CN103060340B - Cloning and function expression method of adversity stress AhMYBL6 gene in peanut - Google Patents
Cloning and function expression method of adversity stress AhMYBL6 gene in peanut Download PDFInfo
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Abstract
The invention relates to a cloning and function expression method of an adversity stress AhMYBL6 gene in peanut. The adversity stress AhMYBL6 gene is synthesized by RT-PCR (reverse transcription-polymerase chain reaction) cloning by the steps of preparation and treatment of materials, extraction of RNA (ribonucleic acid) and synthesis of cDNA (complementary deoxyribonucleic acid). The open reading frame of the gene is 861bp, and 287 amino acids are coded totally. The homology of the amino acid sequence of the gene with the MYb family proteins GmMYB4, GmMYB29 and GmMYB29A2 of the soybean is higher than 60%. The fluorescent quantitative PCR verifies the expression patterns of the AhMYBL6 under low temperature, salt stress and drought stress; the result indicates that the expression levels of the AhMYBL6 are obviously enhanced after various stress treatment for 1 hour, and kept at high levels all the time thereafter; and the expression level of the AhMYBL6 in the leaf subjected to low-temperature treatment is maximally enhanced by 20 times. After the gene is transferred into peanut by transgenic means, compared with the control, the transgenic plant has obvious characteristics of cold tolerance, salt tolerance and drought tolerance. The AhMYBL6 gene can obviously enhance the stress tolerance of the peanut.
Description
Technical field
The invention belongs to biological technical field, relate to the environment stress of cultivating peanut
ahMYBL6the clone of gene and functional expression method.
Background technology
Peanut is pulse family Arachis crop, is rich in grease and albumen, is the important oil crops of China and cash crop.The yield and quality of peanut is subject to arid, saline and alkaline etc. coercing to affect very seriously, and the peanut underproduction rate that the whole nation causes because of arid every year reaches more than 20%.But because the hereditary basis of peanut varieties resource is narrow, lack the GENE SOURCES of height drought resisting, cold-resistant, salt tolerant, utilize conventional breeding method to be difficult to cultivate high resistance kind.Along with molecular biological fast development, the research that utilizes in recent years transgenic technology to improve plant stress tolerance has obtained remarkable achievement, to coercing genes involved and signal transduction pathway, has also had more deep understanding.
MYB albumen is a class transcription factor of finding from plant in recent years, and according to the repeat number of conserved domain, MYB family protein is divided into four subfamily: 1R-MYB, 2R-MYB, 3R-MYB and 4R-MYB.Such transcription factor is at development of plants, and hormone signal transduction, all has critical function in pathogen resistance and abiotic stress resistance.The inductions such as the expression of this family gene is subject to arid, damages to plants caused by sudden drop in temperature, salt stress.Now in the plants such as paddy rice, wheat, Arabidopis thaliana, tobacco, soybean and tomato, found this genoid.
There are some researches show, several
r2R3-MYBgene has participated in the response (Chen of plant to arid, high salt and low temperature adverse circumstance
et al.2006; Agarwal
et al.2006).Ma etc. (2009) overexpression in paddy rice
osMYB3R-
2, transgenic paddy rice improves the resistance of low temperature stress.Pasquali etc. (2008) OsMYB4 of overexpression paddy rice in apple can improve transfer-gen plant to low temperature and arid resistance.Zhang etc. (2012) can improve Arabidopis thaliana to freezing and resistance salt stress by the TaMYB56-B of wheat overexpression in Arabidopis thaliana.Large quantity research shows in various abiotic stress processes, and the accumulation of MYB family protein plays potential provide protection under stress conditions to plant, but has not yet to see the report that such base is trapped in clone and functional study in peanut.
Summary of the invention
In order to improve the degeneration-resistant border ability of peanut growth, strengthen its salt tolerant, drought resisting, low temperature tolerance ability, invented the environment stress gene of cultivating peanut
ahMYBL6clone and functional expression method.
technical scheme
One environment stress of cultivating peanut
ahMYBL6the clone of gene and functional expression method, mainly comprise the following steps:
(1) preparation of material and processing: material is that peanut flower is educated 19(
arachis hypogaeal. cultivar Huayu19).Peanut seed is sprouted in the soil with 2:1 mixing at Nutrition Soil and vermiculite, and after sprouting, growth of seedling condition is 16h illumination/8h dark (28 ℃/22 ℃).The approximately 2 weeks peanut seedlings in tri-leaf period of growing are processed for follow-up abiotic stress.
Subzero treatment, by tri-leaf period peanut seedling as for processing in 4 ℃ of illumination boxs, respectively at the 0h processing, 1h, 3h, 6h, 12h, 24h, 48h and 72h get peanut leaf and root as material; For salt tolerant, with NaCl, process; For drought resisting, with PEG6000, process.Peanut being taken out from soil and carefully the native water on root is rinsed well, then Roots of Peanut is immersed in respectively in 200mM NaCl or weight concentration 20%PEG6000 solution, is 0h in the treatment time, 1h, 3h, 6h, 12h, 24h, 48h and 72h get respectively the root of peanut as material.All material is all stored in-80 ℃ of Ultralow Temperature Freezers standby.
(2) extraction of RNA and cDNA's is synthetic
RNeasy Mini Kit (Qiagen) the separation and Extraction peanut seedling RNA of day root for this experiment.RQ1 RNase-free DNaseI removes the RNA obtaining after DNA pollutes and carries out the synthetic of cDNA again.With M-MLV Reverse Transcriptase (Promega, Madison, WI, USA), carry out the synthetic of cDNA, in 25-μ L reaction system, comprise 2 μ g RNA.Under 42 ° of C conditions, after the reverse transcription reaction of 60 min, reverse transcription product is placed in and places 5 min on ice, afterwards reverse transcription product is saved backup in-20 ℃ of cryogenic refrigerators.
(3) gene clone
By RT-PCR, clone, pcr amplification polysaccharase used is LA Taq
tMdNA polymerase (TaKaRa) adds following composition in 25-μ L system: 2.5 μ L 10 * PCR buffer are (containing MgCl
2); 2.5 μ L 10 mM 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 5 min; (b) 94 ℃, 45 s; 55 ℃, 45 s; 72 ℃, 90 s; Totally 35 cycles; (c) 72 ℃, 10 min.Amplification gene total length the primer is AhMYB6-S:5 '-GAAGCAAAGATGGTGA GA-3 ' and AhMYB6-A:5 '-AGCCCTAATCAAAGACGA-3 '.PCR product reclaims test kit (Axygen) with glue and carries out purifying after 1% agarose gel electrophoresis separation, and purified product connects pMD18-T Easy vector (Takara) order-checking (Sangon, Shanghai).
Of the present invention
ahMYBL6gene open reading frame is 861bp, 287 amino acid of encoding altogether.
Of the present invention
ahMYBL6the aminoacid sequence of gene is found the MYB family protein GmMYB4 of this aminopeptidase gene acid sequence and soybean after analyzing by Blast on NCBI website, GmMYB29, and GmMYB29A2 homology has all reached more than 60%.
Of the present invention
ahMYBL6the nucleotide sequence of gene is SEQUENCE 1 in sequence table.
Of the present invention
ahMYBL6the aminoacid sequence of gene is SEQUENCE 2 in sequence table.
With fluorescent quantitation Real-time RT-PCR couple
ahMYBL6gene carries out functional expression:
Quantitative fluorescent PCR cDNA template used is diluted to 8 ng μ L
-1, the polysaccharase of use is SYBR Premix Ex Taq polymerase (Takara), and the instrument of use is LightCycler 2.0 instrument system (Roche, Germany), and every reaction system adds the cDNA of 2 μ L dilutions.PCR response procedures is as follows: (1) 95 ℃, and 10 s; (2) 95 ℃, 5 s, 40 cycles; (3) 60 ℃, 30 s; (4) 72 ℃, 10 s.PCR reaction finishes rear drafting solubility curve, and it is to increase by 0.5 ℃ in every 10 seconds that temperature increases gradient.
β-actinfor quantitative fluorescent PCR reference gene.
ahMYB6the primer sequence that fluorescence quantitative RT-RCR is used is: QAhMYBL6-S:5 '-ACCACCACTA CAACAACA-3 ' and QAhMYBL6-A:5 '-ATCATCAATTCCTGCCTCT-3 '.
Reference gene
β-actinthe primer sequence is: Qactin-S:5 '-TTGGAATGGGTCAGAAGG ATGC-3 ' and Qactin-A:5 '-AGTGGTGCCTCAGTAAGAAGC-3 '.
unusual effect of the present invention
By quantitative fluorescent PCR, verified
ahMYBL6at low temperature, the expression pattern under salt stress and drought stress, result shows that this gene transcriptional level under three kinds of abiotic stress is all increased significantly (Fig. 2).As can be seen from Figure 2,
ahMYBL6after various Stress treatment 1h, expression amount is all significantly improved, and maintains ever since higher level, in the blade of subzero treatment
ahMYBL6expression amount the highlyest improved more than 20 times (Fig. 2, B).Above result shows
ahMYBL6may play a significant role in to the adaptability of environment stress at peanut.This gene is imported in peanut by transgenosis means, and transfer-gen plant has significantly cold-resistant, salt tolerant and Characteristics of Drought than contrast.Explanation
ahMYBL6gene can significantly improve the resistance of peanut.
Accompanying drawing explanation
MYB family protein aminoacid sequence comparison in Fig. 1 peanut AhMYBL albumen and soybean
The upper albumen numbering of NCBI: GmMYB4, XP_003518022; GmMYB29, NP_001241360; GmMYB29A2, BAA81732.
Fig. 2
ahMYBL6at low temperature, NaCl, PEG6000 processes lower expression pattern analysis
A:
ahMYBL6in the Roots of Peanut of subzero treatment, express and change; B:
ahMYBL6in the peanut leaf of subzero treatment, express and change; C:
ahMYBL6in the Roots of Peanut of processing at 200mM NaCl, express and change; D:
ahMYBL6in the Roots of Peanut of processing at 20% PEG6000, express and change.
Embodiment
Below in conjunction with accompanying drawing and specific embodiment, further illustrate the present invention, but be not limitation of the invention further.
Specific embodiment of the invention process is:
1 experiment material and method
1.1 experiment material
Experiment material is that peanut flower is educated 19(
arachis hypogaeal. cultivar Huayu19).Peanut seed is sprouted in the soil with 2:1 mixing at Nutrition Soil and vermiculite, and after sprouting, growth of seedling condition is 16h illumination/8h dark (28 ℃/22 ℃).The approximately 2 weeks peanut seedlings in tri-leaf period of growing are processed experiment for follow-up abiotic stress.
For subzero treatment, by tri-leaf period peanut seedling as for processing in 4 ℃ of illumination boxs, respectively at the 0h processing, 1h, 3h, 6h, 12h, 24h, 48h and 72h get peanut leaf and root as experiment material; For salt tolerant, with NaCL, process; For drought resisting, with PEG6000, process.Peanut is taken out from soil and carefully the native water on root is rinsed well, then Roots of Peanut is immersed in respectively in 200mM NaCl or 20%PEG6000 solution, the 0h processing, 1h, 3h, 6h, 12h, 24h, 48h and 72h get respectively the root of peanut as experiment material.All material is all stored in-80 ℃ of Ultralow Temperature Freezers standby.
extraction and cDNA synthetic
RNeasy Mini Kit (Qiagen) the separation and Extraction peanut seedling RNA of day root for this experiment.RQ1 RNase-free DNaseI removes the RNA obtaining after DNA pollutes and carries out the synthetic of cDNA again.With M-MLV Reverse Transcriptase (Promega, Madison, WI, USA), carry out the synthetic of cDNA, in 25-μ L reaction system, comprise 2 μ g RNA.Under 42 ° of C conditions, after the reverse transcription reaction of 60 min, reverse transcription product is placed in and places 5 min on ice, afterwards reverse transcription product is saved backup in-20 ℃ of cryogenic refrigerators.
gene clone
By RT-PCR, clone, pcr amplification polysaccharase used is LA Taq
tMdNA polymerase (TaKaRa) adds following composition in 25-μ L system: 2.5 μ L 10 * PCR buffer are (containing MgCl
2); 2.5 μ L 10 mM dNTPs; 1 μ L cDNA template; 0.5 μ L LA polymerase and 17.5 μ L ddH
2o.PCR reaction conditions is: (1) 94 ℃, and 5 min; (2) 94 ℃, 45 s; 55 ℃, 45 s; 72 ℃, 90 s; Totally 35 cycles; (3) 72 ℃, 10 min.Amplification gene total length the primer is AhMYB6-S:5 '-GAAGCAAAGATGGTGA GA-3 ' and AhMYB6-A:5 '-AGCCCTAATCAAAGACGA-3 '.PCR product reclaims test kit (Axygen) with glue and carries out purifying after 1% agarose gel electrophoresis separation, and purified product connects pMD18-T Easy vector (Takara) order-checking (Sangon, Shanghai).
fluorescent quantitation Real-time RT-PCR
Quantitative fluorescent PCR cDNA template used is diluted to 8 ng μ L
-1, the polysaccharase of use is SYBR Premix Ex Taq polymerase (Takara), and the instrument of use is LightCycler 2.0 instrument system (Roche, Germany), and every reaction system adds the cDNA of 2 μ L dilutions.PCR response procedures is as follows: (1) 95 ℃, and 10 s; (2) 95 ℃, 5 s, 40 cycles; (3) 60 ℃, 30 s; (4) 72 ℃, 10 s.PCR reaction finishes rear drafting solubility curve, and it is to increase by 0.5 ℃ in every 10 seconds that temperature increases gradient.
β-actinreference gene for experiment.
ahMYB6the primer sequence of fluorescent quantitation checking use is: QAhMYBL6-S:5 '-ACCACCACTA CAACAACA-3 ' and QAhMYBL6-A:5 '-ATCATCAATTCCTGCCTCT-3 '.
Reference gene
β-actinthe primer sequence is: Qactin-S:5 '-TTGGAATGGGTCAGAAGG ATGC-3 ' and Qactin-A:5 '-AGTGGTGCCTCAGTAAGAAGC-3 '.
experimental result
2.1
ahMYBL6the aminoacid sequence of Nucleotide total length and coding thereof
By pcr amplification and order-checking, obtained goal gene, this gene open reading frame is 861bp, 287 amino acid of encoding altogether.After being analyzed by Blast on NCBI website, the aminoacid sequence of this gene finds the MYB family protein GmMYB4 of this aminopeptidase gene acid sequence and soybean, GmMYB29, and GmMYB29A2 homology has all reached more than 60% (Fig. 1), therefore by this unnamed gene be
ahMYBL6 (Arachis hypogaea MYB Like 6).
the expression pattern analysis of AnMYBL6 under abiotic stress
By quantitative fluorescent PCR, verified
ahMYBL6at low temperature, the expression pattern under salt stress and drought stress, result shows that this gene transcriptional level under three kinds of abiotic stress is all increased significantly (Fig. 2).As can be seen from Figure 2,
ahMYBL6after various Stress treatment 1h, expression amount is all significantly improved, and maintains ever since higher level, in the blade of subzero treatment
ahMYBL6expression amount the highlyest improved more than 20 times (Fig. 2, B).Above result shows
ahMYBL6may play a significant role in to the adaptability of environment stress at peanut.This gene is imported in peanut by transgenosis means, and transfer-gen plant has significantly cold-resistant, salt tolerant and Characteristics of Drought than contrast.Illustrate that this gene can significantly improve the resistance of peanut.
SEQUENCE LISTING
<110> Shandong Peanut Inst.
<120> mono-cultivate peanut clone and the functional expression method of middle environment stress AhMYBL6 gene
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1191
<212> DNA
<213> AhMYBL6
<400> 1
gaagcaaaga tggtgagagc tccttgttgt gaaaagatgg gattgaagaa gggtccatgg 60
acggcagagg aagatcaaat cctcatttct tacatccaaa aacatggcca tggcaattgg 120
cgtgccctcc caaagcaagc agggctgtta aggtgtggaa agagctgcag attaaggtgg 180
ataaattatc tgaggcctga tatcaagaga gggaatttca ccattgaaga agaagaaacc 240
atcatcaagc ttcatgaaat gcttggcaac aggtggtcgg caattgcggc aaaattaccc 300
ggaagaacag acaacgaaat caagaatgtg tggcacacac atttgaagaa gaggctcatg 360
aaaccaaacc aaatgaattc atcatcatca tcagatccca aaagggtatc taataagtca 420
aagatcaaac gttctgattc aaactcaagc accataactg ttcaatcatc ggaaccagca 480
agtctcaata gtaatttccc tgaaatggac acaacttcag catcatgcac caccactaca 540
acaacaacct ctagtgaatt ctcatcagcc acaaatattg gtgaaagcaa gaacgtgaac 600
atgatgaaca tcaagaatga ggagctagga gattcattgg agaacataat tgatgaaagc 660
ttatggacag aggcaggaat tgatgatgaa accaccaaca tgacaatctc aaataatgag 720
ttgccctcac ttcaatatta tgacccagta tttaataatt ctgaggagaa tttaagcttc 780
caaccaagta caaactttga ggatgatggc atggattttt ggtatgacat attcattaga 840
actggggagt caatagaatt gccagagttc tgaaattttt ctcaaaaatt aaaccgtgca 900
tgcatcttgg aacatgaaat tctttttatt agaggattag aatactttga attatgatca 960
tcataattta ttttgaccat catggtaact tgaattgacc gttacaaggc attcaatttg 1020
tacattaatt tctggtgtgg tggggacaga ggatcggatg agggaccaaa agtgagtgaa 1080
acaatgcaca atgacttggt ttaatgtata cttgggccca aaagaatacc aagtctctaa 1140
catttttcag ctttatagat tttactattt tagtcgtctt tgattagggc t 1191
<210> 2
<211> 287
<212> protein
<213> AhMYBL6 proteins encoded
<400> 2
Met Val Arg Ala Pro Cys Cys Glu Lys Met Gly Leu Lys Lys Gly Pro
1 5 10 15
Trp Thr Ala Glu Glu Asp Gln Ile Leu Ile Ser Tyr Ile Gln Lys His
20 25 30
Gly His Gly Asn Trp Arg Ala Leu Pro Lys Gln Ala Gly Leu Leu Arg
35 40 45
Cys Gly Lys Ser Cys Arg Leu Arg Trp Ile Asn Tyr Leu Arg Pro Asp
50 55 60
Ile Lys Arg Gly Asn Phe Thr Ile Glu Glu Glu Glu Thr Ile Ile Lys
65 70 75 80
Leu His Glu Met Leu Gly Asn Arg Trp Ser Ala Ile Ala Ala Lys Leu
85 90 95
Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Val Trp His Thr His Leu
100 105 110
Lys Lys Arg Leu Met Lys Pro Asn Gln Met Asn Ser Ser Ser Ser Ser
115 120 125
Asp Pro Lys Arg Val Ser Asn Lys Ser Lys Ile Lys Arg Ser Asp Ser
130 135 140
Asn Ser Ser Thr Ile Thr Val Gln Ser Ser Glu Pro Ala Ser Leu Asn
145 150 155 160
Ser Asn Phe Pro Glu Met Asp Thr Thr Ser Ala Ser Cys Thr Thr Thr
165 170 175
Thr Thr Thr Thr Ser Ser Glu Phe Ser Ser Ala Thr Asn Ile Gly Glu
180 185 190
Ser Lys Asn Val Asn Met Met Asn Ile Lys Asn Glu Glu Leu Gly Asp
195 200 205
Ser Leu Glu Asn Ile Ile Asp Glu Ser Leu Trp Thr Glu Ala Gly Ile
210 215 220
Asp Asp Glu Thr Thr Asn Met Thr Ile Ser Asn Asn Glu Leu Pro Ser
225 230 235 240
Leu Gln Tyr Tyr Asp Pro Val Phe Asn Asn Ser Glu Glu Asn Leu Ser
245 250 255
Phe Gln Pro Ser Thr Asn Phe Glu Asp Asp Gly Met Asp Phe Trp Tyr
260 265 270
Asp Ile Phe Ile Arg Thr Gly Glu Ser Ile Glu Leu Pro Glu Phe
275 280 285
Claims (6)
1. the environment stress of cultivating peanut
ahMYBL6the cloning process of gene, mainly comprises the following steps:
(1) preparation of material and processing: material selects peanut flower to educate 19, peanut seed is sprouted in the soil with 2:1 mixing at Nutrition Soil and vermiculite, after sprouting, growth of seedling condition is that 16h illumination/8h is dark, temperature is 22 ℃-28 ℃, and the 2 weeks peanut seedlings in tri-leaf period of growing are processed for follow-up abiotic stress;
The subzero treatment of material, by tri-leaf period peanut seedling put into 4 ℃ of illumination boxs and process, get the treatment time to be respectively 0h, 1h, 3h, 6h, 12h, 24h, the peanut leaf of 48h and 72h and root are as material; For salt tolerant, with NaCl, process; For drought resisting, with PEG6000, process; Peanut is taken out from soil and carefully the native water on root is rinsed well, then Roots of Peanut being immersed in respectively to 200mM NaCl or weight concentration is in 20%PEG6000 solution, processing 0h, 1h, 3h, 6h, 12h, 24h, 48h and 72h get respectively the root of peanut as material, and all material is all stored in-80 ℃ of Ultralow Temperature Freezers standby;
(2) extraction of RNA and cDNA's is synthetic
RNeasy Mini Kit separation and Extraction peanut seedling cDNA according to the method for test kit operational manual with day root, removes the RNA obtaining after DNA pollutes and carries out the synthetic of cDNA again with RQ1 RNase-free DNaseI; With M-MLV Reverse Transcriptase, carry out the synthetic of cDNA, in 25-μ L reaction system, comprise 2 μ g RNA, under 42 ° of C conditions, after the reverse transcription reaction of 60 min, reverse transcription product is placed in and places 5 min on ice, afterwards reverse transcription product is saved backup in-20 ℃ of cryogenic refrigerators;
(3)
ahMYBL6gene clone
By RT-PCR, clone, pcr amplification polysaccharase used is LA Taq
tMdNA polymerase adds following composition in 25-μ L system: containing MgCl
22.5 μ L 10 * PCR buffer; 2.5 μ L 10 mM 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 5 min; (b) 94 ℃, 45 s; 55 ℃, 45 s; 72 ℃, 90 s; Totally 35 cycles; (c) 72 ℃, 10 min;
Amplification gene total length the primer is AhMYB6-S:5 '-GAAGCAAAGATGGTGA GA-3 ' and AhMYB6-A:5 '-AGCCCTAATCAAAGACGA-3 ';
PCR product reclaims test kit with Axygen glue and carries out purifying after 1% agarose gel electrophoresis separation, and purified product connects pMD18-T Easy vector order-checking;
Described in it is characterized in that
ahMYBL6the nucleotide sequence of gene is SEQUENCE 1 in sequence table.
2. peanut environment stress according to claim 1
ahMYBL6the cloning process of gene, is characterized in that: described
ahMYBL6the aminoacid sequence of gene is found the MYB family protein GmMYB4 of this aminopeptidase gene acid sequence and soybean after analyzing by Blast on NCBI website, GmMYB29, and GmMYB29A2 homology has all reached more than 60%.
3. peanut environment stress according to claim 1
ahMYBL6the cloning process of gene, is characterized in that: described
ahMYBL6the aminoacid sequence of gene is SEQUENCE 2 in sequence table.
4. according to any peanut environment stress of claims 1 to 3
ahMYBL6gene function expression method, is used fluorescent quantitation Real-time RT-PCR couple
ahMYBL6expression under gene adverse circumstance is analyzed, and its method is as follows:
Quantitative fluorescent PCR cDNA template used is diluted to 8 ng. μ L
-1, the polysaccharase of use is SYBR Premix Ex Taq polymerase, and the instrument of use is LightCycler 2.0 instrument system, and every reaction system adds the cDNA of 2 μ L dilutions;
PCR response procedures is as follows: (a) 95 ℃, and 10 s; (b) 95 ℃, 5 s, 40 cycles; (c) 60 ℃, 30 s; (d) 72 ℃, 10 s; PCR reaction finishes rear drafting solubility curve, and it is to increase by 0.5 ℃ in every 10 seconds that temperature increases gradient,
β-actinreference gene for RT-PCR.
5. according to claim 4 peanut environment stress
ahMYBL6gene function expression method, is characterized in that:
ahMYB6the primer sequence that fluorescence quantitative RT-RCR is used is: QAhMYBL6-S:5 '-ACCACCACTA CAACAACA-3 ' and QAhMYBL6-A:5 '-ATCATCAATTCCTGCCTCT-3 '.
6. according to claim 4 peanut environment stress
ahMYBL6gene function expression method, is characterized in that: described reference gene
β-actinthe primer sequence is: Qactin-S:5 '-TTGGAATGGGTCAGAAGG ATGC-3 ' and Qactin-A:5 '-AGTGGTGCCTCAGTAAGAAGC-3 '.
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| CN104762305B (en) * | 2015-04-28 | 2018-01-19 | 中国科学院遗传与发育生物学研究所 | The gene TaUreG related to Drought-resistance in Wheat and its application |
| CN105483116B (en) * | 2015-10-22 | 2018-08-10 | 山东省花生研究所 | Peanut environment stress AhbHLH1L gene clonings and functional expression method |
| CN105218652A (en) * | 2015-11-04 | 2016-01-06 | 江苏省农业科学院 | Peanut MYB class transcription factor AhMYB31 and application |
| CN105462989A (en) * | 2015-12-28 | 2016-04-06 | 山东省花生研究所 | Cloning and functional expression method of gene AhAP2ER related to drought stress of peanuts |
| CN105949296A (en) * | 2016-07-18 | 2016-09-21 | 江苏省农业科学院 | Peanut MYB transcription factor AhMYB32 and application thereof |
| CN106191278B (en) * | 2016-07-25 | 2019-10-15 | 南京农业大学 | A kind of primer and its application detecting salt stress to grape seedlings extent of injury |
| CN106480068B (en) * | 2016-12-16 | 2019-07-09 | 农业部沼气科学研究所 | Duckweed transcription factor LmMYB gene and its application |
| CN109576282B (en) * | 2018-12-18 | 2021-08-10 | 中国农业大学 | Chinese rose transcription factor RhMYB4 and application thereof in flower organ development regulation |
| CN110195063B (en) * | 2019-06-03 | 2020-08-21 | 华中农业大学 | Application of potato StGLK1 gene in low-temperature saccharification resistance |
| CN112724213B (en) * | 2021-01-13 | 2022-02-18 | 中国农业大学 | Sweet potato anthocyanin synthesis and stress resistance related protein IbMYB4, and coding gene and application thereof |
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