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
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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.
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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.
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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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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 * |
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