CN109735653A - The fluorescent molecule label of rice waxy gene Wx a kind of and its application - Google Patents
The fluorescent molecule label of rice waxy gene Wx a kind of and its application Download PDFInfo
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Abstract
The present invention provides a kind of rice waxy genesWxFluorescent molecule label and its application, its primer includes RWx-FG primer, RWx-FT primer, RWx-R primer, #1 universal primer, #2 universal primer, the sequence of the RWx-FG primer is as shown in SEQ ID NO:1, the sequence of the RWx-FT primer is as shown in SEQ ID NO:2, the sequence of the RWx-R primer is as shown in SEQ ID NO:3, the sequence of the #1 universal primer is as shown in SEQ ID NO:4, and the sequence of the #2 universal primer is as shown in SEQ ID NO:5.Using technical solution of the present invention, test sample only passes through a PCR amplification, does not need electrophoresis detection, and obtains amplification data with Fluorescence Scanner directly on raw sheet；It is easy to operate, quick, it is at low cost, as a result accurately, and realize real stopped pipe operation.
Description
Technical field
The invention belongs to field of molecular biotechnology more particularly to the fluorescent molecule of rice waxy gene Wx a kind of label and
It is applied.
Background technique
Rice is important cereal crops, has more than 100 country's rice cultivation in the world, the population in the whole world 2/3 is with rice
For staple food grain.With the development of social economy and the raising of the level of consumption, people are higher and higher to rice quality and safety requirements, excellent
The soft rice of matter have the characteristics that good food flavor, high resilience, it is cold after be not easy to be hardened and liked by most consumers.Influence rice boiling
One key factor of quality is the starch quality being deposited in endosperm, is divided into straight chain and amylopectin, and amylose is mirror
Determine one of the main indicator of rice quality.Amylose content is high in rice, and gelatinization point also tends to higher, gel consistence reduction,
To make the stickiness, flexibility and glossiness of rice be deteriorated, and then seriously affect the quality and food flavor of rice.Rice waxy gene
(Wx) the amylose synthesis in amylosynthease (GBSSI) catalysis paddy endosperm that coding is incorporated on amylum body.Wx gene
Main allelic variation type it is more complex, in Oryza cultivar have variation type very rich.WxaIt is primarily present in Xian
In rice varieties, the formation of high amylose content is controlled, rice grain amylose content is highest reachable generally all 20% or more
30% or more, the quality and food flavor for resulting in rice reduce.WxbEquipotential type is primarily present in japonica rice variety, amylose
Content is much lower compared to the rice varieties of long-grained nonglutinous rice, generally in 15-18%.This kind of rice is medium partially due to amylose content
Low, rice stickiness, flexibility and glossiness are substantially better than WxaVeriety.
In view of the influence of rice Wx gene pairs rice quality, and its phenotypic evaluation is affected by environment, and receives in Grain Ripening
It just can be carried out after obtaining, difficulty, therefore more and more rice breeding researchs increased to the breed breeding of low amylose content
Person carries out the breeding of rice low amylose kind using Molecular Marker Assisted Selection Technology.As introduced in patent CN1330861A
The method using PCR amplification, but need to carry out after the completion digestion, be transferred to agarose electrophoresis detection, recycle gel at
It as system observation and counts, time-consuming for this method digestion, and restriction endonuclease is with high costs, and complicated for operation, in the process using toxic
Nucleic acid dye.And patent CN108796110A is disclosed and is expanded different fragments size by primer mispairing to distinguish genotype.By
It is smaller in the Fragment Differential expanded, the polyacrylamide gel electrophoresis test strip difference of high resolution must be used.And polypropylene
Acrylamide gel complex manufacturing process, and used reagent acrylamide and methylene diacrylamide are never poisons, to people
There is a great harm for body.After gel electrophoresis, needing to be dyed with silver nitrate and formaldehyde, the above reagent is all toxic reagent and carcinogenic substance,
There is very big injury to environment and human body, not environmentally.
Summary of the invention
Against the above technical problems, it marks and its applies the invention discloses the fluorescent molecule of rice waxy gene Wx a kind of,
Test sample only passes through a PCR amplification, does not need electrophoresis detection, and obtains amplification number with Fluorescence Scanner directly on raw sheet
According to acquisition rice WxaAnd WxbGenotype data；It is easy to operate, quick, it is at low cost, as a result accurately, solve side used at present
The shortcomings that for method because time-consuming for implementation process, process is cumbersome, low efficiency and use noxious material.
In this regard, the technical solution adopted by the present invention are as follows:
A kind of primer of the fluorescent molecule label of rice waxy gene Wx, including RWx-FG primer, RWx-FT primer, RWx-R
Primer, #1 universal primer, #2 universal primer, as shown in SEQ ID NO:1, the RWx-FT draws the sequence of the RWx-FG primer
The sequence of object is as shown in SEQ ID NO:2, and the sequence of the RWx-R primer is as shown in SEQ ID NO:3, the #1 universal primer
Sequence as shown in SEQ ID NO:4, the sequence of the #2 universal primer is as shown in SEQ ID NO:5.
Forward primer RWx-FG:
GAAGGTGACCAAGTTCATGCTTCATCAGGAAGAACATCTGCAAGG；
Forward primer RWx-FT:
GAAGGTCGGAGTCAACGGATTTCATCAGGAAGAACATCTGCAAGT；
Reverse primer RWx-R:GGAAAAACGAGCAATGAAAGATGC.
#1 universal primer: FAM-GAAGGTGACCAAGTTCATGCT；
#2 universal primer: HEX-GAAGGTCGGAGTCAACGGATT.
By the study found that bases G (Wx between the 1st introne of Wx gene and exon 2a) sport T (Wxb), make
Obtaining shear efficiency reduces, and the ability of the granule bound starch synzyme (GBSS) of endosperm reduces, and amylose content declines therewith.
It adopts this technical solution, is based on Penta-primer amplification refractory mutation system
(PARMS), i.e. five primer amplifications are obstructed Wx in technical system and Wx geneaWith WxbTwo the 1st intrones of allele and the 2nd
The difference of bases G and T between exon has developed the codominance fluorescent molecule label PM- that can track amylose content
Wx, i.e. RWx-FG primer, RWx-FT primer, RWx-R primer.It is obstructed system using the amplification that this 5 primers are formed, test sample
Only by a PCR amplification, electrophoresis detection is not needed, and obtains amplification data with Fluorescence Scanner directly on raw sheet, is passed through
Software analysis, obtains the genotype data of rice Wx.Method is simple, easy to operate, quick, at low cost, as a result accurately；And ring
It protects.
The invention discloses a kind of fluorescent molecule of rice waxy gene Wx labels, use rice wax as described above
The primer of the fluorescent molecule label of gene Wx obtains.
Further, the primer pair rice plant for using the fluorescent molecule of rice waxy gene Wx as described above to mark
In gene expanded, by obtained amplimer in the microplate reader comprising tri- kinds of fluorescence detection channels of FAM, HEX and ROX
It is detected, if scanning obtains HEX fluorescence signal, for containing WxbAllele；If scanning obtains FAM fluorescence signal,
For containing WxaAllele.
It adopts this technical solution, 3 are added simultaneously according to the primer and two universal fluorescent primers of Wx gene order design
It is expanded to PCR reaction system, low amylose content WxbAllelic sequences can be according to SNP difference and forward primer
RWx-FT is matched and is expanded and obtain HEX fluorescence signal value；And high amylose content WxaAllelic sequences and forward primer
RWx-FG is matched and is expanded and obtain FAM fluorescence signal value.If rice sample is heterozygous state in the site, two kinds of forward directions are drawn
Object expands simultaneously, and therebetween, scanning result is while having HEX and FAM signal for position in genotyping result figure.
Further, the rice waxy gene Wx fluorescent molecule label the following steps are included:
Step S1 extracts the genomic DNA of rice plant；
Step S2 leads to forward primer RWx-FG, forward primer RWx-FT, reverse primer RWx-R, #1 universal primer, #2
It is added in PCR reaction system simultaneously with primer and carries out amplification acquisition PCR product, wherein the response procedures of PCR are as follows: 94 DEG C,
3min；Then 94 DEG C, 20sec of 10 circulations, 65 DEG C (each cycle annealing temperature is arranged herein reduces by 0.8 DEG C, 10 circulation fortune
Row finish, at this time annealing temperature be 57 DEG C), 1min；Then 30 recycle 94 DEG C, 20sec, 57 DEG C, 1min；
Step S3 detects PCR product in the microplate reader comprising tri- kinds of fluorescence detection channels of FAM, HEX and ROX,
Fluorescence intensity signals value is read, then by fluorescence signal value binding marker information, Genotyping is carried out, obtains genotype results；
Step S4 is analyzed according to fluorescence signal value, if scanning obtains HEX fluorescence signal, for containing WxbEquipotential base
Cause；If scanning obtains FAM fluorescence signal, for containing WxaAllele；If scanning result is while having HEX and FAM letter
Number, position is therebetween in genotyping result figure, then it represents that the sample is heterozygous state.
Further, in step S2, PCR reaction system is the system of 10 μ L: 5 μ L 2 × PARMS master mix,
0.15 μ L10mM RWx-FG labeled primer, 0.15 μ L10mM RWx-FT labeled primer, 0.4 μ L10mM RWx-R is general reversely to be drawn
Object, 1 μ L template DNA, 3.3 μ L ddH2O.Further, wherein PARMS master mix reagent includes #1 universal primer, #2
Universal primer, buffer, DNTP, Taq enzyme, internal standard ROX.
The invention also discloses a kind of application of the fluorescent molecule of rice waxy gene Wx as described above label, applications
In the selection and use of the rice varieties of low amylose content.
Compared with prior art, the invention has the benefit that
(1) technical solution of the present invention is used, test sample only passes through a PCR amplification, do not need electrophoresis detection, and straight
It connects and obtains amplification data with Fluorescence Scanner on raw sheet, by analysis, obtain the genotype data of rice Wx.The technical solution
It can be detected in any period of paddy growth, and easy to operate, quick, it is at low cost, as a result accurately, and realize really
Stopped pipe operation.Method used at present is solved because time-consuming for implementation process, process is cumbersome, low efficiency and using noxious material
Problem, it is more environmentally-friendly.
(2) although fluorescent scanning technique is widely used in molecular labeling, the prior art is used glimmering
Optical scanning detection is also required to certain condition to realize, for example needs to synthesize the specific primer etc. for having fluorophor, at
This height.Using technical solution of the present invention, it is only necessary to general primer is synthesized, it is low-cost, due to using universal fluorescent primer, at
Experimental cost is greatly saved in this reduction.
Detailed description of the invention
Fig. 1 is identification wax allele Wx of the inventionaAnd WxbAmplification schematic diagram.Wherein, #1 is Allele 1
FAM fluorescent universal primer；#2 is 2 HEX fluorescent universal primer of Allele；#3 is that (label draws 1 specific amplification primer of Allele
Object RWx-FG)；#4 is 2 specific amplification primer (labeled primer RWx-FT) of Allele；#5 is general reverse primer (labeled primer
RWx-R)。
The genotypic results figure of fluorescent molecule label 94 parental rice kinds of detection of Fig. 2 embodiment of the present invention.Figure
In, upper left signaling point is HEX fluorescence signal；The signaling point of lower right is FAM fluorescence signal.
Specific embodiment
Preferably embodiment of the invention is described in further detail below.
A kind of fluorescent molecule label of rice waxy gene Wx, uses the fluorescent molecule mark of following rice waxy gene Wx
The primer of note carries out amplification acquisition, identifies wax allele WxaAnd WxbAmplification schematic diagram it is as shown in Figure 1.Primer includes
RWx-FG primer, RWx-FT primer, RWx-R primer, #1 universal primer, #2 universal primer, the sequence of the RWx-FG primer is such as
Shown in SEQ ID NO:1, the sequence of the RWx-FT primer is as shown in SEQ ID NO:2, and the sequence of the RWx-R primer is such as
Shown in SEQ ID NO:3, the sequence of the #1 universal primer is as shown in SEQ ID NO:4, and the sequence of the #2 universal primer is such as
Shown in SEQ ID NO:5.
Forward primer RWx-FG:
GAAGGTGACCAAGTTCATGCTTCATCAGGAAGAACATCTGCAAGG；
Forward primer RWx-FT:
GAAGGTCGGAGTCAACGGATTTCATCAGGAAGAACATCTGCAAGT；
Reverse primer RWx-R:GGAAAAACGAGCAATGAAAGATGC.
#1 universal primer: FAM-GAAGGTGACCAAGTTCATGCT；
#2 universal primer: HEX-GAAGGTCGGAGTCAACGGATT.
The present embodiment is Wx by 94 parts of parental rice materials of detectionaOr WxbType, and combine the phenotypic analysis of detection material
Verify the accuracy of the program.
Specific implementation step is as follows:
(1) oryza sativa genomic dna is extracted
It takes plantation in 94 parts of rice leafs in Nanning respectively, passes through CTAB extraction method (cetyl trimethylammonium bromide method)
Obtain the genomic DNA of rice；
(2) PCR amplification
PCR reaction system is that (reagent purchase is in Wuhan City's scape peptide biology by 10ul:5 μ L 2 × PARMS master mix
Science and Technology Ltd. mainly includes 2 universal fluorescent primers, buffer, DNTP and Taq enzyme, internal standard ROX etc.), 0.15 μ
L10mMRWx-FG labeled primer, 0.15 μ L10mMRWx-FT labeled primer, the 0.4 general reverse primer of μ L10mM RWx-R, 1 μ L
Template DNA, 3.3 μ L ddH2O.PCR response procedures: 94 DEG C, 3min；Then 10 94 DEG C, 20sec, 65 DEG C of circulations (are set herein
Setting each cycle annealing temperature reduces by 0.8 DEG C, and 10 circular flows finish, and annealing temperature is 57 DEG C at this time), 1min；Then 30
A 94 DEG C, 20sec, 57 DEG C of circulation, 1min；
(3) PCR product is used for quickly detecting in the microplate reader comprising tri- kinds of fluorescence detection channels of FAM, HEX and ROX,
Fluorescence intensity signals value is read, fluorescence signal value file is then passed through into SNP decoder (http://www.snpway.com/
Snpdecoder01/) tool, binding marker information carry out Genotyping automatically, obtain genotype results；
(4) it is analyzed according to fluorescence signal value, if scanning obtains HEX fluorescence signal, for WxbAllelotype；
If scanning obtains FAM fluorescence signal, for WxaAllelotype.If be located between the two, for heterozygous.
(5) result and analysis
The Genotyping detection of the molecular labeling of 94 parental rice kinds is detailed as shown in Figure 2.As seen from the figure: detecting
HEX fluorescence signal is that genotype is WxbParent material, and detect that FAM fluorescence signal be genotype is WxaParent
Material.There is FAM fluorescence signal again comprising HEX fluorescence signal between the two positioned at above-mentioned, is heterozygous.In the present embodiment,
The parent material that number the is P119 site is heterozygous.
Meanwhile to corresponding parent material carry out amylose content measurement, and with the testing result of fluorescent marker into
Row joint statistics, the results are shown in Table 1.
1 94 parts of parental rice amylose contents of table and PM-Wx detection and genotyping fluorescence detection result
By the statistical result showed of table 1, molecular labeling is consistent with phenotypic results, and amylose content is lower than 18% sample
Fluorescence signal is HEX, and kind fluorescence signal of the amylose content higher than 20% is FAM, the experimental cultivar that number is P119 by
Belong to heterozygous state in the gene, thus genotyping result show the kind independently of between HEX and FAM two types, and it is straight
Chain content of starch also falls between.As it can be seen that accurate using technical solution of the present invention result.The technical solution can be applied to
The rice varieties of the breeding low amylose content of efficiently and accurately, greatly save time and workload.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
Specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to of the invention
Protection scope.
Sequence table
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gaaggtcgga gtcaacggat ttcatcagga agaacatctg caagt 45
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Claims (5)
1. a kind of rice waxy geneWxFluorescent molecule label primer, it is characterised in that: including RWx-FG primer, RWx-FT
Primer, RWx-R primer, #1 universal primer, #2 universal primer, the sequence of the RWx-FG primer is as shown in SEQ ID NO:1, institute
The sequence of RWx-FT primer is stated as shown in SEQ ID NO:2, the sequence of the RWx-R primer is as shown in SEQ ID NO:3, institute
The sequence of #1 universal primer is stated as shown in SEQ ID NO:4, the sequence of the #2 universal primer is as shown in SEQ ID NO:5.
2. a kind of rice waxy geneWxFluorescent molecule label, it is characterised in that: it uses rice as described in claim 1
Waxy geneWxFluorescent molecule label primer pair rice plant in gene expanded, obtained amplimer is being wrapped
It is detected in microplate reader containing tri- kinds of fluorescence detection channels of FAM, HEX and ROX, if scanning obtains HEX fluorescence signal, for
ContainWx b Allele；If scanning obtain FAM fluorescence signal, for containingWx a Allele.
3. rice waxy gene according to claim 2WxFluorescent molecule label, it is characterised in that: it uses following step
It is rapid to implement:
Step S1 extracts the genomic DNA of rice plant；
RWx-FG primer, RWx-FT primer, RWx-R primer, #1 universal primer, #2 universal primer are added to by step S2 simultaneously
Amplification is carried out in PCR reaction system and obtains PCR product, wherein the response procedures of PCR are as follows: 94 DEG C, 3 min；Then 10 circulations
94 DEG C, 20sec, 65 DEG C, 1 min, for annealing temperature since 65 DEG C, each circulation reduces by 0.8 DEG C；Then 30 recycle 94 DEG C,
20sec, 57 DEG C, 1 min；
Step S3 detects PCR product in the microplate reader comprising tri- kinds of fluorescence detection channels of FAM, HEX and ROX, reads
Fluorescence intensity signals value carries out Genotyping then by fluorescence signal value binding marker information, obtains genotype results；
Step S4 is analyzed according to fluorescence signal value, if scanning obtain HEX fluorescence signal, for containingWx b Allele；Such as
Fruit scanning obtain FAM fluorescence signal, then for containingWx a Allele；If scanning result is while having HEX and FAM signal, table
Show that the sample is heterozygous state.
4. rice waxy gene according to claim 3WxFluorescent molecule label, it is characterised in that: in step S2, PCR
Reaction system be 10 μ L system: 5 μ L 2 × PARMS master mix, 0.15 μ L10 mM RWx-FG labeled primer,
0.15 μ L10 mM RWx-FT labeled primer, the 0.4 general reverse primer of μ L10 mM RWx-R, 1 μ L template DNA, 3.3 μ L
ddH2O；Wherein PARMS master mix reagent includes #1 universal primer, #2 universal primer, buffer, DNTP, Taq enzyme, interior
Mark ROX.
5. a kind of rice waxy gene as described in claim 2 ~ 4 any oneWxFluorescent molecule label application, answer
In the selection and use of the rice varieties of low amylose content.
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| CN110878375A (en) * | 2019-12-18 | 2020-03-13 | 广西壮族自治区农业科学院 | Specific molecular marker for detecting rice cold-resistant gene CTB4a and application thereof |
| CN110885895A (en) * | 2019-11-01 | 2020-03-17 | 广西壮族自治区农业科学院 | Molecular marking method of rice gelatinization temperature gene ALK and special primer thereof |
| CN110885894A (en) * | 2019-11-01 | 2020-03-17 | 广西壮族自治区农业科学院 | Molecular marking method of rice tillering angle gene TAC1 and special primer thereof |
| CN111560458A (en) * | 2020-05-27 | 2020-08-21 | 山东省水稻研究所 | Molecular marker primer for rapidly and efficiently identifying rice phytochrome gene PHYB and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110885895A (en) * | 2019-11-01 | 2020-03-17 | 广西壮族自治区农业科学院 | Molecular marking method of rice gelatinization temperature gene ALK and special primer thereof |
| CN110885894A (en) * | 2019-11-01 | 2020-03-17 | 广西壮族自治区农业科学院 | Molecular marking method of rice tillering angle gene TAC1 and special primer thereof |
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| CN111560458A (en) * | 2020-05-27 | 2020-08-21 | 山东省水稻研究所 | Molecular marker primer for rapidly and efficiently identifying rice phytochrome gene PHYB and application thereof |
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Application publication date: 20190510 |