CN109837355A - The application of Wang grass CYP704B1 gene - Google Patents

Abstract

The application of this invention Ti Gong Wang grass CYP704B1 gene.The present invention also provides the methods of fluorescence quantification PCR primer (SEQ ID NO:2-3) and the Jian Ce Wang grass CYP704B1 gene expression amount established based on the primer for Jian Ce Wang grass CYP704B1 gene expression amount, with this Pan Duan Wang grass to the resistance of sulfonylurea (especially mesosulfuron) herbicide.The method has the advantages that simplicity, high sensitivity, high specificity , Wang grass to important application prospects in the detection and research of the non-target resistance of mesosulfuron.The invention firstly discloses the relationships between Wang grass CYP704B1 gene and sulfonylurea herbicide resistance, Wang grass being Dinged come Jian by detecting the expression quantity of CYP704B1 gene and whether metabolic resistance being generated to herbicide, is and guided by farmland Scientific Usage of Drugs and alleviates drug resistance for timely discovery resistance and further spread with directive significance.

Description

The application of Wang grass CYP704B1 gene

Technical field

The present invention relates to gene engineering technology field, specifically, She Ji Wang grass CYP704B1 gene application.

Background technique

The long-continued not scientific application of herbicide easily leads to the generation of herbicide resistance, China some areas wheat and oil Cai Tian Wang grass produces serious resistance to herbicide, and as the improvement of the generation , Shi Wang grass of non-target resistance is more difficult. Currently, existing research shows that Cytochrome P450 passes through the hydroxylating of dealkylation, cyclohexyl methyl hydroxylation, aromatic ring Herbicide metabolism can be innocuous substance by the modes such as effect, desulfurization oxidation and catalysis ester linkage breaking, and in P450 family The expression quantity increase of certain genes can make originally sensitive weed population generate resistance (Yasuor H, Osuna M to herbicide D,Ortiz A,et al.Mechanism of resistance to penoxsulam in late watergrass [Echinochloa phyllopogon(Stapf)Koss.].Journal of Agricultural and Food Chemistry,2009,57:3653–3660；Yang Q,Deng W,Li X F,et al.Target-site and non- target-site based resistance to the herbicide tribenuron-methyl in flixweed (Descurainia sophiaL.).BMC Genomics,2016,17(1):551；Zhao B C,Fu D,Yu Y,et al.Non-target-site resistance to ALS-inhibiting herbicides in a Sagittaria trifolia L.population.Pesticide Biochemistry Physiology,2017,140:79-84；Pan L, Gao H T,Xia W W.Establishing a herbicide-metabolizing enzyme library in Beckmannia syzigachne to identify genes associated with metabolic resistance.Journal of Experimental Bottany,2016,67:1745-1757)。

Summary of the invention

The application of purpose Shi Ti Gong Wang grass CYP704B1 gene of the invention.

Present inventive concept is as follows: since the expression in the different Wang grass material of related gene of metabolism herbicide is in the presence of poor Different, resistance is different between Zao Cheng Wang grass material, scientifically to administer Kang Yao Wang grass, it is necessary first to study its resistance mechanism.This The primer (as fluorescent quantitation primer) of the expression quantity of the Jian Ce Wang grass CYP704B1 gene provided is invented, itself is without hair clip knot Structure does not form primer dimer, and specific amplified uniquely identifies segment, and amplified production length is 85b_p.Utilize the change of fluorescence signal The variation for changing each of real-time monitoring pcr amplification reaction cyclic amplification product amount, passes through the relationship of Ct value and standard curve Quantitative analysis is carried out to starting template, is quickly judged in different times, the expression quantity of different materials Jian Wang grass CYP704B1, into And Que Dings Wang grass resistance situation, the science that can be used for administers Kang Yao Wang grass.

In order to achieve the object of the present invention, in a first aspect, following any application of this invention Ti Gong Wang grass CYP704B1 gene:

1) identifying different Wang grass material to the application in sulfonylurea herbicide resistance；

2) as different Wang grass material to the application in the marker of sulfonylurea herbicide resistance；

3) application in anti-sulfonylurea herbicide Wang grass material is being identified.

, Suo Shu Wang grass CYP704B1 gene in the present invention are as follows:

I) nucleotide sequence shown in SEQ ID NO:1；

Ii) nucleotide sequence shown in SEQ ID NO:1 be substituted, lack and/or increase one or more nucleotide and Express the nucleotide sequence of identical function protein；

Iii) and i), ii) nucleotide sequence there is 90% or more homology and express the nucleosides of identical function protein Acid sequence.

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

Second aspect, the present invention is provided to the Specific PCR primers of Kuo Zeng Wang grass CYP704B1 gene (i.e. for detecting The primer of Wang grass CYP704B1 gene expression amount), including CYP704B1-F1 and CYP704B1-R1, nucleotide sequence is respectively such as Shown in SEQ ID NO:2 and 3.

The primer CYP704B1-F1/R1 for Jian Ce Wang grass CYP704B1 gene expression amount, can be by the following method It obtains:

(1) Li is analysed in depth Yong the gene order that Wang grass transcript profile is sequenced to obtain CYP704B1；

(2) the alternative primer of quantitative fluorescent PCR is designed, subsequent screening is used for；

(3) Dai Jian Ce Wang grass total serum IgE, reverse transcription cDNA are extracted；

(4) using cDNA obtained by step (3) as template, the primer with step (2) design is that guidance carries out real time fluorescent quantitative PCR amplification filters out the satisfactory primer pair of solubility curve；

(5) using cDNA obtained by step (3) as template, the primer with step (4) screening is that guidance carries out regular-PCR amplification, Gained amplified production is sequenced, verifying primer whether specific amplified target gene CYP704B1, finishing screen selects special inspection Survey the primer CYP704B1-F1/R1 of CYP704B1 gene expression amount.

In step (2), using 7.0 software of Oligo and Primer5.0 software design fluorescence quantification PCR primer, fluorescence is fixed Measure PCR primer design standard are as follows: primer length is greater than 60 in 20-25nt, the of length no more than 300bp of amplified production, annealing temperature DEG C, for annealing temperature gap between 1 DEG C, 3 ends ˊ are not preferably A between single primer, and primer itself does not form hairpin structure, not shape At primer dimer.

SYBR Green I dyestuff and any double-stranded DNA distribute fluorescence after being combined, so if having in reaction system The generation of non-specific amplification or primer dimer will be also detected simultaneously, so as to cause testing result inaccurate, and this The primer CYP704B1-F1/R1 that invention finishing screen is selected itself does not form primer dimer, is only capable of expanding only without hairpin structure One identifies segment, therefore its testing result is credible.

The third aspect, the present invention provide the detection reagent or kit for containing above-mentioned primer.

Further, the kit further includes the fluorescent dye being used cooperatively with the primer, such as SYBR Green I.

Fourth aspect, the present invention provide a kind of method of Jian Ce Wang grass CYP704B1 gene expression amount, which comprises Extract Dai Ce Wang grass total serum IgE, reverse transcription cDNA；Then, using primer CYP704B1-F1 and CYP704B1-R1, it is with cDNA Template carries out real-time fluorescence quantitative PCR amplification；Reference gene is set simultaneously, calculates the relative expression quantity of CYP704B1 gene.

In the present invention, the reference gene is selected from least one of UBQ, CAP and GADPH, for expanding each internal reference base The primer difference of cause is following (SEQ ID NO:4-9):

UBQ-F:5 ˊ-CAAGAAGAAGACGTACACCAAG-3 ˊ

UBQ-R:5ˊ-GACCTTGTAGAACTGGAGGAG-3ˊ；

CAP-F:5ˊ-AAGCCCCAATCAAAATCAACACGAA-3ˊ

CAP-R:5ˊ-AAGAACACCAAGACCCCCTGC-3ˊ；

GADPH-F:5 ˊ-AGGTTATCAATGACAAGTTTGG-3 ˊ

GADPH-R:5 ˊ-ATCAACAGTCTTCTGGGTAGC-3 ˊ.

Optionally, PCR reaction system and response procedures are as follows:

PCR reaction system: 2 μ L, 10 × PCR Buffer of 2.5mM dNTP mixed liquor, 2.5 μ L, 10 μM of upstream and downstream primers 0.5 1 μ L of μ L, cDNA of each 0.5 μ L, 5U/ μ L Taq archaeal dna polymerase, uses ddH₂O polishing is to 25 μ L.

PCR response procedures: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 35 Circulation；72 DEG C of extension 5min.

5th aspect, the present invention provide the above method and are identifying different Wang grass material in sulfonylurea herbicide resistance Using.Wherein, the application includes being identified different Wang grass material to sulfonylureas according to the relative expression quantity of CYP704B1 gene The resistance of class (especially mesosulfuron) herbicide.When the relative expression quantity of CYP704B1 gene is 2 times of sensitive material Show that Dai Ce Wang grass material is resistant material.

6th aspect, the present invention provide a kind of anti-sulfonylurea (especially mesosulfuron) Herbicid resistant phase of grass Yu Wang The DNA molecular marker of pass, on the DNA molecular marker Wei Yu Wang grass CYP704B1 gene, for expanding the DNA molecular marker Primer include CYP704B1-F1 and CYP704B1-R1, nucleotide sequence is respectively as shown in SEQ ID NO:2 and 3.

7th aspect, the present invention provide the application in above-mentioned DNA molecular marker Wang grass molecular mark.

In the specific embodiment of the present invention, for the method for Jian Ce Wang grass CYP704B1 gene expression amount, packet Include following steps:

(1) it extracts after Jian Ce Wang grass sample after mesosulfuron is handled for 24 hours with untreated total serum IgE, reverse transcription is cDNA；

(2) using cDNA obtained by step (1) as template, PCR amplification sequencing is carried out by primer of CYP704B1-F1/R1；

(3) it regard the cDNA sample after gradient dilution (6 gradients, concentration difference is 2 times between gradient) as standard form, it is sharp Real-time fluorescence quantitative PCR is carried out with primer CYP704B1-F1/R1, draws standard curve, threshold value (real-time fluorescence quantitative PCR is set For the fluorescence signal of preceding 15 circulations of reaction as autofluorescent background signal, fluorescence threshold is set as the fluorescence signal of 3-15 circulation 10 times of standard deviation)；

(4) to carry out real-time fluorescence quantitative PCR amplification to reference gene, obtain wait examine the cDNA for surveying Wang grass sample as template Corresponding amplification efficiency e and Ct value (fluorescence signal in each reaction tube reaches recurring number experienced when the threshold value of setting Referred to as Ct value), calculate the expression quantity e of reference gene^-ΔΔt, correction for subsequent Dai Jian Ce Wang grass sample cDNA concentration；

(5) primer CYP704B1-F1/R1 is utilized, the cDNA of Wang grass sample to be detected is carried out simultaneously with step (4) real-time Fluorescent quantitative PCR, Dinging the corresponding Ct value of Wang grass sample according to reaction result Que, (fluorescence signal in each reaction tube reaches Recurring number experienced is referred to as Ct value when the threshold value of setting) and amplification efficiency E, Ji Suan Wang grass sample CYP704B1 gene table Up to amount E^-ΔΔt, further utilize the expression quantity e of reference gene^-ΔΔtBe corrected, acquisition be able to carry out sample room compare it is opposite Expression quantity E^-ΔΔt/e^-ΔΔt；

(6) than the relative expression quantity less with CYP704B1 gene in Wang grass sample, disconnected Wang grass is sentenced to the resistance of herbicide.

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

Total RNA content 800ng,SuperMix (Buffer containing PCR, RNase inhibitor, dNTPs、Anchored Oligo(dT)₁₈Primer) 4 μ L, gDNA Remover, 1 μ L, adds ddH₂O to 20 μ L.

Reverse transcription is the PCR response procedures of cDNA are as follows: 42 DEG C of 15min, 85 DEG C of 5s, 12 DEG C of heat preservations.

In step (2), using the cDNA as template, CYP704B1-F1/R1 is that primer carries out PCR amplification, PCR reactant System are as follows: 2 μ L, 10 × PCR Buffer of dNTP the mixed liquor 2.5 μ L, each 0.5 μ L, 5U/ μ L of 10 μM of upstream and downstream primer of 2.5mM Taq archaeal dna polymerase 0.5 μ L, 1 μ L of cDNA sample to be measured, be added 18 μ L ddH₂O is 25 μ L to reaction system, this operation exists It carries out on ice.

PCR response procedures are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 35 A circulation；72 DEG C of extension 5min.

Step (3), (4), in (5), using the cDNA as template, CYP704B1-F1/R1 or corresponding internal control primer are to draw Object carries out real-time fluorescence quantitative PCR amplification, real-time fluorescence quantitative PCR reaction system are as follows: 2 × SYBR Green, 10 μ L, 10 μM are drawn Object F1 and R1 or each 2 μ L of corresponding internal control primer, 1 μ L of template add ddH₂O to 20 μ L.

Step (3), (4), real-time fluorescence quantitative PCR reacts amplification program in (5) are as follows:

a,95℃4min；

b,95℃20s；

c,60℃1min；

Repeat b-c step, 40 circulations；

D, solubility curve temperature range is 60-95 DEG C, rises to 95 DEG C with 60 DEG C of speed of 0.5 DEG C/min.

Reference gene is UBQ in step (4), using the geometric mean of internal reference gene expression amount as corrected value, to obtain Accurate calibration result.

By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that

The invention firstly discloses Wang grass CYP704B1 gene and sulfonylurea (especially mesosulfuron) herbicide are anti- Relationship between property, expression quantity Yu resistance of the Wang grass to mesosulfuron of CYP704B1 gene are positively correlated.Therefore, can pass through The expression quantity of detection CYP704B1 gene, which carrys out Jian, Dings Wang grass and whether generates metabolic resistance to herbicide, to timely discovery resistance and refers to It leads farmland Scientific Usage of Drugs and alleviates drug resistance and further spread all with directive significance.

The method of Jian Ce Wang grass CYP704B1 expression quantity provided by the invention has simplicity, high sensitivity, high specificity There is important application prospect in advantage , Wang grass drug resistance research, while precisely being administered Wei the drug resistance of Wang grass and providing reference Foundation.

Detailed description of the invention

Fig. 1 is RNA electrophoresis detection figure in the embodiment of the present invention 2.

Fig. 2 is the corresponding solubility curve of detection primer CYP704B1-F1/R1 in the embodiment of the present invention 3.

Fig. 3 is the corresponding solubility curve of detection primer CYP704B1-F2/R2 in the embodiment of the present invention 3.

Fig. 4 is the corresponding solubility curve of detection primer CYP704B1-F3/R3 in the embodiment of the present invention 3.

Fig. 5 is primer CYP704B1-F1/R1, CYP704B1-F2/R2 and CYP704B1-F3/R3 in the embodiment of the present invention 3 The electrophoresis detection figure of amplified production.Wherein, M Marker；1 is CYP704B1-F1/R1 amplified production, and 2 be CYP704B1-F2/ R2 amplified production；3 be CYP704B1-F3/R3 amplified production.

Fig. 6 is the standard curve drawn in the embodiment of the present invention 4.

Fig. 7 is WC10-4 and WC10-10 in the embodiment of the present invention 4 after mesosulfuron processing for 24 hours and when untreated CYP704B1 gene relative expression quantity, abscissa Biao Shi Wang grass title material, ordinate indicate CYP704B1 gene relative expression Amount.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW, Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.

Total RNA extraction reagent box Direct-zol RNA Miniprep respects biology purchased from hundred people of Beijing in following embodiment Science and Technology Ltd., product number: R2050.

Reverse transcription reagent box is purchased from Quan Shi King Company, product number: AT311-03.

Real-time fluorescence quantitative PCR kit Bestar^TMQ PCR MasterMix (SYBR Green) is raw purchased from the Shanghai Milky Way Object Science and Technology Ltd., product number: DBI-2043.

WC10-4 picks up from Jingjiang City, Jiangsu Province, northern six village wastelands, south of a city town, is mesosulfuron sensitive biological type, WC10-10 Ten groups of Jiangsu Province, Hongze County, the Huai'an Huang market town village Yang Hu is picked up from, is mesosulfuron drug-resistant organisms type.Referring to WANG J, LI X,Dan L I,et al.Non-target-site and target-site resistance to AHAS inhibitors in American sloughgrass(Beckmannia syzigachne)[J].Journal of integrative agriculture,2018,17(12):2714-2723.

The acquisition of embodiment 1CYP704B1 gene

The sensitive population WC10-4 and insecticide resistant populations WC10-10 for picking up from Jiangsu Province are chosen in test.

1, transcript profile is sequenced

Min Gan Wang grass and Kang Yao Wang grass are chosen after mesosulfuron is handled for 24 hours each 3 parts with untreated sample, send public affairs Department carries out transcript profile sequencing.

2, the acquisition of CYP704B1 gene

Difference expression gene is screened according to transcription sequencing result, differential expression can determine that by the enrichment of pathway conspicuousness Gene participates in the signal path of jasmonic, and gene information and gene order are obtained from sequencing result.

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

The extraction of 2 total serum IgE of embodiment

Test is chosen and picks up from Jiangsu Province sensitive population WC10-4 and insecticide resistant populations WC10-10.

1. the extraction of total serum IgE

Min Gan Wang grass and Kang Yao Wang grass are chosen after mesosulfuron is handled for 24 hours each 3 parts with untreated sample, according to The specification of kit extracts total serum IgE respectively, obtains corresponding total rna solution.

2.RNA agarose gel electrophoresis

3uL total rna solution is taken, carries out electrophoresis detection, voltage 160V with 1% Ago-Gel, electrophoresis time is 20min, and scanned with gel imaging.As shown in Figure 1,1 duct is Maker from left to right, 2,3 ducts are WC10-4；4,5 ducts are equal For WC10-10, two clear differentiable master tapes (28S and 18S) can be seen in ultraviolet detection, show RNA completely without degradation.

The screening of the primer of 3 Jian Ce Wang grass CYP704B1 gene expression amount of embodiment

1. reverse transcription (synthesis of the first chain of cDNA)

(1) with reverse transcription system shown in tabulation 1:

Table 1

(2) reverse transcription reaction is carried out by following condition in PCR instrument:

42 DEG C of 15min, 85 DEG C of 5s, 12 DEG C of heat preservations.

(3) spare by 3 times of product dilution of (2).

2. design of primers

(1) get Dao Wang grass CYP704B1 gene order is sequenced by transcript profile.

(2) fluorescence quantification PCR primer is designed.

In step (2), using 7.0 software of Oligo and Primer5.0 software Design primers, the primer of design NCBI into Row specific primer sieve, screening obtain 3 pairs of primers, and primer sequence, expanding fragment length and annealing temperature are shown in Table 2:

2 primer sequence of table, expanding fragment length and annealing temperature

Start-stop site difference of 3 pairs of primers on CYP704B1 gene in table 2 is as follows: CYP704B1-F1: start-stop site 60~80bp, CYP704B1-R1: 124~144bp of start-stop site；CYP704B1-F2: 628~647bp of start-stop site, CYP704B1-R2: 707~727bp of start-stop site；CYP704B1-F3: start-stop site 947~960bp, CYP704B1-R3: it rises 1052~1071bp of stop bit point.

3. primer screening

It is screened with 3 pair primers of the real-time fluorescence quantitative PCR to above-mentioned design.

Real-time quantitative PCR system:

Real-time quantitative PCR response procedures are as follows:

A, 95 DEG C of 4min,

B, 95 DEG C of 20s,

C, 60 DEG C of 1min,

Repeat b-c, 40 circulations；

D, solubility curve temperature range is 60-95 DEG C, will rise to 95 DEG C by 60 DEG C with the speed of 0.5 DEG C/min.

Tentatively judging whether primer can be used using solubility curve, only there is single sharp peak for curve in judgment criteria, and The temperature value for peak occur should be near Tm value.By Fig. 2-Fig. 4 (Fig. 2-Fig. 4 respectively corresponds primer: CYP704B1-F1/R1, CYP704B1-F2/R2 and CYP704B1-F3/R3, the results showed that 3 primers meet the standard, can be used for subsequent sequence verification.

4. gene magnification

Using cDNA as template, using CYP704B1-F1/R1, CYP704B1-F2/R2 and CYP704B1-F3/R3 as primer into Row PCR, wherein PCR reaction system are as follows: dNTP mixed liquor 2 μ L, the 10 × PCR Buffer2.5 μ L of 2.5mM, 10 μM upper and lower Taq archaeal dna polymerase 0.5 μ L, the 1 μ L of cDNA sample to be measured of each 0.5 μ L, 5U/ μ L of primer are swum, 18 μ LddH are added₂O is to reactant System is 25 μ L.

PCR response procedures are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 60 DEG C of annealing 45s, 72 DEG C of extension 30s, totally 35 A circulation；72 DEG C of extension 5min.

5. the specificity verification of primer amplification segment

(1) electrophoresis detection

By the corresponding pcr amplification product of primer CYP704B1-F1/R1, CYP704B1-F2/R2 and CYP704B1-F3/R3 It is detected in 2% agarose gel electrophoresis, ultraviolet lamp detection, as shown in figure 5, the mesh of the PCR amplification of primer CYP704B1-F2/R2 Band it is not single；The PCR amplification of CYP704B1-F3/R3 is without purpose band；CYP704B1-F1/R1 amplification purpose band be Single band, amplified fragments size are located at the position 50bp-100bp, no dimer；Screening obtains CYP704B1-F1/R1 primer.

(2) sequence verification

Sequencing result is shown, contains Cytochrome P450-using the segment that CYP704B1-F1/R1 goes out as primer amplification CYP704B1 genetic fragment.

To sum up, primer CYP704B1-F1/R1, can the different Kuo Zeng Wang grass CYP704B1 genetic fragment of Te, be only capable of expanding unique Identify segment, and without dimer, amplified fragments size is 85bp.SYBR Green I dye method shows that solubility curve is good, because This primer can be used for the expression quantity of Jian Ce Wang grass CYP704B1 gene.

4 fluorescence quantitative PCR detection CYP704B1 gene expression amount of embodiment

1, the preparation of cDNA

The RNA of extraction is subjected to reverse transcription into cDNA, is used for subsequent quantitative fluorescent PCR.

2, the production of standard curve and melting curve

1. (the Ct value of the standard items is about 15) as standard items by 3 times of cDNA dilution, and using the dilution as in real time " stoste " of quantitative fluorescent PCR configuration standard product.Sample to be tested is diluted using doubling dilution method, the method is as follows:

" stoste " (standard items are 1.)+1v ddH after 1v dilutes 3 times₂2. O obtains standard items

1v standard items 2.+1v ddH₂3. O obtains standard items

1v standard items 3.+1v ddH₂4. O obtains standard items

1v standard items 4.+1v ddH₂5. O obtains standard items

And so on: multiple is diluted to 6 gradients (1,1/2,1/4,1/8,1/16,1/32), to extract obtained total serum IgE Dilute 3 times be used as loading initial concentrations, using 1 times of the concentration, 1/2 times, 1/4 times, 1/8 times, 1/16 times, 1/32 times as foundation The template of standard curve, the preparation for standard curve.Wherein, v indicates volume.

3, real-time fluorescence quantitative PCR

Using real-time fluorescence quantitative PCR kit RealMasterMix (SYBR Green), in real-time fluorescence quantitative PCR It is run on detector (ABI7500).

PCR reaction composition: 2 × SYBR Green, 10 μ L, 10 μM of primers Fs 1 and R1 or corresponding internal control primer (UBQ-F and UBQ-R) each 2 μ L, 1 μ L of template, adds ddH₂O to 20 μ L, this operation carry out on ice.

Real-time fluorescence quantitative PCR reacts amplification program:

A, 95 DEG C of 4min,

B, 95 DEG C of 20s,

C, 60 DEG C of 1min,

Repeat b-c step, 40 circulations；

D, solubility curve temperature range is 60-95 DEG C, rises to 95 DEG C by 60 DEG C with the speed of 0.5 DEG C/min.

Water is set and compares (ddH₂O replace template) and negative control (not adding RNA template when reverse transcription) carry out test experience mistake Reagent contamination in journey.All samples are done to be repeated three times, to ensure the confidence level of experimental data.

4, data processing

Suitable threshold value is set as 21.74, due to the Ct value of each template in the exponential phase of real-time fluorescence quantitative PCR reaction There are linear relationships with the logarithm of the starting copy number of the template, and starting copy number is more, and Ct value is smaller.Therefore, drafting obtains Standard curve as shown in FIG. 6 dilutes 3 times as loading initial concentration, with 1 times of the concentration, 1/ to extract obtained total serum IgE 2 times, 1/4 times, 1/8 times, 1/16 times, 1/32 times are made standard curve.Ordinate: fluorescence signal, abscissa: with sample cDNA concentration Relative concentration takes negative logarithm.It is computed, the amplification efficiency of primer CYP704B1-F1/R1 is E=2.001；Related coefficient are as follows: R² =0.992.

5, test result analysis

The relative expression quantity data processing of CYP704B1 gene uses 2^-△△CtMethod.First to all test specimens and school Quasi- sample, with the Ct value of the uniform target gene of Ct value of reference gene (UBQ):

△ Ct (test specimen)=Ct (test specimen, CYP704B1 gene)-Ct (test specimen, reference gene)

△ Ct (calibration sample)=Ct (calibration sample, CYP704B1 gene)-Ct (calibration sample, reference gene)

Secondly, with the △ Ct value of the uniform test sample of △ Ct value of calibration sample:

△ △ Ct=△ Ct (test specimen)-△ Ct (calibration sample)

The ratio of last calculation expression, ratio=2 of expression quantity^-△△Ct, resulting experimental data is carried out with SPSS software T-test examines (P < 0.05) and is mapped with Prism software.

(is when Wang grass length to tri-leaf period for 24 hours after mesosulfuron processing by Wang grass material WC10-4 and WC10-10, with 20g a.i.ha^-1Spray mesosulfuron) and untreated sample in CYP704B1 gene relative expression quantity as shown in fig. 7, WC10- The initial expression quantity of CYP704B1 gene is 0.77 times of WC10-4, the expression quantity after mesosulfuron post-processes respectively in 10 It is 17.67 times of WC10-4 respectively.It is significantly higher than sensitivity in the expression quantity of the CYP704B1 gene of Kang Yao Wang grass WC10-10 The expression quantity of bion WC10-4, Zheng Shi Wang grass CYP704B1 gene is positively correlated Yu Wang grass drug resistance, passes through detection The expression quantity of CYP704B1 finds the non-target resistance of Tian Jian Wang grass in time, has the directive function of science to farmland medication, from And the sprawling of such Kang Yao Wang grass seeds group is controlled in time, reduce peasant economy loss.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Bibliography

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

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

[3] Yu Shunwu, Liu Hongyan, Raleigh army analyze Relative gene differential expression using different real time quantitative PCR methods Acta Agronomica Sinica, 2007,33 (7): 1214-1218.

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

Sequence table

<110>Plant Protection institute, Chinese Academy of Agricultral Sciences

< the application of 120>Wang grass CYP704B1 genes

<130> KHP191111085.7

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2036

<212> DNA

< 213>Wang are careless (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 (10)

1. following any application of Wang grass CYP704B1 gene:

1) identifying different Wang grass material to the application in sulfonylurea herbicide resistance；

2) as different Wang grass material to the application in the marker of sulfonylurea herbicide resistance；

3) application in anti-sulfonylurea herbicide Wang grass material is being identified；

Wherein , Suo Shu Wang grass CYP704B1 gene are as follows:

I) nucleotide sequence shown in SEQ ID NO:1；

Ii) nucleotide sequence shown in SEQ ID NO:1 is substituted, lacks and/or increases one or more nucleotide and expression The nucleotide sequence of identical function protein；

Iii) and i), ii) nucleotide sequence there is 90% or more homology and express the nucleotides sequence of identical function protein Column.

2. application according to claim 1, which is characterized in that the sulfonylurea herbicide includes mesosulfuron.

3. being used for the Specific PCR primers of Kuo Zeng Wang grass CYP704B1 gene, which is characterized in that including CYP704B1-F1 and CYP704B1-R1, nucleotide sequence is respectively as shown in SEQ ID NO:2 and 3.

4. detection reagent or kit containing primer described in claim 3.

5. kit according to claim 4, which is characterized in that the kit further includes being used cooperatively with the primer Fluorescent dye；

Preferably, the fluorescent dye is SYBR Green I.

6. the method for Jian Ce Wang grass CYP704B1 gene expression amount, which is characterized in that the described method includes: it is total to extract Dai Ce Wang grass RNA, reverse transcription cDNA；Then, using primer described in claim 3, using cDNA as template, real-time fluorescence quantitative PCR is carried out Amplification；Reference gene is set simultaneously, calculates the relative expression quantity of CYP704B1 gene.

7. according to the method described in claim 6, it is characterized in that, the reference gene in UBQ, CAP and GADPH extremely One few, the primer for expanding each reference gene is distinguished as follows:

UBQ-F:5 ˊ-CAAGAAGAAGACGTACACCAAG-3 ˊ

UBQ-R:5ˊ-GACCTTGTAGAACTGGAGGAG-3ˊ；

CAP-F:5ˊ-AAGCCCCAATCAAAATCAACACGAA-3ˊ

CAP-R:5ˊ-AAGAACACCAAGACCCCCTGC-3ˊ；

GADPH-F:5 ˊ-AGGTTATCAATGACAAGTTTGG-3 ˊ

GADPH-R:5 ˊ-ATCAACAGTCTTCTGGGTAGC-3 ˊ.

8. method according to claim 6 or 7, which is characterized in that PCR reaction system and response procedures are as follows:

PCR reaction system: 2 μ L, 10 × PCR Buffer of 2.5mM dNTP mixed liquor, 2.5 μ L, 10 μM of upstream and downstream primers each 0.5 0.5 1 μ L of μ L, cDNA of μ L, 5U/ μ L Taq archaeal dna polymerase, adds ddH₂O to 25 μ L；

PCR response procedures: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 35 are followed Ring；72 DEG C of extension 5min.

9. any one of claim 6-8 the method is identifying different Wang grass material to answering in sulfonylurea herbicide resistance With.

10. application according to claim 9, which is characterized in that the application includes according to the opposite of CYP704B1 gene Expression quantity identifies different Wang grass material to the resistance of sulfonylurea herbicide；

Preferably, the sulfonylurea herbicide includes mesosulfuron.