CN103160579B - Multiple polymerase chain reaction (PCR) primer used for identifying soybean deletants of Lox1 and Lox3 and method used for identifying soybean deletants of Lox1 and Lox3 - Google Patents
Multiple polymerase chain reaction (PCR) primer used for identifying soybean deletants of Lox1 and Lox3 and method used for identifying soybean deletants of Lox1 and Lox3 Download PDFInfo
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Abstract
The invention discloses a multiple polymerase chain reaction (PCR) primer used for identifying soybean deletants of Lox1 and Lox3. The multiple PCR primer used for identifying the soybean deletants of the Lox1 and the Lox3 comprises two groups of primer pairs which are Lox1F: ATCTTAGCGTGCTTCACCCA; Lox1R: CATCAGCGGCATAAGGATAG; Lox3F: TAACCAGCGTTGGGGAA; and Lox3R: CATTTATAGACGTACGTGCA. The invention further discloses an identifying method which includes that soybean genes of a deletant to be tested are taken as a deoxyribonucleic acid (DNA) template, and the two groups of primer pairs are enabled to be used for performing PCR amplification on the DNA template; if a PCR amplification product contains a 424bp band, the soybean genes to be tested contain the Lox1, and if the PCR amplification product contains a 350bp band, the soybean genes to be tested lack the Lox1; and if the PCR amplification product contains a 474bp band, the soybean genes to be tested contain the Lox3, and if the PCR amplification product does not contain the 474bp band, the soybean genes to be tested lack the Lox3. The multiple PCR primer used for identifying the soybean deletants of the Lox1 and the Lox3 and the identifying method have the advantages of simultaneously improving the screening efficiency for Lox1 deletant material and the screening efficiency for Lox3 deletant material.
Description
Technical field
The present invention relates to a kind of multiple PCR primer and method thereof of identifying soybean Lox1 and 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.Yarmilla etc. (2011), only for a RIL Team-development molecule marker, identify respectively soybean Lox 1, Lox2 and Lox3.Current existing method all can only identify separately three kinds of Lox deletants, and detection efficiency is lower.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of multiple PCR primer and method thereof of identifying soybean Lox1 and Lox3 deletant.
The present invention is achieved through the following technical solutions.
Identify a multiple PCR primer for soybean Lox1 and Lox3 deletant, comprise following two groups of primer pairs:
Lox1F：ATCTTAGCGTGCTTCACCCA；
Lox?1R：CATCAGCGGCATAAGGATAG；
Lox3F：TAACCAGCGTTGGGGGAA?；
Lox3R：CATTTATAGACGTGCGTGCA。
A method of identifying soybean Lox1 and 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 424bp band if state in pcr amplification product, above-mentioned soybean gene to be measured contains Lox1, if there is 350bp band, and above-mentioned soybean gene disappearance Lox1 to be measured; 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 Lox1 deletant is any one in five-pointed star No. 4, mirror 31.
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 Lox1 and Lox3 deletant material simultaneously.
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;
Lox?1R：CATCAGCGGCATAAGGATAG；
Lox3F：TAACCAGCGTTGGGGGAA?；
Lox3R：CATTTATAGACGTGCGTGCA。
Primer all has Primer Premier 5 software designs.
3, reaction system: 2uL 2.5mM dNTP, 0.5uL 10uM primer, 2uL 10*Ex Tap Buffer, 1.4U TaKaRa Tap, 150ng DNA profiling, uses bi-distilled water polishing to 20uL.
4, PCR response procedures: 94 DEG C of sex change 5min; 94 DEG C of sex change 45s; 60 DEG C of annealing 50s; 72 DEG C are extended 50s, 30 circulations.
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.If there is 474bp band in amplified production, illustrate that soybean to be measured contains Lox3, if without this band, disappearance Lox3 is described; If there is 424bp band in amplified production, illustrate that soybean to be measured contains Lox1, if there is 350bp band, disappearance Lox1 is described.
Specific experiment result is with reference to the schematic diagram of the agarose gel electrophoresis shown in Fig. 1.
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 (2)
1. a multiple PCR primer of identifying soybean Lox1 and Lox3 deletant, is characterized in that, comprises following two groups of primer pairs:
Lox1F：ATCTTAGCGTGCTTCACCCA；
Lox1R：CATCAGCGGCATAAGGATAG；
Lox3F：TAACCAGCGTTGGGGGAA；
Lox3R：CATTTATAGACGTGCGTGCA。
2. right to use requires the qualification soybean Lox1 of multiple PCR primer and the method for Lox3 deletant described in 1, it is characterized in that: comprise the following steps:
(1) taking the soybean gene of deletant to be measured as DNA profiling, DNA profiling described in described two groups of primer pairs is carried out to pcr amplification;
(2) have 424bp band if state in pcr amplification product, described soybean gene to be measured contains Lox1, if there is 350bp band, and described soybean gene disappearance Lox1 to be measured; 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.
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| CN201310055956.7A CN103160579B (en) | 2013-02-21 | 2013-02-21 | Multiple polymerase chain reaction (PCR) primer used for identifying soybean deletants of Lox1 and Lox3 and method used for identifying soybean deletants of Lox1 and Lox3 |
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| CN201310055956.7A CN103160579B (en) | 2013-02-21 | 2013-02-21 | Multiple polymerase chain reaction (PCR) primer used for identifying soybean deletants of Lox1 and Lox3 and method used for identifying soybean deletants of Lox1 and Lox3 |
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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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1148455C (en) * | 2000-09-13 | 2004-05-05 | 安徽省农业科学院水稻研究所 | Method for detecting crop poxidase iso enzyme LOX-1, LOX-2 and LOX-3 |
| CN102269731A (en) * | 2011-06-14 | 2011-12-07 | 黑龙江省大豆技术开发研究中心 | Method for rapidly detecting deletion of soybean lipoxygenase |
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2013
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1148455C (en) * | 2000-09-13 | 2004-05-05 | 安徽省农业科学院水稻研究所 | Method for detecting crop poxidase iso enzyme LOX-1, LOX-2 and LOX-3 |
| CN102269731A (en) * | 2011-06-14 | 2011-12-07 | 黑龙江省大豆技术开发研究中心 | Method for rapidly detecting deletion of soybean lipoxygenase |
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),参见第277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277页 * |
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