CN104032019B - The PCR detection method of anti-ALS inhibitor class Chu Cao Ji Wang grass and test kit - Google Patents
The PCR detection method of anti-ALS inhibitor class Chu Cao Ji Wang grass and test kit Download PDFInfo
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Abstract
The invention provides the Specific PCR primers combination for detecting anti-ALS inhibitor class Chu Cao Ji Wang grass, comprise the primer pair (Seq for Te opposite sex Kuo Zeng Wang grass als gene? ID? No.1-4).Adopt the PCR detection method of this combination of primers to have fabulous specificity and susceptibility, prove through specific test, the als gene of Wang grass can be increased out.Because PCR method is simple and efficient to handle, based on the detection to Nucleotide, this season just can sample detection, only needs 2 ~ 4 days from being sampled to out result.Its sensitivity and specificity can realize pressing down the Rapid identification of agent Wang grass processed and the confirmation in mutational site to the doubtful anti-ALS in field, thus in Instructing manufacture practice, the science of resistant weed is administered.
Description
Technical field
The present invention relates to antiweed weeds detection technique, specifically, relate to a kind of PCR detection method and test kit of anti-ALS inhibitor class Chu Cao Ji Wang grass.
Background technology
Acetolactate synthase (acetolactatesynthase, be called for short ALS), also acetohydroxy acid synthase (acetohydroxyacidsynthase is claimed, be called for short AHAS), being key enzyme in the biosynthetic process of α-amino-isovaleric acid in inducing plant and microbe, leucine, Isoleucine, is also a kind of important weedicide target.Exploitation and the use of ALS inhibitor class weedicide start from the beginning of the eighties in last century, and because this type of weedicide has super-active, to the significantly advantage such as people and animal low toxicity, environmental friendliness, is subject to extensive concern, existing more than 50 kind is developed and uses at present.The eighties in last century starts, at winter wheat field, China middle and lower reach of Yangtze River promotes the use of that chlorine sulphur is grand, metsulfuronmethyl, two methiodide sulphurs are grand successively, pyridine sulphur grass amine etc. prevents and kill off gramineous weeds, serves very important effect to gramineous weedss such as control Wang grass processed, Alopecurus.But because such herbicide molecular target is single, life-time service easily causes herbicide resistance, in the last few years, with routine dose spray the weedicide such as mesosulfuron, metsulfuronmethyl cannot the problem of effective Fang Chu Wang grass very outstanding at some wheatlands of China middle and lower reach of Yangtze River, and find, some wheatland anti-mesosulfuron Wang grass also creates cross resistance to ALS inhibitor such as pyridine sulphur grass amine.Wheat weeds produces cross resistance to above-mentioned several ALS inhibitor, make these farmlands can not continue to use such weedicide, and aborning because peasant is to the deficiency in the understanding of herbicide resistance, blindly increase dosage, not only increase cost, make poisoning increased risk, have a strong impact on farmers' income and national food security.Therefore, at present in the urgent need to developing a kind of easy, Herbicide-resistant weeds Testing and appraisal method fast.
The research of China's Herbicide-resistant weeds is started late, traditional Herbicide-resistant weeds detects and usually adopts indoor bioassay method, both the seed of the weeds of the doubtful resistance that field gathers was cultivated in indoor, after certain leaf age herbicide spraying, investigation strain preventive effect and Aboveground Biomass of Young calculate resistance index.This method can evaluate the resistance situation of certain weeds to corresponding weedicide objectively, but also has its limitation, is mainly manifested in following two aspects: one be can not this season, detected at that time, two be take up an area many, the time is long, workload is large.Along with the application of Protocols in Molecular Biology in herbicide resistance study mechanism, increasing resistance weed mechanism obtains clearly.
The display of current existing research report, plant ALS inhibitor being produced to resistance has found 8 SNP site (http://www.weedscience.org/Mutations/MutationDisplayAll.aspx) altogether in its target enzymes ALS conserved regions, and any one site amino acids in these sites sports other amino acid all makes plant produce resistance to corresponding weedicide.These 8 amino acid sites are generally acknowledged at present marks site with Arabidopis thaliana ALS for standard, comprises 122,197,205,376,377,574,653,654 amino acids (Fig. 3).Further research finds Wang grass (Beckmanniasyzigachne (Steud.) Fernald), causes the conserved regions a certain site origination point sudden change that the reason of the ALS inhibitor class weedicide generation resistances such as mesosulfuron is also Code targets enzyme ALS.Therefore, the conserved regions mutational site can detecting its target enzymes ALS by molecular biology method is carried out Jian Ce Wang grass and whether resistance is occurred.
Summary of the invention
The object of this invention is to provide a kind of PCR detection method of anti-ALS inhibitor class Chu Cao Ji Wang grass.
Another object of the present invention is to provide a kind of test kit for detecting anti-ALS inhibitor class Chu Cao Ji Wang grass.
In order to realize the object of the invention, first the present invention provides a kind of Specific PCR primers combination for detecting anti-ALS inhibitor class Chu Cao Ji Wang grass, comprises the primer pair I for Te opposite sex Kuo Zeng Wang grass als gene and primer pair II.The ALS fragment of these two pairs of primer amplifications comprises all 8 SNP site found on the plant of other anti-ALS inhibitor class weedicides at present.Primer sequence is as follows:
Primer pair I:
Forward primer WC-ALS-I F:5 '-CGCCTTACCCAAACCTACT-3 '
Reverse primer WC-ALS-I R:5 '-ATGCGGCTGCTTGTTCTT-3 '
Primer pair II:
Forward primer WC-ALS-II F:5 '-GATAAGGCTGACCTGTTGC-3 '
Reverse primer WC-ALS-II R:5 '-TCACAGTTGACCACACTTC-3 '
The present invention also provides the test kit for detecting anti-ALS inhibitor class Chu Cao Ji Wang grass containing above-mentioned combination of primers.
Preferably, described test kit also comprises dNTPs, Taq DNA polymerase, Mg
2+, at least one in PCR reaction buffer etc.
More preferably, described test kit also comprises standard positive template.
The present invention also provides a kind of PCR detection method of anti-ALS inhibitor class Chu Cao Ji Wang grass, and it utilizes above-mentioned combination of primers or test kit to detect anti-ALS inhibitor class Chu Cao Ji Wang grass.
Preceding method, comprises the following steps:
1) DNA in sample is extracted;
2) with step 1) in extract DNA be template, carry out pcr amplification reaction;
3) PCR primer is analyzed.
PCR reaction system is counted with 20 μ l:
PCR reaction conditions is: 94 DEG C 3 minutes; 94 DEG C 1 minute, 56 DEG C 1 minute, 72 DEG C 1 minute, totally 30 circulations; 72 DEG C 10 minutes.
The als gene sequence (Wang grass als gene sequence of Gen Ju Wang grass of the present invention draws careless Aperaspica-venti (GenBank:JN646110.1) with reference to grass Ah the draping over one's shoulders that sibship is nearer, the conservative region design primer of the als gene sequence of amur foxtail Alopecurusaequalis (GenBank:JQ743908.1), segmentation amplification obtains, nucleotide sequence is as shown in SeqIDNo.5), design Auele Specific Primer to WC-ALS-I F/WC-ALS-I R and WC-ALS-II F/WC-ALS-II R, the PCR detection method of this combination of primers is adopted to have fabulous specificity and susceptibility, prove that (Fig. 2) can Jiang the als gene fragment amplification of Wang grass out through specific test.Because PCR method is simple and efficient to handle, based on the detection to Nucleotide, this season just can sample detection, only needs 2 ~ 4 days from being sampled to out result.Its sensitivity and specificity can realize to the doubtful anti-ALS inhibitor class in field except the careless Rapid identification of agent Wang grass and the confirmation in mutational site, thus in Instructing manufacture practice, the science of resistant weed is administered.
Test kit for detecting anti-ALS inhibitor class Chu Cao Ji Wang grass provided by the invention, can carry out rapid detection to the doubtful resistance Wang grass that field gathers.The traditional detection method of the biological assay of always continuing to use can be substituted, and be suitable for wide popularization and application in the monitoring field of other Herbicide-resistant weeds, practical, the needs of field drug-fastness weeds quick diagnosis can be met.
Accompanying drawing explanation
Fig. 1 is the position on two pairs of primer Wang grass als genes of design in the embodiment of the present invention 1.
Fig. 2 utilizes two pairs of specific detection primers to the pcr amplification result of different Wang grass DNA in the embodiment of the present invention 2; Wherein, M is DNAMarker (DL2000); Odd numbers swimming lane is the result that primer pair WC-ALS-I F/WC-ALS-I R increases, and even numbers swimming lane is the result that primer pair WC-ALS-II F/WC-ALS-II R increases.
Figure 3 is Wang grass ALS aminoacid sequence and Arabidopis thaliana ALS amino acid alignment result, and the mutating acid opposite position that 8 relevant to resistance.
Fig. 4 utilizes two pairs of primer amplification Min Gan Wang grass PCR primer sequencing results in the embodiment of the present invention 2; Wherein, A shows Bian Ma Wang grass ALS is GCC relative to the base of Arabidopis thaliana the 122nd L-Ala; B show Bian Ma Wang grass ALS relative to the base of Arabidopis thaliana the 197th proline(Pro) be CCC, the base of coding the 205th L-Ala is GCC; C show Bian Ma Wang grass ALS relative to the base of Arabidopis thaliana the 376th aspartic acid be GAT, coding the 377th arginic base be CGC; It is TGG relative to the base of Arabidopis thaliana the 574th tryptophane that D shows Bian Ma Wang grass ALS; E show Bian Ma Wang grass ALS relative to the base of Arabidopis thaliana the 653rd Serine be AGC, the base of coding the 654th glycine is GGT.
Fig. 5 utilizes WC-ALS-I F/WC-ALS-I R primer amplification different Kang Yao Wang grass PCR primer sequencing result in the embodiment of the present invention 3; Wherein, A shows Kang Yao Wang grass ALS and sports arginine relative to the 197th of Arabidopis thaliana, and codon is CGC; B shows Kang Yao Wang grass ALS and sports Serine relative to the 197th of Arabidopis thaliana, and codon is TCC; C shows Kang Yao Wang grass ALS and sports Threonine relative to the 197th of Arabidopis thaliana, and codon is ACC.
Fig. 6 is pcr amplification get Dao Wang grass als gene BLAST comparison result on NCBI.
Fig. 7 is responsive in the embodiment of the present invention 4 and the careless preventive effect using after 20g/ha mesosulfuron 21 days of Kang Yao Wang.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (SambrookJ & RussellDW, Molecularcloning:alaboratorymanual, 2001) condition of, or according to manufacturer's specification sheets advising.
Embodiment 1 is for detecting the synthesis of the PCR primer of anti-ALS inhibitor class Chu Cao Ji Wang grass
The als gene sequence (Wang grass als gene sequence of Gen Ju Wang grass draws careless Aperaspica-venti (GenBank:JN646110.1) with reference to grass Ah the draping over one's shoulders that sibship is nearer, the conservative region design primer of the als gene sequence of amur foxtail Alopecurusaequalis (GenBank:JQ743908.1), segmentation amplification obtains, nucleotide sequence is as shown in SeqIDNo.5), design Auele Specific Primer to WC-ALS-I F/WC-ALS-I R and WC-ALS-II F/WC-ALS-II R for Te opposite sex Kuo Zeng Wang grass als gene, relative position on each primer Wang grass als gene is shown in Fig. 1, the ALS fragment of these two pairs of primer amplifications comprises all 8 SNP site (Fig. 3 and Fig. 4) found on the plant of other anti-ALS inhibitor class weedicides at present.Primer sequence is as follows:
Forward primer WC-ALS-I F:5 '-CGCCTTACCCAAACCTACT-3 ' (SeqIDNo.1)
Reverse primer WC-ALS-I R:5 '-ATGCGGCTGCTTGTTCTT-3 ' (SeqIDNo.2)
Forward primer WC-ALS-II F:5 '-GATAAGGCTGACCTGTTGC-3 ' (SeqIDNo.3)
Reverse primer WC-ALS-II R:5 '-TCACAGTTGACCACACTTC-3 ' (SeqIDNo.4)
Primer synthesis is completed by Beijing Liuhe Huada Genomics Technology Co., Ltd.
The PCR detection method of embodiment 2 anti-ALS inhibitor class Chu Cao Ji Wang grass
Experiment material: resistance and susceptibility Wang grass.
Experimental technique:
1, the preparation of Wang grass DNA
Get the fresh blade 200mg of Wang grass, adopt liquid nitrogen grinding, conventional CTAB method extracts each Wang grass leaf DNA.
2, for the Auele Specific Primer in Jian Ce Wang grass ALS mutational site, primer sequence is shown in embodiment 1.
3, for the PCR reaction system in Jian Ce Wang grass ALS mutational site
PCR reaction system, wherein 10 × PCR reaction buffer 2 μ L, 15mMMgCl
20.5 μ L, 2.5mMdNTPs1 μ L, 10 μMs of forwards, reverse primer each 0.5 μ L, Taq DNA polymerase 1U, DNA profiling 1 μ L, all the other are sterilizing distilled water, and final volume is 20 μ L.
4, for the pcr amplification program in Jian Ce Wang grass ALS mutational site
94 DEG C of denaturation 3min; 94 DEG C of sex change 1min, 56 DEG C of annealing 1min, 72 DEG C extend 1min, totally 30 circulations; Last 72 DEG C extend 10min.
5, the qualification of PCR primer
Get 3 μ LPCR products, be separated with 1% (weight/volume) agarose gel electrophoresis, the size result of determination according to amplified production under ultraviolet lamp after ethidium bromide staining.
6, PCR primer order-checking
Pcr amplification product primer WC-ALS-I F and WC-ALS-II F checks order to two amplified fragments forwards respectively.Order-checking is completed by Beijing Liuhe Huada Genomics Technology Co., Ltd.Sequencing result is compared.
Experimental result:
Detection specificity primer pair WC-ALS-I F/WC-ALS-I R and WC-ALS-II F/WC-ALS-II R shows these two pairs of primers to different Wang grass pcr amplification result (Fig. 2) and has good specificity, can Kuo Zeng Chu Wang grass als gene fragment (amplification object clip size is respectively 1078bp, 1049bp), gene fragment carries out BLAST on NCBI, result shows (Fig. 6), with Aperaspica-venti (GenBank:JN646110.1), the plant als gene similarities such as Alopecurusmyosuroides (GenBank:AJ437300.2) are high, show amplified fragments Wei Wang grass als gene.The PCR primer of the sensitivity increased out and anti-medicine Wang grass is checked order respectively, 8 SNP site (Fig. 4) can be found by order-checking peak figure, can be used in detecting the Wang grass with anti-ALS inhibitor.
Embodiment 3PCR method detects field anti-ALS inhibitor Wang grass
Detection method is as follows:
1, prepared by sample collecting and DNA
For verifying the feasibility of PCR detection method, doubtful generation resistance farmland Cai Ji Wang grass plant, after carrying out suitable moisturizing process, post to laboratory.Get the fresh blade 200mg of Wang grass, adopt liquid nitrogen grinding, conventional CTAB method extracts each Wang grass leaf DNA.
2, for the Auele Specific Primer in Jian Ce Wang grass ALS mutational site, primer sequence is shown in embodiment 1.
3, for the PCR reaction system in Jian Ce Wang grass ALS mutational site
With embodiment 2.
4, for the pcr amplification program in Jian Ce Wang grass ALS mutational site
With embodiment 2.
5, the qualification of PCR primer
With embodiment 2.
6, PCR primer order-checking
With embodiment 2.
Experimental result:
Sample picks up from 3 different croplands in somewhere, Jiangsu Province, and there is the history using ALS inhibitor in these farmlands, and current this kind of herbicide effects is bad, suspects that weeds produce resistance.From these 3 farmlands random Cai Ji Wang blade of grass sheet (individual plant collection), gather 20 plant altogether, WC-1# ~ 6# picks up from first farmland, and WC7# ~ 12# picks up from second farmland, and WC13# ~ 20# picks up from the 3rd farmland.According to detection method of the present invention, it is detected.Detected result display (table 1), can both detect the sudden change (Fig. 5) of 197 amino acids, show to produce resistance in 3 farmland Wang grass samples, consistent with variable rate technology.
As can be seen here, the method can detect the generation site of drug-resistant mutation and the amino acid classes of replacement of the doubtful Kang Yao Wang grass picking up from field quickly and accurately, can can provide rapidly and accurately and continue to use ALS inhibitor to carry out Fang Chu Wang grass, be simple, the method fast of a kind of Jian Ce Wang grass insecticide resistant populations.
Table 1PCR method detects Tian Jian Wang grass drug resistance mutant site result
The sudden change of embodiment 4 Wang grass als gene and resistance relation
Experiment material: resistance (WC11-17#, WC11-48#) and susceptibility (WC12-3#, WC11-21#) Wang grass.
Experimental technique:
1, pick up from the seed of different field Wang grass seeds group, after vernalization process, be evenly seeded in the flowerpot that diameter is 12cm, in flowerpot, soil and fertilizer ratio are 3:1 (weight ratio).The Wang sowed is careless at warm indoor cultivation, temperature 15-25 DEG C.
2, plant grows to 2 leaves and starts thinning, and Mei Pen Wang grass plant is uniformly distributed, and every basin stays at most 20 strains.
3, Wang grass grows to 3 foliar spray effective doses is the mesosulfuron of 20g/ha, stays a basin blank, 21 days " Invest, Then Investigate " preventive effects.
4, from insecticide resistant populations WC11-17#, the WC11-48# of surviving after using mesosulfuron, respectively get 5 strains, respectively get 5 strains from the blank of sensitive population WC12-3#, WC11-21#.
5, DNA preparation method is with example 2.
6, for the Auele Specific Primer in Jian Ce Wang grass als gene mutational site, primer sequence is shown in embodiment 1.
7, for Jian Ce Wang grass als gene mutational site PCR reaction system with embodiment 2.
8, for Jian Ce Wang grass als gene mutational site pcr amplification program with embodiment 2.
9, the qualification of PCR primer is with embodiment 2.
10, PCR primer order-checking is with embodiment 2.
Experimental result: sensitive population WC12-3#, WC11-21# be all death in 21 days after using mesosulfuron, and insecticide resistant populations WC11-17#, WC11-48# are all survived after using mesosulfuron.(Fig. 7)
Random selecting 5 plant from each population survival plant, detect its als gene sequence, the results are shown in Table 2.Detected result shows, the ALS of two sensitive population does not undergo mutation, 5 plant of insecticide resistant populations WC11-17# are being mutated into arginine (CGC) relative to 197 of Arabidopis thaliana by proline(Pro) (CCC), and 5 of insecticide resistant populations WC11-48# plant are being mutated into Serine (TCC) relative to 197 of Arabidopis thaliana by proline(Pro) (CCC).This result conforms to the report of existing other plant (http://www.weedscience.org/Mutations/MutationDisplayAll.aspx), and the method rapid detection by PCR to be suddenlyd change the resistance caused due to target.
Table 2PCR method detects responsive and Kang Wang grass ALS mutational site result
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (8)
1. for detecting the Specific PCR primers combination of anti-ALS inhibitor class Chu Cao Ji Wang grass, it is characterized in that, comprise the primer pair I for Te opposite sex Kuo Zeng Wang grass als gene and primer pair II, primer sequence is as follows:
Primer pair I:
Forward primer WC-ALS-I F:5 '-CGCCTTACCCAAACCTACT-3 '
Reverse primer WC-ALS-I R:5 '-ATGCGGCTGCTTGTTCTT-3 ';
Primer pair II:
Forward primer WC-ALS-II F:5 '-GATAAGGCTGACCTGTTGC-3 '
Reverse primer WC-ALS-II R:5 '-TCACAGTTGACCACACTTC-3 '.
2. the test kit for detecting anti-ALS inhibitor class Chu Cao Ji Wang grass containing combination of primers described in claim 1.
3. test kit according to claim 2, is characterized in that, described test kit also comprises dNTPs, Taq DNA polymerase, Mg
2+, at least one in PCR reaction buffer.
4. the test kit according to Claims 2 or 3, is characterized in that, described test kit also comprises standard positive template.
5. anti-ALS inhibitor class is except the PCR detection method of careless agent Wang grass, it is characterized in that, utilizes test kit described in combination of primers described in claim 1 or any one of claim 2-4 to detect anti-ALS inhibitor class except careless agent Wang grass.
6. method according to claim 5, is characterized in that, comprises the following steps:
1) DNA in sample is extracted;
2) with step 1) in extract DNA be template, carry out pcr amplification reaction;
3) PCR primer is analyzed.
7. the method according to claim 5 or 6, is characterized in that, PCR reaction system is counted with 20 μ l:
8. the method according to claim 5 or 6, is characterized in that, PCR reaction conditions is: 94 DEG C 3 minutes; 94 DEG C 1 minute, 56 DEG C 1 minute, 72 DEG C 1 minute, totally 30 circulations; 72 DEG C 10 minutes.
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| CN106119370B (en) * | 2016-07-04 | 2019-10-22 | 山东省农业科学院植物保护研究所 | The PCR detection method and kit of anti-ALS inhibitor class herbicide herba digitariae |
| CN108707616A (en) * | 2018-06-29 | 2018-10-26 | 中国农业科学院植物保护研究所 | With the relevant gene of dayflower Herbicid resistant and its application |
| CN113943829A (en) * | 2021-10-22 | 2022-01-18 | 河北省农林科学院粮油作物研究所 | Specific primer pair and kit for detecting ALS inhibitor herbicide brome, detection method and application |
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