CN106916895A - Two black mustard reiterated DNA sequenceses and its application - Google Patents
Two black mustard reiterated DNA sequenceses and its application Download PDFInfo
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Abstract
The invention discloses two kinds of black mustard genomic dna sequences and its application.The invention provides the complete DNA molecular for identifying brassica plant 1 B gene group, be single strand dna shown in the sequence 1 of sequence table and sequence table sequence 2 shown in single strand dna constitute.The experiment proves that, the acquisition of BNSAT28 and BNSAT68 is easier the identification of the chromosome of Brassica Crops, accurately, has filled up on Brassica Crops Chromosome Identification without the available vacancy of preferable probe.
Description
Technical field
The present invention relates to biological technical field, more particularly to two kinds of black mustard genomic dna sequences and its application.
Background technology
Cruciferae Vegetables in Brassica includes A genome Brassica rapa (AA, n=10) with Chinese cabbage as representative, black
The 1 B gene group of mustard, Brassica nigra (BB, n=8), and the C genome Brassica oleracea that wild cabbage is representative
(CC, n=9), and three different edge tetraploid aggregate species that their Natural doubles are formed：Cabbage type rape class B.napus
(AACC, n=19), mustard B.juncea (AABB, n=18), and brassicacarinata B.carinata (BBCC, n=17),
Constitute famous U triangles.Contain most important edible in world wide, oil is used and seasoning is with vegetables, is also in research category
The type material that genetic diversity and species and polyploidy develop.
Black mustard is the wild species of Brassica genus, and many merits are remained under long-term ecologic adaptation, such as resists various diseases
Opportunistic pathogen (Groat, 2003；Pradhan et al., 2011), and excellent kind oil quality (Chevre et al., 1991；
Pradhan et al.,2011).These are all the important gene sources of Brassica genus breeding, however, the heredity of 1 B gene group and cytology
The shortage of information hinders the transfer and utilization of its gene.Brassica genus spore and ploidy develop research also by 1 B gene group
The limitation of poor information.
With the quick research of A, C genome, the research of black mustard 1 B gene group also will increasingly be taken seriously, black mustard gene
Group sequencing plan also has been turned on, therefore its cytogenetical study also must elder generation's back.Develop stabilization cytological marker and
It is basis to set up convenient and reliable caryogram authentication method.
Vegetables in Brassica chromosome is small, especially the black mustard of 1 B gene group, and form can not possibly be leaned on to recognize all dyeing completely
Body, caryogram is the basic and key of cytogenetical study.At present, the chromosome karyotype analysis on Brassica genus 1 B gene group are fresh
Have been reported that, also rest in chromosome morphology and C banding techniques substantially, poor repeatability, distinguish is influenceed greatly by subjective factor, method
It is cumbersome.
The content of the invention
It is an object of the present invention to provide the complete DNA molecular for identifying brassica plant 1 B gene group.
The complete DNA molecular that the present invention is provided, be it is following 1) or 2)：
1) the single strand dna group shown in single strand dna as shown in the sequence 1 of sequence table and the sequence 2 of sequence table
Into；
2) single strand dna shown in the single strand dna and sequence B shown in sequence A is constituted；
Sequence A is the sequence more than 90% with the homology of sequence 1；
Sequence B is the sequence more than 90% with the homology of sequence 2.
Another object of the present invention is to provide the complete carrier for identifying brassica plant 1 B gene group.
The complete carrier that the present invention is provided, the recombinant vector and expressed sequence of the single strand dna as shown in expressed sequence 1
The recombinant vector composition of the single strand dna shown in 2.
The recombinant vector of the single strand dna shown in expressed sequence 1 is to be cloned into the BNSAT28 sequences shown in sequence 1
The plasmid obtained on PMD-18T carriers.
The recombinant vector of the single strand dna shown in expressed sequence 2 is to be cloned into the BNSAT68 sequences shown in sequence 2
The plasmid obtained on PMD-18T carriers.
The 3rd purpose of the present invention is to provide the kit for identifying brassica plant 1 B gene group.
The kit that the present invention is provided, including above-mentioned complete DNA molecular or above-mentioned complete carrier.
In above-mentioned, the single strand dna shown in the nucleotides of the single strand dna shown in the sequence 1 and the sequence 2
The different fluorophor of nucleotide marker.
Above-mentioned complete DNA molecular or above-mentioned complete carrier or mentioned reagent box are in differentiation or supplementary globe or identification or auxiliary
Application in identification each bar chromosome of brassica plant 1 B gene group is also the scope of protection of the invention.
Above-mentioned complete DNA molecular or above-mentioned complete carrier or mentioned reagent box are being identified or are aiding in identification Brassica genus to be measured to plant
Application in thing in which bar chromosome of the 1 B gene group chromosome containing black mustard is also the scope of protection of the invention；Wherein, it is to be measured
Brassica plant is specially non-black mustard；
Or, above-mentioned complete DNA molecular or above-mentioned complete carrier or mentioned reagent box are in the genome that measuring plants are treated in identification
Whether the application contained in the chromosome or certain chromosome of the 1 B gene group for deriving from black mustard is also the scope of protection of the invention.
In above-mentioned, the brassica plant is black mustard.
The 4th purpose of the present invention be to provide it is a kind of identify or auxiliary identification brassica plant to be measured in from black mustard B
Genome chromosome is the method for which chromosome.
The method that the present invention is provided, comprises the following steps：Made using 2 single strand dnas in above-mentioned complete DNA molecular
It is probe, dual FISH is carried out to brassica plant to be measured and black mustard respectively, according to the brassica plant B to be measured
The signal location of genome chromosome is compared with the signal location of 8 chromosomes of 1 B gene group of the black mustard, if described treat
The signal location for surveying brassica plant 1 B gene group chromosome is consistent with the signal location of the 1 B gene group chromosome of black mustard, then
The brassica plant 1 B gene group chromosome to be measured is or candidate is 1 B gene group this chromosome of black mustard.
The 5th purpose of the present invention is to provide a kind of 8 articles of methods of chromosome for distinguishing black mustard.
The method that the present invention is provided, comprises the following steps：Made using 2 single strand dnas in above-mentioned complete DNA molecular
It is probe, dual FISH is carried out to black mustard, the signal location difference black mustard according to the black mustard 1 B gene group chromosome
8 chromosomes.
Single strand dna shown in the sequence 1 of sequence table and the single strand dna shown in the sequence 2 of sequence table are in conduct
Application in the probe of the 1 B gene group of discriminating black mustard is also the scope of protection of the invention.
Above-mentioned probe is Fluorescence in situ hybridization probe.
This research will set up the chromosome of black mustard using two repetitive dna sequence combined with fluorescent in situ hybridization (FISH) technology
Cytology visable indicia.Clearly distinguish black mustard per item chromosome by signal strength and position relationship.The technology and method
Facility will be provided for Brassica genus spore, genome and cytological analysis equimolecular biological study.
The experiment proves that, (1) BnSAT28 and BnSAT68 is the two classes series connection of rich content in black mustard genome
Repetitive sequence；(2) acquisition of BnSAT28 and BnSAT68 is easier the identification of the chromosome of Brassica Crops, accurately, fills up
The shortage of probe on Brassica Crops Chromosome Identifications；(3) can be to Brassica genus 1 B gene group by BnSAT28 and BnSAT68
Every chromosome in source is accurately distinguished；(4) black mustard can be provided in distant hybridization germplasm innovation is carried out using black mustard
The visual cytological marker of chromosome；(5) black mustard genome genetic map and physical map are assembled into reference to other marks
Row checking and supplement；(6) with reference to other marks to detecting the transposition of 1 B gene group, inversion, gradually ooze and the identification of addition line is provided
It is convenient.
Brief description of the drawings
Fig. 1 is tandem repetitive sequence BnSAT28 and BnSAT68 and 45S rDNA (or CRB) in black mustard mitosis metaphase
FISH results of hybridization twice on chromosome.A and A'：BnSAT28 (red), BnSAT68 (green) and 45S rDNA (Huang)；B and B'：
BnSAT28 (red), BnSAT68 (green) and CRB (Huang), Bar=5 μm.
Fig. 2 is the idiogram that BnSAT28 and BnSAT68 differentiates every chromosome of black mustard.
Specific embodiment
Experimental technique used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
Chinese cabbage ' new No. 2 of Beijing ' (B.rapa, AA, n=10), black mustard ' G1/1 ' (B.nigra, BB, n=8), wild cabbage class are sweet
Blue ' in sweet No. 21 ' (B.oleracea, CC, n=9).
The separation of embodiment 1, repetitive dna sequence BNSAT28 and BNSAT68
With black mustard ' G1/1 ' as material, the base number that acquisition 0.6G is sequenced by random gene group is equivalent to about 0.8 times
Black mustard genome (being that 630Mb is calculated with Genome Size, Johnston et al., 2005).With reference to Nov á k et al.
(2010) and the method for Macas et al. (2010) carries out repetitive sequence analysis to sequence, it was found that two of which genome contains
More rich tandem repetitive sequence BNSAT28 and BNSAT68 is measured, and checking is cloned by PCR.
The nucleotides sequence of BNSAT28 is classified as the sequence 1 in sequence table.
The nucleotides sequence of BNSAT68 is classified as the sequence 2 in sequence table.
Embodiment 2, repetitive dna sequence BNSAT28 and BNSAT68 as detection probe application
First, BNSAT28 and BNSAT68 as detection probe FISH (FISH) test method
1st, chromosome sectioning
1) vegetable seeds to be measured is placed in the culture dish of moistening filter paper, is taken root at room temperature；
2) when tip of a root length 0.5-1cm, cut off root tips and be put into equipped with d2H2In the test tube of O, and the test tube is put into fills
The ice chest of mixture of ice and water is placed 22-24h in refrigerator and is pre-processed；
3) fixer (absolute ethyl alcohol will be put into by the tip of a root of pretreatment:Glacial acetic acid=3:1 (v/v)) in fix 1-7 days；
4) Root apical meristem part is cut and 4% (volumn concentration) cellulase and 1% (volume basis contain
Amount) in pectin enzyme mixation, 37 DEG C for the treatment of 50min soften the tip of a root；
5) by the tip of a root d of softening2H2O is carefully cleaned, and soaks 15min；
6) ethanol is used again：Glacial acetic acid volume ratio 3:1 fixer is fixed, and is transferred on clean slide, in
Broken film-making is squeezed in the glacial acetic acid of 45% (volumn concentration)；
7) slice, thin piece is preserved in -70 DEG C of refrigerators, so can for a long time preserves and not have a strong impact on the result of in situ hybridization,
Obtain chromosome sectioning.
2nd, prepared by probe
1) nick translation label probe reaction system
Nick translation label probe reaction system is as shown in Tables 1 and 2.
Plasmid containing BNSAT28 sequences is that the BNSAT28 sequences shown in sequence 1 are cloned on PMD-18T carriers to obtain
Plasmid.
Plasmid containing BNSAT68 sequences is that the BNSAT68 sequences shown in sequence 2 are cloned on PMD-18T carriers to obtain
Plasmid.
Be cloned into 45S rDNA sequences shown in sequence 3 on PMD-18T carriers and obtain by the plasmid of the sequences of rDNA containing 45S
Plasmid.
CRB sequences shown in sequence 4 are cloned into plasmid containing CRB sequences the plasmid obtained on PMD-18T carriers.
Table 1 is that nick translation marks BNSAT28 probe reaction systems
Table 2 is that nick translation marks BNSAT68 probe reaction systems
Table 3 is that nick translation marks 45S rDNA or CRB probe reaction systems
Use d2H2O adjusts total nick translation label probe reaction system to 50 μ L, often adds a kind of solution to mix, and adds
It is centrifuged afterwards and fully mixes；
2) will be equipped with the test tube of the 50 μ L reaction solutions 14-15 DEG C of water-bath 2h in water-bath；
3) 5 μ L stop buffers (EDTA of 200mM) terminating reactions are added.
Note:
1) 10X buffer solutions:0.5M Tris HCl,pH 7.5,50mM MgCl2
DNTP solution:0.5mM dATP,0.5mM dGTP,0.5mM dCTP
DUTP solution:0.166mM dUTP (digoxin or biotin labeling), 0.333mM dTTP
Above reagent is purchased from Roche Holding Ag (Roche, Indianapolis, IN)
Assist probes 45S rDNA (sequence 3) and CRB (sequence 4) (Brassica genus centromere cytological marker, Lim et
Al., it is 2005) with marked by coumarin (diethylaminocoumarin (DEAC)-dUTP), purchased from Perkinelmer Inc.
(PerkinElmer, Inc.), directly displayed after hybridization it is orange, be not required to secondary antibody detection.
2) part of probe mark most critical is the size of last nick translation product, when the probe size of mark is about
During 200-600bp, the result that in situ hybridization will have been obtained.
3) to determine probe size, the μ L of reaction solution 5 are drawn 4min is denatured in boiling water, and be placed in cooled on ice at once, so
The electrophoresis on small-sized gel sample afterwards.
4) size of nick translation product can add different amounts of DNA enzymatic I to be adjusted by reaction solution.
3rd, probe and chromosomal hybridation
1) take cover glass (slice, thin piece is stored in -80 DEG C of refrigerators) off with blade, by slice, thin piece be placed in graded ethanol (70%, 95%,
100%) wash-out in, each gradient is washed 5min, is dried at room temperature.Slice, thin piece dries a few houres before should hybridizing in the original location.
2) preparation of hybridization solution reaction system
Table 3 is hybridization solution reaction system
3) d is used2H2O adjusts hybridization reaction system to 20 μ L, it is ensured that solution is sufficiently mixed, because 50% dextran sulfate
It is very sticky.
4) by mixed liquor in being denatured 5min on 80 DEG C of dry bath devices, and it is placed at once on ice.Centrifugation is mixed, and obtains hybrid mixed
Liquid.
5) to 70% formamide solution for 150 μ L are added dropwise on dry slice, thin piece being dissolved in 2X SSC, the lid of 22 × 40mm is covered
Slide, is placed on flat board (metal or glass) and toasts 1.5min in 80 DEG C of baking ovens.(10mL is dissolved in 70% formamide of 2X SSC
Solution：7mL formamides, 1mL 20X SSC, 2mL d2H2O)。
6) take cover glass off, be then dipped into the cold ethanol of gradient (70%, 95%, 100%, -20 DEG C, every gradient 5min)
Middle wash-out, air drying.
7) every slice, thin piece adds the above-mentioned hybrid mixed liquid for 4) obtaining of 10 μ L, covers 18 × 18mm cover glasses, close with adhesive
Mounting, is placed in wet box.
Note：Any one box can be used, the wet filter paper of two-layer 3mm on cassette bottom pad, with sticking plaster or other thing branch
Blade.
8) wet box is put into 37 DEG C of insulating boxs and is incubated, minimum six hours or overnight.
Note：
1) phase contrast microscope microscopy can be used, to ensure that cell is in metaphase, and chromosome morphology is preferably, this right and wrong
Often important, if wash-out is dried, after stain colour solid state is very poor to be difficult to obtain gratifying results of hybridization.
2) quality of formamide is critically important, is purchased from sigma companies, and effect is fine.
4th, hybridization signal fluoroscopic examination
1) scrape adhesive off with tweezers, be then put into slice, thin piece in the staining jar for filling 2X SSC, gently shake and wash until lid
Slide falls down from slide.
2) it follow these steps to wash slide：
Table 4 is hybridization solution elution process
Note：Background can be reduced by the wash temperature and time that improve 2X SSC, above-mentioned washing methods is most of
Effect is preferable.
3) it is slide is drained on paper handkerchief (should not be too dry), 100 μ L FITC fluorescein isothiocynates of dropwise addition (according to
Different Reagent Company's dilutions 1:50 to 1:200) cover plate of 22 × 40mm, 37 DEG C of incubation 30min, are covered.
4) by inclining slide or immersing slide in the beaker for filling 1X PBS, take cover plate off, then existed with 1X PBS
Shake at room temperature and wash slide three times, each 5min.
5) it is drained on paper handkerchief, the anti digoxin antibody of 100 μ L rhodamines mark is added dropwise (according to different Reagent Companies
Dilution 1:50 to 1:200) cover plate of 22 × 40mm, 37 DEG C of incubation 30min, are covered.
6) by inclining slide or immersing slide in the beaker for filling 1X PBS, take cover plate off, then existed with 1X PBS
Shake at room temperature and wash slide three times, each 5min.
7) drained on paper handkerchief, the DAPI that 1 μ g/mL of the drop containing anti-quencher (being purchased from Vector) is added dropwise is
4', 6- diamidino -2-phenylindone (4', 6-diamidino-2-phenylindole), cover cover plate.
8) fluorescence microscope detection in situ hybridization result：The probe of rhodamine mark takes on a red color signal under fluorescence, biological
The probe of element mark is in yellowish green chrominance signal under fluorescence, orange with directly displaying for marked by coumarin, and chromosome is the indigo plant of DAPI
Chrominance signal.
2nd, detect
It is right respectively using BNSAT28, BNSAT68,45S rDNA (or CRB) as detection probe according to above-mentioned one method
Black mustard (BB), Chinese cabbage (AA), the individual plant of wild cabbage (CC) different materials carry out the analysis of 2 FISH (FISH), 2 times auxiliary
Help probe respectively 45S rDNA and CRB probes.Each results of hybridization is observing the Metaphase Chromosomes of more than 10
The results of hybridization of cell is defined.
The FISH results of Chinese cabbage (AA) and wild cabbage (CC) show do not have on Chinese cabbage AA genomes and wild cabbage CC genomes
The obvious hybridization signal of BNSAT28 and BNSAT68.
The results of FISH twice of black mustard (BB) as shown in figure 1, have difference respectively on 8 pairs of chromosomes of the BB genomes of black mustard
Hybridization signal feature；Two double-colored FISH of sequence can be clearly distinguished chromosome black mustard 8：Chromosome is according to from length
To short arrangement, No. 1 chromosome：BnSAT28 and BnSAT68 has hybridization signal, two letters of BnSAT28 up and down centromere
Number closer to centromere；No. 2 chromosomes：BnSAT28 has small-signal in the short arm of a chromosome near end；No. 3 chromosomes：
BnSAT28 and BnSAT68 are without hybridization signal；No. 4 chromosomes：The hybridization signal of BnSAT28 and BnSAT68 all in galianconism,
BnSAT68 is closer to centromere one end；No. 5 chromosomes：BnSAT28 signals are located at the short arm of a chromosome, near centromere；No. 6 dyes
Colour solid：, all in galianconism, BnSAT28 is closer to centromere one end for the hybridization signal of BnSAT28 and BnSAT68；No. 7 chromosomes：
BnSAT28 signals are located at the short arm of a chromosome, near centromere；No. 8 chromosomes：BnSAT68 is located at galianconism, and BnSAT28 is located at length
Arm.
20 medium cells are selected, using the softwares of Image-Pro Plus 6.0, to chromosome length and FISH signals
Fluorescence intensity is measured.It is demarcated as 1 in the signal intensity of No. 1 the short arm of a chromosome with BnSAT68, other signal intensities are converted to
It is relative to be worth to table 5, Fig. 2 is drawn out according to table 5.
Table 5 is black mustard Metaphase Chromosomes and signal relative intensity
Fig. 2 is the schematic diagram of BnSAT28 and BnSAT68 on 8 chromosomes of black mustard.
The above results show, are probe using BnSAT28 and BnSAT68, or increase assist probes 45S rDNA or CRB and lead to
Polychrome FISH technology is crossed, once hybridizing just can accurately distinguish 8 chromosomes of Brassica genus 1 B gene group, there is provided
The visual cytological marker of black mustard chromosome.
Sequence table
<110>Beijing City Agriculture and Forestry Institute
<120>Two kinds of black mustard genomic dna sequences and its application
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 703
<212> DNA
<213>Black mustard (B. nigra)
<400> 1
caaagaaatt ctaagagcat ctccagccat agctattttt aactctaaaa tagagaaaat 60
acagtatctg atattagagg cataattttt aatttaaaaa tggctcttta acttgttttt 120
ttgaaataat tgtttttttc taaaatgaaa tacacttatt catatgaata gaatttttat 180
ttcttttgct gcaaaggttt ttaaaattat tttagcttta agaaggtcct ttagaaccct 240
agtatcactg gcaggtctga aaaacacaaa aagaaacacc ggcaagcaag taaaatcatt 300
cgcaggctaa tggcaagcgg agagcaagga ctgccaccgc ttggccaatt ttaaagggat 360
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tacacctaag aaattctatg agcatcgcga gcaatagcta tttttaactc taaaatagag 540
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gttttttgaa ataattgttt ttttctaaaa tgaaatacac ttattcatat gaatagaatt 660
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<211> 1009
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<213>Black mustard (B. nigra)
<400> 2
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tgttttcgaa gagaatcaaa aagcgtgtaa ggaatgttag acaatccttg ttctggatgg 180
ctatcctgaa ttggaactta atcttccaaa aaaaaaacat aatcaaaatc aaaatctata 240
agaaaattca ttggctatag aaatcaccaa atagaggtca ttgtctacaa aatagagaac 300
acatatcaaa cagagaacat taaaatagga tagcttgaag cattttcaat aattttatca 360
agaaagtgtt aaatgtggta tcattataaa acacatatgt tatataggat agcttttggc 420
actttcagga tgggtttcca gaaaaataaa atgagtgtgt tttcttgcat aagtctgttt 480
tcgaagagaa tcaaaagcgt gtaaggaatg ttagacaatc cttgttcagg atggctatcc 540
taaattggaa cttaatcttc caaaaaaaaa cataatcaaa atctataaga aaattcattg 600
gctatagaaa tcaccaaata gaggtcattg tctacaaaat agagaacaca tatcaaacag 660
agaacattaa aataggatag cttgaagcat tttcaataat tctatcaaga aagtgttaaa 720
tgtggtatca ttataaaaca catatgttat ataggatatc ttttggcact ttcaggatgg 780
gtttccaaaa aaaaatgagt gtgttttctt gcataagtct gttttcgaag agaatcaaaa 840
gcgtgtaagg aatgttagac aatccttgtt caggatggct atcctgaatt ggaacttaat 900
cttccaaaaa aaacataatc aaaatctata agaaaattca ttggctatag aaatcaccaa 960
atagagttaa ttgtctacaa aatagagaac attaaaatag catagcttg 1009
<210> 3
<211> 1807
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<213>Artificial sequence
<220>
<223>
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acctggttga tcctgccagt agtcatatgc ttgtctcaaa gattaagcca tgcatgtgta 60
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ttgatggtaa ctactactcg gataaccgta gtaattctag agctaatacg tgcaacaaac 180
cccgacttct ggaagggatg catttattag ataaaaggtc gacgcgggct ctgcccgttg 240
ctctgatgat tcatgataac tcgacggatc gcatggcctt agtgctggcg acgcatcatt 300
caaatttctg ccctatcaac tttcgatggt aggatagtgg cctaccatgg tggtaacggg 360
tgacggagaa ttagggttcg attccggaga gggagcctga gaaacggcta ccacatccaa 420
ggaaggcagc aggcgcgcaa attacccaat cctgacacgg ggaggtagtg acaataaata 480
acaataccgg gctcttcgag tctggtaatt ggaatgagta caatctaaat cccttaacga 540
ggatccattg gagggcaagt ctggtgccag cagccgcggt aattccagct ccaatagcgt 600
atatttaagt tgttgcagtt aaaaagctcg tagttgaacc ttgggatggg tcgcccggtc 660
cgccttcggc gagcaccggt cggcttgtct cttctgtcgg cgatacgctc ctggccttaa 720
ctggccgggt cgtgcctccg gcgctgttac tttgaagaaa ttagagtgct caaagcaagc 780
ctacgctctg tatacattag catgggataa catcatagga tttcgatcct attgtgttgg 840
ccttcgggat cggagtaatg attaacaggg acagtcgggg gcattcgtat ttcatagtca 900
gaggtgaaat tcttggattt atgaaagacg aacaactgcg aaagcatttg ccaaggatgt 960
tttcattaat caagaacgaa agttgggggc tcgaagacga tcagataccg tcctagtctc 1020
aaccataaac gatgccgacc agggatcagc ggatgttgct tttaggactc cgctggcacc 1080
ttatgagaaa tcaaagtttt tgggttccgg ggggagtatg gtcgcaaggc tgaaacttaa 1140
aggaattgac ggaagggcac caccaggagt ggagcctgcg gcttaatttg actcaacacg 1200
gggaaactta ccaggtccag acatagtaag gattgacaga ctgagagctc tttcttgatt 1260
ctatgggtgg tggtgcatgg ccgttcttag ttggtggagc gatttgtctg gttaattccg 1320
ttaacgaacg agacctcagc ctgctaacta gctacgtgga ggcatccctt cacggccggc 1380
ttcttagagg gactatggcc gtttaggcca aggaagtttg aggcaataac aggtctgtga 1440
tgcccttaga tgttctgggc cgcacgcgcg ctacactgat gtattcaacg agttcacacc 1500
ttggccgaca ggcccgggta atctttgaaa tttcatcgtg atggggatag atcattgcaa 1560
ttgttggtct tcaacgagga attcctagta agcgcgagtc atcagctcgc gttgactacg 1620
tccctgccct ttgtacacac cgcccgtcgc tcctaccgat tgaatgatcc ggtgaagtgt 1680
tcggatcgcg gcgacgtggg tggttcgccg tctgcgacgt cgcgagaagt ccactaaacc 1740
ttatcattta gaggaaggag aagtcgtaac aaggtttccg taggtgaacc tgcggaagga 1800
tcattgt 1807
<210> 4
<211> 6010
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 4
tgagatttta gagagtagaa taggtattga gagattaaga gagatttggt gagagattaa 60
gagaagttta gaagagattg tgaaagaaat ggagagatta agtatgatcc atgatgaaca 120
agagagagac tagagtgaga gtaacataat ccgcttaatc ttattaatga gagagtgttt 180
acaatatata ggaggtgcta ctaggggtta agaaaagact aagcatgtga ggtcaaagct 240
aagggagcct ttaatttgaa ccggaccaat gagcctctcc acacgcgtgg tcactatgct 300
aaaagtgaac cttatccttc cagcctgtgc cagcctgtag agacgctcct cctctttcca 360
tcaacggtca tatgccttct ttggtcaagg taaaacatga tctggtcaag taacttcttc 420
ccgcggtaac actcttcacc gcatggtcga taccgtctgt acggccgtac ggccatggta 480
cggacctgta cggaccggta cggacggttc tccctaaggc caatccttct cctctactca 540
acatcacaac ctcttcaacc ctgaacaact ctcctcatat tcctcagctc atctcaacac 600
tccccctcaa gcttgaccat gtggaacaag tcaagcttgg aaggatcatg agctccccct 660
cattaaaaac ctcatcaaag aaaacccatt gggacaaaac tctgatgagg gaaaaagagt 720
aaggacctca tgatcaggag agtgtataga tcagccctta ggcgaaagat caaggagtcc 780
caaccttatg tgaattgact ccattgtctt ttgcctagcc gccttagtga atacgtctgc 840
caactgatcc taactccttg tgtagcacga cagtatcact cccaagacta tcatctgcct 900
cactttatga cagtccactt caatatgctt ggttctctca tggaacaccg agtttgtggc 960
tatgtgaatg gctgcttgat tgtcacaatg catggtcatt ggtgttgctt gctcaatctc 1020
caagtgcttc agtattcctt taatccatac caactcattt gtgagcttta gcattgctct 1080
atactcagcc tcggcacttg agcatgacac caccttctgc ttcttactct tccaagtaac 1140
catgtttcca ccaatgaatg tgcaatagcc ggttgtagat cttctatctg ttctatctcc 1200
agcccaatct gcatcacaat atcccactgc ttcagtgctc ccattacaac ccatccatat 1260
accaagccct tgagttccat tcagatacat gagaactctt tctaccatgc gccagtgatg 1320
ctctcttgga gcttgcatat gctgactcac ttggttcaca gcaaaacaga tgtctggcct 1380
agtgatggtt agatagatca gcttccccac aagcttcctg tatagctttg gatcatgaaa 1440
taccttacta tcttctagct ccccctctcg tggagcttta tacccatcct ccatgggcat 1500
cttggctgtc tttcctccat acgcaccagc ctccttcaac agatccaaga tgtacttcct 1560
ttgtgagatg aacaaccctt cttctgatct gcacagctca attccaagga agtacttcat 1620
ttctcccaag tctttgatgt caaacacaga tttcagaaac tccttggtct ccttgattcc 1680
caccttatca ctgcctgtga taactatgtc atccacatat acaagaagca caacaatgcc 1740
tgcaggtgta gtaagagtga agagagtgtg atcaagctct gatttcctga agcctcttcc 1800
atttagagtt gtgctcagct tgtgatacca tgcccttggt gattgcttta acccatagat 1860
ggccttcttc aatctaagca cattccctgg tttcaccatc ccttctaggc ccggaggtgg 1920
tagcatgtaa acttcatctt ctaactctcc ttggagaaat gcattcttca catccatttg 1980
ccaaagatcc cacccaaggt tagtagccac tgataggact attcgaatgg tgtgtagctt 2040
tgcaactgga gcaaatgtat caatgtagtc ttctccatat gtctgagtga accctcttgc 2100
taccagtcta gtcttcttcc tctcaatcga cccatcagcc ttgtacttga ctgtaaagat 2160
ccatctacta gacacagcct tcttcccttt tggtagttca ctctcatacc atgtatcatt 2220
ctttatcata gctcctgcct cagctcctac tgattccttc cattcttcat cttccattgc 2280
ctcttcatag cttcttggaa tgtgattctc atctaagttt accataaatg cacaatgtgc 2340
ttctggatat tgagcaaagg aacacacggc ctgagtagga tgcttcacag cttgggcatt 2400
gtagtacact ctcgtgttta cccaactgga aggatccctt ctcagccttg tactccttct 2460
caacactggc ccttcttgcg ccatctcttg ttcttcttct acttgtggag tcactgcttc 2520
agtctgttct tcagcttcag ctctagcctc tccttgtctc tgatcttctt cattctgtct 2580
ttcattcagt tcttcccttg ctgatccttc tccactttgg acaggagaat cagttcctct 2640
gtttcctagc tcaccagcct cttctaagcc ttggtcctct agttcagaaa ctgattcact 2700
tcccccctca tgtgcaaagt gggatggttc ttctgctgca gctggagtag gttctggacg 2760
cctactctga tcctgggaca ctccaattcc gagcccctct agaatgtttc tcaagcaagc 2820
agctcgttct gatggctggg atagatcctc caagtcttcc catgtctgct catcatagta 2880
tcctttgtct tccatgaatc tgacatccct tgacactagg actctcctag ctgtcggatc 2940
atagcatttg tatcccttct gtgaagtaga atacccaatc atcatagcct tggtgctctt 3000
tgcttctagt ttgtttctct gctcgcctgg tacaagaaca taacacacgc atccaaaggt 3060
cctcaggtga tccaagactg gcctgctctt gttaagtacc tcaaatggag attgatctgc 3120
taagatcctg gttggtatcc tgttgatcag atagcacgca gtcatcacag catcactcca 3180
atacctcttg ggaacccctt tgtggaacat catagaccgg gacacctcca tgagatgtct 3240
gttcttcctt tcagcaactc cattctgctg tggagtgtat gggcagctag tctggtgaag 3300
aatcccatgt tgagcaaggt gatcacgaaa tgcatgccca gtatactccc caccattatc 3360
agacctgaaa atcttaatct tggcatgata ttggttagtc acataggctt gaaaattttt 3420
aaatgcatca aggaccctat ctttagtttt aatcaatgtt aaccaggtgt atttggattt 3480
ttcatcaatg aatgtgacat aatacttgta atcatcccta gataagcaag gtgcagtcca 3540
aacatcagag tgaattaaat caaagcagtt tgcataaaga gtagatgatc tttgaaaaac 3600
agtcttacag tgtttaccca aaatacaagc ctcacaatca ttatttttaa acatgacacc 3660
tggtaacata atgcttaaag ccctaacatg tggatggcct agcctagcat gccacagagc 3720
atcagtactt aaggaagaaa cagaactaaa cgagaaacaa gaactagaaa taggtgcaag 3780
gtcttcaatc atgtaaagat tccccttggt tgctccttgc ccaatcaact tactgctctt 3840
aatatcctga aacttaacat cattaggact gaaaataaca ttgcattgaa gatcagtagt 3900
gcatttctta actgataaga gattagaagt gaactcaggc atgtaaaaag cttttgaatc 3960
cttattaaac aagttcagtt taccaatgcc tctaatggga attctatctc catttgcaat 4020
cataacatga ccaagtgcag gttctatgtc ttttatcaga ttgttatcac taatcatgtg 4080
atgagatgca ccagaatcaa ttatcaatgg tttatgcaag ctcctagtag tattgattct 4140
agatgtttca ttttcagctt tcaggctctt aactactcct aacaatctat cagttatgct 4200
agtatgagca gtagcagtac atgaggtttt aaaaatgtct aacagcttat cagagatact 4260
aggtaatgta tgagcagagt atgagtatcc aagagtgtgg cctagcattt taccagactc 4320
cttgagggct cggatgaatg cctcaaagtc agccttggtg atcacctcct gagaagttag 4380
agccctgcct tcactttcac ccgccttgac atttggacta gcccctgatg agccagcacc 4440
acttgcttca gcagaaaggt gagccttagc ctctctatcc ttgttgaact tgctcggcct 4500
gaggtgtgga tgtaggatcc agcactgact cttcttatgc ccctgccgct tgcaatgctc 4560
acagcttccc tcatacttct catacttcct tccatctcca cggtacgctg ctatgtttgc 4620
ttgcgcctct tccgctttat gagccgtgga cattcccttc ttgcctccaa acaacccaag 4680
agagccttcc tccttctgaa gttgtgcgca tacctcctcc atggatggga gagtaggtga 4740
tctcaggata tgcttgatca catcctggta gctttcatct agagtcatca atagcccaaa 4800
cacctgatct tgctctctcc tctcaatgag cgattcctca tcagttgtgt tgggtctaag 4860
actctcaagt tcggaccaca acgctccaaa cttccccatg tgcttggtga gatcttctcc 4920
atcttgcctc aagacgctga tggtttgctt cagatcaaac acccgactta tgttggactt 4980
gtttccatac ctcttatgga gcatgtccca aaggcgctta ggagtctcaa agtgcacgta 5040
agcttcaagt atggggagct ccagagaggc atggagcaca gacagcacca tcaaatcctc 5100
ttggacccat ctctttgctt cagctaccgc aagagtcttc ccgtcgcctt cttcttcatc 5160
ttctttggcc actggctccg gaccatcatc tgtgatgtgg ttccacagcc ctagccttcc 5220
aatggtagtc ctcactaact gggaccacat caagtagttc gagcctccct tcagtgcaac 5280
cggaactgtc accagtttgc taaagttggt cgtctccatc tcctcttctt tatcacacaa 5340
gaacaaattc gcaaatgtaa agaaaggaac aaggatctct caataccaaa gccaacctgg 5400
ctctgatacc atgagatttt agagagtaga ataggtattg agagattaag agagatttgg 5460
tgagagatta agagaagttt agaagagatt gtgaaagaaa tggagagatt aagtatgatc 5520
catgatgaac aagagagaga ctagagtgag agtaacataa tccgcttaat cttattaatg 5580
agagagtgtt tacaatatat aggaggtgct actagggttt aagaaaagac taagcatgtg 5640
aggtcaaagc taagggagcc tttaatttga accggaccaa tgagcctctc cacacgcgtg 5700
gtcactatgc taaaagtgaa ccttatcctt ccagcctgtg ccagcctgta gagacgctcc 5760
tcctctttcc atcaacggtc atatgccttc tttggtcaag gtaaaacatg atctggtcaa 5820
gtaacttctt cccgcggtaa cactcttcac cgcatggtcg ataccgtctg tacggccgta 5880
cggccatggt acggacctgt acggaccggt acggacggtt ctccctaagg ccaatccttc 5940
tcctctactc aacatcacaa cctcttcaac cctgaacaac tctcctcata ttcctcagct 6000
catctcaaca 6010
Claims (10)
1. be used to identify the complete DNA molecular of brassica plant 1 B gene group, it includes A, the A for it is following 1) or 2)：
1) single strand dna shown in single strand dna shown in the sequence 1 of sequence table and the sequence 2 of sequence table is constituted；
2) single strand dna shown in the single strand dna and sequence B shown in sequence A is constituted；
Sequence A is the sequence more than 90% with the homology of sequence 1；
Sequence B is the sequence more than 90% with the homology of sequence 2.
2. complete DNA molecular according to claim 1, it is characterised in that：The complete DNA molecular also includes B,
The B for it is following 1) or 2)：
1) single strand dna shown in single strand dna shown in the sequence 3 of sequence table and the sequence 4 of sequence table is constituted；
2) single strand dna shown in the single strand dna and sequence D shown in sequence C is constituted；
Sequence C is the sequence more than 90% with the homology of sequence 3；
Sequence D is the sequence more than 90% with the homology of sequence 4.
3. it is used to identify the complete carrier of brassica plant 1 B gene group, including a,
Single strand dna shown in the recombinant vector and expressed sequence 2 of single strand dnas of a as shown in expressed sequence 1
Recombinant vector is constituted.
4. complete carrier according to claim 3, it is characterised in that：The complete carrier also includes b,
Single strand dna shown in the recombinant vector and expressed sequence 4 of single strand dnas of the b as shown in expressed sequence 3
Recombinant vector is constituted.
5. it is used to identify the kit of brassica plant 1 B gene group, including complete DNA molecular or power shown in claim 1 or 2
Profit requires the complete carrier shown in 3 or 4.
6. the complete carrier or claim shown in the complete DNA molecular or claim 3 or 4 according to claim 1 or 2
Kit described in 5, it is characterised in that：Single strand dna shown in the sequence 1-4 marks different fluorophors.
7. complete DNA molecular described in claim 1 or 2 or the complete carrier shown in claim 3 or 4 or the institute of claim 5 or 6
The kit stated in differentiation or supplementary globe or identification or auxiliary identification each bar chromosome of brassica plant 1 B gene group should
With.
8. complete DNA molecular described in claim 1 or 2 or the complete carrier shown in claim 3 or 4 or the institute of claim 5 or 6
Any bar chromosome of the kit stated 1 B gene group chromosome containing black mustard in identifying or aiding in identification brassica plant to be measured
In application；
Or, complete DNA molecular described in claim 1 or 2 or the complete carrier shown in claim 3 or 4 or claim 5 or 6
Described kit in the genome of measuring plants is treated in identification whether the chromosome containing the 1 B gene group from black mustard or certain
Application in chromosome.
9. the complete carrier or claim shown in the complete DNA molecular or claim 3 or 4 according to claim 1 or 2
Applied described in kit or claim 7 or 8 described in 5 or 6, it is characterised in that：The brassica plant is black mustard.
10. it is a kind of to identify or aid in identifying that the 1 B gene group chromosome in brassica plant to be measured from black mustard is which is dyeed
The method of body, comprises the following steps：Using the single strand dna in complete DNA molecular described in claim 1 or 2 as probe,
Dual FISH is carried out to brassica plant to be measured and black mustard respectively, according to the brassica plant 1 B gene group to be measured
The signal location of chromosome is compared with the signal location of 8 chromosomes of 1 B gene group of the black mustard, if the rape to be measured
The signal location of platymiscium 1 B gene group chromosome is consistent with the signal location of the 1 B gene group chromosome of black mustard, then described to treat
Survey 1 B gene group this chromosome that brassica plant 1 B gene group chromosome is or candidate is black mustard；
Or a kind of 8 methods of chromosome for distinguishing black mustard, comprise the following steps：Using complete DNA described in claim 1 or 2
Single strand dna in molecule carries out dual FISH as probe to black mustard, is contaminated according to the black mustard 1 B gene group
The signal location of colour solid distinguishes 8 chromosomes of black mustard；
Or the single strand dna shown in the single strand dna and the sequence 2 of sequence table shown in the sequence 1 of sequence table is as mirror
Application in the probe of the 1 B gene group of other black mustard；
Or application of the single strand dna shown in the sequence 1-4 of sequence table in as the probe of the 1 B gene group for differentiating black mustard.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116334299A (en) * | 2023-05-26 | 2023-06-27 | 浙江大学海南研究院 | Molecular marker of brassica 'U triangle' B genome specific sequence and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106048057A (en) * | 2016-08-08 | 2016-10-26 | 北京市农林科学院 | Two black mustard genomes DNA sequence and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106048057A (en) * | 2016-08-08 | 2016-10-26 | 北京市农林科学院 | Two black mustard genomes DNA sequence and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| GUI-XIANG WANG ET AL.: "Karyotypes and Distribution of Tandem Repeat Sequences in Brassica nigra Determined by Fluorescence in situ Hybridization", 《CYTOGENETIC AND GENOME RESEARCH》 * |
| LIM ET AL.: "Characterization rDNAs and Tandem Repeats in the Heterochromatin of Brassica rapa", 《MOLECULES AND CELLS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116334299A (en) * | 2023-05-26 | 2023-06-27 | 浙江大学海南研究院 | Molecular marker of brassica 'U triangle' B genome specific sequence and application thereof |
| CN116334299B (en) * | 2023-05-26 | 2023-09-01 | 浙江大学海南研究院 | Molecular marker of brassica 'U triangle' B genome specific sequence and application thereof |
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