CN1778966A - Inspection of wheat polyphenol oxidase activity characteristic and its special primer - Google Patents
Inspection of wheat polyphenol oxidase activity characteristic and its special primer Download PDFInfo
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- CN1778966A CN1778966A CN 200410090421 CN200410090421A CN1778966A CN 1778966 A CN1778966 A CN 1778966A CN 200410090421 CN200410090421 CN 200410090421 CN 200410090421 A CN200410090421 A CN 200410090421A CN 1778966 A CN1778966 A CN 1778966A
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Abstract
The invention presents a measuring method of the activation of wheat polyphenol oxidase and its special primer. The primer provided by this invention, which is used to measure the activation of wheat polyphenol oxidase, is actually a pair of v formed by the nucleotide sequence of the sequence 1 and nucleotide sequence of the sequence 2 in the sequence list. The way of measuring the activation of wheat polyphenol oxidase is to make PCR amplification by taking the genome DNA of wheat waiting for measuring as the templet and the nucleotide sequence of the sequence 1 and nucleotide sequence of the sequence 2 in the sequence list as primer, and then test whether there is a stripe with the size of 198bp in the amplification amplified products. The method of this invention and its special primer will have an important role in the measuring of the activation of wheat polyphenol oxidase as well as in breeding.
Description
Technical field
The present invention relates to a kind of method and primer special thereof that detects activity of wheat polyphenol oxidase.
Background technology
Wheat polyphenol oxidase (Polyphenol oxidase is called for short PPO) is to cause the principal element of wheat goods such as noodles, steamed bun, dumpling and dough at the lay up period browning, 70% of goods colour stabilities such as its decision noodles.Reduce wheat PPO activity by the genetic breeding approach, cultivating the active low wheat breed of PPO is the important goal of Wheat Quality Improvement.
The researchist had done many about the active genetic research of wheat PPO, for example, appliable plant quantitative character key-genes such as Ge Xiuxiu add polygene blending inheritance model winter wheat PPO activity have been carried out genetic analysis, analytical results show the PPO activity be subjected to 2 pairs independently key-gene control.Jimenz and Anderson etc. infer also that by to the active mensuration of substitution line PPO the major gene of control wheat PPO is 1-2, point out simultaneously to have a plurality of allelotrope at the substrate specialization of PPO.But also do not find the position of PPO gene on chromosome of wheat at present.
Demeke etc. have studied the QTL mark of three recombinant inbred strain colonies, find that super close separation all appears in three colonies, and the PPO major gene is positioned on the second section homology group karyomit(e) chain with RFLP mark Xfba314.Udall and Demekes etc. utilize the RFLP labeled analysis to study four recombinant inbred strain colonies, Mares etc. have utilized AFLP labeled analysis DH colony, and above-mentioned two results of study prove that all the active major gene of PPO is positioned on the second homology group karyomit(e).But RFLP mark and AFLP mark are not based on the molecular marking technique of PCR (PCR-based), are difficult to use on wheat breeding.
In addition, Demeke and Morris go out the dna sequence dna of size for the wheat PPO of 2070bp according to the conserved regions design primer amplification of apple, potatoes and other crops PPO gene order.Jukanti etc. and Anderson etc. have also delivered one section PPO gene order.But the also untapped up to now PPO bioactive molecule mark that goes out can be applicable to wheat breeding.
Summary of the invention
The purpose of this invention is to provide a kind of primer that can be used for detecting activity of wheat polyphenol oxidase.
The primer that is used to detect activity of wheat polyphenol oxidase provided by the present invention, name is called Xgwm312, a pair of primer of being made up of the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 1 and sequence 2.
Sequence 1 in the sequence table is by 20 based compositions, and the sequence 2 in the sequence table is by 21 based compositions.
Second purpose of the present invention provides a kind of method that detects activity of wheat polyphenol oxidase.
The method of detection activity of wheat polyphenol oxidase provided by the present invention, be to be template with wheat cdna group DNA to be measured, classify primer as with the nucleotide sequence of sequence in the sequence table 1 and the nucleotides sequence of sequence 2, carry out pcr amplification, detect the band whether the 198bp size is arranged in the amplified production.
As the band of 198bp size is arranged in the amplified production, show the polyphenol oxidase activity height in the wheat to be measured.
Wherein, be example with 20 μ l cumulative volumes, the reaction system of pcr amplification comprises 1 * PCR damping fluid (50mmolL
-1KCl, 10mmolL
-1TrisCl, 0.01% gelatin), MgCl
21.5mM, each 250 μ M, TaqDNA polysaccharase 1U of dNTP (A.T.C.G), every primer 4pmol, template DNA 80ng.Reaction conditions can be: 94 ℃ of sex change 5min at first; 94 ℃ of sex change 1min then, 55 ℃ of annealing 55sec, 72 ℃ are extended 1min, totally 36 circulations; Last 72 ℃ are extended 10min.
Can detect the band whether the 198bp size is arranged in the amplified production by 6% polyacrylamide gel electrophoresis.
The present invention uses the molecular amounts genetic method and the little satellite of wheat (microsatellite or SSR) technology is positioned at wheat polyphenol oxidase gene PPO on the chromosome of wheat 2A long-armed (2AL), and provide the SSR molecule marker Xgwm312 of this gene, this mark and PPO gene close linkage (1.6cM), utilize this mark to carry out molecular marker assisted selection, thereby cultivate the low good wheat breed of polyphenol oxidase activity wheat polyphenol oxidase gene PPO; In addition, also can utilize the activity of polyphenoloxidase in this Markers for Detection wheat, detection method is simple, reliable and stable.Method of the present invention and primer special thereof will play a significant role in the detection of activity of wheat polyphenol oxidase and wheat breeding.
Description of drawings
Fig. 1 is the linkage map of SSR mark and polyphenol oxidase genes PPO
Fig. 2 is the pcr amplification product electrophorogram of primer Xgwm312 to 15 wheat breeds
Embodiment
The acquisition of the SSR molecule marker Xgwm312 of embodiment 1. wheat polyphenol oxidase gene PPO
The active high wheat breed of the PPO that is cultivated with the Chinese Academy of Agricultural Sciences crop ' in excellent 9507 ' wheat breed " CA9632 " hybridization lower with the PPO activity, F
1In generation, plant in the experimental plot, gets vitro anther culture during heading, and carry out doubling monoploids, obtains DH colony, carries out the PPO activation analysis then.
Field test is carried out in crop institute of the Chinese Academy of Agricultural Sciences (Beijing), 3 places of cotton institute of the Chinese Academy of Agricultural Sciences (Anyang) and academy of agricultural sciences, Shandong crop institute (Jinan) respectively, and repeat 2 times in each place, and the sub-district is 3 row districts, long 2 meters of row, 80 of every row sowings.The PPO activity of each DH system of results back test.
With 126 couples of SSR primers (wmc336a, Xgwm136, gdm33c, wmc329, wmc84b, Xgwm357, wmc469, wmc312b, Xgwm448c, Xgwm99, wmc367, Xgwm26, gdm33b, wmc336b, wmc522, wmc177, Xgwm356, wmc191, Xgwm372, Xgwm425, Xgwm448b, Xgwm382, Xgwm312, Xgwm294, Xgwm410, Xgwm374, wmc154, wmc257b, Xgwm261, Xgwm296, Xgwm157, Xgwm539, Xgwm349, wmc419, wmc256a, wmc200a, wmc132c, wmc36a, wmc41, wmc410, wmc532, Xgwm66, Xgwm493, wmc326, wmc308c, Xgwm114c, Xgwm264a, Xgwm285, wmc291, Xgwm376, Xgwm247, Xgwm389, gdm72, wmc533, Xgwm3, wmc170b, Xgwm645, Xgwm383, wmc236, wmc132e, wmc297, wmc308a, wmc238, Xgwm495, wmc349, Xgwm6, Xgwm251, Xgwm149, wmc449, wmc459, wmc285, wmc473, gdm125, wmc308b, wmc457, gdm133a, Xgwm194, Xgwm293, wmc257a, Xgwm186, wmc327b, Xgwm126, gdm133c, wmc537, Xgwm67, wmc118, gdm132b, Xgwm190, gdm68, Xgwm182, Xgwm272, Xgwm114a, wmc215, wmc132a, gdm33a, wmc201, Xgwm570, wmc256b, wmc32, wmc84a, wmc312a, Xgwm169, Xgwm518, Xgwm114g, Xgwm193, gdm132a, Xgwm325, wmc479, wmc17, Xgwm276, Xgwm282, wmc36b, Xgwm264b, Xgwm569, Xgwm297, wmc402b, wmc396, wmc76, Xgwm400, Xgwm577, wmc323, wmc132b, Xgwm111, wmc346, wmc38, wmc438), STS primer (the Glu-A3 of 4 pairs of storage proteins, Glu-B3, Gli-B1 is Glu-D1) with 26 couples of AFLP combination of primers (P41-M32e, P33-M38c, P42-M45c, P42-M45a, P32-M35e, P41-M32a, P42-M45h, P42-M45j, P32-M35b, P33-M38a, P31-M31a, P38-M46d, P42-M45g, P38-M46b, P38-M46a, P42-M45d, P42-M45e, P31-M31c, P42-M45f, P41-M32c, P35-M41d, P42-M45i, P35-M41a, P31-M31e, P35-M41b P31-M31b) carries out genetic analysis to this DH colony, use MAPMAKER3.0 software building linkage map, and carry out (Beijing, 3 places respectively with the composite interval mapping method (CIM) of Cartographer software package, Anyang and Jinan) the active qtl analysis of PPO.
The result as shown in Figure 1, showing has the main PPO of an effect active gene on chromosome of wheat 2A long-armed (2AL), and with microsatellite marker Xgwm312 close linkage (1.6cM).
The active mensuration of embodiment 2. wheat polyphenol oxidases (PPO)
The experiment wheat breed: 1. Soviet Union draws (available from academy of agricultural sciences, Jiangsu crop institute) No. 10,2. agricultural university 152 (available from China Agricultural University), 3. Henan wheat 21 (available from Henan Province's Luohe City research of agricultural science institute), 4. central sill 88375 (available from Tianshui, the Gansu institute of agricultural sciences), 5. raise wheat No. 5 (available from the regional institute of agricultural sciences of going to river in the Jiangsu), 6. Henan wheat 56 (available from academy of agricultural sciences, Henan wheat institute), winter rich 9801 (available from crop institutes of the Chinese Academy of Agricultural Sciences), 8. Shanxi wheat 50 (available from academy of agricultural sciences, Shanxi Province Cotton Research Institute), 9. Gaocheng 8901 (available from Gaocheng, Hebei province research of agricultural science institute), 10. high excellent 503 (available from Chinese Academy of Sciences Shijiazhuang Agricultural Modernization Institute), 11. Ji wheat 38 (available from academy of agricultural sciences, Shijiazhuang City, Hebei Province city), 12.98 in 18 (available from crop institutes of the Chinese Academy of Agricultural Sciences), Linfen 138 13. (available from academy of agricultural sciences, Shanxi Province wheat institute), 14. cloud wheat 46 (available from academy of agricultural sciences, Yunnan Province crop institute), 15. Shandong 928802 (available from academy of agricultural sciences, Shandong Province crop institute).
Above-mentioned 15 winter wheat varieties are planted in Cotton Inst., Chinese Agricultural Academy (Anyang), crop institute of the Chinese Academy of Agricultural Sciences (Beijing) and academy of agricultural sciences, Shandong crop institute (Jinan), field management is carried out according to a conventional method, the results seed is also measured its PPO activity as follows, and concrete steps are:
A) take by weighing 15 seeds and put into about 20ml vial, add the 4.5ml reaction reagent, be placed on the DL device and mix, make sample fully moistening.
Reaction reagent is: and the MOPS of 50mM pH6.5 (3-[N-morpholino] propanesulfonic acid) damping fluid, 10mM L-DOPA (L-3,4-dihydroxyphenyl alanine).
B) sample bottle is placed on 0.5 hour (sample fully is exposed in the air) of vibration on the reciprocating vibration machine.Be placed on again on the DL device and mix rapidly, make the color sample unanimity.Sample is placed on termination reaction on the ice cube at once.
C) be the colorimetric contrast with blank L-DOPA/MOPS solution, get the 1.0cm cuvette, under 475nm, measure the absorbance A of supernatant liquor with spectrophotometer.Calculation result: PPO activity (A475nm/ming
-1* 103)=A/ (30min * 15 a seed gram number) * 10
3
In the formula: A is the absorbancy of supernatant liquor under the 475nm.
Experimental result shows that 1. Soviet Unions draw seed PPO activity No. 10: 10.29 A
475/ g min
-1* 10
3, 2. agricultural university's 152 seed PPO activity: 9.07 A
475/ g min
-1* 10
3, 3. Henan wheat 21 seed PPO activity: 9.34 A
475/ g min
-1* 10
3, 4. central sill 88375 seed PPO activity: 11.83 A
475/ g min
-1* 10
3, 5. raise No. 5 seed PPO of wheat activity: 2.08 A
475/ g min
-1* 10
3, 6. Henan wheat 56 seed PPO activity: 3.09 A
475/ g min
-1* 10
3, 7. rich 9801 seed PPO activity: 2.96A of winter
475/ g min
-1* 10
3, 8. Shanxi wheat 50 seed PPO activity: 2.50 A
475/ gmin
-1* 10
3, 9. Gaocheng 8901 seed PPO activity: 3.29 A
475/ g min
-1* 10
3, 10. high excellent 503 seed PPO activity: 4.60 A
475/ g min
-1* 10
3, 11. Ji wheats, 38 seed PPO activity: 2.88 A
475/ g min
-1* 10
3, 18 seed PPO activity in 12.98: 3.27 A
475/ g min
-1* 10
3, 13. Linfen, 138 seed PPO activity: 9.62 A
475/ gmin
-1* 10
3, 14. cloud wheats, 46 seed PPO activity: 8.61A
475/ g min
-1* 10
3, 15. Shandong, 928802 seed PPO activity: 11.83 A
475/ g min
-1* 10
3The result shows: Soviet Union draws (seed PPO activity: 10.29 A No. 10
475/ gmin
-1* 10
3), agricultural university 152 (seed PPO activity: 9.07 A
475/ g min
-1* 10
3), Henan wheat 21 (seed PPO activity: 9.34 A
475/ g min
-1* 10
3), central sill 88375 (seed PPO activity: 11.83 A
475/ g min
-1* 10
3), Linfen 138 (seed PPO activity: 9.62 A
475/ g min
-1* 10
3), cloud wheat 46 (seed PPO activity: 8.61A
475/ g min
-1* 10
3) and Shandong 928802 (seed PPO activity: 11.83 A
475/ g min
-1* 10
3) the PPO activity apparently higher than other wheat breed.
Embodiment 3.Xgwm312 molecule marker is used to detect the validation verification of activity of wheat polyphenol oxidase
Experiment wheat breed: identical with embodiment 2.
Above-mentioned 15 winter wheat varieties are planted respectively in Cotton Inst., Chinese Agricultural Academy (Anyang), crop institute of the Chinese Academy of Agricultural Sciences (Beijing) and academy of agricultural sciences, Shandong crop institute (Jinan), field management is carried out routinely, extract the genomic dna of each wheat breed behind the results seed, be template with it then, classify primer as with the nucleotide sequence of sequence in the sequence table 1 and the nucleotides sequence of sequence 2, carry out pcr amplification.
Wherein, the reaction system of pcr amplification is: comprise 1 * PCR damping fluid (50mmolL in the 20 μ l cumulative volumes
-1KCl, 10mmolL
-1TrisCl, 0.01% gelatin), MgCl
21.5mM, each 250 μ M, TaqDNA polysaccharase 1U of dNTP (A.T.C.G), every primer 4pmol, template DNA 80ng.Reaction conditions is: 94 ℃ of sex change 5min at first; 94 ℃ of sex change 1min then, 55 ℃ of annealing 55sec, 72 ℃ are extended 1min, totally 36 circulations; Last 72 ℃ are extended 10min.
Can detect the band whether the 198bp size is arranged in the amplified production by polyacrylamide gel electrophoresis.The method of described electrophoresis detection is: electrophoretic buffer is 1 * TBE of pH8.0; Glue is with 6% polyacrylamide gel storage liquid (urea 420.2g, third rare acid amides 60.0g, N ' N-methylene diacrylamide 3.16g, 10 * TBE 50ml, be settled to 1000ml) 60ml, 10% ammonium persulphate (APS), 300 μ l, TEMED 60 μ l, mix the back encapsulating, the glue type is 380mm/300mm/0.4mm; Electrophoretic procedures is 100W prerunning 30min at first, inserts 60 hole application of samples combs, 80W electrophoresis 60min behind the point sample.Carry out silver after electrophoresis finishes and dye program: (1) decolouring is with fixing: offset plate is put into 10% Glacial acetic acid jog 30min; (2) rinsing: with distilled water rinsing 3 times, each 3min; (3) silver dyes: the AgNO of 1L 1%
3The formaldehyde that adds 2ml 37% in the solution is put into offset plate jog 30min; (4) the about 20sec of distilled water rinsing; (5) develop: at the Na of the 1L 30% of precooling
2CO
3Add 2ml formaldehyde and 200 μ l, 1% NaS in the solution
2O
3Solution is put into the offset plate jog and is shown up to target fragment; (6) stop: rapidly offset plate is changed in 10% glacial acetic acid solution over to the color development stopping reaction; (7) waft with distilled water and wash 2min; (8) with after the offset plate seasoning, the record banding pattern is also taken a picture with digital camera.(swimming lane M is the result: pBR332DNA/BsuRI (HaeIII) Marker as shown in Figure 2, the genomic dna that swimming lane 1-15 is respectively with above-mentioned 15 grow wheats is the pcr amplification product of template), four kinds of electrophoresis banding patterns have appearred, be called banding pattern 1,2,3 and 4, its clip size is about 198bp, 216bp, 232bp and 240bp respectively.Wherein, draw (seed PPO activity: 10.29 A No. 10 with Soviet Union
475/ g min
-1* 10
3), agricultural university 152 (seed PPO activity: 9.07 A
475/ g min
-1* 10
3), Henan wheat 21 (seed PPO activity: 9.34 A
475/ g min
-1* 10
3), central sill 88375 (seed PPO activity: 11.83 A
475/ g min
-1* 10
3), Linfen 138 (seed PPO activity: 9.62A
475/ g min
-1* 10
3), cloud wheat 46 (seed PPO activity: 8.61 A
475/ g min
-1* 10
3) and Shandong 928802 (seed PPO activity: 11.83 A
475/ g min
-1* 10
3) genomic dna be that the pcr amplification product of template is banding pattern 1 (198bp), the experimental result of embodiment 2 shows that the PPO activity of this four grow wheat is apparently higher than other wheat breed, thereby having or not with the active size of wheat breed PPO of the 198bp amplified fragments of proof Xgwm312 is closely related, and this fragment occurs showing that the PPO of this wheat breed is active high.Among Fig. 2,1 is that Soviet Union draws (seed PPO activity: 10.29 A No. 10
475/ g min
-1* 10
3), 2 is agricultural university 152 (seed PPO activity: 9.07 A
475/ g min
-1* 10
3), 3 is Henan wheat 21 (seed PPO activity: 9.34 A
475/ g min
-1* 10
3), 4 is central sill 88375 (seed PPO activity: 11.83 A
475/ g min
-1* 10
3), 5 are (the seed PPO activity: 2.08 A of raising wheat No. 5
475/ g min
-1* 10
3), 6 is Henan wheat 56 (seed PPO activity: 3.09 A
475/ g min
-1* 10
3), 7 is rich 9801 (seed PPO activity: 2.96A of winter
475/ g min
-1* 10
3), 8 is Shanxi wheat 50 (seed PPO activity: 2.50A
475/ g min
-1* 10
3), 9 is Gaocheng 8901 (seed PPO activity: 3.29 A
475/ g min
-1* 10
3), 10 is high excellent 503 (seed PPO activity: 4.60 A
475/ g min
-1* 10
3), 11 is Ji wheat 38 (seed PPO activity: 2.88 A
475/ g min
-1* 10
3), 12 is 18 (seed PPO activity: 3.27 A in 98
475/ g min
-1* 10
3), 13 is Linfen 138 (seed PPO activity: 9.62 A
475/ g min
-1* 10
3), 14 is cloud wheat 46 (seed PPO activity: 8.61 A
475/ gmin
-1* 10
3), 15 is Shandong 928802 (seed PPO activity: 11.83 A
475/ g min
-1* 10
3).
Sequence table
<160>2
<210>1
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>1
atcgcatgat?gcacgtagag 20
<210>2
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<230>
<400>2
acatgcatgc?ctacctaatg?g 21
Claims (4)
1, is used to detect the primer of activity of wheat polyphenol oxidase, a pair of primer of forming by the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 1 and sequence 2.
2, a kind of method that detects activity of wheat polyphenol oxidase, be to be template with wheat cdna group DNA to be measured, classify primer as with the nucleotide sequence of sequence in the sequence table 1 and the nucleotides sequence of sequence 2, carry out pcr amplification, detect the band whether the 198bp size is arranged in the amplified production.
3, method according to claim 2 is characterized in that: the cumulative volume of the reaction system of described pcr amplification is 20 μ l, comprises 1 * PCR damping fluid, MgCl
21.5mM, each 250 μ M, TaqDNA polysaccharase 1U of dNTP, every primer 4pmol, template DNA 80ng; Reaction conditions is: 94 ℃ of sex change 5min at first; 94 ℃ of sex change 1min then, 55 ℃ of annealing 55sec, 72 ℃ are extended 1min, totally 36 circulations; Last 72 ℃ are extended 10min.
4, according to claim 2 or 3 described methods, it is characterized in that: described detection amplified production method is for carrying out 6% polyacrylamide gel electrophoresis to amplified production, and silver dyes colour developing then.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101230383B (en) * | 2008-02-22 | 2011-08-24 | 安徽农业大学 | Method for detecting activity of wheat polyphenol oxidase and special primer therefor |
| CN101570791B (en) * | 2009-06-02 | 2012-03-14 | 中国农业科学院作物科学研究所 | Special primer for identifying which variety of Glu-A3 protein subunit is carried in wheat as well as application thereof |
| CN111650136A (en) * | 2020-04-22 | 2020-09-11 | 山东省农业科学院作物研究所 | Detection method for activity of polyphenol oxidase of wheat grains and application of detection method |
| CN112048569A (en) * | 2020-10-26 | 2020-12-08 | 江苏省农业科学院 | Molecular marker coseparated with wheat low polyphenol oxidase activity gene QPpo-5D |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AR024934A1 (en) * | 1999-07-27 | 2002-10-30 | Novartis Ag | CHEMICAL GENES |
-
2004
- 2004-11-18 CN CNB2004100904214A patent/CN100335656C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101230383B (en) * | 2008-02-22 | 2011-08-24 | 安徽农业大学 | Method for detecting activity of wheat polyphenol oxidase and special primer therefor |
| CN101570791B (en) * | 2009-06-02 | 2012-03-14 | 中国农业科学院作物科学研究所 | Special primer for identifying which variety of Glu-A3 protein subunit is carried in wheat as well as application thereof |
| CN111650136A (en) * | 2020-04-22 | 2020-09-11 | 山东省农业科学院作物研究所 | Detection method for activity of polyphenol oxidase of wheat grains and application of detection method |
| CN112048569A (en) * | 2020-10-26 | 2020-12-08 | 江苏省农业科学院 | Molecular marker coseparated with wheat low polyphenol oxidase activity gene QPpo-5D |
| CN112048569B (en) * | 2020-10-26 | 2022-08-02 | 江苏省农业科学院 | Molecular marker coseparated with wheat low polyphenol oxidase activity gene QPpo-5D |
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| Publication number | Publication date |
|---|---|
| CN100335656C (en) | 2007-09-05 |
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