CN109837355B - Application of beckmannia syzigachne CYP704B1 gene - Google Patents

Abstract

The invention provides application of a beckmannia syzigachne CYP704B1 gene. The invention also provides a fluorescent quantitative PCR primer (SEQ ID NO:2-3) for detecting the beckmannia syzigachne CYP704B1 gene expression quantity and a method for detecting the beckmannia syzigachne CYP704B1 gene expression quantity established based on the primer so as to judge the resistance of the beckmannia syzigachne to sulfonylureas (particularly, mesosulfuron-methyl) herbicides. The method has the advantages of simplicity, high sensitivity and strong specificity, and has important application prospect in the detection and research of the non-target resistance of beckmannia syzigachne to mesosulfuron-methyl. The invention discloses the relationship between the beckmannia syzigachzigachne CYP704B1 gene and the resistance of sulfonylurea herbicide for the first time, can identify whether the beckmannia zigachne has metabolic resistance to the herbicide by detecting the expression quantity of the CYP704B1 gene, and has guiding significance for timely finding resistance, guiding farmland scientific medication and relieving further spreading of the resistance to the herbicide.

Description

Application of beckmannia syzigachne CYP704B1 gene

Technical Field

The invention relates to the technical field of genetic engineering, in particular to application of a beckmannia syzigachne CYP704B1 gene.

Background

The long-term continuous unscientific application of the herbicide is easy to cause the generation of weed drug resistance, the wheat and the beckmannia syzigachne in partial areas of China already generate serious resistance to the herbicide, and the control of the beckmannia syzigachne is more difficult along with the generation of non-target resistance. It has been shown that cytochrome P450 metabolizes herbicides to nontoxic substances by dealkylation, cyclomethylhydroxylation, hydroxylation of the aromatic ring, desulfurization oxidation and catalytic ester bond cleavage, whereas increased expression of certain genes in the P450 family renders otherwise susceptible weed species resistant to herbicides (Yasuor H, Osuna MD, Ortiz A, et al. mechanismins of resistance to herbicidal in late watering grass [ Echinochloa phyloprotein (Stapf) Koss. ]. Journal of Agricultural and food chemistry,2009,57: 3653. 3660; Yang Q, Yang W, Li X F, et al. target-site and BMC-site-based herbicide resistance to herbicides-biological sample (biochemical sample, biological sample, 2017,140: 79-84; pan L, Gao H T, Xia W. optimizing a herbicide-synthesizing enzyme library in Beckmann syzigania syziquartz to identification genes associated with a metallurgical plant.journal of Experimental Bottony, 2016,67: 1745-1757).

Disclosure of Invention

The invention aims to provide application of the beckmannia syzigachne CYP704B1 gene.

The invention has the following conception: since the expression of related genes of metabolic herbicide in different beckmannia syzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzi. The primer (namely the fluorescent quantitative primer) for detecting the expression quantity of the beckmannia syzigachne CYP704B1 gene has no hairpin structure, does not form primer dimer, specifically amplifies a unique identification fragment, and has the amplification product length of 85B_p. The change of the quantity of each cyclic amplification product in the PCR amplification reaction is monitored in real time by utilizing the change of a fluorescent signal, the initial template is quantitatively analyzed through the relation between the Ct value and a standard curve, the expression quantity of the beckmannia syzigachzigachzigachzigachzigachgachgachgachgachgachgachgachgachgachgachgachgachgachgachgachgachgachgachgachgachgachgachgachgachgach.

In order to achieve the object of the present invention, in a first aspect, the present invention provides any one of the following uses of a beckmannia syzigachne CYP704B1 gene:

1) application in identifying resistance of different beckmannia syzigachzigachne material to sulfonylurea herbicide;

2) the application of the syzis syzigachne material as a marker for resistance of the syzigarus syzigachne material to sulfonylurea herbicides;

3) application in identifying Slyssyzis quinata material for resisting sulfonylurea herbicide.

In the invention, the beckmannia syzigachzigachne CYP704B1 gene is as follows:

i) 1, SEQ ID NO;

ii) a nucleotide sequence which is obtained by substituting, deleting and/or adding one or more nucleotides into the nucleotide sequence shown in SEQ ID NO. 1 and expresses the same functional protein;

iii) a nucleotide sequence which has more than 90 percent of homology with the nucleotide sequence of i) and ii) and expresses the same functional protein.

In the present invention, the sulfonylurea herbicide includes, but is not limited to, mesosulfuron-methyl.

In a second aspect, the invention provides specific PCR primers for amplifying the beckmannia syzigachne CYP704B1 gene (namely primers for detecting the expression level of the beckmannia syzigachne CYP704B1 gene), which comprise CYP704B1-F1 and CYP704B1-R1, and the nucleotide sequences are respectively shown in SEQ ID NO:2 and SEQ ID NO: 3.

The primer CYP704B1-F1/R1 for detecting the beckmelon sycamzidae CYP704B1 gene expression level can be obtained by the following method:

(1) obtaining a gene sequence of CYP704B1 by sequencing a beckmannia transcriptome to carry out deep analysis;

(2) designing fluorescent quantitative PCR alternative primers for subsequent screening;

(3) extracting total RNA of the beckmannia syzigachne to be detected, and performing reverse transcription to obtain cDNA;

(4) carrying out real-time fluorescent quantitative PCR amplification by taking the cDNA obtained in the step (3) as a template and the primer designed in the step (2) as a guide, and screening out a primer pair with a dissolution curve meeting the requirement;

(5) and (3) performing common PCR amplification by taking the cDNA obtained in the step (3) as a template and the primer screened in the step (4) as a guide, sequencing the obtained amplification product, verifying whether the primer specifically amplifies the target gene CYP704B1 or not, and finally screening out the primer CYP704B1-F1/R1 for specifically detecting the CYP704B1 gene expression level.

In the step (2), the fluorescent quantitative PCR primers are designed by utilizing Oligo 7.0 software and Primer5.0 software, and the design standard of the fluorescent quantitative PCR primers is as follows: the length of the primers is 20-25nt, the length of the amplification product is not more than 300bp, the annealing temperature is more than 60 ℃, the difference of the annealing temperature between single primers is 1 ℃, the 3' end is not suitable to be A, and the primers do not form hairpin structures and primer dimers.

The SYBR Green I dye is combined with any double-stranded DNA and then emits fluorescence, so if non-specific amplification or primer dimer generation exists in a reaction system, the detection is also carried out simultaneously, and the detection result is possibly inaccurate, but the primer CYP704B1-F1/R1 finally screened by the invention has no hairpin structure, does not form primer dimer and only can amplify a unique identification fragment, so the detection result is credible.

In a third aspect, the present invention provides a detection reagent or a kit comprising the above primer.

Further, the kit may further comprise a fluorescent dye, such as SYBR Green I, for use in conjunction with the primer.

In a fourth aspect, the invention provides a method for detecting the expression level of a beckmannia syzigachne CYP704B1 gene, the method comprising: extracting total RNA of beckmannia syzigachne to be detected, and performing reverse transcription to obtain cDNA; then, carrying out real-time fluorescent quantitative PCR amplification by using the primers CYP704B1-F1 and CYP704B1-R1 and taking the cDNA as a template; meanwhile, an internal reference gene is set, and the relative expression quantity of the CYP704B1 gene is calculated.

In the invention, the internal reference gene is selected from at least one of UBQ, CAP and GADPH, and primers for amplifying each internal reference gene are respectively as follows (SEQ ID NO: 4-9):

UBQ-F：5ˊ-CAAGAAGAAGACGTACACCAAG-3ˊ

UBQ-R:5ˊ-GACCTTGTAGAACTGGAGGAG-3ˊ；

CAP-F:5ˊ-AAGCCCCAATCAAAATCAACACGAA-3ˊ

CAP-R:5ˊ-AAGAACACCAAGACCCCCTGC-3ˊ；

GADPH-F：5ˊ-AGGTTATCAATGACAAGTTTGG-3ˊ

GADPH-R：5ˊ-ATCAACAGTCTTCTGGGTAGC-3ˊ。

alternatively, the PCR reaction system and reaction procedure are as follows:

the PCR reaction system consisted of 2. mu.L of 2.5mM dNTP mixture, 2.5. mu.L of 10 × PCR Buffer, 0.5. mu.L of 10. mu.M upstream and downstream primers, 0.5. mu.L of 5U/. mu.L Taq DNA polymerase, 1. mu.L cDNA, and ddH₂The content of O is filled to 25 mu L.

PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension at 72 ℃ for 5 min.

In a fifth aspect, the invention provides application of the method in identifying resistance of different beckmannia syzigachne materials to sulfonylurea herbicides. Wherein, the application comprises identifying the resistance of the beckmannia syzigachne material to sulfonylurea (especially mesosulfuron) herbicide according to the relative expression amount of CYP704B1 gene. When the relative expression amount of the CYP704B1 gene is 2 times of that of the sensitive material, the beckmannia syzigachne material to be tested is indicated to be a resistant material.

In a sixth aspect, the invention provides a DNA molecular marker related to resistance of beckmannia syzigachne to sulfonylurea (especially mesosulfuron-methyl) herbicide, wherein the DNA molecular marker is located on the beckmannia syzigachne CYP704B1 gene, primers for amplifying the DNA molecular marker comprise CYP704B1-F1 and CYP704B1-R1, and nucleotide sequences are respectively shown in SEQ ID NO:2 and SEQ ID NO: 3.

In a seventh aspect, the invention provides an application of the DNA molecular marker in the American beckmannia molecular marker assisted breeding.

In one embodiment of the invention, the method for detecting the expression level of the beckmannia syzigachne CYP704B1 gene comprises the following steps:

(1) extracting the processed 24h and unprocessed total RNA of the beckmannia syzigachne sample to be detected, and carrying out reverse transcription to obtain cDNA;

(2) performing PCR amplification sequencing by taking the cDNA obtained in the step (1) as a template and CYP704B1-F1/R1 as a primer;

(3) taking cDNA samples subjected to gradient dilution (6 gradients with concentration difference of 2 times among the gradients) as standard templates, performing real-time fluorescence quantitative PCR by using primers CYP704B1-F1/R1, drawing a standard curve, and setting a threshold (the fluorescence signal of the first 15 cycles of the real-time fluorescence quantitative PCR reaction is taken as a fluorescence background signal, and the fluorescence threshold is set to be 10 times of the standard deviation of the fluorescence signal of 3-15 cycles);

(4) taking cDNA of a beckmannia syzigachne sample to be detected as a template, carrying out real-time fluorescence quantitative PCR amplification on the reference gene to obtain corresponding amplification efficiency e and Ct value (the number of cycles spent when the fluorescence signal in each reaction tube reaches a set threshold value is called Ct value), and calculating the expression quantity e of the reference gene^-ΔΔtFor subsequent inspectionCorrecting the concentration of the cDNA of the beckmannia syzigachne sample;

(5) performing real-time fluorescence quantitative PCR amplification on the cDNA of the beckmannia syzigachne sample to be detected simultaneously with the step (4) by using the primer CYP704B1-F1/R1, determining the Ct value (the cycle number which is passed when the fluorescence signal in each reaction tube reaches the set threshold value and is called the Ct value) corresponding to the beckmannia syzichne sample and the amplification efficiency E according to the reaction result, and calculating the expression quantity E of the CYP704B1 gene of the beckmannia syzichne sample^-ΔΔtFurther, the expression level e of the reference gene is used^-ΔΔtCorrecting to obtain relative expression amount E capable of comparing samples^-ΔΔt/e^-ΔΔt；

(6) Comparing the relative expression amount of CYP704B1 gene in different beckmannia syzigachne samples, and judging the resistance of the beckmannia syzigachne to herbicide.

In the step (1), the PCR reaction system for reverse transcription into cDNA is as follows:

the total RNA content is 800ng, and the total RNA content is 800ng,

supermix (containing PCR Buffer, RNase inhibitor, dNTPs, adsorbed Oligo (dT)₁₈Primer) 4. mu.L, gDNA Remover 1. mu.L, plus ddH₂O to 20. mu.L.

The procedure of the PCR reaction for reverse transcription into cDNA was: keeping the temperature at 42 ℃ for 15min, 85 ℃ for 5s and 12 ℃.

In the step (2), the cDNA is taken as a template, CYP704B1-F1/R1 is taken as a primer for PCR amplification, the PCR reaction system is 2 mu L of 2.5mM dNTP mixed solution, 2.5 mu L of 10 × PCR Buffer, 0.5 mu L of 10 mu M upstream primer and 10 mu L downstream primer respectively, 0.5 mu L of 5U/mu L Taq DNA polymerase, 1 mu L of cDNA sample to be detected is added, and 18 mu L ddH is added₂O to the reaction system was 25. mu.L, and this operation was performed on ice.

The PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension at 72 ℃ for 5 min.

In the steps (3), (4) and (5), the cDNA is taken as a template, CYP704B1-F1/R1 or a corresponding internal reference primer is taken as a primer to carry out real-time fluorescence quantitative PCR amplification, and the real-time fluorescence quantitative PCR reaction system is 2 × SYBR Green 10 mu L, 10 mu M primers F1 and R1 or a corresponding internal reference primerPrimers 2. mu.L each, template 1. mu.L, plus ddH₂O to 20. mu.L.

The real-time fluorescent quantitative PCR amplification procedure in the steps (3), (4) and (5) is as follows:

a、95℃4min；

b、95℃20s；

c、60℃1min；

repeating steps b-c for 40 cycles;

d. the temperature range of the dissolution curve is 60-95 ℃, and the temperature of 60 ℃ is increased to 95 ℃ at the speed of 0.5 ℃/min.

And (4) taking the reference gene as UBQ in the step (4) and taking the geometric mean of the reference gene expression quantity as a correction value to obtain accurate correction effect.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the invention discloses the relation between the beckmannia syzigachne CYP704B1 gene and the resistance of sulfonylurea (especially mesosulfuron) herbicide for the first time, and the expression level of the CYP704B1 gene is positively correlated with the resistance of the beckmannia syzigachne to the mesosulfuron. Therefore, whether the beckmannia syzigachzigachzigachzigachzigachzigachzigachzigachziq has metabolic resistance to herbicide can be identified by detecting the expression quantity of the CYP704B1 gene, and the method has guiding significance for timely finding resistance, guiding farmland scientific medication and relieving further spread of drug resistance.

The method for detecting the beckmannia syzigachzigachne CYP704B1 expression level has the advantages of simplicity, high sensitivity and strong specificity, has an important application prospect in the research of the drug resistance of beckmannia syzichzichne, and simultaneously provides a reference basis for the accurate treatment of the drug resistance of beckmannia syzichzichzichzichzichzichne.

Drawings

FIG. 1 is an electrophoretic RNA detection scheme of example 2 of the present invention.

FIG. 2 is a graph showing the corresponding dissolution curves of the detection primer CYP704B1-F1/R1 in example 3 of the present invention.

FIG. 3 is a graph showing the corresponding dissolution curves of the detection primer CYP704B1-F2/R2 in example 3 of the present invention.

FIG. 4 is a graph showing the corresponding dissolution curves of the detection primer CYP704B1-F3/R3 in example 3 of the present invention.

FIG. 5 is an electrophoretic image of the amplification products of the primers CYP704B1-F1/R1, CYP704B1-F2/R2 and CYP704B1-F3/R3 in example 3 of the present invention. Wherein M is Marker; 1 is CYP704B1-F1/R1 amplification product, 2 is CYP704B1-F2/R2 amplification product; 3 is CYP704B1-F3/R3 amplification product.

FIG. 6 is a standard curve plotted in example 4 of the present invention.

FIG. 7 shows the relative expression of CYP704B1 gene after 24h of methyldisulfuron treatment and before treatment of WC10-4 and WC10-10 in example 4 of the invention, the abscissa represents the name of beckmark grass material, and the ordinate represents the relative expression of CYP704B1 gene.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

The following examples are provided in which the total RNA extraction kit Direct-zol RNA Miniprep was purchased from Biotech, Inc. of Baimin, Beijing, and the product number is as follows: r2050.

The reverse transcription kit is purchased from the general gold company, and the product number is as follows: AT 311-03.

Bestar of real-time fluorescent quantitative PCR kit^TMq PCR MasterMix (SYBR Green) was purchased from Shanghai Han Biotech Co., Ltd, product number: DBI-2043.

WC10-4 was collected from the wasteland of six villages, south town, north town, Jingjiang city, Jiangsu province, as a metsulfuron-methyl sensitive biotype, WC10-10 was collected from ten groups of Yangtze lake village, Huangji town, Hongze county, Huaian city, Jiangsu province, as a metsulfuron-methyl drug resistant biotype. See WANG J, LIX, Dan L I, et al. non-target-site and target-site resistance to AHAS inhibition in American slope [ J ]. Journal of integrated acquisition, 2018,17(12):2714-2723.

Example 1 acquisition of CYP704B1 Gene

Sensitive population WC10-4 and drug-resistant population WC10-10 collected from Jiangsu province are selected in the test.

1. Transcriptome sequencing

Selecting sensitive beckmannia syzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzizan after being treated.

2. Acquisition of CYP704B1 Gene

Screening the differential expression genes according to a transcription sequencing result, determining the signal path of the differential expression genes participating in jasmonic acid through pathway significant enrichment, and acquiring gene information and a gene sequence from the sequencing result.

The nucleotide sequence of the beckmark grass CYP704B1 gene is shown in SEQ ID NO: 1.

Example 2 extraction of Total RNA

The test selects sensitive population WC10-4 and drug-resistant population WC10-10 collected from Jiangsu province.

1. Extraction of Total RNA

Selecting sensitive beckmannia syzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzigachzichzigachzichzichzichzizuke subjected.

RNA agarose gel electrophoresis

3uL of the total RNA solution is taken, electrophoresis detection is carried out by using 1% agarose gel, the voltage is 160V, the electrophoresis time is 20min, and gel imaging scanning is carried out. As shown in FIG. 1, the pore canal 1 from the left is Maker, and the pore canals 2 and 3 are both WC 10-4; 4,5 pore channels are WC10-10, two clearly distinguishable main bands (28S and 18S) can be seen in ultraviolet detection, and the RNA is complete and has no degradation.

Example 3 screening of primers for detecting the expression level of Slicke syzigachne CYP704B1 Gene

1. Reverse transcription (first Strand cDNA Synthesis)

(1) The reverse transcription system shown in table 1 was prepared:

TABLE 1

(2) The reverse transcription reaction was performed on a PCR instrument under the following conditions:

keeping the temperature at 42 ℃ for 15min, 85 ℃ for 5s and 12 ℃.

(3) Diluting the product of (2) by 3 times for later use.

2. Primer design

(1) The sequence of the beckmannia syzigachne CYP704B1 gene is obtained by transcriptome sequencing.

(2) Designing fluorescent quantitative PCR primers.

In the step (2), Oligo 7.0 software and Primer5.0 software are used for designing primers, the designed primers are subjected to specific primer screening at NCBI, and 3 pairs of primers are obtained by screening, wherein the primer sequences, the lengths of amplified fragments and the annealing temperature are shown in a table 2:

TABLE 2 primer sequences, amplified fragment lengths and annealing temperatures

The start and stop sites of the 3 primer pairs in Table 2 on the CYP704B1 gene are as follows: CYP704B1-F1 has a start-stop site of 60-80 bp, CYP704B 1-R1: the start-stop site is 124-144 bp; CYP704B1-F2, start-stop site 628-647 bp, CYP704B 1-R2: 707-727 bp of start and stop sites; CYP704B1-F3, wherein the start-stop site is 947-960 bp, CYP704B 1-R3: 1052-1071 bp of start and stop sites.

3. Primer screening

And (3) screening the designed 3 pairs of primers by using real-time fluorescent quantitative PCR.

Real-time quantitative PCR system:

the real-time quantitative PCR reaction program is as follows:

a、95℃4min，

b、95℃20s，

c、60℃1min，

repeating b-c for 40 cycles;

d. the temperature of the dissolution curve ranged from 60 ℃ to 95 ℃ and was increased from 60 ℃ to 95 ℃ at a rate of 0.5 ℃/min.

Whether the primer is available or not is preliminarily judged by using a dissolution curve, and the judgment standard is that the curve only has a single sharp peak, and the temperature value of the peak is near the Tm value. The results of the primers shown in FIGS. 2-4 (the primers shown in FIGS. 2-4 are CYP704B1-F1/R1, CYP704B1-F2/R2 and CYP704B1-F3/R3, respectively, show that 3 primers meet the standard and can be used for subsequent sequencing verification.

4. Gene amplification

Taking cDNA as a template, and CYP704B1-F1/R1, CYP704B1-F2/R2 and CYP704B1-F3/R3 as primers to carry out PCR, wherein the PCR reaction system comprises 2 mu L of dNTP mixed liquor with 2.5mM, 10 × PCR buffer2.5 mu L, 0.5 mu L of upstream primer and downstream primer with 10 mu M respectively, 0.5 mu L of Taq DNA polymerase with 5U/mu L, 1 mu L of cDNA sample to be detected, and 18 mu L of ddH is added₂O to the reaction system was 25. mu.L.

The PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 30s for 35 cycles; extension at 72 ℃ for 5 min.

5. Specificity verification of primer amplified fragment

(1) Electrophoretic detection

Detecting PCR amplification products corresponding to the primers CYP704B1-F1/R1, CYP704B1-F2/R2 and CYP704B1-F3/R3 in 2% agarose gel electrophoresis, and detecting by an ultraviolet lamp, wherein as shown in figure 5, target bands of PCR amplification of the primers CYP704B1-F2/R2 are not single; PCR amplification of CYP704B1-F3/R3 does not have a target band; the target band amplified by CYP704B1-F1/R1 is a single band, the size of the amplified fragment is positioned at the position of 50bp-100bp, and no dimer exists; the CYP704B1-F1/R1 primer is obtained by screening.

(2) Sequencing validation

Sequencing results show that the fragment amplified by using CYP704B1-F1/R1 as a primer contains a cytochrome P450-CYP704B1 gene fragment.

In conclusion, the primer CYP704B1-F1/R1 can specifically amplify the beckmark grass CYP704B1 gene fragment, only can amplify a unique identification fragment, has no dimer, and has the amplified fragment size of 85 bp. The SYBR Green I dye method shows that the dissolution curve is good, so the primer can be used for detecting the expression quantity of the beckmannia syzigachne CYP704B1 gene.

Example 4 fluorescent quantitative PCR detection of CYP704B1 Gene expression level

1. Preparation of cDNA

The extracted RNA is reverse transcribed into cDNA for subsequent fluorescent quantitative PCR.

2. Preparation of Standard Curve and melting Curve

The cDNA was diluted 3 times to obtain a standard (Ct value of the standard was about 15), and the diluted solution was used as a "stock solution" for real-time fluorescent quantitative PCR preparation of the standard. Diluting a sample to be tested by adopting a multiple ratio dilution method, wherein the method comprises the following steps:

"stock solution" (Standard ①) +1v ddH after 3-fold dilution at 1v₂O, to Standard ②

1v Standard ② +1v ddH₂O, to Standard ③

1v Standard ③ +1v ddH₂O, to Standard ④

1v Standard ④ +1v ddH₂O, to Standard ⑤

And the analogy is that: the fold dilution is carried out to 6 gradients (1,1/2,1/4,1/8,1/16,1/32), 3 folds of total RNA obtained by extraction is used as the initial loading concentration, and 1 fold, 1/2 fold, 1/4 fold, 1/8 fold, 1/16 fold and 1/32 fold of the total RNA are used as templates for establishing standard curves for preparing the standard curves. Wherein v represents a volume.

3. Real-time fluorescent quantitative PCR

The real-time fluorescent quantitative PCR kit RealMasterMix (SYBR Green) is adopted to run on a real-time fluorescent quantitative PCR detector (ABI 7500).

PCR reaction composition 2. mu.L of 2 × SYBR Green 10. mu.L, 2. mu.L each of 10. mu.M primers F1 and R1 or corresponding internal control primers (UBQ-F and UBQ-R), 1. mu.L of template, and ddH₂O to 20. mu.L, this operation was performed on ice.

Real-time fluorescent quantitative PCR amplification procedure:

a、95℃4min，

b、95℃20s，

c、60℃1min，

repeating steps b-c for 40 cycles;

d. the temperature of the dissolution curve ranged from 60 ℃ to 95 ℃ and increased from 60 ℃ to 95 ℃ at a rate of 0.5 ℃/min.

Set Water control (ddH)₂O instead of template) and negative controls (no RNA template added during reverse transcription) to detect contamination of reagents during the experiment. All ofThe samples were replicated three times to ensure confidence in the experimental data.

4. Data processing

An appropriate threshold value of 21.74 is set in the exponential phase of the real-time fluorescence quantitative PCR reaction, and because the Ct value of each template has a linear relation with the logarithm of the initial copy number of the template, the more the initial copy number is, the smaller the Ct value is. Therefore, a standard curve as shown in FIG. 6 was plotted, and the total RNA extracted was diluted 3-fold to obtain the initial loading concentration, and the standard curves were plotted at 1-fold, 1/2-fold, 1/4-fold, 1/8-fold, 1/16-fold, and 1/32-fold of the concentration. Ordinate: fluorescence signal, abscissa: the negative logarithm was taken as the relative concentration of the sample cDNA. The amplification efficiency of the primer CYP704B1-F1/R1 is calculated to be E-2.001; the correlation coefficient is: r²＝0.992。

5. Analysis of test results

The data of the relative expression amount of CYP704B1 Gene was processed by 2^-△△CtThe method is carried out. First, for all test samples and calibration samples, the Ct value of the reference gene (UBQ) is used to equalize the Ct values of the genes of interest:

Δ Ct (test sample) ═ Ct (test sample, CYP704B1 gene) -Ct (test sample, internal reference gene)

Δ Ct (calibration sample) ═ Ct (calibration sample, CYP704B1 gene) -Ct (calibration sample, internal reference gene)

Next, the Δ Ct values of the test samples were homogenized with the Δ Ct values of the calibration samples:

Δ Ct ═ Δ Ct (test sample) - Δct (calibration sample)

Finally, the ratio of expression is calculated, and the ratio of expression quantity is 2^-△△CtThe obtained experimental data were subjected to T-test (P) using SPSS software<0.05) and plotted using Prism software.

The materials of beckmannia syzigachne WC10-4 and WC10-10 are processed for 24h (from the time of growing to the trefoil stage of beckmannia syzigachne to 20 ga.i.ha)^-1Spraying mesosulfuron) and the relative expression of CYP704B1 gene in untreated samples are shown in FIG. 7, the initial expression of CYP704B1 gene in WC10-10 is 0.77 times of that of WC10-4, and the expression after treatment with mesosulfuron is 17.67 times of that of WC 10-4. I.e. in the presence of drug resistanceThe expression level of the CYP704B1 gene of the beckmannia syzigachzigachzigachzigachzigachziensis WC10-10 is obviously higher than that of sensitive biotype WC10-4, the fact that the expression level of the beckmannia syzichzizachzigachziensis CYP704B1 gene is positively correlated with the drug resistance of the beckmannia syzichziensis is verified, the non-target resistance of the beckmannia zigachzigachziensis in the field is timely discovered by detecting the expression level of the CYP704B1, the scientific guiding effect.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Reference to the literature

[1]Duhoux and Delye,2013.Duhoux A,Delye C.2013.Reference genes tostudy herbicide stress response in Lolium sp.:up-regulation of P450genes inplants resistant to acetolactate-synthase inhibitors.PLoS One 8(5).

[2]Michael W.Plaffl.A new mathematical model for relativequantification in real-time RT-PCR.Nucleic Acids Reaearch,2001,29:2002-2007.

[3] Yunhua, Liuhong Yan, Roily Jun. the relative gene expression differences were analyzed by different real-time quantitative PCR methods, journal of crops, 2007,33(7):1214-1218.

[4]Bustin S A,Benes V,Garson J A,et al.The MIQE guidelines:minimuminformation for publication of quantitative real-time PCRexperiments.Clinical Chemistry,2009,55(4):611.

Sequence listing

<110> institute of plant protection of Chinese academy of agricultural sciences

Application of <120> Slash syzigachne CYP704B1 gene

<130>KHP191111085.7

<160>9

<170>SIPOSequenceListing 1.0

<210>1

<211>2036

<212>DNA

<213> Slash syzigachne (Beckmannia syzigachne)

<400>1

tacactagaa acaaacggca ttttgaacat acatgaggat ttggagttta gccgagataa 60

tcctcacaaa tccccacaaa tacactagaa acaaacaggg tctgagaggg aaaataaacg 120

gaggcccttt ccctccactt aaacgtgcgt atttaaaatg acccccggac agagagcagg 180

catggactcc aatggcgctc ctgccgacct cgcacatcgg tctctccctc taccccaata 240

tatccacact tatgcagata tatatatgtc gccgatcact gtgtgttgtc tcacccatcc 300

ttcctctctc tctccaactc tacatcccct ctctcactag aatcaatcaa tatatcgggt 360

caacaatatg atacctggaa gttacatgat gtgcttccaa tttgtggcat gttgcatctt 420

gctcttgctg tactctcttc gatttaggtc tggcagccct gtgcatgggc caagaagcca 480

cctggtgatc ggctgcctgg ttgccttcta cgagaaccgg cggcggctcc tcgattggta 540

caccgagatg ctgtcggcct cgccgactca gacgatcgtc gttgaccggc tgggcgcgcg 600

ccggactgtg gtaaccgcga acccggtcaa cgtcgagcac atcctcaagg gaaacttcgg 660

caactaccca aaggggaagc ccttcaccga tgtgctcggc gacctgctcg gcaagggtat 720

cttcaacgtc gacggcgcga tgtggttcgc gcagcggaag ctggtgagcc acgagttctc 780

tgcgcgcacg ctccgggagc tggaggtcgc cgtgctcgag gccgaggcgc tcgaccggct 840

ggtgccggct ctggaggcgg ccgcggagcc gggcggcggt gccgtggaca tgcaggatgt 900

cctccgccgc ttcgccttcg acgtcatctg ccgtgtctcg ctaggcgttg accctggatg 960

cctcgatccg gcattgcccg cgccgaggct ggcaactgct ttcgacgccg ccgccgggat 1020

catcgccagg cgtggcgccg cgccggtggc cgccgtttgg aagatcaagc gcgcgctgga 1080

catcggctcg gagcggcggc tacgcgagga gatcaatgtc atccacgagg ccgtcatgga 1140

cctcatccgt acccgcaaga aggagcggtt cctggtcaac ggcgcgagga acgacctgct 1200

gtcacgcatg attgaatgcg gctacgccga cgacgagatc cgggacatgg tgatcagttt 1260

catcatggcc ggccgcgaca cgacttcgtc ggcgctgacc tggttcttct ggctgctcac 1320

gcgccaccgc gacgtggagc gggatgtctt ggaggagatc acaagcatga ggcaacaagg 1380

cggctgcagc aacaacgccg gcgaaggctt cgacctcgac gacttccgcc ggatgcgagt 1440

gctccacgcc gcgctgagcg agacgatgcg gctgtacccg ccggtggcgt gggactccaa 1500

gcacgcagcg gcggcggacg tgctgccgga cggcacccgc gtggggcgcg gcgaccgggt 1560

cacctacttc cagtacggca tggggaggat ggaggccatt tggggctccg acgcgggcga 1620

tttcagcctc gacaggtggc tgacgctgcc gcccgacgtg acgggcgggg gcggcgtgtc 1680

ccccttcaag tacccggtgt tccagggtgg cccgcggacg tgcctcggca gggagatggc 1740

cttcgcgcag atgaagttcg tcgcctgcgc cgtgctccgg cggttcgatc tccgccccgt 1800

ggacgagggc cgcacgccgg tgttcctgcc gctgctcacc tcgcacatgg acggcgggct 1860

caacgtgacg gtgaggagga gggcggagac cgcacgccac ggcctgcatg acggagcaac 1920

agctggaaaa aacccattaa ctatctagtg gtatataacc ctcgtgtaca aaaaggatag 1980

aatattatgt gcggtacact gcatgttctc ttctagctgt acatgcactt tgcaat 2036

<210>2

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

atcctcacaa atccccacaa ataca 25

<210>3

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

gtttaagtgg agggaaaggg c 21

<210>4

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

caagaagaag acgtacacca ag 22

<210>5

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

gaccttgtag aactggagga g 21

<210>6

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

aagccccaat caaaatcaac acgaa 25

<210>7

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

aagaacacca agaccccctg c 21

<210>8

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

aggttatcaa tgacaagttt gg 22

<210>9

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

atcaacagtc ttctgggtag c 21

Claims (5)

1. The application of the beckmark grass CYP704B1 gene comprises the following steps:

1) application in identifying resistance of different beckmannia syzigachzigachne material to sulfonylurea herbicide;

2) the application of the syzis syzigachne material as a marker for resistance of the syzigarus syzigachne material to sulfonylurea herbicides;

wherein the nucleotide sequence of the beckmannia syzigachne CYP704B1 gene is shown as SEQ ID NO. 1;

the sulfonylurea herbicide is mesosulfuron-methyl.

2. A method for identifying resistance of different beckmannia syzigachzigachzigachzigachzigachzigachzigachzigachzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzichzi;

wherein the nucleotide sequence of the beckmannia syzigachne CYP704B1 gene is shown as SEQ ID NO. 1;

the sulfonylurea herbicide is mesosulfuron-methyl.

3. The method of claim 2, wherein the method comprises: extracting total RNA of beckmannia syzigachne to be detected, and performing reverse transcription to obtain cDNA; then, carrying out real-time fluorescent quantitative PCR amplification by using the primers CYP704B1-F1 and CYP704B1-R1 and taking the cDNA as a template; simultaneously setting an internal reference gene, and calculating the relative expression quantity of the CYP704B1 gene;

wherein, the nucleotide sequences of the primers CYP704B1-F1 and CYP704B1-R1 are respectively shown in SEQ ID NO 2 and 3.

4. The method of claim 3, wherein the reference genes are selected from at least one of UBQ, CAP and GADPH, and the primers for amplifying each reference gene are as follows:

UBQ-F：5ˊ-CAAGAAGAAGACGTACACCAAG-3ˊ

UBQ-R: 5ˊ-GACCTTGTAGAACTGGAGGAG-3ˊ；

CAP-F:5ˊ-AAGCCCCAATCAAAATCAACACGAA-3ˊ

CAP-R: 5ˊ-AAGAACACCAAGACCCCCTGC-3ˊ；

GADPH-F：5ˊ-AGGTTATCAATGACAAGTTTGG-3ˊ

GADPH-R：5ˊ-ATCAACAGTCTTCTGGGTAGC-3ˊ。

5. the method of claim 3 or 4, wherein the PCR reaction system and the reaction procedure are as follows:

2 mu L of 2.5mM dNTP mixed solution, 2.5 mu L of 10 × PCR buffer, 0.5 mu L of 10 mu M upstream and downstream primers respectively, 0.5 mu L of 5U/mu L Taq DNA polymerase, 1 mu L cDNA, and ddH₂O to 25 μ L;

PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension at 72 ℃ for 5 min.

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