CN112921069B - Primer group, capture probe and identification method for pear seed dormancy state identification and application of primer group and capture probe - Google Patents
Primer group, capture probe and identification method for pear seed dormancy state identification and application of primer group and capture probe Download PDFInfo
- Publication number
- CN112921069B CN112921069B CN202110324210.6A CN202110324210A CN112921069B CN 112921069 B CN112921069 B CN 112921069B CN 202110324210 A CN202110324210 A CN 202110324210A CN 112921069 B CN112921069 B CN 112921069B
- Authority
- CN
- China
- Prior art keywords
- sequence
- seq
- primer
- seeds
- enzyme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000000523 sample Substances 0.000 title claims abstract description 39
- 235000014443 Pyrus communis Nutrition 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 30
- 230000014284 seed dormancy process Effects 0.000 title claims description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 40
- 238000002965 ELISA Methods 0.000 claims abstract description 18
- 238000009396 hybridization Methods 0.000 claims abstract description 8
- 239000002299 complementary DNA Substances 0.000 claims abstract description 7
- 102000004190 Enzymes Human genes 0.000 claims abstract description 4
- 108090000790 Enzymes Proteins 0.000 claims abstract description 4
- 238000012216 screening Methods 0.000 claims abstract description 4
- 230000005059 dormancy Effects 0.000 claims description 26
- 238000001514 detection method Methods 0.000 claims description 24
- 230000014509 gene expression Effects 0.000 claims description 14
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 10
- 239000012634 fragment Substances 0.000 claims description 9
- 230000000295 complement effect Effects 0.000 claims description 8
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 claims description 7
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 claims description 7
- 229960005156 digoxin Drugs 0.000 claims description 7
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 claims description 7
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 229960002685 biotin Drugs 0.000 claims description 5
- 235000020958 biotin Nutrition 0.000 claims description 5
- 239000011616 biotin Substances 0.000 claims description 5
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 3
- 238000012408 PCR amplification Methods 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 claims 1
- 238000002372 labelling Methods 0.000 claims 1
- 230000003321 amplification Effects 0.000 abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 4
- 230000008105 immune reaction Effects 0.000 abstract 1
- 230000035784 germination Effects 0.000 description 12
- 108020004414 DNA Proteins 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000010586 diagram Methods 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 238000003475 lamination Methods 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 230000007958 sleep Effects 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 101150027068 DEGS1 gene Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Pathology (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a primer group for identifying dormant state of pear seeds, a capture probe, an identification method and application thereof, relates to enzyme-linked immune reaction, and belongs to the field of molecular biology. The method comprises the following steps: screening target genes, extracting RNA, preparing cDNA and synthesized primers, and carrying out target gene specific amplification; and after the probe is synthesized, hybridization and enzyme-linked immunosorbent assay are carried out, and finally, the color after the enzyme-linked immunosorbent assay is detected by using an enzyme-linked immunosorbent assay instrument, so that the dormant state of the seeds is accurately and rapidly identified.
Description
Technical Field
The invention relates to a primer group for identifying dormant state of pear seeds, a capture probe, an identification method and application thereof, and belongs to the technical field of molecular biology.
Background
In nature, seeds are a special storage organ of plants, and dormancy is biological property of the seeds, which is used for adapting to climate change, ensuring germination survival or resisting adverse environmental conditions. In actual production and life, most pear seeds are directly subjected to low-temperature laminated storage without dormancy state identification after being picked. Some pear seeds break dormancy and have germination capacity due to low-temperature weather or falling and rotting, and are uniformly treated without distinguishing. With the extension of the storage time, the viability and germination rate of the seeds are gradually reduced, and the seeds gradually lose germination capacity and even go moldy and rot after long-term storage. Therefore, the method for identifying whether the pear seeds are released from dormancy has important significance for guiding farmers to sow in time, ensuring production and avoiding loss.
However, the quality inspection of pear seeds in the market is mainly germination capacity detection, and the germination rate can be accurately detected by various germination tests, but the related method needs to be subjected to steps of swelling, lamination and the like, the required time is generally up to 2 weeks, and due to the variety and the number of pear seeds, proper environmental conditions are ensured, otherwise, data errors are generated, and the determination of the germination rate of the pear seeds is influenced. Meanwhile, whether the pear seeds are in a dormant state cannot be predicted, and the pear seeds which have broken dormancy during partial picking are subjected to secondary lamination, so that the vitality is reduced, the pear seeds cannot germinate, and the germination rate is low and economic loss is caused.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides a primer group, a capture probe and an identification method for identifying the dormant state of pear seeds and application thereof, can accurately and rapidly identify the dormant state of pear seeds through enzyme-linked immunosorbent assay, and provides an important basis for seed classification treatment.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a primer set for identifying a dormant state of pear seeds, the primer set comprises a primer SHVV-1 and a primer SHVV-2, and the nucleotide sequence of the primer SHVV-1 is shown in SEQ ID NO:1 and SEQ ID NO:2, the nucleotide sequence of the primer SHVV-2 is shown as SEQ ID NO:3 and SEQ ID NO: 4.
In a second aspect, the invention provides a capture probe for identifying the dormant state of pear seeds, which is characterized by comprising a TATATT interval sequence of 6bp and a detection sequence of 23bp, wherein the detection sequence is shown as SEQ ID NO. 5.
In a third aspect, the invention provides a method for identifying dormancy state of pear seeds, which is characterized by comprising the following steps:
extracting RNA from pear seeds of a sample to be detected, screening out target genes a and/or b, and synthesizing cDNA by taking the RNA as a template;
carrying out PCR amplification on cDNA by using the cloning primer set as claimed in claim 1 to obtain a target gene fragment;
the target gene fragment is hybridized with the hybridization mixture containing the capture probe of claim 2, then enzyme-linked immunosorbent assay is carried out, and the dormancy state of the seeds is judged according to the color after the enzyme-linked immunosorbent assay.
With reference to the third aspect, further, the capture probe is labeled with biotin, and the sequence of the capture probe is complementary to the sequence in the middle of the target gene fragment.
Further, the hybridization mixture comprises a digoxin-labeled detection probe, and the sequence of the detection probe is complementary to the sequence of the detection probe.
Further, the nucleotide sequences of the target gene a and the target gene b are shown in SEQ ID NO:9 and SEQ ID NO: shown at 10.
Further, the hybridization mixture comprises a digoxin-labeled detection probe, and the detection sequence is complementary to the detection probe sequence.
Further, after the ELISA, detecting the color after ELISA by using an ELISA meter, if the detection color is yellow-green, the gene expression level in the sample is higher, and the seed breaks dormancy; if colorless, the sleep state is not broken.
In a fourth aspect, the invention provides a primer set for identifying dormant state of pear seeds, a capture probe, and application of any of the above identification methods in identifying dormant state of seeds and classifying seeds.
Compared with the prior art, the invention has the beneficial effects that:
compared with the time length of various germination experiments, the primer group, the capture probe and the identification method for identifying the dormant state of the pear seeds can identify the dormant state of the pear seeds within one day, and the identification efficiency is high; compared with germination experiments, the identification method provided by the invention is not easily influenced by environmental conditions, and from the aspect of molecular biology, the dormancy state of seeds can be accurately and clearly identified, and the accuracy is high; the identification method can realize quantification and standardization, provides possibility for subsequent large-scale application, and has practical value.
Drawings
FIG. 1 is a flow chart of the method for identifying the dormant state of pear seeds by using an ELISA method in an embodiment of the invention;
FIG. 2 is a schematic diagram of the identification of the dormant state of pear seeds according to the embodiment of the present invention;
FIG. 3 is a diagram showing changes in gene expression amounts of target gene a before and after release of dormancy of seeds according to an embodiment of the present invention;
FIG. 4 is a diagram showing changes in gene expression amounts of target gene b before and after release of dormancy of seeds according to an embodiment of the present invention;
FIG. 5 is a schematic diagram showing the color change of the target gene a or b before and after dormancy of seeds by ELISA detection in the embodiment of the invention.
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.
In order to solve the problem that the seed dormancy state identification method is lack in practical production, the method provided by the invention can rapidly and accurately identify the seed dormancy state, shows that the release of seed dormancy involves a large number of gene expression regulation and control changes according to early research, obtains a gene sequence by comparing the results of transcriptome sequencing of seeds in different dormancy states with a genome database, and annotates and analyzes functions of differential genes. Based on this, differential expression genes (Differentially expressed genes, DEGs) at different stages were stratified, the expression level of the genes was counted by using the number of reads (Reads per kilobase transcriptomeper million mapped reads, RPKM) per million reads from a certain gene per thousand base length, and the reliability of the transcriptome data results was confirmed by qPCR experiments. Further screening on the basis, wherein the standard is FPKM > =2, [ log2foldchange ] > =1, the gene of padj < = 0.05 is a differential expression gene, and two corresponding target genes with the expression level highly related to the dormancy degree of seeds are respectively selected according to the consistency of the variation of the expression quantity of the differential gene and the dormancy state, namely a (Chr17.g25338) and b (Chr6.g52718), wherein the nucleotide sequences of the target gene a and the target gene b are shown as SEQ ID NO:9 and SEQ ID NO:10, the expression levels all differed significantly before and after sleep release.
And detecting the expression condition of the a or b gene in the pear seeds by an enzyme-linked immunosorbent assay, namely the content of the gene protein, and identifying whether the pear seeds are released from dormancy or not by using colors. FIG. 1 is a flow chart of the method for identifying seed dormancy according to the ELISA method of the embodiment of the invention; as shown in FIG. 2, a schematic diagram of the identification of seed dormancy status is shown in an embodiment of the present invention.
The invention takes a pear seed dormancy state identification method as an example, and specifically comprises the following steps:
step 1.Rna extraction: and extracting total RNA of pear seeds by using a CTAB method, and detecting the concentration and purity of the total RNA.
Step 2.CDNA preparation: preparing a mixed solution according to a reverse transcription reaction system, fully and uniformly mixing, incubating for 15min at 42 ℃ and incubating for 3min at 95 ℃ and then placing on ice to obtain pear total cDNA.
Table 1: reverse transcription reaction system:
10×Fast RT Buffer | 2μl |
RT Enzyme Mix | 1μl |
FQ-RT Primer Mix | 2μl |
RNase-Free ddH2O | 5μl |
total system | 10μl |
Step 3, target gene specific amplification: according to the known cDNA gene sequence, the gene sequence of mRNA is obtained, a cloning primer (the cloning primer sequence is shown in table 2), a forward primer is marked by Biotin (Biotin), a gene DNA fragment is amplified by a PCR system (the PCR reaction system is shown in table 3, the reaction condition is shown in table 4), and the sizes of products obtained by the primer amplification are about 970bp and 570bp respectively.
Table 2: primer sequences
Table 3: amplification PCR reaction system:
table 4: PCR reaction procedure:
step 4, synthesizing a probe: a biotin-labeled capture probe (the sequence of the capture probe is shown in Table 5) is synthesized, the sequence of which is complementary to the sequence in the middle of the PCR amplified fragment, and the kit further comprises a 6bp TATATT spacer sequence and a 23bp detection sequence (5'-TACATTCGCAATTGAGGCTTCGT-3'), and the detection sequence is complementary to the Digoxin-labeled detection probe sequence. Each probe has a Tm value of 60℃and after synthesis, the probe is dissolved in PBS.
Table 5: capture probe sequences
Step 5, hybridization: mu.l of PBST buffer and 50. Mu.g of streptavidin-coated magnetic beads (Roche, germany) were added per 5. Mu.l of PCR amplification product, and incubated at 94℃for 30min. Mu.l of denaturing solution (0.5 mol/LNaOH,10 mmol/LEDTA) was added; the cells were washed 3 times with PBST buffer, 200. Mu.l of hybridization buffer (5 XSCC buffer, 0.3% Tween-20, 50pmol of gene probe denatured in advance and digoxin-labeled detection probe) was added, hybridized 2 hours at 50℃and washed 3 times with PBST buffer.
Step 6, enzyme-linked immunosorbent assay: PBST buffer containing 1% digoxin alkaline phosphatase (Anti-digoxigenin IgG Fab fragment conjugated with alkaline phosphatase, roche, germany) was added and incubated for 30min at room temperature, after 2 washes with PBST, the reaction was further carried out with PNPP at 37℃for 2h, and finally the reaction was stopped with 3N NaOH.
Step 7, signal detection: the dormancy state of the seeds can be distinguished and judged by distinguishing the colors, the concentration of the reaction color is detected at 405nm by using an enzyme-labeled instrument, if the color is yellow-green, the gene expression quantity in the sample is higher, the seeds break dormancy, and the seeds can be directly dried, stored or transacted; if the seed is colorless, dormancy is not broken, and dormancy germination can be broken only by low-temperature lamination for a certain time.
The changes in gene expression levels of the target gene a and the target gene b before and after the release of dormancy of seeds according to the examples of the present invention are shown in FIGS. 3 and 4, respectively. FIG. 5 is a schematic diagram showing the color change of target genes a and b before and after releasing dormancy of seeds by ELISA detection, wherein the color changes become yellow and deepen sequentially before and after releasing dormancy of seeds.
The method for identifying the dormant state of the pear seeds not only can accurately judge the dormant state of the pear seeds, but also has simple and convenient detection process, and can be used for identifying the dormant state of the seeds first and then determining the subsequent treatment mode in the process of storing and operating the pear seeds. Can help farmers judge and select the treatment mode of corresponding seeds, and timely sow or carry out low-temperature lamination to break dormancy. The method can provide important basis for seed classification treatment, avoids loss caused by secondary layer and has good practicability.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the technical principles of the present invention, and such modifications and variations should also be regarded as being within the scope of the invention.
Sequence listing
<110> university of Yangzhou
<120> primer set for pear seed dormancy state identification, capture probe, identification method and application thereof
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
atggatgtga tggattgctt g 21
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
tcaccttaaa ctttctccaa g 21
<210> 3
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
atggcttcca aggctgtagc t 21
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
tcatgctatc tttgagccgt t 21
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
tacattcgca attgaggctt cgt 23
<210> 6
<211> 6
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
tatatt 6
<210> 7
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
aacgatctca gccttggaat ggga 24
<210> 8
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
caagacatta gccttgattg cagt 24
<210> 9
<211> 966
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
atggatgtga tggattgctt gaatgagagc acgagtgact catcaaataa tggggtaaag 60
gatgcaagtt ctaacaagtt gccttcttct aggtacaagg gagtagtacc acagcccaat 120
ggcagatggg gagctcaaat atatgagaag caccaacgtg tgtggttggg aaccttcaat 180
gaagaagaag aagctgctaa gacctacgac attgccttgc taaagttccg gggacttgat 240
gccatcacaa acttctgtca gagccaaata aaaccctaca tagaagatgg caacgaggcc 300
attttcttgg aatcccattc caaggctgag atcgttgaca tgcttcgaaa acactcgtat 360
gtcaacgagc ttgaaatgta caagcataag ttgctcaacg ccggagttcg tgatggtggt 420
cgtaagcgaa gtaagtgtca cgtcgatcca atcgacgcgt gttatgagag ggagttgctt 480
ttcgagaagg tggcgacgcc aagcgatgta gggaggttga atcgtatggt gataccaaaa 540
caacaagctg agaagcattt tcaggttcat cagagtgtag aactgtgtaa aggagttttg 600
ttgaattttg aggatgagga agggaacgtg tggaggttta ggtattgtta ttggagtagt 660
agtcagagtt atgtgttgac caaaggatgg acgcgttttg tgaaggagaa gaagttgaaa 720
gctggtgatg ttgtgaggat tcagagatcg gcaagggagg ataagaagct gttcattgaa 780
tgtagacata gaaacgtcga tagtctgggg ttcagagaga tgcctgctgg ccgggttgct 840
gagccttcag cggcggttca ggatgatgga gtggtgaggt tgttcggagt taacattatg 900
aaaacatgta aaagttgcat tgtagacaat agcaggtgtc ggaggcttgg agaaagttta 960
aggtga 966
<210> 10
<211> 570
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
atggcttcca aggctgtagc ttccactgcc ctcctcctct ccctcaacct cctcttcttc 60
accttggtca cctccaccaa tgttgactgc ccaccacctg caaaaaaggg acaccaccct 120
aagcacgcaa gtcctgcact accaaacccc aagccctcta agcgtgccac atgcccaata 180
gacactttga aattgggagt atgtgctgac gtgttgaatg gtttggtgca ccttgtcgtt 240
ggtccaccaa agttcccatg ctgcagcctc attgagggcc ttgttgacct tgatgctgct 300
gtgtgccttt gcactgcaat caaggctaat gtcttgggga tccacttaaa cgtccccgtt 360
tcactgagcc tgctcataaa ctactgcggg aagcagaagt acatgcttct aaataagttt 420
ttgttggggg atttttacgt ggacttggac tttgagaacg tcctcttgtc tcccttggta 480
cagattggta acaagctccg agatatattg aaagagatag tttgtcttag gaaataccct 540
ggtaatatca acggctcaaa gatagcatga 570
Claims (3)
1. The primer group for identifying the dormant state of the pear seeds is characterized by comprising a primer SHVV-1 and a primer SHVV-2, wherein the nucleotide sequence of the primer SHVV-1 is shown as SEQ ID NO:1 and SEQ ID NO:2, the nucleotide sequence of the primer SHVV-2 is shown as SEQ ID NO:3 and SEQ ID NO: 4.
2. The pear seed dormancy state identification method is characterized by comprising the following steps:
extracting RNA from pear seeds of a sample to be detected, screening out target genes a and/or b, and synthesizing cDNA by taking the RNA as a template, wherein the nucleotide sequence of the target gene a is shown as SEQ ID NO:9, the nucleotide sequence of the target gene b is shown as SEQ ID NO:10 is shown in the figure;
carrying out PCR amplification on cDNA by using the primer set as set forth in claim 1 to obtain a target gene fragment, and labeling a forward primer with biotin;
the method comprises the steps of hybridizing a target gene fragment with hybridization mixed solution containing a capture probe, and then performing enzyme-linked immunosorbent assay, wherein the enzyme-linked immunosorbent assay reagent comprises a PBST buffer solution containing 1% digoxin alkaline phosphatase and PNPP, detecting the color after enzyme-linked immunosorbent assay by using an enzyme-labeled instrument at 405nm, and if the detection color is yellow-green, indicating that the gene expression level in a sample is higher, and the seed breaks dormancy; if colorless, the dormancy state is not broken;
the hybridization mixture comprises a digoxin-labeled detection probe, and the sequence of the detection probe is complementary to the sequence of the detection probe;
the capture probe consists of a sequence complementary to the middle sequence of the target gene fragment, a TATATT interval sequence of 6bp and a detection sequence of 23bp, wherein the detection sequence is shown as SEQ ID NO.5, and the sequence complementary to the middle sequence of the target gene fragment is shown as SEQ ID NO.7 or SEQ ID NO. 8.
3. Use of the primer set of claim 1 or the identification method of claim 2 for identifying a dormant state of pear seeds.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110324210.6A CN112921069B (en) | 2021-03-26 | 2021-03-26 | Primer group, capture probe and identification method for pear seed dormancy state identification and application of primer group and capture probe |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110324210.6A CN112921069B (en) | 2021-03-26 | 2021-03-26 | Primer group, capture probe and identification method for pear seed dormancy state identification and application of primer group and capture probe |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112921069A CN112921069A (en) | 2021-06-08 |
CN112921069B true CN112921069B (en) | 2023-12-08 |
Family
ID=76176148
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110324210.6A Active CN112921069B (en) | 2021-03-26 | 2021-03-26 | Primer group, capture probe and identification method for pear seed dormancy state identification and application of primer group and capture probe |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112921069B (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106973581A (en) * | 2017-04-07 | 2017-07-25 | 扬州大学 | A kind of method for releasing pears seed dormancy |
CN106987592A (en) * | 2017-04-07 | 2017-07-28 | 扬州大学 | A kind of method for suppressing pears seed gibberellin synthetic gene expression |
-
2021
- 2021-03-26 CN CN202110324210.6A patent/CN112921069B/en active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106973581A (en) * | 2017-04-07 | 2017-07-25 | 扬州大学 | A kind of method for releasing pears seed dormancy |
CN106987592A (en) * | 2017-04-07 | 2017-07-28 | 扬州大学 | A kind of method for suppressing pears seed gibberellin synthetic gene expression |
Non-Patent Citations (5)
Title |
---|
Transcription factor TCP20 regulates peach bud endodormancy by inhibiting DAM5/DAM6 and interacting with ABF2;Qingjie Wang等;J Exp Bot;第71卷(第4期);第1585–1597页 * |
Transcriptome sequencing and analysis of major genes involved in calcium signaling pathways in pear plants (Pyrus calleryana Decne.);Yuanyuan Xu 等;BMC Genomics(第16期);第738-750页 * |
利用酶联免疫反应鉴定梨树S基因型;王春雷等;江苏农业学报;第31卷(第05期);正文第3段,1.2方法 * |
杜梨种子休眠与萌发过程中酶活性变化;李军霞等;北方园艺(第11期);第21-24页 * |
梨花芽休眠相关miRNA的鉴定和差异表达分析;马鑫瑞等;园艺学报;第45卷(第11期);第22-38页 * |
Also Published As
Publication number | Publication date |
---|---|
CN112921069A (en) | 2021-06-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN113637789B (en) | Wheat stripe rust resistance gene YrTD121 linked KASP molecular marker, primer, kit and application | |
WO2023208078A1 (en) | Genome structure variation for regulating tomato fruit soluble solid content, related product, and application | |
CN103160937A (en) | Method for conducting enrichment library construction and SNP analysis on gene of complex genome of higher plant | |
CN103571822B (en) | A kind of multipurpose DNA fragmentation enriching method analyzed for new-generation sequencing | |
CN111235295A (en) | KaSP molecular marker of wheat powdery mildew resistance gene Pm21 derived from haynaldia villosa and application thereof | |
WO2019085546A1 (en) | Method for constructing microbial 16s rdna single-molecule level sequencing library | |
US12084652B2 (en) | Methods and compositions for processing samples containing nucleic acids | |
CN105506747B (en) | It is enriched with construction method and its application in the libraries RNA of primary transcript information | |
CN109207611A (en) | One kind SNP marker relevant to sheep heat character and its detection kit and application | |
CN106497916A (en) | A kind of construction method in the NK cell polygenic variations library for high-flux sequence detection and its application | |
CN112921069B (en) | Primer group, capture probe and identification method for pear seed dormancy state identification and application of primer group and capture probe | |
CN117683927A (en) | Functional KASP molecular marker of rice blast resistance gene and application thereof | |
CN110878376B (en) | SSR molecular marker primer for identifying dendrobium huoshanense and application thereof | |
KR102226016B1 (en) | Marker for identification of chloroplast derived from Ilex integra and Primer set for identification of Ilex x wandoensis | |
CN106167825A (en) | Microsatellite marker that a kind of yellow catfish growing characteristic is relevant and detection thereof and application | |
CN107447022B (en) | SNP molecular marker for predicting corn heterosis and application thereof | |
CN110964790B (en) | Method for detecting tea-goat PIGY gene CNV marker and application thereof | |
CN113755628A (en) | Mixed sample detection method for detecting purity of white radish seeds based on mSNP technology | |
CN114058732A (en) | Primer combination for efficiently identifying different alleles of grain weight gene GW5, identification method and application | |
CN113963745A (en) | Method for constructing plant development molecule regulation network and application thereof | |
CN117004743B (en) | SNP (Single nucleotide polymorphism) marker related to multi-lamb character of black goats on cloud and application of SNP marker | |
CN112251516A (en) | Method for detecting standard weight day age of pig by using No. 7 chromosome of pig and kit thereof | |
KR20220090737A (en) | SNP marker composition for discriminating Korean native chicken or new breed chicken and uses thereof | |
WO2020199127A1 (en) | Design of sequencing primers and pcr-based method for sequencing whole genome | |
CN117965787B (en) | SNP (Single nucleotide polymorphism) marker and primer set for identifying authenticity of pineapple Josapine and MD2 hybrid and application of SNP marker and primer set |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |