CN101235380A - Peanut delta12-fatty acid dehydrogenase mutant gene and its coding protein and clone method - Google Patents
Peanut delta12-fatty acid dehydrogenase mutant gene and its coding protein and clone method Download PDFInfo
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Abstract
The invention firstly discloses nucleotide sequence of mutant gene delta12-fatty acid dehydrogenase gene (AhFAD2') which leads oleic acid content of peanut variety E11 and E16 to increase and shortened protein sequence which is coded. The nucleotide sequence also provides a clone method of the peanut fatty acid dehydrogenase mutant gene. The expression analysis of mRNA of gene AhFAD2' of the invention shows that the gene can express in function.
Description
Technical field
The invention belongs to fatty acid metabolism engineering field, be specifically related to cultivate peanut fatty acid dehydrogenase mutant gene and an encoded protein matter and a cloning process.
Background technology
Peanut is important oil crops, is rich in quality protein and fat, is the Dietotherapy health good merchantable brand that people's body-building increases the longevity.Oleaginousness is 45-51% in the Semen arachidis hypogaeae, and wherein about 80% is made up of oleic acid and linolic acid.Oleic acid is being brought into play special effect in the lipid metabolism of human body, it can reduce bad cholesterol (low-density lipoprotein, LDL), but keep useful cholesterol (high-density lipoprotein (HDL), HDL) level, thus atherosclerosis slowed down, effectively prevent the generation of cardiovascular disordeies such as coronary heart disease.And linolic acid is a polyunsaturated fatty acid, also can reduce useful content of cholesterol though can reduce bad cholesterol, thereby unfavorable to health.And the height of oleic acid and linoleic acid ratio has determined the quality trait of peanut.Oleic acid and linoleic ratio height, resistance of oxidation are just strong, are difficult for oxidative rancidity under the situation of summer high temperature, and the shelf cycle is longer, and anti-energy storage power is also stronger.Therefore, the peanut varieties that oleic acid content is high has better commodity.Improve oleic relative content, increase the ratio of oleic acid/linolic acid (O/L), become the important content of present oil crops quality-improving breeding.The used peanut varieties E11 oleic acid content of the present invention is 76.6%, linoleic acid content 5.2%; The E16 oleic acid content is 78.1%, linoleic acid content 3.7%.The oleic acid content of E11 and E16 is compared with common strain peanut, and oleic acid content is doubled, and belongs to high oleic acid kind.
Δ
12-fatty acid dehydrogenase is that the oleic acid dehydrogenation forms linoleic key enzyme, at oleic Δ
12Add on the position that two keys form linolic acid.1993, Ray et al discovered Δ in the common peanut strain
12The activity of-fatty acid dehydrogenase is ten times in the high oleic acid peanut strain.2000, Y.Lopez studies show that, at Δ
12Two single nucleotide polymorphism are arranged in the coding region sequence of-fatty acid dehydrogenase gene, may be the quantitative trait locus of the high O/L value of control.The present invention has identified Δ from high oleic acid peanut varieties (E11 and E16)
12Δ in the-fatty acid dehydrogenase gene, itself and common peanut strain
12-fatty acid dehydrogenase gene sequence is compared, and base mutation has taken place, and causes the coding region premature termination, thereby peanut fatty acid dehydrogenase activity is reduced greatly even loses.
Summary of the invention
The objective of the invention is to disclose the Δ of cultivating peanut
12The mutant nucleotide sequence of-fatty acid dehydrogenase gene and encoded protein matter thereof, the sudden change of this gene makes the fatty acid dehydrogenase loss of activity, makes oleic acid change linoleic approach into and is obstructed, oleic acid content increases in the peanut thereby make.Simultaneously, the present invention also will provide the cloning process of this peanut fatty acid dehydrogenase mutant gene.
Peanut Δ provided by the present invention
12-fatty acid dehydrogenase mutant gene, it is from peanut (Arachis hypogaea), called after AhFAD2 ' gene, nucleotide sequence SEQ ID NO.1:
1 actcacttct?cttctctctg?ttggaattgc?tttcacggag?ctttaacaac?acaacaatgg
61 gagctggagg?gcgtgtcact?aagattgaag?ctcaaaagaa?gcctctttca?agggttccac
121 attcaaaccc?tccattcagt?gttggccaac?tcaagaaagc?aattccacca?cattgctttg
181 aacgttctct?tttcatatca?ttctcatatg?ttgtctatga?tctcttaatg?gcctacttac
241 tcttctacat?tgccaccact?tatttccaca?agcttccata?cccattttcc?ttccttgctt
301 ggccaatcta?ttgggccatc?caaggctgca?ttctcaccgg?tgtttgggtg?attgctcatg
361 agtgtggcca?ccatgccttc?agcaagtacc?aacttgttga?tgacatggtt?ggtttgaccc
421 ttcactcttg?tctattagtt?ccttatttct?catggaaaat?cagccaccgc?cgccaccact
481 ccaacacagg?ttccctcaga?ccgcgacgaa?gtgtttgtcc?cgaaaccaaa?atcaaaggta
541 tcatggtata?acaagtacat?gaacaatcca?ccagggaggg?ctatttccct?tttcatcaca
601 ctcacactag?gatggccctt?gtacttggcc?ttcaatgttt?ctggcagacc?ctatgataga
661 tttgcaagcc?actatgaccc?ttatgctccc?atatactcta?acagggaaag?gcttctaatt
721 tatgtctcag?attcatctgt?ctttgctgta?acatatctgc?tatatcacat?agcaactttg
781 aaaggtttgg?gttgggtggt?atgtgtttat?ggggtgccat?tgctcattgt?gaatgggttt
841 ctagttacca?taacctattt?gcagcacaca?catgcatcat?tgcctcacta?tgattcatcc
901 gaatgggact?ggttaagagg?agcattggca?acagtggaca?gagattatgg?gatactgaat
961 aaggcatttc?atcatataac?tgatacgcat?gtggctcatc?atttgttctc?aacaatgcct
1021 cattaccatg?caatggaagc?aaccaatgca?ataaagccaa?tattgggtga?ttactaccaa
1081 tttgatggca?ccccagttta?caaagcattg?tggagagaag?ccaaagagtg?cctctatgtg
1141 gagccagatg?atggagcttc?tcagaagggt?gtttattggt?acaagaacaa?gttctgatgc
1201 atagtcagag?ttggaaacgt?ttgtgttaaa?ttagaaactt?aagtacttta?gtaacttgta
1261 atggtcatca?acaaaaataa?agaatgtgtg?tgtggatttg?c
The above-mentioned one fatty acid dehydrogenase mutant gene encoded protein matter of cultivating peanut, it is from peanut (Arachis hypogaea), called after AhFAD2 ' protein, it has aminoacid sequence SEQ ID NO.2 as follows:
Met?Gly?Ala?Gly?Gly?Arg?Val?Thr?Lys?Ile?Glu?Ala?Gln?Lys?Lys?Pro
1 5 10 15
Leu?Ser?Arg?Val?Pro?His?Ser?Asn?Pro?Pro?Phe?Ser?Val?Gly?Gln?Leu
20 25 30
Lys?Lys?Ala?Ile?Pro?Pro?His?Cys?Phe?Glu?Arg?Ser?Leu?Phe?Ile?Ser
35 40 45
Phe?Ser?Tyr?Val?Val?Tyr?Asp?Leu?Leu?Met?Ala?Tyr?Leu?Leu?Phe?Tyr
50 55 60
Ile?Ala?Thr?Thr?Tyr?Phe?His?Lys?Leu?Pro?Tyr?Pro?Phe?Ser?Phe?Leu
65 70 75 80
Ala?Trp?Pro?Ile?Tyr?Trp?Ala?Ile?Gln?Gly?Cys?Ile?Leu?Thr?Gly?Val
85 90 95
Trp?Val?Ile?Ala?His?Glu?Cys?Gly?His?His?Ala?Phe?Ser?Lys?Tyr?Gln
100 105 110
Leu?Val?Asp?Asp?Met?Val?Gly?Leu?Thr?Leu?His?Ser?Cys?Leu?Leu?Val
115 120 125
Pro?Tyr?Phe?Ser?Trp?Lys?Ile?Ser?His?Arg?Arg?His?His?Ser?Asn?Thr
130 135 140
Gly?Ser?Leu?Arg?Pro?Arg?Arg?Ser?Val?Cys?Pro?Glu?Thr?Lys?Ile?Lys
145 150 155 160
Gly?Ile?Met?Val
165 170 175
The cloning process of above-mentioned peanut fatty acid dehydrogenase mutant gene AhFAD2 ' comprises the steps:
(1) select for use the peanut seed of E11 or E16 to place mortar, add the 0.5ml plant RNA under the room temperature and extract reagent (can use the RNAplant reagent of sky) as Time Inc., after fully grinding, be transferred in the 1.5ml EP pipe, vibration is to thorough mixing, extracted total RNA is identified total RNA quality with the denaturing formaldehyde gel electrophoresis, measures rna content then on spectrophotometer;
(2) according to the peanut FAD2 partial sequence (881bp) that has increased, the design primer:
GSP1：5-AGTGGTGGCGGCGGTGGCTGATTTTC-3
GSP2：5-GGCAACAGTGGACAGAGATTATGGG-3
Total RNA with acquisition is a template, through the synthetic 5 '-RACE-Ready cDNA of reverse transcription and 3 '-RACE-Ready cDNA, carries out pcr amplification with high-fidelity Taq enzyme,
The PCR program of 5 '-RACE is as follows: 94 ℃ of pre-sex change 2min, and 94 ℃ of sex change 30s, 72 ℃ are extended 2min, after 5 circulations, 94 ℃ of sex change 30s, 70 ℃ of renaturation 30s, 72 ℃ are extended 2min, after 5 circulations, 94 ℃ of sex change 30s, 68 ℃ of renaturation 30s, 72 ℃ are extended 2min, after 26 circulations, 72 ℃ of 15min;
The PCR program of 3 '-RACE is as follows: 94 ℃ of pre-sex change 2min, and 94 ℃ of sex change 30s, 68 ℃ of renaturation 30s, 72 ℃ are extended 2min, after 27 circulations, 72 ℃ of 15min;
The PCR product reclaims rear clone to the pMD18-T carrier; Entrust Shanghai Ying Jun company order-checking back to obtain 5 ' terminal sequence and the 3 ' terminal sequence of AhFAD2 '.
(3) according to the 5 ' terminal sequence and the 3 ' terminal sequence of the AhFAD2 ' gene that has obtained, design the two ends primer:
FADf5：5′-ACTCACTTCTCTTCTCTCTG-3′
FADf3：5′-GCAAATCCACACACACATTC-3′
The 5 '-RACE-Ready cDNA that obtains with step (2) is a template, carries out pcr amplification with high-fidelity Taq enzyme, and the PCR program is as follows: 94 ℃ of pre-sex change 4 minutes, and 94 ℃ of sex change 30s, 59 ℃ of renaturation 50s, 72 ℃ of extension 2min, after 32 circulations, 72 ℃ of 15min.The PCR product is cloned the most at last, checking order has obtained the peanut Δ
12The full length cDNA sequence of-fatty acid dehydrogenase mutant gene.
The invention discloses the Δ of cultivating peanut
12-fatty acid dehydrogenase mutant gene (AhFAD2 ' gene) and coded protein.This gene is from high oleic acid peanut (Arachis hypogaea), and the fatty acid dehydrogenase of this genes encoding brachymemma causes fatty acid dehydrogenase to lose activity, and be terminated thereby make oleic acid change linoleic approach into, so oleic acid content improves in the peanut.
Embodiment
The present invention is described in further detail below in conjunction with specific examples.
The seed (no more than 0.1 gram) of selecting peanut high-oleic acid kind E11 or E16 for use is in mortar, add the 0.5ml plant RNA under the room temperature and extract reagent (sky is the RNAplant reagent of Time Inc.), fully grind, be transferred in the 1.5mL EP pipe, vibration is to thorough mixing, extracted total RNA is identified total RNA quality with the denaturing formaldehyde gel electrophoresis, measures rna content then on spectrophotometer.Total RNA with acquisition is a template, through the synthetic 5 '-RACE-ReadycDNA and 3 ' of reverse transcription-RACE-Ready cDNA, utilizes the RACE technology to obtain the 5 ' terminal sequence and the 3 ' terminal sequence of fatty acid dehydrogenase mutant gene.5 ' terminal sequence and 3 ' terminal sequence design two ends primer according to fatty acid dehydrogenase mutant gene
FADf5：5′-ACTCACTTCTCTTCTCTCTG-3′；
FADf3：5′-GCAAATCCACACACACATTC-3′
Adopt the RT-PCR method to carry out the cDNA clone, the PCR response procedures is: 94 ℃ of pre-sex change 4 minutes, and 94 ℃ of sex change 30s, 59 ℃ of renaturation 50s, 72 ℃ are extended 2min, after 32 circulations, 72 ℃ of 15min.The PCR product is cloned the most at last, checking order has obtained the cDNA sequence of peanut fatty acid dehydrogenase mutant gene AhFAD2 '.
The fatty acid dehydrogenase gene AhFAD2 ' of sudden change compares with the FAD2B that has registered (number of registration AF272950) gene order, base mutation has taken place, there is the insertion of base A at the 442bp place after initiator codon, thereby make the coding region premature termination, the fatty acid dehydrogenase albumen of the brachymemma of having encoded.
AhFAD2 ' total length 1301bp of the present invention, its first open reading frame is positioned at the 57-551 place.The DNAssist software analysis shows AhFAD2 ' 164 amino acid of encoding altogether.The open reading frame of FAD2B (number of registration AF272950) gene contains 1140bp, 379 amino acid of encoding.AhFAD2 ' gene is compared with the FAD2B gene, and there is the insertion of base A at the 442bp place after initiator codon, thereby makes the coding region premature termination, the fatty acid dehydrogenase albumen of the brachymemma of having encoded.All known fatty acid dehydrogenases all have 3 Histidines bunch, may relate to the formation of enzymic activity position.Because AhFAD2 ' gene coding region premature termination, cause the 3rd Histidine frame disappearance, may make fatty acid dehydrogenase reduced activity even forfeiture, be obstructed thereby make in the peanut oleic acid dehydrogenation form linoleic approach, so oleic acid content improves in the peanut.
Carry out the expression that sxemiquantitative RT-PCR analyzes peanut root, stem, leaf and seed with AhFAD2 ' the two ends primer that designs in the example, with 18S rRNA gene (
Deng, 2003) expression as confidential reference items, the result shows that AhFAD2 ' gene all has expression in the peanut different tissues.
The foregoing description shows the peanut fatty acid dehydrogenase mutant gene AhFAD2 ' that clones among the present invention, is the major cause that causes that oleic acid content of peanuts improves.The inventor has cloned the fatty acid dehydrogenase gene AhFAD2 ' of a sudden change from dicotyledons peanut (Arachis hypogaea).The mRNA expression analysis shows that AhFAD2 ' has expression in the peanut root, stem, blade and the seed that detect.Δ
12-fatty acid dehydrogenase is that catalysis oleic acid forms linoleic key enzyme in the fatty acid metabolism.If peanut seed Δ
12-fatty acid dehydrogenase gene nucleotide sequence generation base is inserted or deletion mutantion, Δ
12-fatty acid dehydrogenase forms and will be obstructed, thereby reduces linoleic synthesizing, and reaches a large amount of oleic purposes of accumulation.
Sequence that the present invention relates to and mark apportion are as follows:
(1) information of SEQ ID NO.1
(i) sequence signature:
(A) length: 1301bp
(B) type: Nucleotide
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: Nucleotide
(iii) sequence description: SEQ ID NO.1
(2) information of SEQ ID NO.2
(i) sequence signature:
(A) length: 164a.a
(B) type: amino acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: protein
(iii) sequence description: SEQ ID NO.2
Nucleotide sequence SEQ ID NO.1:
1 actcacttct?cttctctctg?ttggaattgc?tttcacggag?ctttaacaac?acaacaatgg
61 gagctggagg?gcgtgtcact?aagattgaag?ctcaaaagaa?gcctctttca?agggttccac
121 attcaaaccc?tccattcagt?gttggccaac?tcaagaaagc?aattccacca?cattgctttg
181 aacgttctct?tttcatatca?ttctcatatg?ttgtctatga?tctcttaatg?gcctacttac
241 tcttctacat?tgccaccact?tatttccaca?agcttccata?cccattttcc?ttccttgctt
301 ggccaatcta?ttgggccatc?caaggctgca?ttctcaccgg?tgtttgggtg?attgctcatg
361 agtgtggcca?ccatgccttc?agcaagtacc?aacttgttga?tgacatggtt?ggtttgaccc
421 ttcactcttg?tctattagtt?ccttatttct?catggaaaat?cagccaccgc?cgccaccact
481 ccaacacagg?ttccctcaga?ccgcgacgaa?gtgtttgtcc?cgaaaccaaa?atcaaaggta
541 tcatggtata?acaagtacat?gaacaatcca?ccagggaggg?ctatttccct?tttcatcaca
601 ctcacactag?gatggccctt?gtacttggcc?ttcaatgttt?ctggcagacc?ctatgataga
661 tttgcaagcc?actatgaccc?ttatgctccc?atatactcta?acagggaaag?gcttctaatt
721 tatgtctcag?attcatctgt?ctttgctgta?acatatctgc?tatatcacat?agcaactttg
781 aaaggtttgg?gttgggtggt?atgtgtttat?ggggtgccat?tgctcattgt?gaatgggttt
841 ctagttacca?taacctattt?gcagcacaca?catgcatcat?tgcctcacta?tgattcatcc
901 gaatgggact?ggttaagagg?agcattggca?acagtggaca?gagattatgg?gatactgaat
961 aaggcatttc?atcatataac?tgatacgcat?gtggctcatc?atttgttctc?aacaatgcct
1021 cattaccatg?caatggaagc?aaccaatgca?ataaagccaa?tattgggtga?ttactaccaa
1081 tttgatggca?ccccagttta?caaagcattg?tggagagaag?ccaaagagtg?cctctatgtg
1141 gagccagatg?atggagcttc?tcagaagggt?gtttattggt?acaagaacaa?gttctgatgc
1201 atagtcagag?ttggaaacgt?ttgtgttaaa?ttagaaactt?aagtacttta?gtaacttgta
1261 atggtcatca?acaaaaataa?agaatgtgtg?tgtggatttg?c
Aminoacid sequence SEQ ID NO.2:
Met?Gly?Ala?Gly?Gly?Arg?Val?Thr?Lys?Ile?Glu?Ala?Gln?Lys?Lys?Pro
1 5 10 15
Leu?Ser?Arg?Val?Pro?His?Ser?Asn?Pro?Pro?Phe?Ser?Val?Gly?Gln?Leu
20 25 30
Lys?Lys?Ala?Ile?Pro?Pro?His?Cys?Phe?Glu?Arg?Ser?Leu?Phe?Ile?Ser
35 40 45
Phe?Ser?Tyr?Val?Val?Tyr?Asp?Leu?Leu?Met?Ala?Tyr?Leu?Leu?Phe?Tyr
50 55 60
Ile?Ala?Thr?Thr?Tyr?Phe?His?Lys?Leu?Pro?Tyr?Pro?Phe?Ser?Phe?Leu
65 70 75 80
Ala?Trp?Pro?Ile?Tyr?Trp?Ala?Ile?Gln?Gly?Cys?Ile?Leu?Thr?Gly?Val
85 90 95
Trp?Val?Ile?Ala?His?Glu?Cys?Gly?His?His?Ala?Phe?Ser?Lys?Tyr?Gln
100 105 110
Leu?Val?Asp?Asp?Met?Val?Gly?Leu?Thr?Leu?His?Ser?Cys?Leu?Leu?Val
115 120 125
Pro?Tyr?Phe?Ser?Trp?Lys?Ile?Ser?His?Arg?Arg?His?His?Ser?Asn?Thr
130 135 140
Gly?Ser?Leu?Arg?Pro?Arg?Arg?Ser?Val?Cys?Pro?Glu?Thr?Lys?Ile?Lys
145 150 155 160
Gly?Ile?Met?Val
165 170 175
Claims (3)
1. the Δ of cultivating peanut
12-fatty acid dehydrogenase mutant gene has following nucleotide sequence:
1 actcacttct?cttctctctg?ttggaattgc?tttcacggag?ctttaacaac?acaacaatgg
61 gagctggagg?gcgtgtcact?aagattgaag?ctcaaaagaa?gcctctttca?agggttccac
121 attcaaaccc?tccattcagt?gttggccaac?tcaagaaagc?aattccacca?cattgctttg
181 aacgttctct?tttcatatca?ttctcatatg?ttgtctatga?tctcttaatg?gcctacttac
241 tcttctacat?tgccaccact?tatttccaca?agcttccata?cccattttcc?ttccttgctt
301 ggccaatcta?ttgggccatc?caaggctgca?ttctcaccgg?tgtttgggtg?attgctcatg
361 agtgtggcca?ccatgccttc?agcaagtacc?aacttgttga?tgacatggtt?ggtttgaccc
421 ttcactcttg?tctattagtt?ccttatttct?catggaaaat?cagccaccgc?cgccaccact
481 ccaacacagg?ttccctcaga?ccgcgacgaa?gtgtttgtcc?cgaaaccaaa?atcaaaggta
541 tcatggtata?acaagtacat?gaacaatcca?ccagggaggg?ctatttccct?tttcatcaca
601 ctcacactag?gatggccctt?gtacttggcc?ttcaatgttt?ctggcagacc?ctatgataga
661 tttgcaagcc?actatgaccc?ttatgctccc?atatactcta?acagggaaag?gcttctaatt
721 tatgtctcag?attcatctgt?ctttgctgta?acatatctgc?tatatcacat?agcaactttg
781 aaaggtttgg?gttgggtggt?atgtgtttat?ggggtgccat?tgctcattgt?gaatgggttt
811 ctagttacca?taacctattt?gcagcacaca?catgcatcat?tgcctcacta?tgattcatcc
901 gaatgggact?ggttaagagg?agcattggca?acagtggaca?gagattatgg?gatactgaat
961 aaggcatttc?atcatataac?tgatacgcat?gtggctcatc?atttgttctc?aacaatgcct
1021 cattaccatg?caatggaagc?aaccaatgca?ataaagccaa?tattgggtga?ttactaccaa
1081 tttgatggca?ccccagttta?caaagcattg?tggagagaag?ccaaagagtg?cctctatgtg
1141 gagccagatg?atggagcttc?tcagaagggt?gtttattggt?acaagaacaa?gttctgatgc
1201 atagtcagag?ttggaaacgt?ttgtgttaaa?ttagaactt?aagtacttta?gtaacttgta
1261 atggtcatca?acaaaaataa?agaatgtgtg?tgtggatttg?c
2. the Δ of cultivating peanut
12-fatty acid dehydrogenase mutant gene encoded protein matter has following aminoacid sequence:
Met?Gly?Ala?Gly?Gly?Arg?Val?Thr?Lys?Ile?Glu?Ala?Gln?Lys?Lys?Pro
1 5 10 15
Leu?Ser?Arg?Val?Pro?His?Ser?Asn?Pro?Pro?Phe?Ser?Val?Gly?Gln?Leu
20 25 30
Lys?Lys?Ala?Ile?Pro?Pro?His?Cys?Phe?Glu?Arg?Ser?Leu?Phe?Ile?Ser
35 40 45
Phe?Ser?Tyr?Val?Val?Tyr?Asp?Leu?Leu?Met?Ala?Tyr?Leu?Leu?Phe?Tyr
50 55 60
Ile?Ala?Thr?Thr?Tyr?Phe?His?Lys?Leu?Pro?Tyr?Pro?Phe?Ser?Phe?Leu
65 70 75 80
Ala?Trp?Pro?Ile?Tyr?Trp?Ala?Ile?Gln?Gly?Cys?Ile?Leu?Thr?Gly?Val
85 90 95
Trp?Val?Ile?Ala?His?Glu?Cys?Gly?His?His?Ala?Phe?Ser?Lys?Tyr?Gln
100 105 110
Leu?Val?Asp?Asp?Met?Val?Gly?Leu?Thr?Leu?His?Ser?Cys?Leu?Leu?Val
115 120 125
Pro?Tyr?Phe?Ser?Trp?Lys?Ile?Ser?His?Arg?Arg?His?His?Ser?Asn?Thr
130 135 140
Gly?Ser?Leu?Arg?Pro?Arg?Arg?Ser?Val?Cys?Pro?Glu?Thr?Lys?Ile?Lys
115 150 155 160
Gly?Ile?Met?Val
165 170 175
3. the Δ of cultivating peanut
12The cloning process of-fatty acid dehydrogenase mutant gene comprises the steps:
(1) select for use the peanut seed of E11 or E16 to place mortar, add the 0.5ml plant RNA under the room temperature and extract reagent, after fully grinding, be transferred in the 1.5ml EP pipe, vibration is to thorough mixing, extracted total RNA is identified total RNA quality with the denaturing formaldehyde gel electrophoresis, measures rna content then on spectrophotometer;
(2) according to the peanut FAD2 partial sequence (881bp) that has increased, the design primer:
GSP1：5-AGTGGTGGCGGCGGTGGCTGATTTTC-3
GSP2：5-GGCAACAGTGGACAGAGATTATGGG-3
Total RNA with acquisition is a template, through the synthetic 5 '-RACE-Ready cDNA of reverse transcription and 3 '-RACE-Ready cDNA, carries out pcr amplification with high-fidelity Taq enzyme,
The PCR program of 5 '-RACE is as follows: 94 ℃ of pre-sex change 2min, and 94 ℃ of sex change 30s, 72 ℃ are extended 2min, after 5 circulations, 94 ℃ of sex change 30s, 70 ℃ of renaturation 30s, 72 ℃ are extended 2min, after 5 circulations, 94 ℃ of sex change 30s, 68 ℃ of renaturation 30s, 72 ℃ are extended 2min, after 26 circulations, 72 ℃ of 15min;
The PCR program of 3 '-RACE is as follows: 94 ℃ of pre-sex change 2min, and 94 ℃ of sex change 30s, 68 ℃ of renaturation 30s, 72 ℃ are extended 2min, after 27 circulations, 72 ℃ of 15min;
The PCR product reclaims rear clone to the pMD18-T carrier, and the order-checking back obtains 5 ' terminal sequence and the 3 ' terminal sequence of AhFAD2 ';
(3) according to the 5 ' terminal sequence and the 3 ' terminal sequence of the AhFAD2 ' gene that has obtained, design the two ends primer:
FADf5：5′-ACTCACTTCTCTTCTCTCTG-3′
FADf3：5′-GCAAATCCACACACACATTC-3′
The 5 '-RACE-Ready cDNA that obtains with step (2) is a template, carry out pcr amplification with high-fidelity Taq enzyme, the PCR program is as follows: 94 ℃ of pre-sex change 4 minutes, 94 ℃ of sex change 30s, 59 ℃ of renaturation 50s, 72 ℃ are extended 2min, after 32 circulations, 72 ℃ of 15min, the PCR product is cloned the most at last, checking order has obtained the peanut Δ
12The full length cDNA sequence of-fatty acid dehydrogenase mutant gene.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525931A (en) * | 2013-10-16 | 2014-01-22 | 山东省花生研究所 | Peanut high-oleic-acid character gene selection method |
| CN107058334A (en) * | 2016-11-18 | 2017-08-18 | 山东省花生研究所 | One cultivate peanut the genes of transcription factor AhJ11 FAR1 5 clone and functional expression method |
| CN114324653A (en) * | 2021-12-28 | 2022-04-12 | 广东省农业科学院作物研究所 | Identification method of peanut variety |
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2007
- 2007-02-02 CN CNA200710003083XA patent/CN101235380A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525931A (en) * | 2013-10-16 | 2014-01-22 | 山东省花生研究所 | Peanut high-oleic-acid character gene selection method |
| CN107058334A (en) * | 2016-11-18 | 2017-08-18 | 山东省花生研究所 | One cultivate peanut the genes of transcription factor AhJ11 FAR1 5 clone and functional expression method |
| CN107058334B (en) * | 2016-11-18 | 2021-04-13 | 山东省花生研究所 | Cloning and functional expression method of peanut transcription factor AhJ11-FAR1-5 gene |
| CN114324653A (en) * | 2021-12-28 | 2022-04-12 | 广东省农业科学院作物研究所 | Identification method of peanut variety |
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