CN112921069A - Primer group for identifying dormant state of pear seeds, capture probe, identification method and application of primer group - Google Patents
Primer group for identifying dormant state of pear seeds, capture probe, identification method and application of primer group Download PDFInfo
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Abstract
The invention discloses a primer group for identifying a dormant state of pear seeds, a capture probe, an identification method and application thereof, relates to enzyme-linked immunosorbent assay, and belongs to the field of molecular biology. The method comprises the following steps: screening target genes, then carrying out RNA extraction, preparing cDNA and synthesizing primers to carry out target gene specific amplification; and (3) synthesizing a probe, performing hybridization and enzyme-linked immunosorbent assay, and finally detecting the color of the enzyme-linked immunosorbent assay by using an enzyme-linked immunosorbent assay to accurately and quickly identify the dormant state of the seeds.
Description
Technical Field
The invention relates to a primer group for identifying a dormant state of pear seeds, a capture probe, an identification method and application thereof, belonging to the technical field of molecular biology.
Background
The seeds are the unique organs of plants in nature, and the dormancy refers to the biological characteristics of the seeds generated for adapting to climatic change and ensuring germination and survival or resisting adverse environmental conditions. In actual production and life, most of pear seeds are directly stored at low temperature without being identified in a dormant state after being picked. Part of pear seeds break dormancy and have germination capacity due to low-temperature weather or dropping and rotting, and the like, but are uniformly treated without distinguishing and distinguishing. With the prolonged storage time, the viability and germination rate of the seeds are gradually reduced, and the seeds gradually lose germination capacity and even become mildewed and rotten 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, most of the quality tests of pear seeds on the market currently detect the germination capacity, the germination rate is tested by multiple germination tests, although the related methods can accurately detect the germination rate, the steps of imbibition, lamination and the like are required, the required time is generally 2 weeks, and due to the fact that the pear seeds are numerous in variety and quantity, proper environmental conditions are guaranteed, otherwise, data errors are generated, and the determination of the germination rate of the pear seeds is influenced. Meanwhile, because whether the pear seeds are in a dormant state cannot be predicted, the vitality of part of the pear seeds which break the dormancy during picking is reduced due to secondary lamination, 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, an identification method and application for identifying the dormant state of pear seeds, can accurately and quickly identify the dormant state of the pear seeds through enzyme-linked immunosorbent assay, and provides an important basis for seed classification treatment.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a primer group for identifying a dormant state of pear seeds, wherein the primer group comprises a primer SHVV-1 and a primer SHVV-2, and the nucleotide sequence of the primer SHVV-1 is shown as SEQ ID NO: 1 and SEQ ID NO: 2, and the nucleotide sequence of the primer SHVV-2 is shown as SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
In a second aspect, the invention provides a capture probe for identifying a dormant state of pear seeds, which is characterized in that the capture probe comprises a TATATT spacer sequence of 6bp and a detection sequence of 23bp, and the detection sequence is shown as SEQ ID NO. 5.
In a third aspect, the invention provides a pear seed dormancy state identification method, which is characterized by comprising the following steps:
extracting RNA from pear seeds of a sample to be detected, screening target genes a and/or b, and synthesizing cDNA by taking the RNA as a template;
performing PCR amplification on the cDNA by using the cloning primer set according to claim 1 to obtain a target gene fragment;
and (3) hybridizing the target gene fragment with the hybridization mixed solution containing the capture probe of claim 2, performing enzyme-linked immunosorbent assay, and judging the dormancy state of the seeds according to the color after the enzyme-linked immunosorbent assay.
In combination with the third aspect, further, the capture probe is labeled with biotin, and the capture probe sequence is complementary to the middle sequence 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 detection sequence.
Further, the nucleotide sequences of the target gene a and the target gene b are shown as 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 enzyme-linked immunosorbent assay, detecting the color of the enzyme-linked immunosorbent assay by using an enzyme-linked immunosorbent assay instrument, wherein if the detected color is yellow green, the expression level of the gene in the sample is high, and the seed breaks dormancy; if the color is not changed, the dormant state is not broken.
In a fourth aspect, the invention relates to a primer group and a capture probe for identifying the dormant state of pear seeds, and an application of any one of the identification methods in identifying the dormant state of seeds and classifying the seeds.
Compared with the prior art, the invention has the following beneficial effects:
compared with the time length of various germination experiments, the primers, the capture probes, the identification method and the application thereof for identifying the dormant state of the pear seeds can identify the dormant state of the pear seeds within one day, and have high identification efficiency; compared with a germination experiment, the identification method provided by the invention is not easily influenced by environmental conditions, can accurately and clearly identify the dormant state of the seeds from the perspective of molecular biology, and has high accuracy; 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 enzyme-linked immunosorbent assay for identifying the dormant state of pear seeds according to the embodiment of the invention;
FIG. 2 is a schematic diagram illustrating the identification of the dormant state of pear seeds according to an embodiment of the present invention;
FIG. 3 is a schematic diagram showing the changes in the expression level of a target gene a before and after dormancy breaking in an example of the present invention;
FIG. 4 is a schematic diagram showing the changes in the expression level of a target gene b before and after dormancy breaking in an example 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 breaking by ELISA detection.
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
In order to solve the problem, the method for identifying the dormant state of the pear seeds can quickly and accurately identify the dormant state of the pear seeds, the dormancy release of the seeds is shown to relate to a large amount of gene expression regulation and control changes according to early research, transcriptome sequencing is carried out on the seeds in different dormant states, and the result is compared with a genome database to obtain a gene sequence, and the annotation and the function analysis of a differential gene are carried out. On the basis, Differentially Expressed Genes (DEGs) in different periods are analyzed, the expression amount of the genes is counted by using the value of Reads per kilokaline length of a certain gene in each million Reads (RPKM), and the reliability of the result of the transcriptome data is confirmed by utilizing a qPCR experiment. On the basis of the above, the genes with the standard of FPKM > 2, [ log2foldchange ] > <1, padj < 0.05 are differential expression genes, and two corresponding target genes with expression levels highly correlated with the degree of dormancy of seeds, namely a (Chr17.g25338) and b (Chr6.g52718), are screened according to the consistency of the expression level change of the differential genes and the dormancy state, 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 were all significantly different before and after dormancy release.
And detecting the expression condition of the gene a or b 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 color. FIG. 1 is a flow chart of the enzyme-linked immunosorbent assay for identifying the dormant state of the seeds according to the embodiment of the invention; fig. 2 is a schematic diagram illustrating the identification of the seed dormancy state according to 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 (3) extracting the total RNA of the pear seeds by using a CTAB method, and detecting the concentration and purity of the total RNA.
Step 2.cDNA preparation: and (3) preparing a mixed solution according to a reverse transcription reaction system, fully and uniformly mixing, incubating at 42 ℃ for 15min, incubating at 95 ℃ for 3min, and placing on ice to obtain the 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 |
| general System | 10μl |
And 3, specific amplification of the target gene: according to the known cDNA gene sequence, obtaining the gene sequence of mRNA, designing clone primers (the clone primer sequence is shown in table 2), marking the forward primer by Biotin (Biotin), amplifying the gene DNA fragment by using a PCR system (the PCR reaction system is shown in table 3, the reaction conditions are shown in table 4), and obtaining the product sizes of 970bp and 570bp respectively by the primer amplification.
Table 2: primer sequences
Table 3: and (3) amplification of a PCR reaction system:
table 4: PCR reaction procedure:
step 4, probe synthesis: synthesizing a biotin-labeled capture probe (the sequence of the capture probe is shown in Table 5), wherein the sequence of the capture probe is complementary to the middle sequence of the PCR amplified fragment, and the capture probe also comprises a 6bp TATATT spacer sequence and a 23bp detection sequence (5'-TACATTCGCAATTGAGGCTTCGT-3'), and the detection sequence is complementary to a Digoxin (Digoxin) labeled detection probe sequence. The Tm value of each probe was 60 ℃ and the probe was dissolved in PBS after synthesis.
Table 5: capture probe sequence
Step 5, hybridization: each 5. mu.l of PCR amplification product was incubated with 100. mu.l of PBST buffer and 50. mu.g of streptavidin-coated magnetic beads (Roche, Germany) at 94 ℃ for 30 min. Add 50. mu.l denaturing solution (0.5mol/LNaOH, 10 mmol/LEDTA); washing with PBST buffer 3 times, adding 200. mu.l hybridization buffer (5 XSCC buffer, 0.3% Tween-20, 50pmol of pre-denatured gene probe and digoxigenin-labeled detection probe), hybridizing at 50 ℃ for 2h, and washing with PBST buffer 3 times.
Step 6, enzyme-linked immunoassay: PBST buffer containing 1% Anti-digoxigenin IgG Fab fragment conjugated with alkaline phosphatase, Roche, Germany was added and incubated at room temperature for 30min, washed 2 times with PBST, reacted with PNPP at 37 ℃ for 2h, and finally stopped with 3N NaOH.
And step 7, signal detection: distinguishing and judging the dormant state of the seeds by distinguishing colors, detecting the color concentration of reaction at 405nm by using an enzyme-labeling instrument, and if the color is yellow green, indicating that the gene expression level in a sample is higher and the seeds break dormancy, and directly drying, storing or trading; if the seed is colorless, dormancy is not broken, and low-temperature stratification for a certain time is needed to break dormancy germination.
FIGS. 3 and 4 are schematic diagrams showing changes in the expression levels of the target gene a and the target gene b before and after dormancy breaking, respectively, according to the embodiment of the present invention. FIG. 5 is a schematic diagram of the color change of target genes a and b before and after the dormancy of seeds is resolved by enzyme-linked immunosorbent assay, wherein the color changes to yellow and deepen sequentially before and after the dormancy of seeds is resolved.
The pear seed dormancy state identification method provided by the invention not only can accurately judge the pear seed dormancy state, but also has simple and convenient detection process, and can be used for identifying the seed dormancy state and then determining a subsequent treatment mode in the pear seed harvesting, storing and operating processes. Can help peasant household to judge and select the treatment mode of corresponding seed, in time sow or carry out low temperature and stratify and break dormancy. The method can provide important basis for the classification treatment of the seeds, avoids the loss caused by secondary stratification and has good practicability.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
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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 (9)
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, and the nucleotide sequence of the primer SHVV-2 is shown as SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
2. The capture probe for identifying the dormant state of the pear seeds is characterized by comprising a TATATT spacer sequence of 6bp and a detection sequence of 23bp, wherein the detection sequence is shown as SEQ ID NO. 5.
3. 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 target genes a and/or b, and synthesizing cDNA by taking the RNA as a template;
performing PCR amplification on the cDNA by using the cloning primer set according to claim 1 to obtain a target gene fragment;
and (3) hybridizing the target gene fragment with the hybridization mixed solution containing the capture probe of claim 2, performing enzyme-linked immunosorbent assay, and judging the dormancy state of the seeds according to the color after the enzyme-linked immunosorbent assay.
4. The method for identifying the dormant state of pear seeds as claimed in claim 3, wherein the capture probe is labeled by biotin, and the sequence of the capture probe is complementary to the middle sequence of the target gene segment.
5. The method for identifying the dormant state of pear seeds as claimed in claim 3, wherein the hybridization mixture comprises a digoxin-labeled detection probe, and the sequence of the detection probe is complementary to that of the detection sequence.
6. The method for identifying the dormant state of pear seeds as claimed in claim 3, wherein 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.
7. The method for identifying the dormant state of pear seeds as claimed in claim 3, wherein the hybridization mixture comprises a digoxin-labeled detection probe, and the detection sequence is complementary to the detection probe sequence.
8. The pear seed dormancy state identification method according to claim 3, wherein after enzyme-linked immunosorbent assay, the enzyme-linked immunosorbent assay is used for detecting the color after enzyme-linked immunosorbent assay, and if the detected color is yellow-green, it indicates that the gene expression level in the sample is high, and the dormancy of the seeds is broken; if the color is not changed, the dormant state is not broken.
9. Use of the primer set of claim 1, the capture probe of claim 2, the identification method of claim 3 for identifying the dormant state of a seed and the classification of a seed.
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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 |
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2021
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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 |
|---|
| QINGJIE WANG等: "Transcription factor TCP20 regulates peach bud endodormancy by inhibiting DAM5/DAM6 and interacting with ABF2", J EXP BOT, vol. 71, no. 4, pages 1585 * |
| YUANYUAN XU 等: "Transcriptome sequencing and analysis of major genes involved in calcium signaling pathways in pear plants (Pyrus calleryana Decne.)", BMC GENOMICS, no. 16, pages 738 - 750 * |
| 李军霞等: "杜梨种子休眠与萌发过程中酶活性变化", 北方园艺, no. 11, pages 21 - 24 * |
| 王春雷等: "利用酶联免疫反应鉴定梨树S基因型", 江苏农业学报, vol. 31, no. 05, pages 3 * |
| 马鑫瑞等: "梨花芽休眠相关miRNA的鉴定和差异表达分析", 园艺学报, vol. 45, no. 11, pages 22 - 38 * |
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