CN105349623A - HRM (high-resolution melt) detection method for herbicide-resistant gene OsmALS and application of method - Google Patents
HRM (high-resolution melt) detection method for herbicide-resistant gene OsmALS and application of method Download PDFInfo
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Abstract
The invention provides an HRM (high-resolution melt) detection method for herbicide-resistant gene OsmALS and application of the method, belongs to the technical field of plant biology, and particularly relates to development and application of a gene-specific molecular marker of rice gene OsmALS resistant to imidazolinone herbicides. In order to avoid false selection or death of required materials due to improper herbicide usage or non-uniform herbicide spraying, mutation of the gene-specific molecular marker 548 of the rice gene OsmALS resistant to imidazolinone herbicides has a promising prospect of application in production practice, and the marker can be used to improve the utilization efficiency of this gene in molecular marker assisted selective breeding, gene pyramiding breeding and transgenic breeding.
Description
Technical field
The invention belongs to plant biotechnology field, be specifically related to a kind of development and application of Rice Resistance imidazolinone herbicide gene OsmALS gene specific molecule marker.
Background technology
Amino acid is requisite material in plant materials, and he except working in the protein synthesis of plant, in elementary and secondary metabolism, also play important function.If the Amino acid synthesis of plant is obstructed, plant metabolism will be caused disorderly, thus it is even dead that plant-growth is badly damaged, so the key enzyme in the Amino acid synthesis of plant is the focus of weedicide research and development always.Acetolactate synthestase (acetolactatesynthase, ALS), also claims acetohydroxy acid synthetase (acetohydroxyacidsynthase, AHAS), is a key enzyme in branched-chain amino acid synthesis.In α-amino-isovaleric acid and leucic synthesis, catalysis two molecule pyruvic acid generates acetylactis and carbonic acid gas, and in the synthesis of Isoleucine, catalysis a part pyruvic acid and a part batanone acid generate 2-aldehyde-base-2-hydroxybutyric acid and carbonic acid gas.If ALS activity is suppressed, branched-chain amino acid biosynthesis block will be caused, and then affect protein synthesis and plant-growth.
In the last few years, the key areas carrying out molecular designing, exploitation ultra-high efficiency weedicide kind has been become using ALS as target, the ALS inhibitor class weedicide now developed has 5 large classes more than 50 to plant: sulfonylurea (Sulfonylureas, SU), imidazolone type (imidazolinones, IMI), triazolo pyrimidine class (triazolopyrimidines, TP), pyrimidine salicylic acid (pyrimidinylthio-benzoates, PTB), sulfonamides (sulfonylamino-carbonyl-triazolinones, SCT).These ALS inhibitor class weedicides have the features such as selectivity is strong, broad weed-killing spectrum, low toxicity are efficient, and wherein representative is imidazolinone herbicide.This type of weedicide is mainly used in the important kind of the leguminous crops such as soybean, to the important kind non-selectivity of the gramineous crops such as corn, wheat, paddy rice, and in use, because the long residual of its soil often causes rear stubble various crop to be injured.Along with the seed selection success of resistance crop, this problem is resolved.At present, resistance crop and imidazolinone herbicide are used and formed a kind of " Clearfield production system ", namely plant anti-imidazolone crop and use some special weeds that imidazolinone herbicide can not be prevented and treated to prevent and treat other weedicides, as the red rice of anti-imidazolone rice terrace, the bird rape of Rapeseed Field and other cruciferous weed etc.
After obtaining ALS antiweed mutant material, by conventional breeding methods such as hybridizing, backcross, the resistant gene of this antiweed new lines can be imported other to the nonresistant rice varieties of imidazolinone herbicide or strain, raising importing target variety or strain are to the tolerance of imidazolinone herbicide.If after resistant gene is imported restorer, the true hybrid of F1 just has anti-imidazolinone herbicide characteristic, all can be killed, improve seed production efficiency and seed purity by herbicide spraying by disposable for various types of pseudostationary.But resistance trait is dominant character, F1 generation shows as imidazolinone-resistance, be separated from F2 generation, need to start often to identify for herbicide spraying in the seed selection of field, if doses improper use or spray uneven cause most probably falsely dropping or required material dead.In addition, in anti-herbicide gene research process, sometimes need the material cannot do not preserved anti-ly, with making comparative research.For this reason, if codominant molecule marker with resistant gene can be found, thus carry out marker assisted selection, distinguish the different genotype of resistant gene in breeding material, then can instruct field seed selection, accelerate breeding process.
Summary of the invention
In order to whether can easier, more fast and effeciently detect in rice material containing anti-imidazolinone herbicide gene OsmALS mutant, and apply it in resistance breeding process, the invention provides a kind of based on HRM technology, for detecting the method for the 548th amino acids sudden change of Rice Resistance imidazolinone herbicide gene OsmALS, 548th amino acids sudden change of described OsmALS gene includes but not limited to sport Cys or Met by Trp, or replacement, the disappearance of base occurs in this position or adds one or several Nucleotide.Described sudden change can be point mutation, also can be DNA disappearance or insertion mutation.Described sudden change can be obtained by the mode of chemomorphosis or site-directed point mutation, and the method for chemomorphosis comprises the mutagenesis caused with mutagenic compound process such as EMS; The method of site-directed point mutation includes but not limited to the directed mutagenesis method such as ZFN directed mutagenesis method, TALEN directed mutagenesis method and/or CRISPR/Cas9.To the exploitation of this site base mutation detection method, this gene can be improved in molecular marker assisted selection breeding, gene pyramiding breeding, and the efficiency applied in transgenic breeding.
Particularly, 548th amino acids of above-mentioned rice Os als gene is sported the point mutation of Met (ATG) by Trp (TGG), the DNA sequence dna of the OsmALS gene after this point mutation is as shown in SEQIDNO:1, and aminoacid sequence is as shown in SEQIDNO:2.Specifically detecting primer by designing, detecting the base existence of this position in candidate material, especially by its HRM solubility curve of analysis thus whether understand plant endogenous containing this sudden change, and described in sport and isozygoty or a kind of detection method of heterozygous state.
The present invention by taking turns experiment more, and go back the specific primer that optimization detects for the HRM of the 548th sudden change of OsmALS for a pair, described primer numbers and sequence are: ALS-22415F:5 '-AAGTATGTATGCGCCCT-3 '; ALS-22494R:5 '-GTGTTGAACAACCAACA-3 '.
HRM detection method of the present invention adopts 10 μ L reaction systems, comprise 1 μ LPCR template, 0.5UrTaqDNA polysaccharase (Takara, Japan), 1 × PCR damping fluid (containing Mg2+), 250 μMs of dNTP, 0.1 μM of forward and reverse primer and 0.1 μ L20 × EvaGreen fluorescence dye (Biotium, USA).Pcr amplification program is 95 DEG C of 3min; 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 10s, 40 circulations; 72 DEG C of 5min, and then 95 DEG C of sex change 1min, 25 DEG C of renaturation 1min.In order to avoid evaporating, system must drip 15 μ L mineral oil and cover.
The experimental procedure of HRM detection method provided by the present invention is: a) extract sample to be tested DNA; B) prepare the PCR reaction system comprising sample to be tested DNA, primer pair and EvaGreen fluorescence dye and carry out pcr amplification; And c) HRM analysis scan is carried out to PCR primer.
Present invention also offers a kind of method extracting genomic dna in vegetable cell, tissue or organ, described method comprises: get the materials such as appropriate vegetable cell, tissue or organ, directly add extracting solution (10mMTris-HCl, 0.5mMEDTA, 0.15MKCl) grinding, after boiling water bath 10min, stratification gets supernatant, namely obtain its genomic dna, can be used as the pcr template in HRM detection.
Present invention also offers a kind of rice Os mALS gene the 548th abrupt climatic change PCR kit based on HRM technology, it is characterized in that described test kit comprises primer pair: forward primer: AAGTATGTATGCGCCCT (SEQIDNO:3); And reverse primer: GTGTTGAACAACCAACA (SEQIDNO:4), for detecting the Trp548Met sudden change of ALS.
More specifically, test kit provided by the present invention comprises PCR reagent and fluorescence dye further.
Present invention also offers a kind of using method of test kit, the using method of described test kit comprises the steps: to extract sample to be tested DNA; Prepare the PCR reaction system comprising sample to be tested DNA, primer pair, PCR kit and EvaGreen fluorescence dye and carry out pcr amplification; HRM analysis scan is carried out with to PCR primer.
Particularly, the method for detection Rice Resistance imidazolinone herbicide gene OsmALS gene the 548th site mutation molecule marker of the present invention is as follows: carry out pcr amplification by the genomic dna using primer pair ALS-22415F and ALS-22494R to treat test material; After the collection of high resolving power melting curve being carried out to aforementioned pcr amplification product with high resolving power melting curve analysis instruments such as LightScanner96, what obtained is consistent specific peak type high resolving power melting curve with positive control Rice Resistance imidazolinone herbicide gene OsmALS, can determine that this material has the 548th sudden change of paddy rice als gene.
The nucleotide sequence of described primer pair ALS-22415F and ALS-22494R is as follows:
ALS-22415F：5’-AAGTATGTATGCGCCCT-3’(SEQIDNO:3)；
ALS-22494R：5’-GTGTTGAACAACCAACA-3’(SEQIDNO:4)；
Described is that the high resolving power melting curve of specific peak type is specifically as shown in the red curve of Fig. 2 with Rice Resistance imidazolinone herbicide gene OsmALS; When practical application, only need to detect the high resolving power melting curve of sample consistent with the high resolving power melting curve of paddy rice non-transgenic antiweed material D3-10;
The exploitation of the HRM detection method of the 548th sudden change of described Rice Resistance imidazolinone herbicide gene OsmALS gene, comprises the following steps:
(1) by Multiple Sequence Alignment Software tool (as DNAMAN), (Nucleotide corresponding to coding region the 548th amino acids compares, the 548th amino acids catastrophe of examination OsmALS gene to account for the OsmALS gene of als gene (LOC_Os02g0510200) and paddy rice non-transgenic antiweed material to the yellow China of wild-type;
(2) utilize the SNP information that step (1) obtains, at this 36bp place, SNP upstream design gene specific upstream primer, at this 44bp place, SNP downstream design gene specific downstream primer, primer pair base sequence is as follows:
ALS-22415F：5’-AAGTATGTATGCGCCCT-3’(SEQIDNO:3)；
ALS-22494R：5’-GTGTTGAACAACCAACA-3’(SEQIDNO:4)；
(3) to carry the STb gene of the imidazolinone herbicide resistant material of Rice Resistance imidazolinone herbicide gene OsmALS for template, carry out pcr amplification, after the high resolving power melting curve analysis instruments such as the PCR primer LightScanner96 obtained carry out the collection of high resolving power melting curve, obtaining is the high resolving power melting curve of specific peak type with Rice Resistance imidazolinone herbicide gene OsmALS, is Rice Resistance imidazolinone herbicide gene OsmALS gene the 548th mutation specific molecule marker;
Described Rice Resistance imidazolinone herbicide gene OsmALS gene specific molecule marker 548 suddenlys change in the application differentiating rice material imidazolinone herbicide resistant gene, is particularly suitable for the application at the imidazolinone herbicide resistant gene differentiating introgressive line different genotype; Preferably comprise following steps:
(1) increase: utilize the genome of primer ALS-22415F and ALS-22494R to paddy rice introgressive line material to be detected to carry out PCR;
(2) detect: the high resolving power solubility curve of the PCR primer obtained with high resolving power melting curve analysis instruments acquisition step (1) such as LightScanner96, detect that the peak type of high resolving power melting curve is consistent with paddy rice non-transgenic antiweed material, then paddy rice introgressive line material to be detected carries homozygous imidazolinone herbicide resistant gene OsmALS; If detect the peak type of high resolving power melting curve and paddy rice non-transgenic antiweed material inconsistent, but the peak type of high resolving power melting curve is dual hump, then paddy rice introgressive line material to be detected carries the imidazolinone herbicide resistant gene OsmALS of heterozygous; If the high resolving power melting curve peak type detected is consistent with the receptor parent peak type of transformation, then paddy rice introgressive line material to be detected does not carry imidazolinone herbicide resistant gene OsmALS.
Principle of the present invention: SNP (SingleNucleotidePolymorphism, single nucleotide polymorphism) refers to the DNA sequence polymorphism of same site not between isoallele caused by single nucleotide difference.SNP quantity in genome is many, and distribution is wide, and the exploitation for molecule marker provides important resource.The detection realizing SNP by high resolving power melting curve small segment method (SmallAmplicon) extends the practicality that SNP site detects widely, it is no longer limited to restriction enzyme site, not with the size of DNA fragmentation for detection means, the high resolving power melting curve gathered under the speed of 0.1 DEG C/s can distinguish the difference of single base significantly.Based on this principle, the present invention obtains by designing specially amplimer the amplification small segment including SNP to be detected, then utilize high resolving power melting curve analysis instrument to gather the high resolving power melting curve of PCR primer, verify and obtain molecule marker provided by the present invention.The present invention obtains 2 specificity SNP (AT, as Fig. 1) that OsmALS gene order only occurs by the method for Multiple Sequence Alignment, be just in time the resistance locus Met548 being positioned at imidazolinone herbicide resistant gene OsmALS.Based on this, the present invention designs primer in the upstream and downstream of these 2 SNP and obtains amplified fragments, LightScanner96 instrument is utilized to gather the high resolving power solubility curve of amplified production, every paddy rice introgressive line material carrying homozygous imidazolinone herbicide resistant gene OsmALS, detect that the peak type of high resolving power melting curve is consistent with paddy rice non-transgenic antiweed material, and every paddy rice introgressive line material carrying the imidazolinone herbicide resistant gene OsmALS of heterozygous, detect that the peak type of high resolving power melting curve is rendered as dual hump, otherwise the peak type of high resolving power melting curve then detected, both not consistent with paddy rice non-transgenic antiweed material, also dual hump is not rendered as.Namely high resolving power melting curve detected result presents (as Fig. 2, Fig. 3) with the form at peak type proterties and Zuo Feng/right peak.
The present invention has following advantage and effect relative to prior art:
(1) there is false positive hardly in molecule marker provided by the invention: traditional Herbicid resistant material generally screens by spraying corresponding weedicide, if but doses improper use or spray uneven cause most probably falsely dropping or required material dead.And in Molecular Detection, traditional pcr amplification in conjunction with restriction enzyme detection method with electrophoretic band " with or without " carry out interpretation, often false positive is higher.Because before enzyme is cut, if PCR primer does not carry out purifying, easily cause restriction enzyme enzymic activity to reduce, cause cannot cutting and causing false positive; If PCR primer carries out purifying, easily cause reclaiming the amount of amplified fragments extremely low even without, affect enzyme and cut the interpretation of rear electrophoresis result and cause false positive.Molecule marker provided by the invention has simplicity and susceptibility in detection, the PCR primer being added with saturated fluorescence dyestuff directly carries out the collection of high resolving power melting curve, reach " zero loss ", and the existence of PCR system composition does not affect fluorescence signal acquisition, therefore there is false positive in high resolving power melting curve detected result hardly.
(2) molecule marker provided by the invention in actual applications, low cost, high-throughput: at present, high resolving power melting curve analysis method is the middle high-throughput SNP detection method being only second to DNA chip, the high resolving power getting final product disposable collection 96 PCR primer samples in 10min melts, and the restriction enzyme digestion and electrophoresis detection method obviously than traditional is simple and efficient; And the PCR system of this molecule marker only needs 10 μ l, it is the half of regular-PCR system, even if therefore need the saturated fluorescence dyestuff of 0.04 yuan/system, its cost, also not higher than regular-PCR, is specially adapted in the production practice of large-scale molecular assisted selection.
(3) molecule marker provided by the invention can reduce human cost and time cost prepared by pcr template significantly: in actual applications, traditional regular-PCR is very high to the requirement of pcr template purity in conjunction with the SNP detection method of restriction enzyme, often need the extracting method using CTAB etc. to waste time and energy, to avoid the impurities left such as protein, RNA.Molecule marker of the present invention is less demanding to pcr template, and the extracting solution obtained by simple crushing " tube method " namely can be used as pcr template, substantially increases the simplicity of Molecular Detection.
Therefore, in order to avoid doses improper use or spray uneven and cause falsely dropping or required material dead, imidazolinone herbicide resistant gene OsmALS gene specific molecule marker 548 provided by the present invention suddenlys change and to have broad application prospects in production practice, utilize this mark can improve this gene in molecular marker assisted selection breeding, gene pyramiding breeding, and the efficiency utilized in transgenic breeding.
Accompanying drawing explanation
Fig. 1 example describes Auele Specific Primer ALS-22415F and ALS-22494R amplified fragments sequence (80bp), and shows the base difference that the yellow China of parental wildtype accounts for (HHZ) and non-transgenic Herbicide Resistant Rice material D3-10.
Fig. 2 passes through the HRM analytical results of parent HHZ, D3-10 and Hybrids F1, and example describes three kinds of genotype (grey-wild homozygous TG/TG of SNP (TG → AT); Redness-mutant homozygous type AT/AT; Blueness-heterozygous TG/AT) the characteristic trait of melting curve.
Fig. 3 example describe molecule marker (ALS-22415F and ALS-22494R) that this experiment develops in conjunction with HRM and rice herbicide resistance trait be divided into from.
Fig. 4 example illustrates that the HRM genotyping result of molecule marker (ALS-22415F with ALS-22494R) conforms to sequencing result.
Embodiment
Below in conjunction with implementation column and accompanying drawing, further detailed description is done to the present invention, but embodiments of the present invention are not limited thereto.Method therefor if no special instructions, is conventional molecular biology, tissue culture technique and the method described in agronomy handbook.Such as, concrete steps can be see: " MolecularCloning:ALaboratoryManual (3
rdedition) " (Sambrook, J., Russell, DavidW., 2001, ColdSpringHarbor), " PlantPropagationbyTissueCulture " (EdwinF.George, MichaelA.Hall, Geert-JanDeKlerk, 2008, Springer).
In the present invention, the 548th amino acids sudden change of Rice Resistance imidazolinone herbicide gene OsmALS, includes but not limited to sport Cys or Met by Trp, or replacement, the disappearance of base occurs in this position or adds one or several Nucleotide.In specific embodiment of the invention part, contriver is only sported the situation of Met (ATG) by Trp (TGG) for the 548th amino acids of the endogenous OsmALS gene of paddy rice imidazolinone herbicide resistant material D3-10, carried out the exploitation of HRM detection method and detected the explanation of application.Those skilled in the art should know, and after being sported the HRM detection feasibility of Met by Trp at the 548th that discloses OsmALS, other sudden change situations that this HRM detection system may be used for the 548th amino acids of OsmALS equally detect.
The analysis of OsmALS gene specific molecule marker and design of primers thereof and detection in embodiment 1.D3-10 material
(1) analysis in OsmALS specificity list base difference (SNP) site:
By Multiple Sequence Alignment Software tool DNAMAN, als gene is accounted for and with the imidazolinone herbicide resistant material D3-10 (Chinese patent: CN201210037789.9 of yellow Hua Zhanwei genetic background to the Huang China published, rice herbicide resistant protein and the application in plant breeding thereof) coding region carry out nucleotide sequence comparison, examination OsmALS specific single base (SNP) difference site.OsmALS gene and the yellow China of imidazolinone herbicide sensitive varieties with the paddy rice imidazolinone herbicide resistant material D3-10 of gene specific SNP account for the sequence alignment result of the ALS of (HHZ) as shown in Figure 1.Wherein, the Nucleotide highlighted with distinct colors is OsmALS gene specific list base difference (SNP) identified.Concrete, 548th amino acids of the endogenous als gene of this paddy rice imidazolinone herbicide resistant material D3-10 sports Met (ATG) by Trp (TGG), its DNA sequence dna is as shown in SEQIDNO:1, and aminoacid sequence is as shown in SEQIDNO:2.
(2) primer is designed:
At the upstream and downstream design gene-specific amplification primer of this SNP site, it is 4 right to devise altogether, the base sequence of described primer pair and the length of pcr amplification product as shown in table 1.
Table 1. primer sequence table
(3) paddy rice is selected to represent material:
Rice material D3-10, carries homozygous imidazolinone herbicide resistance OsmALS gene, elects positive control as;
The yellow China of rice varieties accounts for, for imidazolinone herbicide sensitive material (document " Zhou Shaochuan etc. the good character that the yellow China of early-mid-late rice dual-purpose type grain quality rice Core Germplasms accounts for and breeding achievement analysis [J]. Chinese agriculture science and technology Leader; 2008; 10 (3): 77-83 " in disclose), be negative control;
Carry the rice material of the OsmALS gene of heterozygous: with D3-10 and Huang Huazhan (HHZ) each other Parent hybridize, first filial generation seed (F1) is through 2% " hundred ridges lead to " (BASF AG's production, effective constituent: 240g/L AC 263222) " soak the true hybrid survived after 24 hours.
(4) pcr amplification, obtains the fragment containing OsmALS gene specific SNP
Utilize above-mentioned 4 groups of primer pairs, with the STb gene of above-mentioned rice material (using simple crushing " tube method " extracting to obtain) for template, carry out standard PCR amplification, the fragment obtained that increases uses LightScanner96 instrument to carry out high resolving power melting curve collection analysis, found that in above-mentioned 4 primer pairs, only have the high resolving power melting curve peak type feature of primer pair ALS-22415F and ALS-22494R obvious, the somatotype poor effect of other three primer pairs.This primer pair ALS-22415F and ALS-22494R carries out detecting and analyzes the result that obtains as shown in Figure 2.
The STb gene preferred process of simple crushing " tube method " extracting rice material is as follows:
Get 2-4cm blade, directly add extracting solution (10mMTris-HCl, 0.5mMEDTA, 0.15MKCl) grinding, after boiling water bath 10min, stratification gets the DNA profiling that supernatant (1 μ L) can do pcr amplification.
Pcr amplification reaction system is as follows:
10×PCRBuffer(Mg2+plus):1μL
dNTPs(2.5mMeach):0.1μL
Primer ALS-22415F (10 μMs): 0.1 μ L
Primer ALS-22494F (10 μMs): 0.1 μ L
20×EveGreen：0.1μL
TakaRaTaqTM(5U/μL):0.1μL
DNA profiling (2-3ng/ μ L): 1 μ L
DdH2O: complement to 10 μ L.
In order to prevent system from evaporating, 15 μ L-20 μ L mineral oil need be dripped by force at pcr amplification and covering.
PCR Thermal cycling conditions is as follows: 94 DEG C 3 minutes; 94 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 10 seconds, 40 circulations; 72 DEG C 2 minutes; 95 DEG C 1 minute; 12 DEG C were transferred to 4 DEG C of Refrigerator stores after 1 minute.
After PCR reaction terminates, PCR primer is all transferred to 96 hole white background black surround semi-conductor PCR.Conveniently temperature contrast between follow-up correction 96 orifice bore, each hole adds 1 μ L temperature internal reference (InternalTemperatureControls) and adds 15 μ L-20 μ L mineral oil coverings.
Described temperature internal reference base sequence is as follows:
InTemF：5＇-ATCGTGATTTCTATAGTTATCTAAGTAGTTGGCATTAATAATTTCATTTT-3＇(SEQIDNO:11)；
InTemR：5＇-AAAATGAAATTATTAATGCCAACTACTTAGATAACTATAGAAATCACGAT-3＇(SEQIDNO:12)；
After oligonucleotide InTemF and the InTemR synthesis of above-mentioned complementation, the amount of 1OD is dissolved with 800 μ LddH2O, and then reaction system is as follows:
Saturated NaCl:1 μ L
InTemF：1μL
InTemR：1μL
DdH2O: complement to 10 μ L.
Under 95 DEG C of conditions, sex change is after 3 minutes, naturally cools to room temperature renaturation, namely can be used as temperature internal reference.
Wherein, the curve (grey, redness and blueness) of 3 kinds of different colours marks in Fig. 2 represents Huang Huazhan (HHZ), D3-10 respectively, and both hybridize the high resolving power melting curve peak type characteristic trait of first filial generation (F1 generation).That is, the high resolving power melting curve of red-label is homozygous OsmALS gene specific molecule marker W548M, and the high resolving power melting curve of blue markings is heterozygous OsmALS gene specific dual hump type.The result display of Fig. 2, OsmALS gene specific molecule marker W548M can screen homozygous OsmALS gene, can screen heterozygous OsmALS again.In paddy rice introgressive line, backcross progeny (BCnF1) all carries heterozygous OsmALS gene, and the high resolving power melting curve of its PCR primer presents dual hump peak type.
Embodiment 2: resistant gene OsmALS gene specific molecule marker differentiates the application of different genotype plant in paddy rice introgressive line selfing segregating population.
In the selfing segregating population of Resistance genes of vice OsmALS introgressive line, although the plant of not carrying resistant gene OsmALS can be removed by spraying " hundred ridges lead to (effective constituent: 240g/L AC 263222) ", and remain resistant plant and carry homozygous or heterozygous OsmALS gene, utilize molecule marker W548M to be distinguished rapidly.
First filial generation (F1) the selfing segregating population F2 of above-mentioned " embodiment 1 ", clean after soaking for 2% " hundred ridges lead to (effective constituent: 240g/L AC 263222) " 24 hours by seed, the plant of plantation survival is carries resistant gene OsmALS two kinds of genotypic rice materials.Random selecting 48 plant, according to the method for embodiment 1, carry out DNA profiling preparation, pcr amplification and the collection of high resolving power melting curve.According to high resolving power melting curve peak type proterties, two of resistant gene OsmALS kind of a genotype can be made a distinction.As shown in Figure 3, the plant presenting the dual hump peak type high resolving power melting curve of grey mark has 32, and the plant presenting the red-label high resolving power melting curve consistent with D3-10 has 16, i.e. heterozygous and homozygously present 2:1 segregation ratio, meets Mendel's law of segregation.As shown in Figure 4, these 16 plant presenting red-label high resolving power melting curve peak type, its PCR primer sequencing result also shows 2 specificity SNP all with OsmALS, consistent with the sequencing result of D3-10 (A, B), and be different from the sequencing result of Huang Huazhan (HHZ).Visible, this resistant gene OsmALS gene specific molecule marker is differentiated to be applied in different genotype plant etc. in paddy rice introgressive line selfing segregating population.
Embodiment 3: the detection application of molecule marker W548M in anti-herbicide gene OsmALS transformation conventional fine grain quality rice and restorer work
The transformation workload of conventional fine grain quality rice and restorer is large, cenospecies harvest yield is few, therefore introgressive line is often very precious, wherein the removal of pseudostationary, if doses improper use or spray uneven cause most probably falsely dropping or required material dead.Based on this worry, inventor uses resistant gene OsmALS gene specific molecule marker W548M to detect the introgressive line of this gene always.At present, the introgressive line BC4F1 plant 5 that it is genetic background that inventor has successfully obtained with " Huang Huazhan " take 6 kinds of conventional fine grain quality rices such as " ivory perfume (or spice) accounts for ", " peasants who dig gold's silk seedling " as the BC2F1 plant 22 of genetic background; 116 strains are amounted to a series of (BC1F1-BC4F1) introgressive line plant that 20 kinds of recoveries such as " bright extensive 63 ", " blue or green precious " are genetic background.The PCR primer of these introgressive line plant, its high resolving power melting curve peak type all presents dual hump peak type, is the true hybrid carrying heterozygous OsmALS.Visible, this OsmALS gene specific molecule marker obtains important application in molecular mark.
SEQUENCELISTING
<110> Shenzhen Crop Molecular Design Breeding Institute
Shenzhen Xingwang Biological Seed Industry Co., Ltd.
Hunan Wang Hua biological husbantry Science and Technology Ltd.
Xingwang Investment Pty Ltd.
The HRM detection method of <120> anti-herbicide gene OsmALS and application
<130>
<160>12
<170>PatentInversion3.3
<210>1
<211>1935
<212>DNA
<213> paddy rice (OryzasativaL.ssp.indica)
<400>1
atggctacgaccgccgcggccgcggccgccaccttgtccgccgccgcgacggccaagacc60
ggccgtaagaaccaccagcgacaccacgtccttcccgctcgaggccgggtgggggcggcg120
gcggtcaggtgctcggcggtgtccccggtcaccccgccgtccccggcgccgccggccacg180
ccgctccggccgtgggggccggccgagccccgcaagggcgcggacatcctcgtggaggcg240
ctggagcggtgcggcgtcagcgacgtgttcgcctacccgggcggcgcgtccatggagatc300
caccaggcgctgacgcgctccccggtcatcaccaaccacctcttccgccacgagcagggc360
gaggcgttcgcggcgtccgggtacgcgcgcgcgtccggccgcgtcggggtctgcgtcgcc420
acctccggccccggggcaaccaacctcgtgtccgcgctcgccgacgcgctgctcgactcc480
gtcccgatggtcgccatcacgggccaggtcccccgccgcatgatcggcaccgacgccttc540
caggagacgcccatagtcgaggtcacccgctccatcaccaagcacaattaccttgtcctt600
gatgtggaggacatcccccgcgtcatacaggaagccttcttcctcgcgtcctcgggccgt660
cctggcccggtgctggtcgacatccccaaggacatccagcagcagatggctgtgccagtc720
tgggacacctcgatgaatctaccggggtacattgcacgcctgcccaagccacccgcgaca780
gaattgcttgagcaggtcttgcgtctggttggcgagtcacggcgcccgattctctatgtc840
ggtggtggctgctctgcatctggtgatgaattgcgccggtttgttgagctgaccggcatc900
ccagttacaaccactctgatgggcctcggcaatttccccagtgatgatccgttgtccctg960
cgcatgcttgggatgcatggcacggtgtacgcaaattatgcggtggataaggctgacctg1020
ttgcttgcatttggcgtgcggtttgatgatcgtgtgacagggaaaattgaggcttttgca1080
agcagggccaagattgtgcacattgacattgatccagcggagattggaaagaacaagcaa1140
ccacatgtgtcaatttgcgcagatgttaagcttgctttacagggcttgaatgctctgcta1200
gaccagagcacaacaaagacaagttctgattttagtgcgtggcacaatgagttggaccag1260
cagaagagggagtttcctctggggtacaagacttttggtgaagagatcccaccgcaatat1320
gctattcaggtgctggatgagctgacgaaaggggaggcaatcatcgctactggtgttgga1380
cagcaccagatgtgggcggcacaatattacacctacaagcggccacggcagtggctgtct1440
tcggctggtctgggcgcaatgggatttgggctgcctgctgcagctggtgcttctgtggct1500
aacccaggtgtcacagttgttgatattgatggggatggtagcttcctcatgaacattcag1560
gagttggcattgatccgcattgagaacctcccggtgaaggtgatggtgttgaacaaccaa1620
catttgggtatggttgtgcaaatggaggataggttttacaaggcaaatagggcgcataca1680
tacttgggcaacccagaatgtgagagtgagatatatccagattttgtgactattgctaaa1740
gggttcaatattcctgcagtccgtgtaacaaagaagagtgaagtccgtgccgccatcaag1800
aagatgctcgataccccagggccatacttgttggatatcatcgtcccacaccaggagcat1860
gtgctgcctatgatcccaagtgggggcgcattcaaggacatgatcctggatggtgatggc1920
aggactgtgtattaa1935
<210>2
<211>644
<212>PRT
<213> paddy rice (OryzasativaL.ssp.indica)
<400>2
MetAlaThrThrAlaAlaAlaAlaAlaAlaThrLeuSerAlaAlaAla
151015
ThrAlaLysThrGlyArgLysAsnHisGlnArgHisHisValLeuPro
202530
AlaArgGlyArgValGlyAlaAlaAlaValArgCysSerAlaValSer
354045
ProValThrProProSerProAlaProProAlaThrProLeuArgPro
505560
TrpGlyProAlaGluProArgLysGlyAlaAspIleLeuValGluAla
65707580
LeuGluArgCysGlyValSerAspValPheAlaTyrProGlyGlyAla
859095
SerMetGluIleHisGlnAlaLeuThrArgSerProValIleThrAsn
100105110
HisLeuPheArgHisGluGlnGlyGluAlaPheAlaAlaSerGlyTyr
115120125
AlaArgAlaSerGlyArgValGlyValCysValAlaThrSerGlyPro
130135140
GlyAlaThrAsnLeuValSerAlaLeuAlaAspAlaLeuLeuAspSer
145150155160
ValProMetValAlaIleThrGlyGlnValProArgArgMetIleGly
165170175
ThrAspAlaPheGlnGluThrProIleValGluValThrArgSerIle
180185190
ThrLysHisAsnTyrLeuValLeuAspValGluAspIleProArgVal
195200205
IleGlnGluAlaPhePheLeuAlaSerSerGlyArgProGlyProVal
210215220
LeuValAspIleProLysAspIleGlnGlnGlnMetAlaValProVal
225230235240
TrpAspThrSerMetAsnLeuProGlyTyrIleAlaArgLeuProLys
245250255
ProProAlaThrGluLeuLeuGluGlnValLeuArgLeuValGlyGlu
260265270
SerArgArgProIleLeuTyrValGlyGlyGlyCysSerAlaSerGly
275280285
AspGluLeuArgArgPheValGluLeuThrGlyIleProValThrThr
290295300
ThrLeuMetGlyLeuGlyAsnPheProSerAspAspProLeuSerLeu
305310315320
ArgMetLeuGlyMetHisGlyThrValTyrAlaAsnTyrAlaValAsp
325330335
LysAlaAspLeuLeuLeuAlaPheGlyValArgPheAspAspArgVal
340345350
ThrGlyLysIleGluAlaPheAlaSerArgAlaLysIleValHisIle
355360365
AspIleAspProAlaGluIleGlyLysAsnLysGlnProHisValSer
370375380
IleCysAlaAspValLysLeuAlaLeuGlnGlyLeuAsnAlaLeuLeu
385390395400
AspGlnSerThrThrLysThrSerSerAspPheSerAlaTrpHisAsn
405410415
GluLeuAspGlnGlnLysArgGluPheProLeuGlyTyrLysThrPhe
420425430
GlyGluGluIleProProGlnTyrAlaIleGlnValLeuAspGluLeu
435440445
ThrLysGlyGluAlaIleIleAlaThrGlyValGlyGlnHisGlnMet
450455460
TrpAlaAlaGlnTyrTyrThrTyrLysArgProArgGlnTrpLeuSer
465470475480
SerAlaGlyLeuGlyAlaMetGlyPheGlyLeuProAlaAlaAlaGly
485490495
AlaSerValAlaAsnProGlyValThrValValAspIleAspGlyAsp
500505510
GlySerPheLeuMetAsnIleGlnGluLeuAlaLeuIleArgIleGlu
515520525
AsnLeuProValLysValMetValLeuAsnAsnGlnHisLeuGlyMet
530535540
ValValGlnMetGluAspArgPheTyrLysAlaAsnArgAlaHisThr
545550555560
TyrLeuGlyAsnProGluCysGluSerGluIleTyrProAspPheVal
565570575
ThrIleAlaLysGlyPheAsnIleProAlaValArgValThrLysLys
580585590
SerGluValArgAlaAlaIleLysLysMetLeuAspThrProGlyPro
595600605
TyrLeuLeuAspIleIleValProHisGlnGluHisValLeuProMet
610615620
IleProSerGlyGlyAlaPheLysAspMetIleLeuAspGlyAspGly
625630635640
ArgThrValTyr
<210>3
<211>17
<212>DNA
<213> synthetic
<400>3
aagtatgtatgcgccct17
<210>4
<211>17
<212>DNA
<213> synthetic
<400>4
gtgttgaacaaccaaca17
<210>5
<211>17
<212>DNA
<213> synthetic
<400>5
agaacctccctgtgaag17
<210>6
<211>16
<212>DNA
<213> synthetic
<400>6
cgccctattcgccttg16
<210>7
<211>16
<212>DNA
<213> synthetic
<400>7
ctgtgaaggtgatggt16
<210>8
<211>16
<212>DNA
<213> synthetic
<400>8
ttgcccaagtatgtat16
<210>9
<211>16
<212>DNA
<213> synthetic
<400>9
caacatttgggtatgg16
<210>10
<211>16
<212>DNA
<213> synthetic
<400>10
ttcgccttgtaaaacc16
<210>11
<211>50
<212>DNA
<213> synthetic
<400>11
atcgtgatttctatagttatctaagtagttggcattaataatttcatttt50
<210>12
<211>50
<212>DNA
<213> synthetic
<400>12
aaaatgaaattattaatgccaactacttagataactatagaaatcacgat50
Claims (11)
1. a HRM detection method for OsmALS gene the 548th amino acids sudden change, it is characterized in that described HRM detection system comprises pair of primers, the sequence of described primer is: forward primer: AAGTATGTATGCGCCCT; And reverse primer: GTGTTGAACAACCAACA.
2. HRM detection method according to claim 1, wherein said OsmALS gene the 548th amino acids sudden change includes but not limited to sport Cys or Met by Trp, or replacement, the disappearance of base occurs in this position or adds one or several Nucleotide.
3. the HRM detection method described in claim 1 or 2, is characterized in that also comprising PCR reagent and EvaGreen fluorescence dye in described detection system.
4. HRM detection method according to claim 3, is characterized in that said method comprising the steps of:
A) sample to be tested DNA is extracted;
B) prepare the PCR reaction system comprising sample to be tested DNA, primer pair, PCR kit and EvaGreen fluorescence dye and carry out pcr amplification; With
C) HRM analysis scan is carried out to PCR primer.
5. HRM detection method according to claim 4, the method of the extraction sample to be tested DNA described in its kind is: sampling, directly add extracting solution grinding, after boiling water bath 10min, stratification is got supernatant and is sample to be tested DNA, and wherein said extracting solution comprises 10mMTris-HCl, 0.5mMEDTA and 0.15MKCl.
6. HRM detection method according to claim 4, wherein said PCR reaction system comprises 1 μ LPCR template, 0.5UrTaqDNA polysaccharase, 1 × PCR damping fluid, 250 μMs of dNTP, 0.1 μM of forward and reverse primer and 0.1 μ L20 × EvaGreen fluorescence dye.
7. HRM detection method according to claim 4, wherein said pcr amplification condition is: 95 DEG C of 3min; 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 10s, 40 circulations; 72 DEG C of 5min, and then 95 DEG C of sex change 1min, 25 DEG C of renaturation 1min.
8., based on the PCR kit for rice Os mALS gene the 548th amino acids abrupt climatic change of HRM technology, it is characterized in that described test kit comprises a primer pair, the sequence of described primer pair is: forward primer: AAGTATGTATGCGCCCT; And reverse primer: GTGTTGAACAACCAACA.
9. test kit according to claim 8, wherein said test kit also comprises PCR reagent and fluorescence dye.
10. the using method of arbitrary described test kit of claim 8-9, is characterized in that described using method comprises the steps:
A) sample to be tested DNA is extracted;
B) prepare the PCR reaction system comprising sample to be tested DNA, primer pair, PCR kit and EvaGreen fluorescence dye and carry out pcr amplification; With
C) HRM analysis scan is carried out to PCR primer.
Arbitrary described test kit of 11. claim 8-9 is detecting the application in the sudden change of OsmALS gene the 548th amino acids, described sudden change includes but not limited to sport Cys or Met by Trp, or replacement, the disappearance of base occurs in this position or adds one or several Nucleotide.
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| CN106636027A (en) * | 2017-02-08 | 2017-05-10 | 上海市农业科学院 | Wheat protein with herbicide resisting activity as well as coding gene and application thereof |
| CN107043761A (en) * | 2017-02-08 | 2017-08-15 | 上海市农业科学院 | Rice protein, its encoding gene and application with antiweed activity |
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