CN103525931A - Peanut high-oleic-acid character gene selection method - Google Patents
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Abstract
The invention discloses a peanut high-oleic-acid character gene selection method. The method comprises the steps of extracting leaf gene groups of different peanut breeds, and amplifying AhFAD2A and AhFAD2B genes from the gene groups by using a specificity primer; cutting and recycling specificity amplification stripes and sequencing to obtain the sequence of the gene; translating the obtained sequence, comparing and analyzing, and searching the point mutation or inserting site to judge the oleic acid content and specific value of oleic acid/linoleic acid (O/L) of the breed. The method is simple to operate, variety of peanuts germ plasm can be analyzed, the oleic acid content of single plant of peanut can be determined in the seedling stage, the breeding process can be shortened and the breeding effect of oleic acid can be improved by combining comprehensive agronomic character selection of the single plant.
Description
Technical field
The present invention relates to the system of selection of peanut high-oleic acid character gene, belong to peanut biological technical field.
Background technology
China is maximum in the world peanut production, consumption and export State.Peanut has consequence and effect in Chinese national economy.Oleic acid is the main fatty acid in peanut grease, account between the 39%-49% of Fatty Acid In Peanut total amount, in human body lipid metabolism, can reduce bad cholesterol, but keep the level of useful cholesterol, thereby slow down atherosclerosis, effectively prevent the generation of the cardiovascular and cerebrovascular diseases such as coronary heart disease.In addition, oleic acid content is high, and resistance of oxidation is strong, and product is difficult for oxidative rancidity, and shelf-life is longer, and resistance to energy storage power is also strong.Therefore, the breeder of the main oil crops in the world (soybean, rape, peanut) is using cultivating high oleic acid new variety as main breeding objective.
In peanut breeding process, the interior qualities such as oleic acid only have by seed test chemical could determine its concrete content, therefore at segregating generation, is difficult to the quality traits such as oleic acid to be selected.Near infrared seed not damaged selects the application of technology to solve the problem that segregating generation is selected interior quality proterties such as oleic acid, but this technology only can predict gathering in the crops the oleic acid content of rear seed, the material of the super-high oil acid content that some field Comprehensive Traits is poor is easily lost.Invent the genetic marker relevant to oleic acid content, in seedling stage, determine the oleic acid content of individual plant, in conjunction with comprehensive agronomy proterties, select simultaneously, can greatly improve the effect of high oleic acid breeding.
Genetic research shows, peanut high-oleic acid proterties by two pairs of recessive genes (
ol1with
ol2) co-controlling.Research show Δ 12 fatty acid dehydrogenase genes (
fAD2) be the key gene of controlling arachic acid proterties.
fAD2by two non-allelic genes that are positioned in different genes group
fAD2Awith
fAD2Bcommon coding,
ol1suddenlyd change
fAD2Agene,
ol2suddenlyd change
fAD2Bgene.The sudden change of these two genes causes the variation of enzymatic structure, enzymic activity or expression regulation, jointly causes the generation of high oleic acid proterties.
In order to understand the hereditary basis of controlling peanut high-oleic acid proterties in depth, improve the Breeding Efficiency of peanut breeding, breeder has developed CAPS, Real-time PCR, AS-PCR(Allele-Specific PCR for the peanut varieties of some high O/L ratios) etc. marking method, but these methods only limit to the mutant of particular type, cannot analyze rich and varied peanut germplasm.This research is by different peanut varieties (being)
ahFAD2gene checks order and compare of analysis, searches point mutation or insertion point, finds the gene locus associated with high oleic acid proterties, for peanut high-oleic acid breeding provides important theoretical foundation.
Summary of the invention
The object of this invention is to provide conveniently peanut high-oleic acid character gene system of selection, in seedling stage, determine the oleic acid content of individual plant, in conjunction with comprehensive agronomy proterties, select simultaneously, improve the effect of high oleic acid breeding.
For achieving the above object, the present invention adopts following steps:
Extract the blade genome of different peanut varieties (being), adopt Auele Specific Primer to increase from genome
ahFAD2Awith
ahFAD2Bgene.Specific amplified band is cut after glue recovery, carried out sequencing, obtain the sequence of gene.Obtained sequence is translated, and compare of analysis, searches point mutation or insertion point, judges accordingly the height of kind (being) oleic acid content and oleic acid/linolic acid (O/L) ratio.
Specifically, described method is as follows:
(1) adopt thick formulation to extract the blade genome of different peanut varieties (being);
(2) adopt Auele Specific Primer to increase from genome
ahFAD2Awith
ahFAD2Bgene.Specific amplified band is cut after glue reclaims and connected T carrier, and after transformed competence colibacillus cell, screening positive clone is sent to order-checking company and carries out sequencing, has obtained the sequence of gene;
(3) obtained sequence is translated, compare of analysis, searches point mutation or insertion point, judges accordingly the height of kind (being) oleic acid content and oleic acid/linolic acid (O/L) ratio.
Preferably, the described Auele Specific Primer of step (2) is FAD2A-F/FAD2A-R and FAD2B-F/FAD2B-R, respectively amplification
ahFAD2Awith
ahFAD2Bgene.FAD2A-F is 5'-GATTACTGATTATTGACTT-3', and FAD2A-R is 5'-CTCTGACTATGCATCAG-3'; FAD2B-F is 5'-CAGAACCATTAGCTTTG-3', and FAD2B-R is 5'-CTCTGACTATGCATCAG-3'.
Preferably, the described gene amplification of step (2) is by PCR method, and the polysaccharase used that increases is LA Taq
tMdNA polymerase (TaKaRa) adds following composition in 25 μ L systems: 2.5 μ L 10 * PCR buffer are (containing MgCl
2); 2.5 μ L 10mM dNTPs; 1 μ L cDNA template; 0.5 μ L LA polymerase and 17.5 μ L ddH
2o.PCR reaction conditions is: (a) 94 ℃, and 5min; (b) 94 ℃, 30s; 58 ℃, 1min40s; 72 ℃, 90s; Be total to 30cycles; (c) 72 ℃, 10min.PCR product reclaims test kit (Axygen) with glue and carries out purifying after 1% agarose gel electrophoresis separation, purified product connects pMD18-T Easy vector (Takara), transform after TOP10 competent cell, screening positive clone is sent to order-checking company (Sangon, Shanghai) order-checking.
Preferably, in the common peanut kind that in step (3), order-checking obtains
ahFAD2Amrna length is 1228bp,
ahFAD2Bgene is 1220bp, and open reading frame is 1140bp, 379 amino acid of encoding.By what obtain in comparison different varieties (being)
ahFAD2Awith
ahFAD2Bthe nucleotide sequence of gene and aminoacid sequence, search point mutation or insertion point, judges accordingly the height of kind (being) oleic acid content and oleic acid/linolic acid (O/L) ratio.
We have cloned 3152 portions of peanuts of different oleic acid contents
ahFAD2Awith
ahFAD2Bgene, shows in conjunction with oleic acid content and oleic acid/linolic acid (O/L) ratio Analysis
ahFAD2Agene exist G448A point mutation and
ahFAD2Bit is superelevation oleic acid (more than 70%) material or kind that gene exists 442A to select the peanut inserting, and O/L value surpasses 7.0.Only having point mutation or selecting the peanut inserting is high oleic acid (more than 49%) material or kind, and O/L value is between 1.5-7.0.Therefore,
ahFAD2Awith
ahFAD2Bit is the marker gene of high oleic acid proterties.
Embodiment
embodiment 1
To 3 peanut varieties (being) flowers educate 30, Shandong spends 12 and E11, gets 1 of peanut Newborn Leaves (about l0mg), is placed in 1.5mL EP pipe, adds liquid nitrogen, with the little grinding rod of plastics by the rapid grind into powder of blade; Add 400 μ L extracting solutions (the Tris-HCl solution (PH8.0) that comprises concentration 200mmol/L, the sodium chloride solution of concentration 250mmol/L, the EDTA solution of concentration 25mmol/L, the SDS solution of concentration 0.5% is used after high pressure steam sterilization.Before use, adding concentration is 1% beta-mercaptoethanol solution.), concussion mixes, standing 10min; The liquor kalii acetici (pH5.0 or uncomfortable pH) that adds 200 μ L 5mol/L, concussion mixes, standing 15min; The centrifugal 2min of 12000r/min rotating speed, gets supernatant liquor 400 μ L and moves into another l.5mL in EP pipe, adds equal-volume Virahol; After mixing gently under room temperature standing 2 minutes, the centrifugal 5min of 12000r/min rotating speed, abandoned supernatant liquor, and precipitation is washed 1-2 time with 75% alcohol again, the centrifugal 5min of 12000r/min rotating speed; At room temperature uncap and place 15min, add 10 μ L distilled water, dissolution precipitation, obtains peanut leaf genome crude extract.The genome product extracting detects through 1% agarose gel electrophoresis, can see obvious genome band, shows that genome extracts successfully.Adopt primers F AD2A-F/FAD2-R and the FAD2B-F/FAD2-R of gene specific to increase respectively by PCR method
ahFAD2Awith
ahFAD2Bgene.FAD2A-F is 5'-GATTACTGATTATTGACTT-3', and FAD2B-F is 5'-CAGAACCATTAGCTTTG-3', and FAD2-R is 5'-CTCTGACTATGCATCAG-3'.The polysaccharase used that increases is LA Taq
tMdNA polymerase (TaKaRa) adds following composition in 25 μ L systems: 2.5 μ L 10 * PCR buffer(are containing MgCl
2); 2.5 μ L 10mM dNTPs; 1 μ L cDNA template; 0.5 μ L LA polymerase and 17.5 μ L ddH
2o.PCR reaction conditions is: (a) 94 ℃, and 5min; (b) 94 ℃, 30s; 58 ℃, 1min40s; 72 ℃, 90s; Be total to 30cycles; (c) 72 ℃, 10min.PCR product reclaims test kit (Axygen) with glue and carries out purifying after 1% agarose gel electrophoresis separation, purified product connects pMD18-T Easy vector (Takara), transform after TOP10 competent cell, screening positive clone is sent to order-checking company (Sangon, Shanghai) order-checking.The nucleotide sequence of sequencing result and aminoacid sequence are compared, find in these 3 kinds (being),
ahFAD2Awith
ahFAD2Bneitherly there is transgenation.
ahFAD2Amrna length is 1228bp,
ahFAD2Bmrna length is 1220bp, and open reading frame is 1140bp, 379 amino acid of encoding.This 3 kinds (being) flower educates 30, Shandong spends 12, the seed benevolence oleic acid content of E11 is respectively 45.5%, 44.1%, 43.5%, O/L ratio and is respectively 1.40,1.35,1.30.
embodiment 2
3 peanut varieties flowers are educated to 19, flower educate 23 and Shandong spend 14, get 1 of peanut Newborn Leaves (about l0mg), be placed in 1.5mL EP pipe, add liquid nitrogen, with plastics little grinding rods by the rapid grind into powder of blade; Add 400 μ L extracting solutions (the Tris-HCl solution (PH8.0) that comprises concentration 200mmol/L, the sodium chloride solution of concentration 250mmol/L, the EDTA solution of concentration 25mmol/L, the SDS solution of concentration 0.5% is used after high pressure steam sterilization.Before use, adding concentration is 1% beta-mercaptoethanol solution.), concussion mixes, standing 10min; The liquor kalii acetici (pH5.0 or uncomfortable pH) that adds 200 μ L 5mol/L, concussion mixes, standing 15min; The centrifugal 2min of 12000r/min rotating speed, gets supernatant liquor 400 μ L and moves into another l.5 in mL EP pipe, adds equal-volume Virahol; After mixing gently under room temperature standing 2 minutes, the centrifugal 5min of 12000r/min rotating speed, abandoned supernatant liquor, and precipitation is washed 1-2 time with 75% alcohol again, the centrifugal 5min of 12000r/min rotating speed; At room temperature uncap and place 15min, add 10 μ L distilled water, dissolution precipitation, obtains peanut leaf genome crude extract.The genome product extracting detects through 1% agarose gel electrophoresis, can see obvious genome band, shows that genome extracts successfully.Adopt primers F AD2A-F/FAD2-R and the FAD2B-F/FAD2-R of gene specific to increase respectively by PCR method
ahFAD2Awith
ahFAD2Bgene.FAD2A-F is 5'-GATTACTGATTATTGACTT-3', and FAD2B-F is 5'-CAGAACCATTAGCTTTG-3', and FAD2-R is 5'-CTCTGACTATGCATCAG-3'.The polysaccharase used that increases is LA Taq
tMdNA polymerase (TaKaRa) adds following composition in 25 μ L systems: 2.5 μ L 10 * PCR buffer(are containing MgCl
2); 2.5 μ L 10mM dNTPs; 1 μ L cDNA template; 0.5 μ L LA polymerase and 17.5 μ L ddH
2o.PCR reaction conditions is: (a) 94 ℃, and 5min; (b) 94 ℃, 30s; 58 ℃, 1min40s; 72 ℃, 90s; Be total to 30cycles; (c) 72 ℃, 10min.PCR product reclaims test kit (Axygen) with glue and carries out purifying after 1% agarose gel electrophoresis separation, purified product connects pMD18-T Easy vector (Takara), transform after TOP10 competent cell, screening positive clone is sent to order-checking company (Sangon, Shanghai) order-checking.The nucleotide sequence of sequencing result and aminoacid sequence are compared, find in these 3 kinds,
ahFAD2Bthere is no producer sudden change, mrna length is 1220bp, and open reading frame is 1140bp, 379 amino acid of encoding.And
ahFAD2Asudden change becomes its allelotrope
ol1, the bases G at the 448bp place of gene opening code-reading frame sports A(G448A), cause corresponding amino acid to sport l-asparagine (N by aspartic acid (D), D150N), mrna length is still 1228bp, and open reading frame is 1140bp, 379 amino acid of encoding.These 3 kind flowers educate 19, flower educate 23 and Shandong spend 14 seed benevolence oleic acid content to be respectively 54.02%, 49.3%, 51.0%, O/L ratio to be respectively 1.97,1.54,1.57.
embodiment 3
3 peanut varieties (being) E18, flower are educated No. 32 and E16, get 1 of peanut Newborn Leaves (about l0mg), be placed in 1.5mL EP pipe, add liquid nitrogen, use the little grinding rod of plastics by the rapid grind into powder of blade; Add 400 μ L extracting solutions (the Tris-HCl solution (PH8.0) that comprises concentration 200mmol/L, the sodium chloride solution of concentration 250mmol/L, the EDTA solution of concentration 25mmol/L, the SDS solution of concentration 0.5% is used after high pressure steam sterilization.Before use, adding concentration is 1% beta-mercaptoethanol solution.), concussion mixes, standing 10min; The liquor kalii acetici (pH5.0 or uncomfortable pH) that adds 200 μ L 5mol/L, concussion mixes, standing 15min; The centrifugal 2min of 12000r/min rotating speed, gets supernatant liquor 400 μ L and moves into another l.5 in mL EP pipe, adds equal-volume Virahol; After mixing gently under room temperature standing 2 minutes, the centrifugal 5min of 12000r/min rotating speed, abandoned supernatant liquor, and precipitation is washed 1-2 time with 75% alcohol again, the centrifugal 5min of 12000r/min rotating speed; At room temperature uncap and place 15min, add 10 μ L distilled water, dissolution precipitation, obtains peanut leaf genome crude extract.The genome product extracting detects through 1% agarose gel electrophoresis, can see obvious genome band, shows that genome extracts successfully.Adopt primers F AD2A-F/FAD2-R and the FAD2B-F/FAD2-R of gene specific to increase respectively by PCR method
ahFAD2Awith
ahFAD2Bgene.FAD2A-F is 5'-GATTACTGATTATTGACTT-3', and FAD2B-F is 5'-CAGAACCATTAGCTTTG-3', and FAD2-R is 5'-CTCTGACTATGCATCAG-3'.The polysaccharase used that increases is LA Taq
tMdNA polymerase (TaKaRa) adds following composition in 25 μ L systems: 2.5 μ L 10 * PCR buffer(are containing MgCl
2); 2.5 μ L 10mM dNTPs; 1 μ L cDNA template; 0.5 μ L LA polymerase and 17.5 μ L ddH
2o.PCR reaction conditions is: (a) 94 ℃, and 5min; (b) 94 ℃, 30s; 58 ℃, 1min40s; 72 ℃, 90s; Be total to 30cycles; (c) 72 ℃, 10min.PCR product reclaims test kit (Axygen) with glue and carries out purifying after 1% agarose gel electrophoresis separation, purified product connects pMD18-T Easy vector (Takara), transform after TOP10 competent cell, screening positive clone is sent to order-checking company (Sangon, Shanghai) order-checking.The nucleotide sequence of sequencing result and aminoacid sequence are compared, find in these 3 kinds,
ahFAD2Awith
ahFAD2Ball producer sudden changes.
ahFAD2Athe bases G at the 448bp place of gene opening code-reading frame sports A(G448A), cause corresponding amino acid to sport l-asparagine (N, D150N) by aspartic acid (D), mrna length is still 1228bp, open reading frame is 1140bp, 379 amino acid of encoding.
ahFAD2Bthere is the insertion of base A at gene order 442bp place after initiator codon, and mrna length increases 1bp, causes coding region premature termination, and the albumen of the brachymemma of encoding (164 amino acid), has lost a Histidine frame that embrane-associated protein is conservative.These 3 kinds (being) E18, flower are educated No. 32 and the seed benevolence oleic acid content of E16 is respectively 83.7%, 77.8%, 73.21%, O/L ratio and is respectively 41.85,12.3,7.0.
Claims (4)
1. a peanut high-oleic acid character gene system of selection, comprises the following steps:
(1) adopt thick formulation to extract the blade genome of different peanut varieties (being);
(2) adopt Auele Specific Primer to increase from genome
ahFAD2Awith
ahFAD2Bgene;
Specific amplified band is cut after glue reclaims and connected T carrier, and after transformed competence colibacillus cell, screening positive clone is sent to order-checking company and carries out sequencing, has obtained the sequence of gene;
(3) obtained sequence is translated, compare of analysis, searches point mutation or insertion point, judges accordingly the height of kind (being) oleic acid content and oleic acid/linolic acid (O/L) ratio;
fAD2Agene exist G448A point mutation and
fAD2Bit is superelevation oleic acid (more than 70%) material or kind that gene exists 442A to select the peanut inserting, and O/L value surpasses 7.0; Only having point mutation or selecting the peanut inserting is high oleic acid (more than 49%) material or kind, and O/L value is between 1.5-7.0, and not having point mutation or selecting the peanut inserting is common peanut, and oleic acid content is below 49%, and O/L value is less than 1.5.
2. a kind of peanut high-oleic acid character gene according to claim 1 system of selection, is characterized in that, the described Auele Specific Primer of step (2) is FAD2A-F/FAD2A-R and FAD2B-F/FAD2B-R, respectively amplification
ahFAD2Awith
ahFAD2Bgene;
FAD2A-F is 5'-GATTACTGATTATTGACTT-3', and FAD2A-R is 5'-CTCTGACTATGCATCAG-3'; FAD2B-F is 5'-CAGAACCATTAGCTTTG-3', and FAD2B-R is 5'-CTCTGACTATGCATCAG-3'.
3. a kind of peanut high-oleic acid character gene according to claim 1 system of selection, is characterized in that, the described gene amplification of step (2) is by PCR method, and the polysaccharase used that increases is LA Taq
tMdNA polymerase (TaKaRa) adds following composition in 25 μ L systems: 2.5 μ L 10 * PCR buffer are (containing MgCl
2); 2.5 μ L 10mM dNTPs; 1 μ L cDNA template; 0.5 μ L LA polymerase and 17.5 μ L ddH
2o;
PCR reaction conditions is: (a) 94 ℃, and 5min; (b) 94 ℃, 30s; 58 ℃, 1min40s; 72 ℃, 90s; Be total to 30cycles; (c) 72 ℃, 10min;
PCR product reclaims test kit (Axygen) with glue and carries out purifying after 1% agarose gel electrophoresis separation, purified product connects pMD18-T Easy vector (Takara), transform after TOP10 competent cell, screening positive clone is sent to order-checking company (Sangon, Shanghai) order-checking.
4. a kind of peanut high-oleic acid character gene according to claim 1 system of selection, is characterized in that, in the common peanut kind that in step (3), order-checking obtains
ahFAD2Amrna length is 1228bp,
ahFAD2Bgene is 1220bp, and open reading frame is 1140bp, and 379 amino acid of encoding, by what obtain in comparison different varieties (being)
ahFAD2Awith
ahFAD2Bthe nucleotide sequence of gene and aminoacid sequence, search point mutation or insertion point, judges accordingly the height of kind (being) oleic acid content and oleic acid/linolic acid (O/L) ratio.
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| CN105505930A (en) * | 2015-09-11 | 2016-04-20 | 河南省农业科学院 | Peanut delta 12 fatty acid dehydrogenase AhFAD2-1B-m gene promoter and its preparation method and use |
| CN105505930B (en) * | 2015-09-11 | 2018-05-25 | 河南省农业科学院 | Peanut Δ12Promoter of fatty acid dehydrogenase AhFAD2-1B-m genes and its preparation method and application |
| CN105385778A (en) * | 2015-12-26 | 2016-03-09 | 山东省农业科学院生物技术研究中心 | Primers and method for detecting SNP gene typing of AhFAD2B genes of peanuts in high throughput |
| CN105519432A (en) * | 2016-01-15 | 2016-04-27 | 福建农林大学 | Method for breeding high-oleic acid recessive mutant Ahfad2a-1 strains of peanuts |
| CN108753803A (en) * | 2018-07-16 | 2018-11-06 | 河北省农林科学院粮油作物研究所 | A kind of high oleic acid peanut mutator AhFAD2B-814 and application |
| CN108893551A (en) * | 2018-07-16 | 2018-11-27 | 河北省农林科学院粮油作物研究所 | It is a kind of detect peanut high-oil acid content molecule labelling method and application |
| CN108753803B (en) * | 2018-07-16 | 2019-04-23 | 河北省农林科学院粮油作物研究所 | A kind of high oleic acid peanut mutated gene AhFAD2B-814 and application |
| CN108893551B (en) * | 2018-07-16 | 2021-09-14 | 河北省农林科学院粮油作物研究所 | Molecular marking method for detecting high oleic acid content of peanuts and application |
| CN109295253A (en) * | 2018-11-16 | 2019-02-01 | 山东省花生研究所 | High oleic acid peanut is quickly chosen seeds kit |
| CN111944828A (en) * | 2020-07-14 | 2020-11-17 | 深圳大学 | Preparation method of peanut mutant, peanut mutant gene, protein coded by peanut mutant gene and application of peanut mutant gene |
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