CN103146819B - Specific primer for identification of soybean Lox3 and identification method - Google Patents
Specific primer for identification of soybean Lox3 and identification method Download PDFInfo
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- CN103146819B CN103146819B CN201310055876.1A CN201310055876A CN103146819B CN 103146819 B CN103146819 B CN 103146819B CN 201310055876 A CN201310055876 A CN 201310055876A CN 103146819 B CN103146819 B CN 103146819B
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Beans For Foods Or Fodder (AREA)
Abstract
The invention discloses a specific primer for identification of deletant of soybean Lox3. The specific primer comprises the following primer pair: Lox3F: TAACCAGCGTTGGGGGAA and Lox3R: CATTTATAGACGTGCGTGCA. The invention further discloses a method for identifying the deletant of soybean Lox3. According to the method, the soybean gene of to-be-detected deletant is used as a DNA template, and PCR amplification is carried out on the DNA template by using the primer pair; if a PCR amplification product contains a 474 bp band, the to-be-detected soybean gene contains Lox3, and if the PCR amplification product does not contain the 474 bp band, deletion of Lox3 in the to-be-detected soybean gene exists. The invention has the following beneficial effects: time and cost are saved, identification results are accurate and reliable, and screening efficiency of a Lox3 deletant material can be substantially improved.
Description
Technical field
The present invention relates to identify Auele Specific Primer and the method thereof of soybean Lox3 deletant, be mainly used in the technical field of molecule marker.
Background technology
Soybean fat oxidase (Lox) is a kind of containing nonheme iron, water-soluble globular protein.Lox is by suitable in catalysis unsaturated fatty acids, along Isosorbide-5-Nitrae-pentadiene structure, form hydrogen peroxide derivative, continue again to change into the rudimentary objectionable impurities such as aldehyde and ketone, thereby produce beany flavor and other volatile matter, hindered the widespread use of soybean in food.The hydroperoxide that Lox catalysis produces, directly the nutritive ingredient in food is combined, and reduces eating quality and the nutritive value of food.Pyroprocessing can alleviate the awake taste of soybean beans, but can cause protein denaturation, and protein processing characteristics and utilization ratio are reduced.In addition,, by methods such as microwave irradiation, change medium pH, organic solvent extraction and the hydrolysis of aldehyde lytic enzyme, also can reduce or eliminate soybean beany flavor.Aforesaid method all can increase soybean prod cost in various degree, and usually can cause soy-protein solubleness to decline, and reduces food value.Therefore, by genetic breeding means seed selection soybean Lox deletant material, to reduce or to eliminate the impact of Lox activity significant.Wang Ruolan etc. (2008) study and think, soybean Lox deletant sample changes slower than normal kind in the quality of storage period, and Lox disappearance can improve the stability of soybean storage quality to a certain extent.Wang Jinlong etc. (2000) research thinks, soybean Lox disappearance does not affect protein, fat and fatty acid content, but may cause that in seed, proline content reduces, and on other amino acid without impact.Proline(Pro) does not belong to essential amino acid, therefore Lox disappearance can not cause that soybean nutritional quality reduces.Julian etc. (2010) adopt probe technique to identify respectively Lox2 and Lox3, but this technology exists the problems such as technical sophistication, expense are higher.Screen soybean Lox deletant by molecular marking technique, can accelerate non-odor soybean breed breeding process, and then fundamentally improve soybean prod beany flavor, improve the storage quality of soybean.LOX gene in current existing LOX deletant is carried out to sequencing analysis, show that Lox1 deletant lacks the fragment of 1 74bp in the 8th exon of its encoding gene, causes Lox1 protein translation to cross early stopping; Lox3 deletant lacks 1 mononucleotide " G " in the 1st exon of its encoding gene, thereby causes phase shift mutation.
Summary of the invention
The technical problem to be solved in the present invention is to provide Auele Specific Primer and the method thereof of qualification soybean Lox3 deletant.
The present invention is achieved through the following technical solutions.
Identify an Auele Specific Primer for soybean Lox3 deletant, comprise following two groups of primer pairs:
Lox3F：TAACCAGCGTTGGGGGAA?；
Lox3R：CATTTATAGACGTGCGTGCA。
A method for above-mentioned Auele Specific Primer qualification soybean Lox3 deletant, comprises the following steps:
(1) taking the soybean gene of deletant to be measured as DNA profiling, above-mentioned two groups of above-mentioned DNA profilings of primer pair are carried out to pcr amplification;
(2) have 474bp band if state in pcr amplification product, above-mentioned soybean gene to be measured contains Lox3, if without 474bp band, and above-mentioned soybean gene disappearance Lox3 to be measured.
Further, the soybean of above-mentioned Lox3 deletant is any one in five-pointed star No. 1, five-pointed star No. 4, mirror 31.
Beneficial effect of the present invention:
Auele Specific Primer provided by the invention and authenticate technology thereof, not only save time and expense, and qualification result accurately and reliably, can significantly improve the screening efficiency to Lox3 deletant material.
Brief description of the drawings
Fig. 1 is the schematic diagram of case study on implementation 1 agarose gel electrophoresis.
Embodiment
According to drawings and embodiments the present invention is described in further detail below.
In following case study on implementation 1, choose altogether seven kinds of soybean varieties and identify, these seven kinds of general types that soybean varieties provides by Hebei Prov. Academy of Agricultural &. Forest Sciences.
1, five-pointed star No. 1
Authorization numbering: beans are examined No. 2001002 in Ji
Cultivar origin: Food and Oil Crops Inst., Hebei Agriculture and Forestry Academy is bred as for 1997, and parental combination is Ji beans 9/Century.
2, five-pointed star No. 2
Authorization numbering: state examines beans 2004005
Seed selection unit: Food and Oil Crops Inst., Hebei Agriculture and Forestry Academy
Cultivar origin: No. 9 × Century of Ji beans
3, five-pointed star No. 3
Seed selection unit: Food and Oil Crops Inst., Hebei Agriculture and Forestry Academy
Cultivar origin: No. 9 × Century of Ji beans
4, five-pointed star No. 4
Authorization (registration) numbering: beans are examined No. 2009005 in Ji
Seed selection unit: Food and Oil Crops Inst., Hebei Agriculture and Forestry Academy
Cultivar origin: Ji beans 12 × Suzuyutaka
5, mirror 31
Being provided by Hebei Prov. Academy of Agricultural &. Forest Sciences, is commercial variety
6, middle yellow 13
Variety certification numbering: state examines beans 2001008
Cultivar origin: beans No. 8/middle 90052-76 in Henan is commercial variety
Seed selection unit: Crop Breeding Cultivation Inst., Chinese Agriculture Academy
Case study on implementation 1:
1, DNA extraction: (1) 0.1g soyflour adds 0.7mLSDS extracting solution (0.1M Tris-HCl(PH=8.5), 0.1M NaCl, 0.05M EDTA (PH=8.0), 2% SDS) cracking 45min under 65 DEG C of constant temperature, therebetween repeatedly concussion.
(2) under 4 DEG C of 12000rpm conditions, centrifugal 10min.
(3) get supernatant liquor and add isopyknic phenol: chloroform: primary isoamyl alcohol (25:24:1) turns upside down and rotates to the not bery fast layering of two-phase.
(4) under 4 DEG C of 12000rpm conditions, centrifugal 10min.
(5) get supernatant liquor and add isopyknic phenol: chloroform: primary isoamyl alcohol (25:24:1) turns upside down rotation for several times.
(6) under 4 DEG C of 12000rpm conditions, centrifugal 10min.
(7) getting supernatant liquor adds isopyknic Virahol to leave standstill 30min in-20 DEG C of refrigerators.
(8) under 4 DEG C of 12000rpm conditions, centrifugal 10min.
(9) by twice of 70% washing with alcohol.
(10) under 4 DEG C of 12000rpm conditions, centrifugal 10min.
(11) precipitation natural air drying.
2, primer Lox1F:ATCTTAGCGTGCTTCACCCA LOX1R:CATCAGCGGCATAAGGATAG.
Primer all has Primer Premier 5 software designs.
3, reaction system: 2uL 2.5mM dNTP, 0.5uL 10uM upstream and downstream primer, 2uL 10*Ex Tap Buffer, 1U TaKaRa Tap, 150ng DNA profiling, uses bi-distilled water polishing to 20uL.
4, PCR response procedures: sex change: 94 DEG C of 5min, 94 DEG C of 45s, annealing: 0.4 DEG C of 20 circulation 50s of 65 DEG C of each cycle down, extend: 72 DEG C of 45s, sex change: 94 DEG C of 45s, annealing: 61.5 DEG C of 50s14 circulations, extend: 72 DEG C of 45s, extend: 72 DEG C of 8min.
5, agarose gel electrophoresis: 1.5% sepharose, buffer system is 1 × TAE solution, under 100V constant voltage, electrophoresis 50min, ethidium bromide (EB) dyeing, gel imaging system scans and preserves.Have 474bp band if state in pcr amplification product, above-mentioned soybean gene to be measured contains Lox3, if without 474bp band, and above-mentioned soybean gene disappearance Lox3 to be measured.
Fig. 1 is the schematic diagram of case study on implementation 1 agarose gel electrophoresis, with reference to Fig. 1, wherein, M:marker 1: No. 1 (disappearance Lox2,3) 2 of five-pointed star: No. 2 (disappearance Lox2) 3 of five-pointed star: No. 3 (disappearance Lox2) 4 of five-pointed star: No. 4 (disappearance Lox1,2,3) 5 of five-pointed star: mirror 31(disappearance Lox1,2,3) 6: middle yellow 13
Fig. 1 has clearly illustrated in five-pointed star No. 1, five-pointed star No. 4, the mirror 31 of these seven kinds of different varietiess as Lox1 deletant.
Specific experiment result is with reference to the schematic diagram of the agarose gel electrophoresis shown in Fig. 1.
The present invention, the Auele Specific Primer providing and authenticate technology thereof, not only save time and expense, and qualification result accurately and reliably, can significantly improve the screening efficiency to Lox1 deletant material.
Above-described embodiment is only explanation technical conceive of the present invention and feature, and its object is to allow the personage who is familiar with this art can understand content of the present invention and be implemented, and can not limit the scope of the invention with this.All equivalences that spirit is done according to the present invention change or modify, and all should be encompassed in protection scope of the present invention.
Claims (3)
1. an Auele Specific Primer of identifying soybean Lox3 deletant, is characterized in that, comprises following primer pair:
Lox3F：TAACCAGCGTTGGGGGAA?；
Lox3R：CATTTATAGACGTGCGTGCA。
2. right to use requires a method for the Auele Specific Primer qualification soybean Lox3 deletant described in 1, it is characterized in that, comprises the following steps:
(1) taking the soybean gene of deletant to be measured as DNA profiling, DNA profiling described in described primer pair is carried out to pcr amplification;
(2) have 474bp band if state in pcr amplification product, described soybean gene to be measured contains Lox3, if without 474bp band, and described soybean gene disappearance Lox3 to be measured.
3. the method for qualification soybean Lox1 deletant according to claim 2, is characterized in that, the soybean of described Lox1 deletant is any one in five-pointed star No. 1, five-pointed star No. 4, mirror 31.
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| CN201310055876.1A CN103146819B (en) | 2013-02-21 | 2013-02-21 | Specific primer for identification of soybean Lox3 and identification method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310055876.1A CN103146819B (en) | 2013-02-21 | 2013-02-21 | Specific primer for identification of soybean Lox3 and identification method |
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| CN103146819A CN103146819A (en) | 2013-06-12 |
| CN103146819B true CN103146819B (en) | 2014-08-20 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113848202B (en) * | 2021-09-03 | 2024-03-01 | 东北农业大学 | Soybean lipoxygenase and screening and identifying method of 7S and 11S globulin subunit deletion hybrid offspring |
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Non-Patent Citations (2)
| Title |
|---|
| LIU Nan-nan et al.Role of LOX3 Gene in Alleviating Adverse Effects of Drought and Pathogens in Rice.《Rice Science》.2008,第15卷(第4期),第276－282页. |
| Role of LOX3 Gene in Alleviating Adverse Effects of Drought and Pathogens in Rice;LIU Nan-nan et al;《Rice Science》;20081231;第15卷(第4期);第276－282页 * |
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