CN107058334A - One cultivate peanut the genes of transcription factor AhJ11 FAR1 5 clone and functional expression method - Google Patents
One cultivate peanut the genes of transcription factor AhJ11 FAR1 5 clone and functional expression method Download PDFInfo
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- 241001553178 Arachis glabrata Species 0.000 title claims abstract 16
- 102100022366 Fatty acyl-CoA reductase 1 Human genes 0.000 title abstract 6
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Abstract
The present invention relates to biological gene engineering field, the specifically clone of the genes of peanut transcription factor AhJ11 FAR1 5 and functional expression method.Preparation and processing, RNA extraction and cDNA synthesis, RACE clone technology of the present invention by material, have synthesized AhJ11 FAR1 5.The present invention is also analyzed the expression under the gene drought stresses of AhJ11 FAR1 5 by using fluorescent quantitation RT PCR, and finally show that the genes of AhJ11 FAR1 5 play a significant role in adaptability of the peanut to drought stress.The present invention imports the gene in peanut by transgenic approach, and transfer-gen plant has obvious Characteristics of Drought than control group, illustrates that the genes of AhJ11 FAR1 5 can significantly improve Drought Resistance in Peanut.
Description
【Technical field】
The present invention relates to biological gene engineering field, specifically one cultivates peanut transcription factor AhJ11-FAR1-5 genes
Clone and functional expression method.
【Background technology】
China is Peanut big producer, and cultivated area occupies second place of the world, and total output accounts for Peanut total output
More than 40%, rank first in the world.But, the further development of peanut industry is threatened by drought.According to statistics, China is every
Year peanut underproduction caused by arid is up to 30%~50%.In addition to yield declines, arid is it is also possible to cause aspergillus flavus poison
A series of consequences such as plain pollution, quality decline.In addition, with the increase of the arid climate frequency of occurrences, if under environment stress
Keep even increasing the yield of peanut, a direction as scientific research.
With the development of science and technology, using transgenic technology improve plant stress tolerance research achieve it is huge enter
Step.After stress signal is produced, peanut can start a series of corresponding signals, finally be transmitted to related gene, start the gene table
Up to assist peanut to adapt to or resist adverse circumstance.Transcription factor is the key factor for regulating and controlling these gene expressions.Transcription factor refers to
Can with eukaryotic gene promoter region cis-acting elements occur specific effect DBP, by them it
Between and the interaction between other GAP-associated protein GAPs, the expression of controlling gene.
FARl obtains (Hudson et in 1999 as the positive regulatory factor of phyA signal paths by map based cloning
a1.1999).Found in subsequent research, the transcription factor contains the transposase MuRA of one and corn Mutator families
(Hudsonet a1.2003) and the similar sequences of transposase Jittery (Lisch, 2002；Xu et al.2004).As
One origin is evolved in the gene new family of transposons, and FARl can stably be present in arabidopsis gene group, and not examine
Measure significant inverted terminal repeat sequence (TIR) and other transposons similar structures (Hudsonet a1.2003).FARl
The transposons similar with Mutator possesses similar structure, including N-terminal C2H2 Zinc finger domains, the swivel base daughter nucleus that middle part speculates
Core structure domain and C-terminal SWIM Zinc finger domains.Wherein N-terminal C2H2 domains have DNA binding activity, and C-terminal domain has
Transcriptional activation activity.Based on this evolutionary process, FARl becomes a kind of new transcription factor, identification FBS that can be special
(FHY3-binding motif, CACGCGC) cis-acting elements (Linet a1.2007), therefore, it is possible to containing in promoter
The gene specific for having FBS sequences is combined, and regulates and controls their expression, exercises biological function.Up to the present, FARl in peanut
The research report of gene is less, and the research report on its function is less.
【The content of the invention】
The purpose of the present invention not enough is cultivated peanut transcription factor AhJ11-FAR1-5 bases there is provided one for of the prior art
The clone of cause and functional expression method, significantly improve the drought-resistant ability of peanut.
Cultivated peanut the invention provides one the cloning process of transcription factor AhJ11-FAR1-5 genes, mainly including following step
Suddenly：
(a) preparation and processing of material, it includes sprouting, growth and the drought stress processing of peanut seed.Specific step
It is rapid as follows：Material selects peanut varieties J11, and peanut seed is sprouted in the culture of Hoagland nutrient solutions, sprouts and growth of seedling bar
Part is 14h illumination/10h dark, and temperature is 26-28 DEG C, and growth is used for follow-up drought stress for 12 days and handled；
Drought stress is immersed in 15%PEG6000 solution with PEG6000 processing, Roots of Peanut, processing 0h, 6h, 12h,
Take the root of peanut as material when 18h, 24h, 36h and 48h, all material is stored in standby in -80 DEG C of ultra low temperature freezers.
(b) RNA of peanut seedling extraction and cDNA synthesis
It is comprised the following steps that：With TAKARA MiniBEST Universal RNA Extraction Kit kits
Method separation and Extraction peanut seedling RNA, carries out cDNA synthesis after obtained RNA is removed into DNA pollution, is tried with SMART-RACE again
Agent cassette method carries out cDNA synthesis, afterwards saves backup reverse transcription product in -20 DEG C of low temperature refrigerators, vessel used above are all
Need to be by removing RNase processing.0.1% DEPC of vessel soaks 12 hours, then autoclaving removal.Solution reagent is used
0.1% DEPC processing (37 DEG C place 12 hours after autoclave sterilization), the reagent of non-refractory is directly prepared with DEPC-H2O.
(c) cloned by RACE
Comprise the following steps that:
RACE used kits are Clontech SMART-RACE kits, and RACE amplification gene total length the primers are
3-GPS-AhJ11-FAR1-5:5’-CGTGCTGAGGGTGCAGAAATGA-3’、5-1-AhJ11-FAR1-5:5’-
TGTTGCCCGCCGAGAAAGATG-3’、5-2-AhJ11-FAR1-5:5’-CGTAGCAACGCCCTTGATCTGTT-3’、5-3-
AhJ11-FAR1-5:5 '-ATCCGACATCATCTCACCATCCAC-3 ' and NGPS-AhJ11-FAR1-5：5’-
GTATAATCAGTCACTGGGAAG-3。
RACE clone products use UNIQ-10 PCR Purification Kit after the separation of 1% agarose gel electrophoresis
Kit (Shanghai life work) recovery purifying.(Beijing is the limited public affairs of golden biochemical technology entirely to purified product with pGEM-T Easy carriers
Department) connection after convert into competence Escherichia coli (Beijing is golden biochemical technology Co., Ltd entirely), LB medium cultures, at random
10 positive colonies of picking are enlarged culture, and whether bacterium solution PCR amplifications pre-detection has Insert Fragment and be sequenced.
Total length pcr amplification product is designed after the result of sequencing is spliced, with UNIQ-10 PCR Purification
Kit kits, purified product is converted into competence Escherichia coli after being connected with pGEM-T Easy carriers, LB culture mediums
Culture, random 10 positive colonies of picking are enlarged culture, and whether bacterium solution PCR amplifications pre-detection has Insert Fragment and be sequenced,
Amplimer is AhJ11-FAR1-5-S1：5 '-CGCAGTGGTTTCCAATGGATTT-3 ' and AhJ11-FAR1-5-S2：5’-
GCCACACCTGGGTTGGTGGACCCC-3’。
Further, total length PCR amplification polymerase used is TAKARA PCR MIX, added in 20 μ L systems with
Lower composition：10 μ LTAKARA PCR MIX, the 1 total cDNA of μ L, 0.5 μ L AhAP2ER-S1,0.5 μ L AhAP2ER-S2 and 8 μ L without
Bacterium distilled water；
Total length pcr amplification reaction condition：a)94℃5min；(b)94℃1min；57℃1min；72℃4min；Altogether
30cycles；(c)72℃10min.
Further, AhJ11-FAR1-5 gene opens reading frame of the present invention is 2727bp, and 908 ammonia are encoded altogether
Base acid.
Further, the amino acid sequence of AhJ11-FAR1-5 genes of the present invention passes through on NCBI websites
The FAR1-5 albumen (XP_016184350.1) of the gene amino acid sequence and Arachis ipaensis is found after Blast analyses
Similitude be 94%, Arachis duranens GAP-associated protein GAPs (XP_015950391.1)) similitude be 68%, see Fig. 1.
Further, the nucleotide sequence of AhJ11-FAR1-5 genes of the present invention is SEQUENCE1 in sequence table.
Further, the amino acid sequence of AhJ11-FAR1-5 genes of the present invention is SEQUENCE2 in sequence table.
Further, present invention also offers the functional expression side of the peanut transcription factor AhJ11-FAR1-5 genes
Method, it is analyzed the expression under AhJ11-FAR1-5 genes arid using fluorescence quantitative RT-RCR, and its method is as follows：
CDNA templates used in fluorescence quantitative RT-RCR are diluted to 8ng/ μ L, and polymerase is SYBR Green, use
Instrument is 7500FAST quantitative real time PCR Instruments, the cDNA for adding 2 μ L to dilute per reaction system；
PCR response procedures are as follows：(a) 95 DEG C, 10s；(b) 95 DEG C, 5s；(c) 60 DEG C, 30s；(d) 72 DEG C, 10s；40 are followed
Ring；Solubility curve is drawn, temperature increase gradient is to increase by 0.5 DEG C per 10s, and Actin is RT-PCR reference gene.
The primer sequence of described AhJ11-FAR1-5 fluorescent quantitations is：AhJ11-FAR1-5-R：5’-
AGGACTACTTCAAGATGTG-3 ' and AhJ11-FAR1-5-F:5’-GCTGTTACTTCATTATTACGA-3’.
Described reference gene Actin the primer sequences are：Actin-F:5’-GAGGAGAAGCAGAAGCAAGTTG-
3 ' and Actin-R:5’-AGACAGCATATCGGCACTCATC-3’.
The present invention demonstrates expression pattern of the AhJ11-FAR1-5 genes under drought stress by quantitative fluorescent PCR, ties
Fruit shows that transcriptional level is increased significantly under the gene drought stress.Figure it is seen that the gene is in drought stress processing
Relative expression quantity is constantly in the trend of rising afterwards.Result above shows AhJ11-FAR1-5 genes in peanut to drought stress
Played a significant role in adaptability.The gene is imported in peanut by transgenic approach, transfer-gen plant has obvious anti-
Non-irrigated characteristic, illustrates that AhJ11-FAR1-5 genes can significantly improve Drought Resistance in Peanut.
【Brief description of the drawings】
Fig. 1 is that peanut AhJ11-FAR1-5 albumen is compared with FAR1-5 albuminoids amino acid sequence in other peanuts；
Fig. 2 is expression pattern analysis of the peanut AhJ11-FAR1-5 genes under drought stress.
【Embodiment】
The technology path of the present invention is described in further details below in conjunction with specific embodiment, but is not to the present invention
Further restriction.
1.1 implement material：
Material prepares and processing：Material selects peanut varieties J11, and peanut seed is sprouted in the culture of Hoagland nutrient solutions,
Sprout and growth of seedling condition is 14h illumination/10h dark, temperature is 26 DEG C, and growth is used at follow-up drought stress for 12 days
Reason.Drought stress is with PEG6000 processing.Roots of Peanut is immersed in 15%PEG6000 solution, in processing 0h, 6h, 12h, 18h,
Take the root of peanut as material when 24h, 36h and 48h, all material is stored in standby in -80 DEG C of ultra low temperature freezers.
1.2 RNA extraction and cDNA synthesis
Vessel used are all needed by removing RNase processing.Vessel are soaked 12 hours with 0.1% DEPC, and then high pressure is gone out
Bacterium removes.Solution reagent is with 0.1% DEPC processing (37 DEG C place 12 hours after autoclave sterilization), and the reagent of non-refractory is direct
Use DEPC-H2O is prepared.Total RNAs extraction presses TAKARA MiniBEST Universal RNA Extraction Kit operation
It is required that single-stranded cDNA synthesis, referring in particular to Clontech SMART-RACE kit methods.Reverse transcription product is in -20 DEG C of low temperature
Saved backup in refrigerator.
1.3 AhJ11-FAR1-5 gene clonings
Cloned by RACE, RACE used kits try for Clontech SMART-RACE kit methods reference
Agent box explanation.RACE amplification gene total lengths the primer is 3-GPS-AhJ11-FAR1-5:5’-
CGTGCTGAGGGTGCAGAAATGA-3’、5-1-AhJ11-FAR1-5:5’-TGTTGCCCGCCGAGAAAGATG-3’、5-2-
AhJ11-FAR1-5:5’-CGTAGCAACGCCCTTGATCTGTT-3’、5-3-AhJ11-FAR1-5:5’-
ATCCGACATCATCTCACCATCCAC-3 ' and NGPS-AhJ11-FAR1-5：5’-GTATAATCAGTCACTGGGAAG-3.
RACE clone products use UNIQ-10 PCR Purification Kit after the separation of 1% agarose gel electrophoresis
Kit (Shanghai life work) recovery purifying.(Beijing is the limited public affairs of golden biochemical technology entirely to purified product with pGEM-T Easy carriers
Department) connection after convert into competence Escherichia coli (Beijing is golden biochemical technology Co., Ltd entirely), LB medium cultures, at random
10 positive colonies of picking are enlarged culture, bacterium solution PCR amplifications pre-detection whether have Insert Fragment and be sequenced (Sangon,
Shanghai)。
Total length pcr amplification product is designed after the result of sequencing is spliced, PCR amplifications polymerase used is TAKARA
PCR MIX, add following component 10 μ LTAKARA PCR MIX, 1 μ L total cDNA, 0.5 μ L AhAP2ER- in 20 μ L systems
S1,0.5 μ L AhAP2ER-S2,8 μ L aseptic double-distilled waters.Reaction condition：94℃5min→(94℃1min→57℃1min→72
℃4min)30cycles→72℃10min.UNIQ-10 PCR Purification Kit kits (Shanghai life work) reclaim pure
Change.Purified product converts big to competence after being connected with pGEM-T Easy carriers (Beijing is golden biochemical technology Co., Ltd entirely)
In enterobacteria (Beijing is golden biochemical technology Co., Ltd entirely), LB medium cultures, random 10 positive colonies of picking are expanded
Whether big culture, bacterium solution PCR amplifications pre-detection has Insert Fragment and is sequenced (Sangon, Shanghai).
Amplimer：AhJ11-FAR1-5-S1：5 '-CGCAGTGGTTTCCAATGGATTT-3 ' and AhJ11-FAR1-5-
S2：5’-GCCACACCTGGGTTGGTGGACCCC-3’.
1.4 quantitative fluorescent PCR
Functional expression is carried out to AhAP2ER genes with quantitative fluorescent PCR：CDNA templates are diluted to used in quantitative fluorescent PCR
8ng/ μ L, polymerase is SYBR Green, and instrument is 7500FAST quantitative real time PCR Instruments (ABI company), is often reacted
System adds the cDNA that 2 μ L dilute；PCR response procedures are as follows：95℃10s；95 DEG C of 5s, 60 DEG C of 30s, 72 DEG C of 10s, 40 circulations；
Solubility curve is drawn, temperature raises 0.5 DEG C per 10s.Relative expression quantity is calculated with 2- Δ Δs CT.Actin is RT-PCR internal reference
Gene.
The primer sequence of AhJ11-FAR1-5 gene fluorescence quantitative RT-PCRs is：AhJ11-FAR1-5-R：5’-
AGGACTACTTCAAGATGTG-3 ' and AhJ11-FAR1-5-F:5’-GCTGTTACTTCATTATTACGA-3’.
Reference gene Actin the primer sequences are：Actin-F:5′-GAGGAGAAGCAGAAGCAAGTTG-3′；With
Actin-R:5′-AGACAGCATATCGGCACTCATC-3′。
2 result of the tests
The amino acid sequence of 2.1 AhJ11-FAR1-5 nucleotides total lengths and its coding
Expanded and be sequenced by PCR, obtained target gene, the gene open reading frame is 2727bp, and 908 are encoded altogether
Amino acid.Fig. 1 is shown the amino acid sequence of the gene is analyzed on NCBI websites by Blast after find the aminopeptidase gene
The similitude of acid sequence and Arachis ipaensis FAR1-5 albumen (XP_016184350.1) is 94%, Arachis
The similitude of duranens GAP-associated protein GAPs (XP_015950391.1) is 68%, therefore is AhJ11-FAR1-5 by the unnamed gene
(Arachis hypogaea J11 FAR1-5-like transcription factor)。
Fig. 2 is expression pattern analysis of the peanut AhJ11-FAR1-5 genes under drought stress.The present invention is fixed by fluorescence
Amount PCR demonstrates expression patterns of the AhJ11-FAR1-5 under drought stress, as a result shows：Water is transcribed under the gene drought stress
Averagely it is increased significantly.Figure it is seen that relative expression quantity is constantly in the trend of rising after drought stress processing.More than
As a result show that AhJ11-FAR1-5 genes play a significant role in adaptability of the peanut to drought stress.By the gene by turning
Gene means are imported in peanut, and transfer-gen plant has obvious Characteristics of Drought than control, illustrates AhJ11-FAR1-5 gene energy
Significantly improve Drought Resistance in Peanut.
It is described above, only it is highly preferred embodiment of the present invention, any formal limitation not is made to the present invention, appoints
What those skilled in the art, without departing from the scope of the technical proposal of the invention, using in the method for the disclosure above
Appearance makes many possible variations and modification to technical solution of the present invention, belongs to the scope of claims protection.
Claims (9)
1. a cloning process for cultivating peanut transcription factor AhJ11-FAR1-5 genes, it is characterised in that comprise the following steps：
(a) preparation and processing of material, includes sprouting, growth and the drought stress processing of peanut seed；
(b) RNA of peanut seedling extraction and cDNA synthesis；
(c) cloned by RACE
RACE used kits are Clontech SMART-RACE kits, and RACE amplification gene total lengths the primer is 3-
GPS-AhJ11-FAR1-5:5’-CGTGCTGAGGGTGCAGAAATGA-3’、5-1-AhJ11-FAR1-5:5’-
TGTTGCCCGCCGAGAAAGATG-3’、5-2-AhJ11-FAR1-5:5’-CGTAGCAACGCCCTTGATCTGTT-3’、5-3-
AhJ11-FAR1-5:5 '-ATCCGACATCATCTCACCATCCAC-3 ' and NGPS-AhJ11-FAR1-5：5’-
GTATAATCAGTCACTGGGAAG-3’；
RACE clone products are after the separation of 1% agarose gel electrophoresis, with UNIQ-10 PCRPurification Kit reagents
Box is purified, and is converted after purified product connection pGEM-T Easy carriers to competence Escherichia coli, in LB medium cultures,
Random picking expands culture, then enters performing PCR augmentation detection and be sequenced；
Total length pcr amplification product is designed after the result of sequencing is spliced, is tried with UNIQ-10PCRPurification Kit
Agent box is purified, and purified product is converted into competence Escherichia coli after being connected with pGEM-T Easy carriers, is trained with LB culture mediums
Support, random 10 positive colonies of picking are enlarged culture afterwards, whether have Insert Fragment simultaneously using bacterium solution PCR amplifications pre-detection
Sequencing, amplimer is AhJ11-FAR1-5-S1：5 '-CGCAGTGGTTTCCAATGGATTT-3 ' and AhJ11-FAR1-5-S2：
5’-GCCACACCTGGGTTGGTGGACCCC-3’。
2. the cloning process of peanut transcription factor AhJ11-FAR1-5 genes according to claim 1, it is characterised in that：Total length
PCR amplifications polymerase used is TAKARA PCR MIX, and total length pcr amplification reaction system includes 10 μ LTAKARA PCR MIX, 1
The total cDNA of μ L, 0.5 μ L AhJ11-FAR1-5-S1,0.5 μ L AhJ11-FAR1-5-S2 and 8 μ L aseptic double-distilled waters；
Total length pcr amplification reaction condition is as follows：(a)94℃5min；(b)94℃1min；57℃1min；72℃4min；Altogether
30cycles；(c)72℃10min.
3. the cloning process of peanut transcription factor AhJ11-FAR1-5 genes according to claim 1 or claim 2, it is characterised in that
AhJ11-FAR1-5 gene opens reading frame is 2727bp, and 908 amino acid are encoded altogether.
4. the cloning process of peanut transcription factor AhJ11-FAR1-5 genes according to claim 3, it is characterised in that：
The nucleotide sequence of AhJ11-FAR1-5 genes is SEQUENCE1 in sequence table.
5. the cloning process of peanut transcription factor AhJ11-FAR1-5 genes according to claim 4, it is characterised in that：
The amino acid sequence of AhJ11-FAR1-5 genes is SEQUENCE2 in sequence table.
6. include the functional expression method of peanut transcription factor AhJ11-FAR1-5 genes as claimed in claim 1.
7. the functional expression method of peanut transcription factor AhJ11-FAR1-5 genes according to claim 6, it is characterised in that：
The expression under transcription factor AhJ11-FAR1-5 genes arid is analyzed using fluorescence quantitative RT-RCR, its method is as follows：
CDNA templates used in fluorescence quantitative RT-RCR are diluted to 8ng/ μ L, using SYBR Green polymerases；
PCR response procedures are as follows：(a) 95 DEG C, 10s；(b) 95 DEG C, 5s；(c) 60 DEG C, 30s；(d) 72 DEG C, 10s；40 circulations；
Solubility curve is drawn, temperature increase gradient is to increase by 0.5 DEG C per 10s, and RT-PCR reference gene is Actin.
8. the functional expression method of peanut transcription factor AhJ11-FAR1-5 genes according to claim 6, it is characterised in that：
The primer sequence of fluorescence quantitative RT-RCR is：AhJ11-FAR1-5-R：5 '-AGGACTACTTCAAGATGTG-3 ' and AhJ11-
FAR1-5-F:5’-GCTGTTACTTCATTATTACGA-3’。
9. the clone of peanut transcription factor AhJ11-FAR1-5 genes and functional expression method according to claim 7, it is special
Levy and be：The Actin the primers sequence is：Actin-F:5 '-GAGGAGAAGCAGAAGCAAGTTG-3 ' and Actin-R:
5’-AGACAGCATATCGGCACTCATC-3’。
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| 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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