CN102242201B - Methods for detecting drug resistance of cockspur grass to quinclorac - Google Patents
Methods for detecting drug resistance of cockspur grass to quinclorac Download PDFInfo
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- CN102242201B CN102242201B CN 201110154159 CN201110154159A CN102242201B CN 102242201 B CN102242201 B CN 102242201B CN 201110154159 CN201110154159 CN 201110154159 CN 201110154159 A CN201110154159 A CN 201110154159A CN 102242201 B CN102242201 B CN 102242201B
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Abstract
The invention provides new gene fragments and primers for detecting the drug resistance of cockspur grass to quinclorac and methods for detection by the gene fragments and primers. Whether cockspur grass has drug resistance to the quinclorac can be accurately detected by the methods.
Description
Technical field
The invention belongs to and utilize gene fragment or sequence to carry out weeds resistance detection range, especially, belong to and utilize the barnyard grass grass that the drug-fast gene fragment of quinclorac and primer are detected the barnyard grass grass to the quinclorac resistance and utilize the drug-fast method of these gene tests.
Background technology
358 biotypes by the existing 197 kinds of weeds (115 kinds dicotyledonous, 82 kinds of unifacial leaves) in May, 2011 whole world have produced resistance to 19 class chemical herbicides, almost the cover all kinds significant herbicide.The resistance weeds have become the serious threat of weeds improvement and agriculture production, are extremely paid close attention in the whole world by serious economy and the safety problem of its initiation.
Barnyard grass grass (Echinochloa crusgalli) is global rice field malignant weed, is first of the 15 kinds of serious harm weeds in China farmland.And the quinclorac (Quincloric) of BASF Aktiengesellschaft in 1988 exploitation is the specifics of preventing and kill off dogstail such as barnyard grass grass, from 1990 since China promotes the use of, preventive effect is remarkable, but, because more than ten years use continuously, engender resistance barnyard grass grass in China north and south rice district, and be on the rise.Stretching of resistance barnyard grass grass brought many negative effects with wildness, as reducing the input of species diversity, increase agricultural chemicals, pollutes the home environment of crop, reduces the yield and quality of paddy rice etc.
GH3 (Gretchen Hagen 3) gene is the early stage responsive genes of plant hormone (Primary-response genes) that clones from soybean the earliest, afterwards Arabidopis thaliana (19 GH3 family members are arranged), paddy rice (the similar gene that 13 GH3 are arranged), tobacco (at least 1 GH3 gene), capsicum (1 GH3 gene)), find many GH3 genes or its similar gene among Indian mustard (the similar gene that 2 GH3 are arranged) and the bryophyte P.patens (3 GH3 family members are arranged), the GH3 that has exists with the form of gene family, for example in the arabidopsis gene group, at the 5th karyomit(e) 5 GH3 genes are arranged, and these gene directions as one man arranged in series is together.Growth hormone can induce the most gene in the GH3 gene family to express fast, instantaneously.This rapid answer is one of mechanism of regulating growth hormone running balance in the plant materials.Plant GH3 albumen has plant hormone amino acid compounds such as IAA and becomes enzyme and adenosine to be combined to enzymic activity.
Fast, accurate, high-throughout weeds resistance detection method, be a key link of resistance control of weeds.Weeds resistance detection method mainly contains (1), the whole strain plant identification method in greenhouse at present.It is a kind of major technique that international weeds resistance management organization is recommended, and advantage is simple and easy to do, good reproducibility.Shortcoming is that greenhouse space is big, and the test period is grown, can not identify this season weeds.(2), the morphological structure of tissue or organ identification method relatively, comprise seed or seedling detection method, the detection method of tillering, pollen germination method, leaf disk dipping technique mensuration etc., its advantage is simple and easy to do, shortcoming is inaccurate.(3), cell or organoid level determination, comprise the leaf chlorophyll fluorometry, exsomatize chloroplast(id) determination techniques, photosynthetic rate assay method, advantage is accurately, shortcoming be complicated, to technology and equipment requirement height.(4), the Physiology and biochemistry identification method, mainly be the enzyme assay method, shortcoming is: known weeds of suitable target enzyme only.(5), DNA analysis technical evaluation method, mainly be the mutational site of target gene sequence relatively.Shortcoming is, only the weeds of suitable mutant target gene.(6), the identification method of protein level, mainly be the detection of the specific protein of ELISA method antagonism weeds.Shortcoming is only to be fit to the resistance weeds of known specific protein.
It is with the obvious advantage that the marker gene method detects the weeds resistance, at first it can realize on a large scale, the sample size detection of high energy throughput, thereby can grasp weeds resistance level and the scope in big area farmland rapidly.Secondly the weeds grass that this season rice field is taken place is realized detecting, and grasps its resistance level.The 3rd is that sense cycle is short, to go out the result fast, only needs several days just can get the result.Its shortcoming is that instrument is had relatively high expectations, and quantitative real time PCR Instrument is more expensive.
Summary of the invention
A marker gene can only detect weeds resistance a kind of or a class agricultural chemicals.So the marker gene of excavation agricultural chemicals is development and the only way of improving the marker gene method.The marker gene of agricultural chemicals should possess: in the weeds plant of different resistance levels, marker gene there are differences at the expression amount of transcriptional level, this species diversity is to distinguish the drug-fast quantitative targets of weeds, and there are the better linearity relation in its expression amount and resistance level.In order further to verify result's accuracy.Marker gene also should have other feature, and such as behind corresponding pesticide-treated weeds, in the weeds plant of different resistance levels, marker gene has different expression characteristics.
It is a kind of new weeds resistance detection method at the expression amount of transcriptional level that the personnel of group of the present invention have proposed by detecting marker gene.The GH3 gene is that plant hormone stores metabolic gene, plays an important role aspect the plain concentration of normal growth in keeping cell.Homologous gene and the called after ec-GH31 of GH3 gene cloned in group of the present invention from anti-quinclorac barnyard grass grass.In the ecGH31 gene title, be the acronym of barnyard grass grass Latin Echinochloa crusgalli by world name custom EC.GH3 is from the barnyard grass grass and the gene GH3 dna homolog, and 1 refers to first gene of cloning in the GH3 gene family of barnyard grass grass.
Its expression amount in anti-, sense quinclorac barnyard grass grass plant body is obviously different, with the dichloroquinoline acid treatment prevent and treat optimum period anti-, feel the barnyard grass plant after, have visibly different expression curve.Above feature makes the GH3 gene become the marker gene that detects the careless anti-quinclorac level of barnyard grass, provides possibility for setting up a kind of weeds resistance detection novel method.
Adopt the RACE method, from the anti-quinclorac barnyard grass grass from Zhejiang, cloned growth hormone IAA metabolism related gene and called after ec-GH31.It is 1839bp that this gene is read frame length, 612 amino acid of encoding.Find that based on Real-time PCR experimental result there is obvious characteristic difference in this gene expression amount in the anti-quinclorac barnyard grass grass of different levels, so propose the barnyard grass grass to the drug-fast detection method of quinclorac.
On the one hand, the invention provides for detection of the barnyard grass grass and belong to gene order to the dichloroquinoline acid resistance.A kind ofly be used for detecting the barnyard grass grass to the drug-fast gene order of quinclorac, wherein this sequence is SED IN NO:29.
Preferably, described gene order, wherein, this gene order comprises the primer according to described continuous row design.Preferably, wherein said primer sequence is IN NO:31 and SED IN NO:32; Or SED IN NO:33 and SED IN NO:34.Preferably, described detection is for detecting this gene order at the expression amount of transcriptional level.Preferably, the method that wherein detects is the Real-Time PCR method.
Preferably, described detection may further comprise the steps: produce sample at barnyard grass grass to be detected and the sampling of contrast barnyard grass grass; Produce sample at barnyard grass grass to be detected with after contrasting the barnyard grass grass to use the dichloroquinoline acid treatment in the different time sampling; Detect the GH3 gene of barnyard grass grass sample at the expression amount of transcriptional level; The GH3 expression of gene amount of GH3 expression of gene amount and control treatment is compared; Result relatively carries out barnyard grass grass to be detected to the resistance evaluation of quinclorac.Preferably, detecting the employed primer of contrast barnyard grass grass is sequence SED IN NO:33 and SED IN NO:34.Preferred, wherein, detecting the employed primer of barnyard grass grass to be detected is sequence SED IN NO:31 and SED IN NO:32.
On the other hand, the invention provides a kind of detection barnyard grass grass to the drug-fast method of quinclorac, comprising: produce sample at barnyard grass grass to be detected and the sampling of contrast barnyard grass grass; Produce sample at barnyard grass grass to be detected with after contrasting the barnyard grass grass to use the dichloroquinoline acid treatment in the different time sampling; Detect the GH3 gene of barnyard grass grass sample at the expression amount of transcriptional level; The GH3 expression of gene amount of GH3 expression of gene amount and control treatment is compared; Result relatively carries out barnyard grass grass to be detected to the resistance evaluation of quinclorac.
Preferably, be contrast with barnyard grass grass actin, barnyard grass grass GH3 gene to be detected the relative expression quantity of transcriptional level greater than 100 be anti-quinclorac type.Preferred, wherein, when the quinclorac that uses conventional amount used after the medication window phase is handled the barnyard grass grass, the expression amount rising amplitude of GH3 is less than 1.5 times in the barnyard grass cursive script to be detected in 1 hour, the expression amount rising amplitude of GH3 is 0.2～1 times in its body in 2 hours to 5 days; The expression amount rising amplitude of GH3 in the 6th day its body is 2～3 times the anti-quinclorac type that is.
Preferably, wherein, be contrast with barnyard grass grass actin, barnyard grass grass GH3 gene to be detected the relative expression quantity of transcriptional level less than 50 be sense quinclorac kind.Preferably, wherein, use the quinclorac of conventional amount used after the medication window phase is handled the barnyard grass grass, the expression amount rising amplitude of GH3 is greater than 4.5 times in the barnyard grass cursive script to be detected in 1 hour, and the expression amount rising amplitude of GH3 is 0.3～1.5 times in its body in 2 hours to 3 days; The expression amount rising amplitude of GH3 is at 2～3 times in the 4th and the 6th day its body, in the 5th day its body the expression amount rising amplitude of quinclorac greater than 16 times be sense quinclorac type.
Preferably, wherein, actin is contrast with the barnyard grass grass, and barnyard grass grass GH3 gene to be detected is the intermediate type of quinclorac sensitivity and resistance at the relative expression quantity of transcriptional level between 50～100.Preferably, wherein, use the quinclorac of conventional amount used after the medication window phase is handled the barnyard grass grass, in 1 hour in the barnyard grass cursive script to be detected the expression amount rising amplitude of GH3 between in 1.5～4.5 times be the intermediate type of quinclorac sensitivity and resistance.
In above all embodiments, the GH3 of described barnyard grass grass is SED IN NO:29, and the primer of sequences Design according to this.Preferably, wherein, the method that detects described barnyard grass grass GH3 to be detected genetic expression is Real-Time PCR, and primer is sequence SED IN NO:31 and SED IN NO:32.Preferred, wherein, the method that detects described contrast barnyard grass grass GH3 genetic expression is Real-Time PCR, and primer is sequence SED IN NO:33 and SED IN NO:34.In addition, this method also comprises following feature:
(1), barnyard grass grass with following feature belongs to that quinclorac is had drug-fast barnyard grass grass: (1) be confidential reference items with barnyard grass grass actin (Ct), the careless ecGH31 gene of barnyard grass at the relative expression quantity of transcriptional level greater than 100.(2) quinclorac that uses conventional amount used is after the medication window phase is handled the barnyard grass grass, and the expression amount rising amplitude of ecGH31 is less than 1.5 times in its body in 1 hour, and the expression amount rising amplitude of ecGH31 is 0.2～1 times in its body in 2 hours to 5 days.The expression amount rising amplitude of ecGH31 in the 6th day its body is 2～3 times.
Or (two), the barnyard grass grass with following feature belong to the barnyard grass grass to the quinclorac sensitivity: be confidential reference items with barnyard grass grass actin (Ct) (a), the careless ecGH31 gene of barnyard grass at the relative expression quantity of transcriptional level less than 50.(b) quinclorac that uses conventional amount used is after the medication window phase is handled the barnyard grass grass, and the expression amount rising amplitude of ecGH31 is greater than 4.5 times in its body in 1 hour, and the expression amount rising amplitude of ecGH31 is 0.3～1.5 times in its body in 2 hours to 3 days.The expression amount rising amplitude of ecGH31 is at 2～3 times in the 4th and the 6th day its body, and the expression amount rising amplitude of quinclorac is greater than 16 times in the 5th day its body.
Or (three), the barnyard grass grass with following feature belong to the intermediate type to quinclorac sensitivity and resistance: (1) be confidential reference items with barnyard grass grass actin (Ct), the careless ecGH31 gene of barnyard grass at the relative expression quantity of transcriptional level between 50～100.(2) quinclorac that uses conventional amount used is after the medication window phase is handled the barnyard grass grass, in 1 hour in its body the expression amount rising amplitude of ecGH31 between in 1.5～4.5 times.
Perhaps, the combination of three aspects or satisfy the detection method of above three conditions simultaneously more than.
Description of drawings
Fig. 1 is the total RNA electrophoresis proof diagram of barnyard grass grass in an embodiment of the invention;
Fig. 2 is the middle segment RT-PCR of barnyard grass grass electrophoresis proof diagram in an embodiment of the invention;
Fig. 3 is ecGH35RACE-PCR product in an embodiment of the invention (about 1800bp) electrophorogram;
Fig. 4 is ecGH35RACE bacterium colony PCR electrophoresis result figure in an embodiment of the invention;
Fig. 5 is ecGH313RACE-PCR product in an embodiment of the invention (about 1100bp) electrophorogram;
Fig. 6 is ecGH313RACE bacterium colony PCR electrophoresis result figure in an embodiment of the invention;
Fig. 7 is that three fragment positions concern and the primer location signal in an embodiment of the invention;
Fig. 8 is three interior nest PCR electrophoresis result figure of checking ecGH31 total length in an embodiment of the invention;
Fig. 9 is the sibship figure of the GH3 family member of ecGH31 and Arabidopis thaliana in an embodiment of the invention;
Figure 10 is ec-GH3-R gene amplification tracing analysis figure in an embodiment of the invention;
Figure 11 is that ec-GH3-R gene melting point curve is analyzed (Tm=80.5 ℃) figure in an embodiment of the invention;
Figure 12 is ec-GH3-S gene amplification tracing analysis figure in an embodiment of the invention;
Figure 13 is that ec-GH3-S gene melting point curve is analyzed (Tm=80.5 ℃) figure in an embodiment of the invention;
Figure 14 is ec-GH3 gene amplification efficiency analysis figure in an embodiment of the invention;
Figure 15 is ec-actin-R gene amplification tracing analysis figure in an embodiment of the invention;
Figure 16 is that ec-actin-R gene melting point curve is analyzed (Tm=84 ℃) figure in an embodiment of the invention;
Figure 17 is ec-actin-S gene amplification tracing analysis figure in an embodiment of the invention;
Figure 18 is that ec-actin-S gene melting point curve is analyzed (Tm=84 ℃) figure in an embodiment of the invention;
Figure 19 is ec-actin gene amplification efficiency analysis figure in an embodiment of the invention.
Embodiment
For further the present invention will be described in detail, describe with specific embodiment now, the limited row that these explanations only are concrete modes of the present invention are exhausted, the present invention do not done any restriction.
The clone of embodiment 1:ecGH31 gene
Material is prepared
Choose through greenhouse whole strain plant identification method (referring to Moss, S.R. (1995) Techniques for determining herbicide resistance.Proceedings of the Brighton Crop Protection Conference-Weeds, 547-556.) the anti-quinclorac barnyard grass grass seed identified, placed under 25 ℃ on the moistening filter paper vernalization 3-5 days, choose the careless seedling replanting of the neat barnyard grass that germinates in artificial soil, be cultured to the 3-5 leaf phase, standby.
Kill barnyard grass king (in the Xinyi triumphant agricultural chemical industry company limited) with 50% and be made into 0.5g/L concentration (effective constituent), even spraying was won blade in 0.5 hour and is immersed liquid nitrogen rapidly and preserve.
The clone of segment in the middle of the embodiment 1.1
The degenerated primer design is with synthetic:
In NCBI, find out the homologous gene dna sequence dna of the GH3 of Arabidopis thaliana, paddy rice, corn, tobacco, grape, in the online comparison in DDBJ website, design degenerated primer and synthetic (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd is synthetic): the degenerated primer sequence is: SED IN NO:1 and SED IN NO:2.
Total tissue RNA is extracted:
Carry out (Beijing Quanshijin Biotechnology Co., Ltd) by the explanation of TransZOL total RNA extraction reagent box.Handle water dissolution RNA with DEPC, and carry out the mensuration of concentration and purity, detect with the 12g/L agarose gel electrophoresis.
1. the extraction of the total RNA of barnyard grass grass
The total RNA of barnyard grass blade of grass sheet of extraction, purifying detects with the 12g/L agarose gel electrophoresis, and electrophoresis result is seen Fig. 1.Can see 28S rRNA and 18S rRNA two bands, the two content ratio is approximately 2: 1, illustrates that the RNA that carries is complete, does not have degraded (Fig. 1).The content of RNA and purity can satisfy next step test requirements document.
Reverse transcription synthesizes cDNA article one chain:
Press cDNA article one chain synthetic agent test kit (TransScript II First-Strand cDNA Synthesis SuperMix) (Beijing Quanshijin Biotechnology Co., Ltd, lot identification mark: #D11030) the synthetic cDNA article one chain of reverse transcription is carried out in explanation, wherein with Anchored Olig (dT)
20(primer sequence is Primer: SED IN NO:39) be primer.Segment in the middle of the pcr amplification:
The degenerated primer (SED IN NO:1 and SED IN NO:2) of doing template and design with article one chain of the synthetic cDNA of reverse transcription carry out RT-PCR (primerSTAR HS (premix), Takara), the PCR condition is 35 circulations, condition is: 98 ℃, 10sec; 57 ℃, 5sec; 72 ℃, 48sec.The result carries out 1% agarose gel electrophoresis, reclaims test kit EasyPure Quick Gel Extraction Kit (Beijing Quanshijin Biotechnology Co., Ltd with glue; Lot identification mark: #E20205) reclaim 600bp size segment.
Connection and conversion and the screening of blue hickie:
With the test kit that contains pEASY-Blunt Cloning Vector (Beijing Quanshijin Biotechnology Co., Ltd) purpose segment (through the 600bp size after the RT-PCR amplification) is connected and conversion Trans-T1 (DH5 α) competent cell (Beijing Quanshijin Biotechnology Co., Ltd, lot identification mark: #E200331), by blue hickie (carrier and Trans-T1 (DH5 α) competent cell in the test kit of the Cloning of the pEASY-Blunt to the effect that Vector of blue hickie screening) screening transformant.
Order-checking:
Universal primer M13F, M13R (SED IN NO:3 SED IN NO:4) check order (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd synthesizes)
As a result 1
(1) clone of ecGH31 gene
2. the middle segment of barnyard grass grass obtains.Obtaining size through the RT-PCR amplification is the purpose fragment (Fig. 2) of 600bp.
3. segment dna sequence dna in the middle of the barnyard grass grass: the intermediate segment sequence (SED IN NO:5) that obtaining the ecGH31 gene through blue hickie screening and order-checking
(1) RACE (Rapid Amplification ofcDNAEnds, the terminal method of rapid amplifying cDNA) method 5 ' clone with 3 ' segment
5 ' RACE and 3 ' RACE template adopt GeneRacer Kit (Wei Tejin (Invitrogen), lot identification mark: L1500-01) synthesize.Test kit provides joint primer primer order to be respectively:
The outside primer (GeneRacer outer primer) of 5 ' Wei Tejin: SED IN NO:35
The outside primer (GeneRacer Outer Primer) of 3 ' Wei Tejin: SED IN NO:36
The inner primer (GeneRacer Inner primer) of 5 ' Wei Tejin: SED IN NO:37
The inner primer (GeneRacer Inner Primer) of 3 ' Wei Tejin: SED IN NO:38
According to middle segment DNA (600bp) sequence Primer5 software design gene specific primer (SED IN NO:6, SED IN NO:7; SED IN NO:9, SED IN NO:10)
5 ' RACE primer is SED IN NO:6, SED IN NO:7.
The condition of 5 ' RACE PCR:
The first round
94℃2min
5cycles：98℃ 10sec 72℃ 1min
5cycles：98℃ 10sec 70℃ 1min
25cycles：98℃ 10sec 66℃ 30sec 68℃ 1min
Second takes turns
94℃ 2min
25cycles：98℃ 10sec 66℃ 30sec 68℃ 1min
After the electrophoresis checking, the PCR product is through tailing, connect pUCm-T carrier (Shanghai biotechnology company limited), transformed competence colibacillus cell JM109 (Shanghai biotechnology company limited) then, adopt M13F and M13R to carry out colony PCR amplification, select bacterium colony about the about 1800bp of size check order (prompt basic (Shanghai) trade Co., Ltd in the English Weihe River)
As a result 2
4.ecGH31 5 ' RACE sequence of gene
The electrophoresis result of ecGH31 gene 5 ' RACE amplified production shows, obtains the cDNA fragment (Fig. 3) of a 1800bp.The PCR product connects the pUCm-T carrier through adding the polyA tail, and transformed competence colibacillus cell JM109 (Shanghai biotechnology company limited) adopts M13F and M13R to carry out colony PCR amplification, electrophoresis result such as Fig. 4 then.
Select the bacterium colony about the about 1800bp of size to check order, the results are shown in SED IN NO:8:
3 ' RACE primer: SED IN NO:9, SED IN NO:10
3 ' RACE PCR condition:
The first round
94℃ 2min
5cycles：98℃ 10sec 72℃ 1min
5cycles：98℃ 10sec 70℃ 1min
25cycles：98℃ 10sec 66℃ 30sec 68℃ 1min
Second takes turns
94℃ 2min
25cycles：98℃ 10sec 66℃ 30sec 68℃ 1min
The PCR product is through tailing, connect pUCm-T carrier (Shanghai biotechnology company limited), transformed competence colibacillus cell JM109 (Shanghai biotechnology company limited) then, adopt M13F and M13R to carry out colony PCR amplification, select bacterium colony about the about 1100bp of size check order (prompt basic (Shanghai) trade Co., Ltd in the English Weihe River).
As a result 3
3 ' RACE sequence of ecGH31 gene
The electrophoresis result of ecGH31 gene 3 ' RACE amplified production shows, obtains the cDNA fragment (Fig. 5) of an about 1100bp of size.
The PCR product connects the pUCm-T carrier through adding the polyA tail, and transformed competence colibacillus cell JM109 adopts M13F and M13R to carry out colony PCR amplification, electrophoresis result such as Fig. 6 then.Select the bacterium colony about the about 1100bp of size to check order result following (SED IN NO:11).
6.ecGH31 the full length sequence of gene
Theoretical splicing back complete sequence is seen (SED IN NO:24): sequence total length: 2574bp, the dna sequence dna institute deduced amino acid of the theoretical splicing of coding region: 668-2143=1475bp is seen (SED IN NO:25), 491aa.
(2) checking of ecGH31 total length segment
(1) three-stage process checking total length ecGH31 gene strategy is seen Fig. 7
5 end RACE segments, middle segment and 3 end RACE segments are spliced into the theoretical segment (SED IN NO:24) of total length with DANStar software, be template with this theoretical segment, be divided into three sections, use Primer primer5 software respectively, design three pairs of nidos (nest) primer, carry out PCR, interior PCR product is connected to (Shanghai biotechnology company limited) on the pUCm-T carrier, through the screening of blue hickie, with M13F and M13R primer (SED IN NO:3 SED IN NO:4) check order (Shanghai life worker).
Verify used primer:
The primer that first part's checking is used:
SED IN NO：12(143-164)；
SED IN NO：13(1034-1057)
First round size: 915bp
SED IN NO：14′(166-189)
SED IN NO：15(862-886)
Second takes turns size: 721bp
The primer that the second section checking is used:
SED IN NO：16(665-689)
SED IN NO：17(1410-1433)
First round size: 759bp
SED IN NO：18(747-767)
SED IN NO：19(1384-1409)
Second takes turns size: 663bp
The primer that the third part checking is used:
SED IN NO：201317-1341)
SED IN NO：21(2209-2237)
First round size: 921bp
SED IN NO：22(1381-1401)
SED IN NO：23(2006-2031)
Second takes turns size: 651bp
The PCR reaction conditions is following, and (reaction conditions of two-wheeled PCR is the same.Different is reactant and product, but condition is the same.First round reaction result be the amplification three bigger segments, second take turns amplification be with first round product be template amplification than small pieces):
First round PCR reaction
Reaction conditions: 94 ℃ of 2min; 65 ℃ of 1min of 35cycles:94 ℃ of 30sec
Second takes turns the PCR reaction
Reaction conditions: 94 ℃ of 2min; 65 ℃ of 1.0min of 35cycles:94 ℃ of 30sec
(2) ecGH31 gene verification
(1) three of ecGH31 ORF district nest inner primer PCR results
The ecGH31 full-length cDNA of theoretical splicing is divided into 5 ends, middle and three parts of 3 ends.Three segments are separately through two wheel PCR, obtain expecting the segment of size through electrophoresis, and wherein the size of 5 dististyles disconnected 1 is about the 720bp size, and the size of middle segment 2 is about 660bp, and the size of 3 dististyles disconnected 3 is about 650bp (Fig. 8).
(3) three of the ecGH31ORF district sequencing fragment results:
First part's sequencing result (5 end) (SED IN NO:26):
Second section sequencing result (centre) (SED IN NO:27):
Third part sequencing result (3 end) (SED IN NO:28):
(3) total length after the ecGH31 checking
Splicing back complete sequence following (SED IN NO:29): sequence total length: 2532bp, the DNA institute deduced amino acid following (SED IN NO:30) after coding region: the 263-2101=1839bp checking: 612aa
ClusterW analyzes the GH3 family member's of discovery and Arabidopis thaliana sibship (see figure 9) far away
Real-time PCR
(1) method for extracting total RNA
1. reagent: total RNA extraction reagent box (the great basic bio tech ltd in Hangzhou)
2. instrument: pipettor (Eppendorf), high speed freezing centrifuge (Sigma company), vibrator, ultraviolet spectrophotometer (Eppendorf company)
(2) reverse transcription experiment
1. reagent: SuperScript
TMII RTase (Invitrogen)
2. instrument: pcr gene amplification instrument (Bio-Rad)
(3) gene expression dose difference detects (Real-Time PCR)
1. reagent:
Premix Ex Taq
TM(Perfect Real Time) (TaKaRa company) (Catalog No.DRR041A)
2. instrument: iQ
TM5 multiple real time fluorescence quantifying PCR instrument (U.S. Bio-Rad company)
3. adopt Primer Premier 6.0 and Beacon designer software to carry out the design of fluorescent primer, be responsible for synthesizing by Shanghai biotechnology company limited then, primer sequence is as follows:
Table: Real-Time PCR Primers (RT-PCR) and condition
(4) Real-Time pcr amplification reaction condition
Reaction conditions
95℃，1min；
45 circulations: 95 ℃, 10sec; 60-65 ℃, 25sec (collection fluorescence)
Melting point curve is analyzed 95 ℃ of 55 ℃ of to
(5) data statistics of Real-Time pcr gene differential expression
Each sample triplicate, relative expression's level of each gene is with 2
(Ct internal control gene-Ct goal gene)Carry out statistical study.
The result
(2) based on the ecGH31 gene transcription level expression characteristic of Real-time PCR
1: each is analyzed the fluorescent primer specific amplification
By the melting point curve analysis, show that these gene primer amplifications have stronger specificity
2: anti-, as to feel the ec-GH31 gene transcription level of barnyard grass grass relative expression's characteristics
The relative expression quantity of ec-GH3 gene is 105.89 in the anti-quinclorac barnyard grass grass plant body, and after the dichloroquinoline acid treatment, expression amount raises rapidly in 1 hour, but is no more than 1.5 times.Behind the medicine in 2 hours to 5 days its bodies the relative expression quantity of GH3 gene float about 0.5 times all less than before the dispenser, the 6th day, be increased to 2.57 times (seeing Table 1).
Table 1: anti-quinclorac barnyard grass grass plant ec-GH3 gene relative expression component analysis
Relative expression quantity to ec-GH3 gene in the responsive barnyard grass grass of the quinclorac plant body is 47.43, and after the dichloroquinoline acid treatment, expression amount raises rapidly in 1 hour, and greater than 4.5 times.Behind the medicine in 2 hours to 5 days its bodies the relative expression quantity of GH3 gene about 1 times, float, be increased to 16.8 times on the 5th day, the 6th day, be increased to 2.11 times (seeing Table 2).
Table 2: to the responsive barnyard grass grass of quinclorac plant ec-GH3 gene relative expression component analysis
Claims (4)
1. one kind is detected the barnyard grass grass to the drug-fast method of quinclorac, it is characterized in that, comprise: get the sample of generation at barnyard grass grass to be detected and the careless actin of contrast barnyard grass, namely behind barnyard grass grass to be detected and contrast barnyard grass grass actin processing quinclorac, get the sample of generation at different time; Detect the GH3 gene of barnyard grass grass sample at the expression amount of transcriptional level; The GH3 expression of gene amount of GH3 expression of gene amount and control treatment is compared; Result relatively carries out barnyard grass grass to be detected to the resistance evaluation of quinclorac, wherein, and described barnyard grass grass
The GH3 gene isShown in the SEQ ID NO:29; Detect described barnyard grass grass to be detected
GH3The method of genetic expression is Real-Time PCR, and employed primer sequence is shown in SEQ ID NO:31 and the SEQ ID NO:32; Detect described contrast barnyard grass grass
GH3The method of genetic expression is Real-Time PCR, and employed primer is that sequence is shown in SEQ ID NO:33 and the SEQ ID NO:34; Wherein, actin is the confidential reference items contrasts with contrast barnyard grass grass, barnyard grass grass to be detected
GH3Gene the relative expression quantity of transcriptional level greater than 100 be anti-quinclorac type; Barnyard grass grass to be detected
GH3Gene the relative expression quantity of transcriptional level less than 50 be sense quinclorac kind; Barnyard grass grass to be detected
GH3Gene is the intermediate type of quinclorac sensitivity and resistance at the relative expression quantity of transcriptional level between 50～100.
2. method according to claim 1 is characterized in that, when the quinclorac that uses conventional amount used after the medication window phase is handled the barnyard grass grass, in 1 hour in the barnyard grass cursive script to be detected
GH3Expression amount rising amplitude less than 1.5 times, in 2 hours to 5 days in its body
GH3Expression amount rising amplitude be 0.2～1 times; In the 6th day its body
GH3Expression amount rising amplitude be 2～3 times for anti-quinclorac type.
3. method according to claim 1 is characterized in that, uses the quinclorac of conventional amount used after the medication window phase is handled the barnyard grass grass, in 1 hour in the barnyard grass cursive script to be detected
GH3Expression amount rising amplitude greater than 4.5 times, in 2 hours to 3 days in its body
GH3Expression amount rising amplitude be 0.3～1.5 times; In the 4th and the 6th day its body
GH3Expression amount rising amplitude at 2～3 times, in the 5th day its body the expression amount rising amplitude of quinclorac greater than 16 times be sense quinclorac type.
4. method according to claim 1 is characterized in that, uses the quinclorac of conventional amount used after the medication window phase is handled the barnyard grass grass, in 1 hour in the barnyard grass cursive script to be detected
GH3Expression amount rising amplitude between 1.5～4.5 times be the intermediate type of the responsive and resistance of quinclorac.
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