31 2015-04-22 中南民族大学 Plant vascular bundle development gene sm-Nvas and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
齐海军;孙云轩;李昂;陈双越;高慧;金东淳;: "人参组培过程中SERK和WUS基因的表达特性", 延边大学农学学报, vol. 39, no. 01, pages 16 - 22 *

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