CN105462989A - Cloning and functional expression method of gene AhAP2ER related to drought stress of peanuts - Google Patents
Cloning and functional expression method of gene AhAP2ER related to drought stress of peanuts Download PDFInfo
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Abstract
The invention relates to the biotechnology field and in particular relates to a cloning and functional expression method of a gene AhAP2ER related to drought stress of peanuts. The gene AhAP2ER in drought stress response is synthesized through preparation and treatment of materials, extraction of RNA (ribonucleic acid), synthesis of cDNA (complementary deoxyribonucleic acid) and RACE (rapid-amplification of cDNA ends) cloning technology. Expression of the gene AhAP2ER under the condition of drought stress is also analyzed by using fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction), thus finally drawing a conclusion that the gene AhAP2ER plays an important role in the adaptability of the peanuts to drought stress. The transgenic plants have obvious drought resistance characteristic compared with a control group by transferring the gene to the peanuts by transgenic means, indicating that the gene AhAP2ER can conduce to obviously improving the drought resistance of the peanuts.
Description
[technical field]
The present invention relates to biological gene engineering field, specifically one to cultivate peanut the clone of drought stress AhAP2ER gene and functional expression method.
[background technology]
China is Peanut big producing country, and ultimate production more than 1,500 ten thousand tons, accounts for more than 40% of Peanut ultimate production, rank first in the world.But, peanut industry further develop the threat being subject to drought.The peanut underproduction that China causes because of arid every year according to statistics reaches 30% ~ 50%.Except production declining, arid also likely causes a series of consequence of degradation under aflatoxin pollution of peanuts, quality.But because the hereditary basis of peanut varieties resource is narrow, lack corresponding gene source, utilize traditional breeding way to be difficult to cultivate high resistance kind.Along with the fast development of modern biology, the research of stress-related genes in recent years achieves remarkable achievement, and the research simultaneously utilizing transgenic technology to improve plant stress tolerance also achieves progress.After stress signal produces, peanut can start a series of corresponding signal, is finally transmitted to genes involved, starts this genetic expression and adapts to assist peanut or resist adverse circumstance.Transcription factor is the important factor of these genetic expressions of regulation and control.Transcription factor also referred to as trans-acting factor, refer to can with the DBP of cis-acting elements generation specific effect in eukaryotic gene promoter region, by between them and and other associated protein between interaction, the expression of regulatory gene.Transcription factor has become an important research direction of plant stress-resistance research.
A DNA binding domain be made up of 58 or 59 amino acid is all contained in AP2/EREBP family, and this section of sequence high conservative, a class transcription factor specific to higher plant.The DNA binding domain comprised according to it and DNA bond type are divided into 5 subtribes: DREB subtribe, AP2 subtribe, RAV subtribe, ERF subtribe and AL079349.Such transcription factor all has critical function in plant-growth and abiotic stress resistance.Now in Arabidopis thaliana, soybean, pea, wheat, find this genoid.There are some researches show, several AP2 genoid take part in the response (Liuetal.2013 of plant to arid; Nakashimaetal.2009).Lu Yan etc. (2007) find that this genoid improves the drought resistance of transgenic wheat; Oh etc. (2005) find that transgenic paddy rice significantly improves drought resistance.Large quantity research shows that, in various abiotic stress process, the accumulation of AP2/EREBP family protein plays potential provide protection to plant under stress conditions, but at present such base to be trapped in peanut the report of clone and functional study less, especially AP2 subtribe is less.
[summary of the invention]
The object of the invention is for deficiency of the prior art, provide one to cultivate peanut the clone of drought stress AhAP2ER gene and functional expression method, significantly improve the drought-resistant ability of peanut.
The invention provides a cloning process of cultivating peanut drought stress AhAP2ER gene, mainly comprise the following steps:
The preparation of (a) material and process: Material selec-tion peanut varieties J11, peanut seed is cultivated at Hoagland nutrient solution and is sprouted, sprouting and growth of seedling condition are that 16h illumination/8h is dark, and temperature is 26-28 DEG C, grows 14 days for follow-up drought stress process;
Drought stress PEG6000 process, Roots of Peanut is immersed in 20%PEG6000 solution, and get the root of peanut as material when processing 0h, 6h, 12h, 18h, 24h, 48h and 72h, all material is all stored in-80 DEG C of Ultralow Temperature Freezers for subsequent use.
The extraction of (b) RNA and the synthesis of cDNA
With the MiniBESTUniversalRNAExtractionKit kit method separation and Extraction peanut seedling RNA of TAKARA, cDNA synthesis is carried out again after the RNA obtained is removed DNA pollution, cDNA synthesis is carried out with SMART-RACE kit method, saved backup in-20 DEG C of cryogenic refrigerators by reverse transcription product afterwards, above vessel used all need through removing RNA ferment treatment.The vessel DEPC of 0.1% soaks 12 hours, and then autoclaving is removed.Solution reagent with 0.1% DEPC process (37 DEG C place 12 hours after autoclave sterilization), the reagent of non-refractory is directly prepared with DEPC-H2O.
C () is cloned by RACE, RACE used kit is the SMART-RACE test kit of Clontech, and RACE amplification gene total length the primer is GPS-AhAP2ERf-1:5 ' CGTTTACCCGTTGACTCCATTC-3 ', GPS-AhAP2ERr-1:5 '-TACCGACGCAAAGACGCAAGGTA-3 ' and NGPS-AhAP2ERr-1:5 '-GTATAATCAGTCACTGGGAAG-3 '.
RACE clone products reclaims purifying with UNIQ-10PCRPurificationKit test kit (the raw work in Shanghai) after 1% agarose gel electrophoresis is separated.Purified product is converted into after being connected with pGEM-TEasy carrier (Beijing Quan Shijin biochemical technology company limited) in competence E.coliTOP10 (Beijing Quan Shijin biochemical technology company limited), LB culture medium culturing, random picking 10 positive colonies carry out enlarged culturing, and whether bacterium liquid pcr amplification pre-detection has Insert Fragment and check order.
The result of order-checking is carried out splice rear design total length pcr amplification product, use UNIQ-10PCRPurificationKit kits, purified product is converted in competence E.coliTOP10 after being connected with pGEM-TEasy carrier, LB culture medium culturing, random picking 10 positive colonies carry out enlarged culturing, whether bacterium liquid pcr amplification pre-detection has Insert Fragment and checks order, and amplimer is AhAP2ER-S1:5 '-AAACGCTCGCACACTG-3 ' and AhAP2ER-S2:5 '-CATTTGTATTACCATGAGAATGC-3 '.
Further, described total length pcr amplification polysaccharase used is TAKARAPCRMIX, in 20 μ L systems, add following composition: 10 μ LTAKARAPCRMIX, the total cDNA of 1 μ L, 0.5 μ LAhAP2ER-S1,0.5 μ LAhAP2ER-S2 and 8 μ L aseptic double-distilled waters;
Total length pcr amplification reaction condition: (a) 94 DEG C of 5min; (b) 94 DEG C of 1s; 55 DEG C of 1s; 72 DEG C of 2min; 30cycles altogether; (c) 72 DEG C of 10min; (d) 1% agarose electrophoresis detect.
Further, AhAP2ER gene open reading frame of the present invention is 1266bp, 422 amino acid of encoding altogether.
Further, the aminoacid sequence of AhAP2ER gene of the present invention finds that the homology such as AP2 proteinoid At2g41710, Kidney bean AP2 proteinoid 001G131300g, mung bean AP2 proteinoid At2g41710, clover AP2 proteinoid of this gene amino acid sequence and soybean all reaches more than 85% (Fig. 1) after being analyzed by Blast on NCBI website.
Further, the nucleotide sequence of AhAP2ER gene of the present invention is SEQUENCE1 in sequence table.
Further, the aminoacid sequence of AhAP2ER gene of the present invention is SEQUENCE2 in sequence table.
Further, present invention also offers the functional expression method of described peanut drought stress AhAP2ER gene, it uses fluorescence quantitative RT-RCR to analyze the expression under AhAP2ER gene arid, and its method is as follows:
Fluorescence quantitative RT-RCR cDNA template used is diluted to 8ng/ μ L, polysaccharase be SYBRGreen, the instrument of employing is 7500FAST quantitative real time PCR Instrument, every reaction system add 2 μ L dilute cDNA;
PCR response procedures is as follows: (a) 95 DEG C, 10s; (b) 95 DEG C, 5s; (c) 60 DEG C, 30s; (d) 72 DEG C, 10s; 40 circulations; Draw solubility curve, it is that every 10s increases by 0.5 DEG C that temperature increases gradient, and Actin is the reference gene of RT-PCR.
The primer sequence of described AhAP2ER fluorescent quantitation checking is: AhAP2ER-R:5 '-ACCTACTGAGCCATACCA-3 ' and AhAP2ER-F:5 '-TTGTAAGCAGCGGATTGT-3 '.
Described reference gene Actin the primer sequence is: Actin-F:5 '-GAGGAGAAGCAGAAGCAAGTTG-3 ' and AhAP2ER-F:5 '-AGACAGCATATCGGCACTCATC-3 '.
The present invention demonstrates the expression pattern of AhAP2ER gene under drought stress by quantitative fluorescent PCR, and under result shows this gene drought stress, transcriptional level is all increased significantly (Fig. 2).As can be seen from Figure 2, this gene expression amount in drought stress process 24h slowly improves, and improves fast, and maintain higher level after 24h.Above result shows that AhAP2ER gene plays a significant role in the adaptability of peanut to drought stress.Imported in peanut by transgenic approach by this gene, transfer-gen plant has obvious Characteristics of Drought, illustrates that AhAP2ER gene can significantly improve Drought Resistance in Peanut.
[accompanying drawing explanation]
Fig. 1 is that peanut AhAP2ER albumen compares with AP2 proteinoid aminoacid sequence in other pulse families
Fig. 2 is the expression pattern analysis of peanut AhAP2ER gene under drought stress.
[embodiment]
Below in conjunction with specific embodiment, technological line of the present invention is described in further details, but is not limitation of the invention further.
1.1 implement material:
Material preparation and process: Material selec-tion peanut varieties J11, peanut seed is cultivated at Hoagland nutrient solution and is sprouted, and sprouting and growth of seedling condition are that 16h illumination/8h is dark, and temperature is 26 DEG C, grows 14 days for follow-up drought stress process.Drought stress PEG6000 process.Roots of Peanut is immersed in 20%PEG6000 solution, and get the root of peanut as material when processing 0h, 6h, 12h, 18h, 24h, 48h and 72h, all material is all stored in-80 DEG C of Ultralow Temperature Freezers for subsequent use.
The extraction of 1.2RNA and the synthesis of cDNA
Vessel used all need through removing RNA ferment treatment.The vessel DEPC of 0.1% soaks 12 hours, and then autoclaving is removed.Solution reagent with 0.1% DEPC process (37 DEG C place 12 hours after autoclave sterilization), the reagent of non-refractory is directly prepared with DEPC-H2O.The operational requirement strand cDNA that Total RNAs extraction presses the MiniBESTUniversalRNAExtractionKit of TAKARA synthesizes, the concrete SMART-RACE kit method with reference to Clontech.Reverse transcription product saves backup in-20 DEG C of cryogenic refrigerators.
1.3AhAP2ER gene clone
Cloned by RACE, RACE used kit is the SMART-RACE kit method reference reagent box explanation of Clontech.RACE amplification gene total length the primer is GPS-AhAP2ERf-1:5 ' CGTTTACCCGTTGACTCCATTC-3 ', GPS-AhAP2ERr-1:5 '-TACCGACGCAAAGACGCAAGGTA-3 ' and NGPS-AhAP2ERr-1:5 '-GTATAATCAGTCACTGGGAAG-3 '.
RACE clone products reclaims purifying with UNIQ-10PCRPurificationKit test kit (the raw work in Shanghai) after 1% agarose gel electrophoresis is separated.Purified product is converted into after being connected with pGEM-TEasy carrier (Beijing Quan Shijin biochemical technology company limited) in competence E.coliTOP10 (Beijing Quan Shijin biochemical technology company limited), LB culture medium culturing, random picking 10 positive colonies carry out enlarged culturing, whether bacterium liquid pcr amplification pre-detection has Insert Fragment and check order (Sangon, Shanghai).
The result of order-checking is carried out splice rear design total length pcr amplification product, pcr amplification polysaccharase used is TAKARAPCRMIX, following composition 10 μ LTAKARAPCRMIX is added in 20 μ L systems, the total cDNA of 1 μ L, 0.5 μ LAhAP2ER-S1,0.5 μ LAhAP2ER-S2,8 μ L aseptic double-distilled waters.Reaction conditions: 94 DEG C of 5min → (94 DEG C of 1s → 55 DEG C 1s → 72 DEG C 2min) 30cycles → 72 DEG C 10min → 1% agarose electrophoresiss detect.UNIQ-10PCRPurificationKit test kit (the raw work in Shanghai) reclaims purifying.Purified product is converted into after being connected with pGEM-TEasy carrier (Beijing Quan Shijin biochemical technology company limited) in competence E.coliTOP10 (Beijing Quan Shijin biochemical technology company limited), LB culture medium culturing, random picking 10 positive colonies carry out enlarged culturing, whether bacterium liquid pcr amplification pre-detection has Insert Fragment and check order (Sangon, Shanghai).
Amplimer: AhAP2ER-S1:5 '-AAACGCTCGCACACTG-3 '; AhAP2ER-S2:5 '-CATTTGTATTACCATGAGAATGC-3 '
1.4 quantitative fluorescent PCR
With quantitative fluorescent PCR, functional expression is carried out to AhAP2ER gene: quantitative fluorescent PCR cDNA template used is diluted to 8ng/ μ L, polysaccharase be SYBRGreen, instrument be 7500FAST quantitative real time PCR Instrument (ABI company), every reaction system add 2 μ L dilute cDNA; PCR response procedures is as follows: 95 DEG C of 10s; 95 DEG C of 5s, 60 DEG C of 30s, 72 DEG C of 10s, 40 circulations; Draw solubility curve, the every 10s of temperature raises 0.5 DEG C.Relative expression quantity is calculated with 2-Δ Δ CT.Actin is the reference gene of RT-PCR.
The primer sequence of AhAP2ER gene fluorescence quantitative RT-PCR is: AhAP2ER-R:5 '-ACCTACTGAGCCATACCA-3 ' and AhAP2ER-F:5 '-TTGTAAGCAGCGGATTGT-3 '.
Reference gene Actin the primer sequence is: Actin-F:5 '-GAGGAGAAGCAGAAGCAAGTTG-3 '; With Actin-R:5 '-AGACAGCATATCGGCACTCATC-3 '
2 test-results
2.1AhAP2ER the aminoacid sequence of Nucleotide total length and coding thereof
By pcr amplification and order-checking, obtain goal gene, this gene open reading frame is 1266bp, 422 amino acid of encoding altogether.Fig. 1 finds that the homology such as AP2 proteinoid At2g41710, Kidney bean AP2 proteinoid 001G131300g, mung bean AP2 proteinoid At2g41710, clover AP2 proteinoid of this gene amino acid sequence and soybean all reaches more than 85% after showing and being analyzed by Blast on NCBI website by the aminoacid sequence of this gene, therefore is AhAP2ER (ArachishypogaeaAP2-likeethylene-responsivetranscriptionf actor) by this unnamed gene.
Fig. 2 is the expression pattern analysis of peanut AhAP2ER gene under drought stress.The present invention demonstrates the expression pattern of AhAP2ER under drought stress by quantitative fluorescent PCR, and result shows: under this gene drought stress, transcriptional level is all increased significantly.As can be seen from Figure 2, this gene expression amount in drought stress process 24h slowly improves, and improves fast, and maintain higher level after 24h.Above result shows that AhAP2ER gene plays a significant role in the adaptability of peanut to drought stress.Imported in peanut by transgenic approach by this gene, transfer-gen plant has obvious Characteristics of Drought than contrast, illustrates that AhAP2ER gene can significantly improve Drought Resistance in Peanut.
The above; it is only most preferred embodiment of the present invention; not any pro forma restriction is done to the present invention; any those of ordinary skill in the art; do not departing under technical solution of the present invention ambit; utilize the method content of above-mentioned announcement to make many possible variations and modification to technical solution of the present invention, all belong to the scope of claims protection.
Claims (9)
1. a cloning process of cultivating peanut drought stress AhAP2ER gene, is characterized in that, comprise the following steps:
The preparation of (a) material and process
Material selec-tion peanut varieties J11, peanut seed is cultivated at Hoagland nutrient solution and is sprouted, and sprouting and growth of seedling condition are that 16h illumination/8h is dark, and temperature is 26-28 DEG C, grows 14 days for follow-up drought stress process;
Drought stress PEG6000 process, Roots of Peanut is immersed in 20%PEG6000 solution, and get the root of peanut as material when processing 0h, 6h, 12h, 18h, 24h, 48h and 72h, all material is all stored in-80 DEG C of Ultralow Temperature Freezers for subsequent use;
The extraction of (b) RNA and the synthesis of cDNA
With the MiniBESTUniversalRNAExtractionKit kit method separation and Extraction peanut seedling RNA of TAKARA, cDNA synthesis is carried out again after the RNA obtained is removed DNA pollution, carry out cDNA synthesis with SMART-RACE kit method, afterwards reverse transcription product is saved backup in-20 DEG C of cryogenic refrigerators;
C () is cloned by RACE
RACE used kit is the SMART-RACE test kit of Clontech, and RACE amplification gene total length the primer is GPS-AhAP2ERf-1:5 ' CGTTTACCCGTTGACTCCATTC-3 ', GPS-AhAP2ERr-1:5 '-TACCGACGCAAAGACGCAAGGTA-3 ' and NGPS-AhAP2ERr-1:5 '-GTATAATCAGTCACTGGGAAG-3 ';
RACE clone products carries out purifying with UNIQ-10PCRPurificationKit test kit after 1% agarose gel electrophoresis is separated, purified product is converted into competence E.coliTOP10 after connecting pGEM-TEasy carrier, in LB culture medium culturing, random picking enlarged culturing, then carry out pcr amplification detection and check order;
The result of order-checking is carried out splice rear design total length pcr amplification product, use UNIQ-10PCRPurificationKit kits, purified product is converted in competence E.coliTOP10 after being connected with pGEM-TEasy carrier, LB culture medium culturing, random picking 10 positive colonies carry out enlarged culturing, whether bacterium liquid pcr amplification pre-detection has Insert Fragment and checks order, and amplimer is AhAP2ER-S1:5 '-AAACGCTCGCACACTG-3 ' and AhAP2ER-S2:5 '-CATTTGTATTACCATGAGAATGC-3 '.
2. the cloning process of peanut drought stress AhAP2ER gene according to claim 1, it is characterized in that: total length pcr amplification polysaccharase used is TAKARAPCRMIX, it adds following composition in 20 μ L systems: 10 μ LTAKARAPCRMIX, the total cDNA of 1 μ L, 0.5 μ LAhAP2ER-S1,0.5 μ LAhAP2ER-S2 and 8 μ L aseptic double-distilled waters;
Total length pcr amplification reaction condition: (a) 94 DEG C of 5min; (b) 94 DEG C of 1s; 55 DEG C of 1s; 72 DEG C of 2min; 30cycles altogether; (c) 72 DEG C of 10min; (d) 1% agarose electrophoresis detect.
3. according to the cloning process of peanut drought stress AhAP2ER gene described in claim 1 to 2, described AhAP2ER gene open reading frame is 1266bp, 422 amino acid of encoding altogether.
4. the cloning process of peanut drought stress AhAP2ER gene according to claims 1 to 3, is characterized in that: the aminoacid sequence of described AhAP2ER gene finds that the homology such as AP2 proteinoid At2g41710, Kidney bean AP2 proteinoid 001G131300g, mung bean AP2 proteinoid At2g41710, clover AP2 proteinoid of this gene amino acid sequence and soybean all reaches more than 85% after being analyzed by Blast on NCBI website.
5. the cloning process of peanut drought stress AhAP2ER gene according to Claims 1-4, is characterized in that: the nucleotide sequence of described AhAP2ER gene is SEQUENCE1 in sequence table.
6., according to clone and the functional expression method of peanut drought stress AhAP2ER gene described in claim 1 to 5, it is characterized in that: the aminoacid sequence of described AhAP2ER gene is SEQUENCE2 in sequence table.
7. according to clone and the functional expression method of peanut drought stress AhAP2ER gene described in claim 1 to 6, it is characterized in that: use fluorescence quantitative RT-RCR to analyze the expression under AhAP2ER gene arid, its method is as follows:
Fluorescence quantitative RT-RCR cDNA template used is diluted to 8ng/ μ L, polysaccharase be SYBRGreen, the instrument of employing is 7500FAST quantitative real time PCR Instrument, every reaction system add 2 μ L dilute cDNA;
PCR response procedures is as follows: (a) 95 DEG C, 10s; (b) 95 DEG C, 5s; (c) 60 DEG C, 30s; (d) 72 DEG C, 10s; 40 circulations; Draw solubility curve, it is that every 10s increases by 0.5 DEG C that temperature increases gradient, and Actin is the reference gene of RT-PCR.
8. the clone of peanut drought stress AhAP2ER gene and functional expression method according to claim 7, is characterized in that: the primer sequence of described AhAP2ER fluorescent quantitation checking is: AhAP2ER-R:5 '-ACCTACTGAGCCATACCA-3 ' and AhAP2ER-F:5 '-TTGTAAGCAGCGGATTGT-3 '.
9. the clone of peanut drought stress AhAP2ER gene and functional expression method according to claim 7, is characterized in that: described reference gene Actin the primer sequence is: Actin-F:5 '-GAGGAGAAGCAGAAGCAAGTTG-3 ' and AhAP2ER-F:5 '-AGACAGCATATCGGCACTCATC-3 '.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN113584052A (en) * | 2021-08-24 | 2021-11-02 | 山东省花生研究所 | Peanut transcription factor AhbHLH10 gene and cloning and functional expression method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060340A (en) * | 2013-01-30 | 2013-04-24 | 山东省花生研究所 | Cloning and function expression method of adversity stress AhMYBL6 gene in peanut |
| CN103555738A (en) * | 2013-10-25 | 2014-02-05 | 山东省花生研究所 | Cloning and function-expressing methods of peanut adversity stress AhROLP1 gene |
-
2015
- 2015-12-28 CN CN201510992654.1A patent/CN105462989A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060340A (en) * | 2013-01-30 | 2013-04-24 | 山东省花生研究所 | Cloning and function expression method of adversity stress AhMYBL6 gene in peanut |
| CN103555738A (en) * | 2013-10-25 | 2014-02-05 | 山东省花生研究所 | Cloning and function-expressing methods of peanut adversity stress AhROLP1 gene |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN113584052A (en) * | 2021-08-24 | 2021-11-02 | 山东省花生研究所 | Peanut transcription factor AhbHLH10 gene and cloning and functional expression method thereof |
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