CN117757984B - DNA probe, kit and method for identifying sorghum halepense - Google Patents
DNA probe, kit and method for identifying sorghum halepense Download PDFInfo
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- CN117757984B CN117757984B CN202410196295.8A CN202410196295A CN117757984B CN 117757984 B CN117757984 B CN 117757984B CN 202410196295 A CN202410196295 A CN 202410196295A CN 117757984 B CN117757984 B CN 117757984B
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- 240000002439 Sorghum halepense Species 0.000 title claims abstract description 50
- 108020003215 DNA Probes Proteins 0.000 title claims abstract description 20
- 239000003298 DNA probe Substances 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 title claims abstract description 9
- 210000003763 chloroplast Anatomy 0.000 claims abstract description 28
- 241000209072 Sorghum Species 0.000 claims abstract description 10
- 235000011684 Sorghum saccharatum Nutrition 0.000 claims abstract description 10
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- 241000196324 Embryophyta Species 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 230000000155 isotopic effect Effects 0.000 claims description 4
- 239000000700 radioactive tracer Substances 0.000 claims description 3
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- 102000004190 Enzymes Human genes 0.000 claims description 2
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- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The application relates to a DNA probe, a kit and a method for identifying sorghum halepense, wherein the DNA probe has a nucleotide sequence shown as SEQ ID NO. 1. According to the alignment result of the genome of the sorghum halepense and the sorghum chloroplast, a difference site exists in the 4 kb region. Through this one site, sanger sequencing was performed on the PCR amplified products and the sequencing results were aligned, which showed that the presence of an 11 bp insertion site at this site on the sorghum halepense was able to distinguish sorghum halepense from sorghum halepense. The locus is stably existing between the sorghum halepense and the sorghum halepense, an oligonucleotide fluorescent probe is designed according to the locus, molecular biological methods such as RPA and the like can be applied to detect the sorghum halepense and sorghum halepense leaf tissues, and molecular identification can be realized by whether fluorescent signals exist or not, so that the sorghum halepense and the sorghum halepense can be reliably and effectively distinguished.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a DNA probe, a kit and a method for identifying sorghum halepense.
Background
The sorghum halepense is a grass plant of the grass family, has strong fertility, strong stress resistance and plant toxicity, is often mixed in fields to invade crop resources and provide hosts for other harmful organisms, has morphological characteristics very similar to that of sorghum halepense, is a malignant weed which is difficult to control in the agricultural planting industry, and is one of external invasive plants which are important to manage in China.
The molecular identification technology is a technology for identifying molecular level information based on biological genome difference, and compared with subjectivity and complexity of traditional morphological feature identification, the molecular identification technology can make the distinction between species considerable and simplified in the form of sequence data so as to obtain more reliable identification results more efficiently.
Chloroplast genome (cpDNA) molecular markers are of great importance in the field of plant science, in particular in phylogenetic, evolutionary biology, species identification, population genetics, ecology and protective biology. The chloroplast genome is typically a single circular molecule, ranging in size from 120-160 kb, containing a series of genes encoding photosynthesis, electron transfer and other biosynthetic pathways, with high genetic stability, low recombination rates and maternal genetic properties. The characteristics enable chloroplast genome to be a high-efficiency and reliable molecular marker, can provide high-resolution information, and is particularly suitable for analyzing distant related plants and evaluating population structures, gene flows and historical dynamics. By comparing chloroplast genomes of different species or populations, researchers can more deeply understand how plants adapt to various ecological environments and evolution mechanisms behind, thereby providing scientific basis for biodiversity protection and sustainable utilization.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a DNA probe, a kit and a method for identifying sorghum halepense;
A DNA probe for the identification of sorghum halepense, which is a nucleotide sequence shown in SEQ ID No. 1.
Wherein, the DNA probe is marked with a tracing mark;
wherein the tracer label is one of isotopic label and non-isotopic label.
Wherein the non-isotopic label is an enzyme or a colored group.
A method of fixing sorghum, comprising the steps of:
s1, extracting chloroplast genome DNA of a plant to be detected;
S2, carrying out PCR amplification by taking a chloroplast genome of the plant as a template, and detecting whether a chloroplast specific gene exists or not by using the DNA probe;
S3, judging the plant to be the sorghum halepense if the chloroplast specific gene exists through detection.
A kit for the identification of sorghum halepense comprising the DNA probe described above.
The beneficial effects of the invention are as follows: according to the alignment results of the sorghum halepense and sorghum chloroplast genome, a difference site exists in the 4 kb region. Through the one locus, we can distinguish false sorghum, red english pose and other 9 sorghum, and the locus is an ideal locus of molecular markers. The sequence 200 bp at the upstream and downstream of the difference site is taken as a reference sequence to select a homologous sequence to design a primer. The DNA of sorghum halepense and sorghum halepense is used as templates for PCR amplification, single bright bands consistent with the expected size can be amplified in 11 templates, and the existence of differential site fragments obtained by chloroplast genome sequence analysis can be proved, and the amplification and subsequent identification can be carried out.
Sanger sequencing of the PCR amplified products and comparison of the sequencing results showed that the presence of an 11 bp insertion site at this site in the sorghum halepense can distinguish sorghum halepense from sorghum halepense. The locus is stably existing between the sorghum halepense and the sorghum halepense, an oligonucleotide fluorescent probe is designed according to the locus, molecular biological methods such as RPA (Recombinase Polymerase Amplification) and the like can be applied to detect the sorghum halepense and sorghum halepense leaf tissues, and molecular identification can be realized by whether fluorescent signals exist or not, so that the sorghum halepense and the sorghum halepense can be reliably and effectively distinguished.
Drawings
The invention will be described in further detail with reference to the drawings and the detailed description.
FIG. 1 is a schematic representation of PCR amplified electrophoresis bands of sorghum chloroplast genome molecular markers;
FIG. 2 is a schematic representation of specific sequences of the differential sites.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
1. Test equipment
High-speed refrigerated centrifuge: thermo fisher, micro 21R;
constant temperature water bath: refined macro, DK-S24;
Ultra-micro spectrophotometer: thermoFisher, nanoDrop2000,2000;
PCR instrument: thermo fisher Applied Biosystems VeritiPro;
gel electrophoresis apparatus: sixthly, DYY-8C;
Gel imaging system: VILBER, FUSION FX7.
2. Test reagent
Plant genome DNA extraction kit: root, DP305;
2.X Phanta Max Master Mix (Dye Plus): nupraise, P525;
D2000 molecular weight standard: root of Tiangen, MD114.
3. Test method
1. Sample grinding: sample tissue was ground to a uniform powder using liquid nitrogen.
DNA extraction: referring to the flow of the specification, the DNA of plant leaf tissues of sorghum halepense and sorghum halepense of various varieties is extracted and the concentration is detected.
3. Chloroplast genome acquisition: and (3) sequencing the chloroplast genome of the sample, removing low-quality data, and assembling CLEAN READS to obtain complete chloroplast genome data.
4. Sequence alignment: the sorghum halepense chloroplast genome was aligned using MAFFT software.
PCR primer design: according to the alignment result of the sorghum halepense and sorghum chloroplast genome, selecting a difference site which can distinguish the sorghum halepense and sorghum halepense, extracting a difference site and a sequence of 200 bp at the upper and lower streams thereof, designing primers by using Primer-BLAST, screening to obtain a Primer pair JGL 4K-1F=SEQ ID NO. 2= CATTGAATTAGCAAGACATC, JGL K-1R=SEQ ID NO. 3= GGGTCTCTTTATTGTCACTC, and synthesizing by the Shanghai stock of bioengineering (Shanghai).
And 6, PCR amplification verification: the PCR reaction system was configured according to Table 1, and PCR amplification was performed according to the PCR program set in Table 2.
Table 1 PCR System
TABLE 2PCR procedure
7. Gel electrophoresis: the band size of the PCR products was detected using 1.5% agarose gel electrophoresis.
8. Sequencing: PCR products with the size of the electrophoresis band consistent with the expected result were sent to the engineering and bioengineering (Shanghai) Co., ltd for sequencing.
9. Sequence alignment: the dna man was used to align the sorghum halepense genomic difference sites.
10. Probe design:
Probe sequence = SEQ ID No.1:5'-CAGATCCGGTTCAATTTCACTAAGGCTAAGGGTA (THF) AAAAAAAAGCAGCC-3'.
4. Test results
1. Chloroplast genome sequencing results
Sequencing of chloroplast genome of sorghum halepense and other sorghum varieties was uploaded to Genbank to obtain the search numbers shown in Table 3.
TABLE 3 Genbank accession number for the genomic sequence of chloroplast of samples
2. Differential site selection and amplification verification
And (3) amplifying and verifying about 200 bp fragments at the upstream and downstream of the difference site, wherein as shown in figure 1, the agarose nucleic acid electrophoresis detection result meets the expectations, and target fragments are amplified from all varieties of sorghum halepense and sorghum halepense.
In fig. 1, the electrophoresis bands are Marker, JGL (sorghum halepense), HYZ, JXL1, JXL2, JN1, JN2, JN4, JN7, JN11, JN104, LZ19, respectively, from the left, and the band size is about 250 bp.
3. Differential site sequencing and comparison
After alignment analysis of the sequences of fragments upstream and downstream of the differential site, the differential site can be selected as shown in FIG. 2.
4. Design probe
According to the design principle of a probe, designing an oligonucleotide probe with the length of 45-50 nt at a pseudosorghum chloroplast gene specific site, marking a FAM fluorescent group at the 5' end, modifying with a C3-Spacer at the 3' end, and inserting a d-Spacer (THF) at a position, which is more than or equal to 35 nt away from the 5' end, in the middle of the probe.
The probe design results are shown in Table 4;
TABLE 4 DNA probe sequences
5. Method for identifying sorghum halepense
1. Extracting chloroplast genome DNA of a plant to be detected;
2. PCR amplification is carried out by taking chloroplast genome of the plant as a template, and the DNA probe is used for detecting whether chloroplast specific genes exist or not;
3. through inspection, if chloroplast specific genes exist, the plant is judged to be sorghum halepense.
It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Claims (6)
1. A DNA probe for identifying sorghum halepense, which is characterized in that the DNA probe is a nucleotide sequence shown as SEQ ID NO. 1.
2. A DNA probe for use in the identification of sorghum halepense according to claim 1, wherein said DNA probe is labeled with a tracer label.
3. A DNA probe for use in the identification of sorghum as claimed in claim 2, wherein the tracer label is one of isotopically labelled and non-isotopically labelled.
4. A DNA probe for use in the identification of sorghum as claimed in claim 3, wherein the non-isotopic label is an enzyme or a coloured group.
5. A method of setting sorghum halepense, comprising the steps of:
s1, extracting chloroplast genome DNA of a plant to be detected;
S2, carrying out PCR amplification by taking chloroplast genome of the plant as a template, and detecting whether chloroplast specific genes exist or not by using the DNA probe according to any one of claims 1 to 4;
s3, judging the plant to be the sorghum halepense if the chloroplast specific gene exists.
6. A kit for the identification of sorghum halepense, comprising a DNA probe according to any one of claims 1 to 4.
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| Application Number | Priority Date | Filing Date | Title |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002057471A2 (en) * | 2001-01-09 | 2002-07-25 | Monsanto Technology Llc | Maize chloroplast aldolase promoter compositions and methods for use thereof |
| CN101748191A (en) * | 2008-12-12 | 2010-06-23 | 福建出入境检验检疫局检验检疫技术中心 | PCR identification method for sorghum halepense and similar species and kit |
| CN101748174A (en) * | 2008-12-12 | 2010-06-23 | 福建出入境检验检疫局检验检疫技术中心 | DNA rapid extraction method for single-seed of sorghum halepense and similar species |
| CN104178579A (en) * | 2014-09-12 | 2014-12-03 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Primer pair, probe, nucleic acid composition and method for detecting Sorghum halepense |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2011248754A1 (en) * | 2010-04-29 | 2012-11-15 | Pioneer Hi-Bred International, Inc. | Detection of Johnsongrass and its hybrid seed |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002057471A2 (en) * | 2001-01-09 | 2002-07-25 | Monsanto Technology Llc | Maize chloroplast aldolase promoter compositions and methods for use thereof |
| CN101748191A (en) * | 2008-12-12 | 2010-06-23 | 福建出入境检验检疫局检验检疫技术中心 | PCR identification method for sorghum halepense and similar species and kit |
| CN101748174A (en) * | 2008-12-12 | 2010-06-23 | 福建出入境检验检疫局检验检疫技术中心 | DNA rapid extraction method for single-seed of sorghum halepense and similar species |
| CN104178579A (en) * | 2014-09-12 | 2014-12-03 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Primer pair, probe, nucleic acid composition and method for detecting Sorghum halepense |
Non-Patent Citations (3)
| Title |
|---|
| Development of PCR-based chloroplast DNA markers that characterize domesticated cowpea (Vigna unguiculata ssp.unguiculata var. unguiculata) and highlight its crop-weed complex;Felek Y et al.;《Plant Systematics and Evolution》;20061024;第262卷;75-87 * |
| 假高粱的分子生物学检测方法研究;魏莎莎 等;《上海市植物生理学会成立三十周年庆祝活动暨第三届上海市植物生理学青年学术研讨会论文集》;20091113;1 * |
| 基于rDNA ITS分析的假高粱鉴定方法;郭琼霞 等;《福建农业学报》;20051130;第21卷(第1期);32-34 * |
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