CN111154907A - Primer, method and application for detecting target gene repeated-amplification type glyphosate-resistant eleusine indica population - Google Patents
Primer, method and application for detecting target gene repeated-amplification type glyphosate-resistant eleusine indica population Download PDFInfo
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Abstract
The invention discloses a Primer for detecting a target gene repeated-amplification glyphosate-resistant eleusine indica species group, which comprises an upstream Primer CT-F and a downstream Primer Anti-Sense Primer CT-R, wherein the base sequence of the upstream Primer CT-F is shown as SEQ ID NO: 1, and the base sequence of the Anti-sense Primer CT-R of the downstream Primer is shown as SEQ ID NO: 2, respectively. The method is based on molecular biology detection, and is simple, convenient, rapid, time-saving, labor-saving, high in sensitivity and high in accuracy. And the application of the primer in preparing a kit or a biological agent for detecting the target gene repeated glyphosate-resistant goosegrass herb population.
Description
Technical Field
The invention belongs to the technical field of glyphosate-resistant goosegrass detection, and particularly relates to a primer, a method and application for identifying a target gene repeated-amplification glyphosate-resistant goosegrass population.
Background
Goosegrass (Eleusine indica (L.) gartn) is an annual grass plant and is widely distributed worldwide. The goosegrass herb has strong adaptability and reproductive capacity, grows in farmlands, lawns, nurseries, orchards, roadside and other places, is one of ten world malignant weeds, and seriously threatens the malignant weeds for the safe production of crops all over the world.
Glyphosate [ N- (phosphocarboxymethyl) -glycine ] (glyphosate) is the first broad-spectrum biocidal herbicide in the world with high efficiency, low toxicity and low residue, and is known as the most great herbicide in the 20 th century. The use of glyphosate for prevention and control of eleusine indica is the simplest, economic and effective means for broad farmers. With the long-term, continuous and excessive use of the glyphosate, the eleusine indica generates the drug resistance to the glyphosate. In China, the glyphosate-resistant eleusine indica is firstly found in the Guangdong Huizhou area, and then rapidly occurs and spreads in orchards, vegetable fields, sugarcane fields, corn fields and other farmlands in the Guangdong and south China, so that the glyphosate-resistant eleusine indica is seriously abused and abused in production due to the lack of a rapid and accurate glyphosate-resistant eleusine indica detection technology and scientific guidance. Farmers often prevent and kill off by increasing the use amount of glyphosate, the production cost is increased, simultaneously the yield and the quality of crops are influenced, and the pollution to the ecological environment of farmlands is serious.
At present, the main gene target enzyme EPSPS of the drug resistance of eleusine indica to glyphosate is subjected to gene mutation or gene duplication. At present, the glyphosate-resistant eleusine indica species found in south China are mainly target gene duplication.
The traditional resistance identification of the target gene duplication type glyphosate-resistant eleusine indica population mainly comprises three steps:
1) material culture: collecting seeds in the mature period of the field goosegrass herb, airing and storing. And (3) filling soil without goosegrass seeds and herbicide residues in pots with the diameter of not less than 10cm, thinning the seedlings when the seedlings grow to the 2-leaf stage, and keeping about 10 plants growing all the time in each pot.
2) And (3) biological determination: when the plants grow to 4-5 leaf stage, spraying stems and leaves with glyphosate for at least 5 doses, wherein each dose is not less than 4 times, and identifying the drug resistance index of the population by using a dose response curve.
3) Target gene detection: the method is mainly used for detecting whether the EPSPS of the glyphosate target gene in the eleusine indica is multiplied or not by adopting two methods of southern blot and fluorescent quantitative PCR (qPCR). The Southern blot identification comprises 6 steps of genome DNA extraction, enzyme digestion, agarose electrophoresis, membrane transfer, hybridization, development and the like; the qPCR identification comprises four steps of total RNA extraction-reverse transcription-PCR-data analysis and the like. The Southern blot result is visual, but the steps are more, time-consuming and labor-consuming; qPCR has high requirements on RNA quality and primers, is easy to generate false positive, and has low accuracy.
Polyacrylamide gel electrophoresis: polyacrylamide gel electrophoresis, abbreviated as page (polyacrylamide gel electrophoresis), is a common electrophoretic technique using polyacrylamide gel as a support medium for separating proteins and tube nucleotides. The polyacrylamide gel is a gel with a net-shaped three-dimensional structure formed by polymerizing and crosslinking acrylamide and methylene bisacrylamide under the action of a catalyst. Under the action of an electric field, the charged particles can migrate in the polyacrylamide gel, and the migration rate is related to the size, configuration and charged charge of the charged particles.
Silver staining method: silver staining is a highly sensitive method for detecting proteins (or nucleic acids) in polyacrylamide gel electrophoresis. Silver nitrate (silver ions) on the protein band is reduced to metallic silver with formaldehyde under alkaline conditions to deposit silver particles on the protein (or nucleic acid) band.
At present, no one adopts the combination of common PCR and polyacrylamide gel electrophoresis and silver staining method to detect the target gene repeated type glyphosate-resistant eleusine indica population.
Disclosure of Invention
The first purpose of the invention is to provide a primer for detecting a target gene duplication type glyphosate-resistant eleusine indica species, and the primer has strong specificity.
The second purpose of the invention is to provide a method for rapidly detecting the target gene re-amplification type glyphosate-resistant eleusine indica population by using the primer, which is based on the molecular biological method for detection, and has the advantages of simplicity, rapidness, time and labor saving, high sensitivity and high accuracy.
The third purpose of the invention is to provide the application of the primer in the preparation of the anti-glyphosate goosegrass herb population or the related biological agent for detecting the target gene duplication.
The first object of the present invention can be achieved by the following technical solutions: a Primer for detecting a target gene repeated-amplification glyphosate-resistant eleusine indica species comprises an upstream Primer CT-F and a downstream Primer Anti-Sense Primer CT-R, wherein the base sequence of the upstream Primer CT-F is shown as SEQ ID NO: 1, and the base sequence of the Anti-sense Primer CT-R of the downstream Primer is shown as SEQ ID NO: 2, respectively.
The research of the application finds that compared with the EPSPS gene characteristics of sensitive eleusine indica, the 5' end untranslated region of the glyphosate-resistant eleusine indica correspondingly has an insertion mutation of a 12bp CT repetitive sequence after the EPSPS gene of the glyphosate-resistant eleusine indica is repeatedly increased.
The primer designed in the application can amplify a sequence of about 130bp upstream and downstream of a CT insertion mutation site of a 5 'end untranslated region of an EPSPS gene of a target gene complex glyphosate-resistant goosegrass colony (if no CT insertion mutation exists, the length of the amplified fragment is about 120bp, and if the CT insertion mutation occurs, the amplified fragment is about 130bp), and can be used for judging whether glyphosate resistance based on the insertion mutation of the CT repetition sequence of the 5' end untranslated region of the EPSPS gene is generated in the goosegrass to be detected or not by combining the high-resolution characteristic of polyacrylamide gel electrophoresis.
Specifically, the base sequences of the primers in the group are as follows:
CT-F:5’-GCGGCGCACGCCTCAGCTCA-3’
CT-R:5’-GTCGAGGTTGGTTTGGCTGC-3’。
the second object of the present invention can be achieved by the following technical solutions: a rapid detection method for a target gene duplication type glyphosate-resistant eleusine indica species group comprises the following steps:
(1) selecting a goosegrass sample to be detected and a sensitive goosegrass population, and respectively extracting genomic DNAs of the goosegrass sample and the sensitive goosegrass population;
(2) designing the primer according to the existence of a 12bp CT repetitive sequence insertion in the 5' untranslated region of the target gene duplication type glyphosate-resistant eleusine indica EPSPS gene;
(3) selecting the genome DNA in the step (1), and performing PCR amplification by using the primer designed in the step (2) to obtain a PCR product;
(4) separating the PCR product by polyacrylamide gel electrophoresis, and observing the position of an amplified band after developing by a silver staining method;
(5) identifying whether the target gene is the target gene repeated-amplification type glyphosate-resistant goosegrass herb population according to the electrophoresis result, and if the position of the amplified strip of the goosegrass herb sample to be detected is about 12bp above the amplified strip of the sensitive goosegrass herb population, judging the goosegrass herb sample to be detected as the target gene repeated-amplification type glyphosate-resistant goosegrass herb population; and if the position of the amplification strip of the sample to be detected is parallel to the position of the amplification strip of the sensitive eleusine indica colony, judging that the non-target gene complex amplification type glyphosate-resistant eleusine indica colony.
In the method for rapidly detecting the target gene repeated glyphosate-resistant eleusine indica population:
during PCR amplification in the step (3), a PCR reaction system is as follows: 2 mu L of 2mM dNTP mixed solution, 2.5 mu L of 10 XPCR Buffer, 0.5 mu L of each of 10 mu M CT-F and CT-R primers, 0.5 mu L of 5U/mu L high-fidelity Taq DNA polymerase, 1 mu L of extracted DNA sample to be detected, and 18 mu L of sterile distilled water is added until the reaction system is 25 mu L.
During PCR amplification in the step (3), the PCR reaction program is as follows: heating at 95 deg.C for 5min to denature DNA; entering a reaction cycle, wherein in each cycle, cracking at 95 ℃ for 30sec, annealing at 58 ℃ for 30sec, and extending at 68 ℃ for 20sec for 30 cycles; and finally, extending for 5min at 72 ℃ to ensure that the product is completely extended.
And (3) the mass percentage of the polyacrylamide in the polyacrylamide gel electrophoresis is 8%.
Researches show that a CT sequence is inserted into an EPSPS gene 5' untranslated region in a gene duplication type glyphosate-resistant eleusine indica population, after genomic DNA is extracted from eleusine indica leaves to be detected, a common PCR combined high-resolution polyacrylamide gel electrophoresis and silver staining method is adopted, and whether field eleusine indica generates drug resistance on glyphosate through EPSPS gene duplication can be rapidly, accurately and intuitively identified. In the embodiment of the invention, the method detects the CT areas of 47 plants of 8 gene duplication type (8 goosegrass populations in different places are collected) glyphosate-resistant goosegrass populations, so that the universality of CT insertion mutation in the gene duplication type glyphosate-resistant goosegrass populations is determined, and the reliability of the method is further determined.
The third object of the present invention can be achieved by the following technical solutions: the primer is applied to preparation of a kit or a biological agent for detecting a target gene repeated glyphosate-resistant goosegrass herb population.
The molecular biology method for rapidly detecting the target gene repeated type glyphosate-resistant goosegrass herb population has the characteristics of high sensitivity, strong specificity and the like, and compared with the prior art, the molecular biology method has the beneficial results that:
(1) the method is simple and easy to implement: the detection method is used for testing through a molecular biology technical means, traditional detection steps such as culture materials, medicament screening and resistance index determination are not needed, the leaves of a population to be detected can be directly obtained, the detected strips are compared with the standard strips of a sensitive population after the strips are detected by the method, and rapid identification is carried out.
(2) The detection is efficient: compared with the traditional medicament screening of the resistant eleusine indica population, the method does not need the fussy processes of culturing materials, spraying pesticides, weighing dry weight or wet weight, calculating resistance index and the like, greatly shortens the detection time, and can complete the detection in about 6 hours in the whole process.
(3) The sensitivity is high: the method adopts silver nitrate dyeing technology to detect polyacrylamide gel electrophoresis results, the products after silver dyeing are clear, and the nucleic acid genotype with 12bp difference can be judged.
(4) The accuracy is high: the mutation sites detected by the detection method are obviously different in sensitive and resistant goosegrass herb flora genes, and the detection accuracy is high.
(5) The invention firstly uses molecular biology technology to detect the glyphosate-resistant goosegrass seed group at home and abroad, and the method is quick, simple and convenient, and has important practical significance for guiding farmers to correctly and scientifically use the medicine, reducing cost and reducing environmental pollution.
Drawings
FIG. 1 shows the comparison of the molecular characteristics of the EPSPS gene of the glyphosate resistant (R) and sensitive (S) type Eleusine indica in example 1;
FIG. 2 is the polyacrylamide gel electrophoresis analysis of the features of the CT insertion mutation site of the Eleusine indica (EPSPS) gene of anti (R) type and sensitive (S) type in example 2;
FIG. 3 shows the analysis of CT insertion mutation and gene duplication of the Eleusine indica EPSPS gene by polyacrylamide gel electrophoresis (A) and fluorescence quantification (qPCR) in example 3.
Detailed Description
The method of the present invention is further illustrated by the following examples. The following examples and drawings are illustrative only and are not to be construed as limiting the invention. Unless otherwise specified, the reagent raw materials used in the following examples are biochemical reagent raw materials which are conventionally commercially available or commercially available, and the laboratory instruments used are laboratory conventional instruments, and unless otherwise specified, the methods and apparatuses used in the following examples are those conventionally used in the art.
Example 1
The research of the application finds that compared with the EPSPS gene sequence of the glyphosate target enzyme in the sensitive eleusine indica (GenBank access number: KX018288), a CT repeated sequence insertion mutation of 12bp exists in the untranslated region at the 5' end of the EPSPS gene in the target gene repetitive glyphosate-resistant eleusine indica (as shown in figure 1), and the mutation occurs in eleusine indica populations collected in 6 different places, which indicates that the molecular characteristics have broad spectrum in the target gene repetitive-increase glyphosate-resistant eleusine indica population.
Therefore, a group of primers can be designed based on the basic principle of PCR primer design (the length is between 15 and 30bp, and the Tm value is between 50 and 72 ℃) and used for detecting the insertion mutation of the CT repetitive sequence of 12bp in the untranslated region at the 5' end of the EPSPS gene in the target gene repetitive glyphosate-type eleusine indica.
Thus obtaining a group of primers for detecting the target gene repeated-amplification type glyphosate-resistant eleusis indica population, wherein the primers comprise an upstream Primer CT-F and a downstream Primer Anti-Sense Primer CT-R, and the base sequence of the upstream Primer CT-F is shown as SEQ ID NO: 1, and the base sequence of the Anti-sense Primer CT-R of the downstream Primer is shown as SEQ ID NO: 2, respectively.
Specifically, the base sequences of the primers in the group are as follows:
CT-F:5’-GCGGCGCACGCCTCAGCTCA-3’
CT-R:5’-GTCGAGGTTGGTTTGGCTGC-3’。
example 2
The method for rapidly detecting the target gene repeated type glyphosate-resistant eleusine indica population by using the primer in the embodiment 1 comprises the following steps:
(1) selecting a goosegrass sample to be detected and a sensitive goosegrass population (wherein the sensitive goosegrass population is collected and stored in a laboratory, collected on a grassland without glyphosate pollution before glyphosate-resistant goosegrass is found, or collected on other grasslands without glyphosate pollution as the sensitive goosegrass population, stored in the laboratory and provided to the public for verification test), and respectively extracting the genomic DNAs of the two;
(2) according to the existence of a 12bp CT repetitive sequence insertion in the 5' untranslated region of the EPSPS gene of the grass goosegrass herb with the target gene duplication type glyphosate resistance, 1 pair of primers (the primers designed in the example 1) are designed on the CT region of the EPSPS gene containing the grass goosegrass herb, and the primers used in the PCR reaction are as follows:
CT-F:5’-GCGGCGCACGCCTCAGCTCA-3’
CT-R:5’-GTCGAGGTTGGTTTGGCTGC-3’
(3) respectively selecting a goosegrass sample to be detected and the genome DNA of the sensitive goosegrass population, and carrying out PCR amplification by using the primers;
the PCR reaction system is as follows: 2 mu L of 2mM dNTP mixed solution, 2.5 mu L of 10 XPCR Buffer, 0.5 mu L of each of 10 mu M CT-F and CT-R primers, 0.5 mu L of 5U/mu L high-fidelity Taq DNA polymerase, 1 mu L of extracted DNA sample to be detected, and 18 mu L of sterile distilled water is added until the reaction system is 25 mu L.
The PCR reaction procedure was as follows: heating at 95 deg.C for 5min to denature DNA; entering a reaction cycle, wherein in each cycle, cracking at 95 ℃ for 30sec, annealing at 58 ℃ for 30sec, and extending at 68 ℃ for 20sec for 30 cycles; and finally, extending for 5min at 72 ℃ to ensure that the product is completely extended.
(4) After 50. mu.L of the PCR product was mixed with 10. mu.L of 6 × Loading Buffer, the mixture was separated on 8% polyacrylamide gel electrophoresis (PAGE), and the position of the amplified band was observed. The 8% PAGE gel formulation was as follows: urea 2.4g, 10 XTBE 2mL, 40% acrylamide 4mL, TEMED 9. mu.L, 10% ammonium persulfate 0.8 mL. Wherein, 10 XTBE is 108g Tris +55g boric acid +40mL EDTA (0.5M), and the volume is up to 1000 mL; 40% acrylamide: 38g of acrylamide and 2g of methylene bisacrylamide are added to the total volume of 100 mL.
PAGE electrophoresis conditions:
buffer 0.5 × TBE, voltage 150V, time 150 min.
The silver staining solution and the developing solution have the following formula:
0.1% silver nitrate: 1g silver nitrate + dd H2O, and metering to 1000 mL.
Developing solution: 30g NaOH +0.38g sodium tetraborate +8mL formaldehyde to a volume of 2000 mL.
Silver staining experiment procedure:
① after electrophoresis, carefully taking off the gel with a rubber shovel, and washing the gel with distilled water for 30 s;
② discarding distilled water, and dyeing with 0.1% silver nitrate dyeing solution for 10 min;
③ discarding the staining solution, washing the gel with distilled water for 1 min;
④ discarding distilled water, and developing for 2 min;
⑤ changing the developing solution, continuing to dye for 2min, and repeating the steps for 1-2 times until the color development of the gel is clear;
⑥ discarding the developing solution, washing the gel with distilled water for 30s, and imaging under a gel imager.
(5) Identifying whether the plant is a target gene duplication type glyphosate-resistant eleusine indica population: identifying whether the EPSPS gene of the goosegrass herb sample to be detected has 12bp CT insertion mutation or not according to the electrophoresis result; if the position of the eleusine indica sample amplification strip to be detected is about 12bp above the sensitive eleusine indica species group amplification strip, judging that the anti-glyphosate eleusine indica species group with CT insertion mutation (namely the target gene repeated amplification type anti-glyphosate eleusine indica species group) is present; and if the position of the to-be-detected eleusine indica sample amplification strip is parallel to the position of the sensitive eleusine indica species group amplification strip, judging that the glyphosate sensitive eleusine indica species group without CT insertion mutation (namely the non-target gene repeated amplification type glyphosate-resistant eleusine indica species group) is judged. As shown in particular in fig. 2.
47 plants of 8 eleusine indica populations which survive after spraying are collected in the south China, and detection shows that 45 glyphosate-resistant eleusine indica populations with EPSPS genes with CT insertion mutation sites are present, the EPSPS genes are increased repeatedly, and 2 glyphosate-sensitive eleusine indica populations without glyphosate-resistant mutation sites (No. 28 and No. 36 plants in figure 3) have no increased repeatedly. The result shows that the EPSPS gene duplication and the CT insertion mutation in the glyphosate-resistant goosegrass are in one-to-one correspondence, so that the accuracy and the reliability of the gene duplication type goosegrass are judged by detecting the CT insertion mutation, and the goosegrass with the EPSPS gene duplication has the CT insertion mutation from the current result.
Therefore, the primers in example 1 and the PCR reaction system in example 2 can be made into a kit or a biological agent, and the method in example 2 is adopted to detect whether an unknown goosegrass sample has a CT insertion mutation of 12bp in the 5' untranslated region of the glyphosate-resistant goosegrass EPSPS gene, namely whether the unknown goosegrass sample is a target gene duplication type glyphosate-resistant goosegrass population.
In conclusion, the primers, the detection method, the kit, the biological agent and the like designed in the invention can accurately and quickly detect the target gene re-amplification type glyphosate-resistant goosegrass herb population, provide a simple, convenient and quick detection technology with low cost for scientific research and production practice, and also provide theoretical basis and technical guidance for early warning, epidemic monitoring and reasonable medication of the resistant weeds.
Sequence listing
<110> institute for plant protection of academy of agricultural sciences of Guangdong province
<120> primer, method and application for detecting target gene repeated-increase type glyphosate-resistant eleusine indica species group
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<170>SIPOSequenceListing 1.0
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
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Claims (6)
1. A primer for detecting a target gene repeated-amplification type glyphosate-resistant eleusine indica species group is characterized in that: the Primer comprises an upstream Primer, namely SensePrimer CT-F and a downstream Primer, namely Anti-Sense Primer CT-R, wherein the base sequence of the upstream Primer, namely SensePrimer CT-F is shown as SEQ ID NO: 1, and the base sequence of the Anti-sense Primer CT-R of the downstream Primer is shown as SEQ ID NO: 2, respectively.
2. A rapid detection method for a target gene duplication type glyphosate-resistant eleusine indica species group is characterized by comprising the following steps:
(1) selecting a goosegrass sample to be detected and a sensitive goosegrass population, and respectively extracting genomic DNAs of the goosegrass sample and the sensitive goosegrass population;
(2) designing a primer in claim 1 according to the existence of a 12bp CT repetitive sequence insertion in a 5' untranslated region of a target gene duplication type glyphosate-resistant eleusine indica EPSPS gene;
(3) selecting the genome DNA in the step (1), and performing PCR amplification by using the primer designed in the step (2) to obtain a PCR product;
(4) separating the PCR product by polyacrylamide gel electrophoresis, and observing the position of an amplified band after developing by a silver staining method;
(5) identifying whether the target gene is the target gene repeated-amplification type glyphosate-resistant goosegrass herb population according to the electrophoresis result, and if the position of the amplified strip of the goosegrass herb sample to be detected is about 12bp above the amplified strip of the sensitive goosegrass herb population, judging the goosegrass herb sample to be detected as the target gene repeated-amplification type glyphosate-resistant goosegrass herb population; and if the position of the amplification strip of the sample to be detected is parallel to the position of the amplification strip of the sensitive eleusine indica colony, judging that the non-target gene complex amplification type glyphosate-resistant eleusine indica colony.
3. The method for rapidly detecting the glyphosate-resistant eleusine indica species group with the repeated increase of the target gene as claimed in claim 2, which is characterized in that: during PCR amplification in the step (3), a PCR reaction system is as follows: 2 mu L of 2mM dNTP mixed solution, 2.5 mu L of 10 XPCR Buffer, 0.5 mu L of each of 10 mu M CT-F and CT-R primers, 0.5 mu L of 5U/mu L high-fidelity Taq DNA polymerase, 1 mu L of extracted DNA sample to be detected, and 18 mu L of sterile distilled water is added until the reaction system is 25 mu L.
4. The method for rapidly detecting the glyphosate-resistant eleusine indica species group with the repeated increase of the target gene as claimed in claim 2, which is characterized in that: during PCR amplification in the step (3), the PCR reaction program is as follows: heating at 95 deg.C for 5min to denature DNA; entering a reaction cycle, wherein in each cycle, cracking at 95 ℃ for 30sec, annealing at 58 ℃ for 30sec, and extending at 68 ℃ for 20sec for 30 cycles; and finally, extending for 5min at 72 ℃ to ensure that the product is completely extended.
5. The method for rapidly detecting the glyphosate-resistant eleusine indica species group with the repeated increase of the target gene as claimed in claim 2, which is characterized in that: the volume percentage of the polyacrylamide in the polyacrylamide gel electrophoresis in the step (4) is 8%.
6. Use of the primer of claim 1 in the preparation of a kit or a biological agent for detecting a target gene-replicating glyphosate-resistant goosegrass herb population.
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| CHUN ZHANG等: "Alterations in the 5"untranslated region of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene influence EPSPS overexpression in glyphosate-resistant Eleusine indica", 《PEST MANAGEMENT SCIENCE》 * |
| 张纯等: "牛筋草EPSPS基因启动子对草甘膦的响应", 《分子植物育种》 * |
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