CN107058334B - Cloning and functional expression method of peanut transcription factor AhJ11-FAR1-5 gene - Google Patents
Cloning and functional expression method of peanut transcription factor AhJ11-FAR1-5 gene Download PDFInfo
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Abstract
The invention relates to the field of biological genetic engineering, in particular to a cloning and functional expression method of a peanut transcription factor AhJ11-FAR1-5 gene. The AhJ11-FAR1-5 is synthesized by the preparation and treatment of materials, the extraction of RNA, the synthesis of cDNA and the RACE cloning technology. The invention also analyzes the expression of the AhJ11-FAR1-5 gene under drought stress by using fluorescent quantitative RT-PCR, and finally obtains that the AhJ11-FAR1-5 gene plays an important role in the adaptability of peanuts to the drought stress. The gene is introduced into the peanut by a transgenic means, and a transgenic plant has obvious drought resistance compared with a control group, which shows that the AhJ11-FAR1-5 gene can obviously improve the drought resistance of the peanut.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the field of biological genetic engineering, in particular to a cloning and functional expression method of a peanut transcription factor AhJ11-FAR1-5 gene.
[ background of the invention ]
China is a world large country for peanut production, the planting area is the second place in the world, and the total production accounts for more than 40% of the total production of peanuts in the world and is the first place in the world. However, further development of the peanut industry is threatened by drought. According to statistics, the yield reduction of peanuts caused by drought in China reaches 30% -50% every year. Besides the yield reduction, drought can also cause a series of consequences such as peanut aflatoxin pollution and quality reduction. Furthermore, with the increasing frequency of drought climate, it is a direction of scientific research if the yield of peanuts is maintained or even increased under adversity stress.
With the development of science and technology, the research of improving the stress tolerance of plants by using a transgenic technology makes great progress. When a stress signal is generated, peanuts can start a series of corresponding signals, and finally, the signals are transmitted to related genes, so that the expression of the genes is started to assist the peanuts to adapt or resist the adverse environment. Transcription factors are important factors in regulating the expression of these genes. Transcription factors are DNA binding proteins that specifically interact with cis-acting elements in the promoter region of eukaryotic genes, and regulate gene expression through interactions between them and other related proteins.
FARl was obtained by map-based cloning in 1999 as a positive regulator of the phyA signaling pathway (Hudson et a 1.1999). In subsequent studies, it was found that the transcription factor contained a sequence similar to the transposase MuRA (Hudsonet a1.2003) and transposase Jitter y of the maize Mutat family (Lisch, 2002; Xu et al 2004). As a new family of genes evolved from transposons, FARl could stably exist in the arabidopsis genome and no significant Terminal Inverted Repeats (TIR) and other transposon-like structures were detected (Hudsonet a 1.2003). Similar transposons to FARl and Mutator share a similar structure, including an N-terminal C2H2 zinc finger domain, a central putative transposon core domain, and a C-terminal SWIM zinc finger domain. Wherein the N-terminal C2H2 domain has DNA binding activity and the C-terminal domain has transcription activation activity. Based on the evolution process, FARl becomes a novel transcription factor, can specifically recognize cis-acting elements (Linet a1.2007) of FBS (FHY3-binding motif, CACGCGCGC), and therefore can be specifically combined with genes containing FBS sequences on promoters, regulate the expression of the genes and perform biological functions. So far, few studies on the FARl gene in peanuts have been reported, and few studies on the function of the FARl gene have been reported.
[ summary of the invention ]
The invention aims to provide a cloning and functional expression method of a peanut transcription factor AhJ11-FAR1-5 gene aiming at the defects in the prior art, and the drought resistance of peanuts is obviously improved.
The invention provides a cloning method of a peanut transcription factor AhJ11-FAR1-5 gene, which mainly comprises the following steps:
(a) preparation and treatment of materials, including germination, growth and drought stress treatment of peanut seeds. The method comprises the following specific steps: selecting a peanut variety J11 as a material, culturing and germinating peanut seeds in a Hoagland culture solution, wherein the conditions of germination and seedling growth are 14h of light/10 h of darkness and 26-28 ℃, and the temperature is used for subsequent drought stress treatment after 12 days of growth;
treating drought stress with PEG6000, soaking peanut root in 15% PEG6000 solution, taking peanut root as material at 0h, 6h, 12h, 18h, 24h, 36h and 48h, and storing all materials in an ultra-low temperature refrigerator at-80 deg.C for use.
(b) RNA extraction and cDNA Synthesis of peanut seedlings
The method comprises the following specific steps: separating and extracting peanut seedling RNA by using a method of a MiniBEST Universal RNA Extraction Kit of TAKARA, removing DNA pollution from the obtained RNA, then performing cDNA synthesis, performing cDNA synthesis by using a SMART-RACE Kit method, and then storing a reverse transcription product in a low-temperature refrigerator at the temperature of-20 ℃ for later use, wherein all the vessels need to be treated by removing RNase. The ware was soaked with 0.1% DEPC for 12 hours and then autoclaved to remove. The solution reagent was treated with 0.1% DEPC (autoclaved after 12 hours at 37 ℃), and the reagent that did not resist high temperature was prepared directly from DEPC-H2O.
(c) Cloning by RACE
The method comprises the following specific steps:
the kit used by RACE is Clontech SMART-RACE kit, and primers used by RACE amplification gene full length 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-FAR 1-5: 5' -GTATAATCAGTCACTGGGAAG-3.
The RACE clone was isolated by 1% agarose gel electrophoresis and recovered and purified using UNIQ-10PCR Purification Kit (Shanghai Biotech). Connecting the purified product with pGEM-T Easy vector (Beijing is all gold Biochemical technology Co., Ltd.), transforming into competent Escherichia coli (Beijing is all gold Biochemical technology Co., Ltd.), culturing in LB culture medium, randomly selecting 10 positive clones for amplification culture, and detecting whether insert fragment exists or not by PCR amplification of bacterial liquid and sequencing.
Splicing sequencing results, designing a full-length PCR amplification product, purifying by using a UNIQ-10PCR Purification Kit, connecting the purified product with a pGEM-T Easy vector, converting into competent escherichia coli, culturing by using an LB (lysogeny broth) culture medium, randomly selecting 10 positive clones for amplification culture, carrying out PCR amplification on a bacterial liquid to detect whether an insert fragment exists or not and sequencing, wherein an amplification primer is AhJ11-FAR 1-5-S1: 5'-CGCAGTGGTTTCCAATGGATTT-3' and AhJ11-FAR 1-5-S2: 5'-GCCACACCTGGGTTGGTGGACCCC-3' are provided.
Further, the polymerase used for full-length PCR amplification is TAKARA PCR MIX, and the following components are added into a 20-mu-L system: 10 μ LTAKARA PCR MIX, 1 μ L total cDNA, 0.5 μ L AhAP2ER-S1, 0.5 μ L AhAP2ER-S2 and 8 μ L sterile double distilled water;
full-length PCR amplification reaction conditions: a) 5min at 94 ℃; (b) 1min at 94 ℃; 1min at 57 ℃; 4min at 72 ℃; 30cycles in total; (c) 10min at 72 ℃.
Furthermore, the AhJ11-FAR1-5 gene open reading frame is 2727bp, and 908 amino acids are coded.
Furthermore, the amino acid sequence of the AhJ11-FAR1-5 gene disclosed by the invention is found to have 94% of similarity with the FAR1-5 protein (XP _016184350.1) of Arachis ipaensis and 68% of similarity with the Arachis durans related protein (XP _015950391.1) after being analyzed by Blast on an NCBI website, and the similarity is shown in figure 1.
Furthermore, the nucleotide SEQUENCE of the AhJ11-FAR1-5 gene is SEQUENCE1 in the SEQUENCE table.
Furthermore, the amino acid SEQUENCE of the AhJ11-FAR1-5 gene is SEQUENCE2 in the SEQUENCE table.
Furthermore, the invention also provides a functional expression method of the peanut transcription factor AhJ11-FAR1-5 gene, which uses fluorescent quantitative RT-PCR to analyze the expression of the AhJ11-FAR1-5 gene under drought, and the method comprises the following steps:
diluting cDNA template used by fluorescent quantitative RT-PCR to 8 ng/muL, using SYBR Green as polymerase, using 7500FAST fluorescent quantitative PCR instrument as instrument, adding 2 muL diluted cDNA into each reaction system;
the PCR reaction procedure was as follows: (a)95 ℃ for 10 s; (b)95 ℃ for 5 s; (c) 30s at 60 ℃; (d)72 ℃ for 10 s; 40 cycles; and (3) drawing a dissolution curve, wherein the temperature increase gradient is 0.5 ℃ every 10s, and Actin is an internal reference gene of the RT-PCR.
The primer sequence for AhJ11-FAR1-5 fluorescence quantification is as follows: AhJ11-FAR 1-5-R: 5'-AGGACTACTTCAAGATGTG-3' and AhJ11-FAR1-5-F: 5'-GCTGTTACTTCATTATTACGA-3'.
The primer sequence used by the reference gene Actin is as follows: Actin-F: 5'-GAGGAGAAGCAGAAGCAAGTTG-3' and Actin-R: 5'-AGACAGCATATCGGCACTCATC-3'.
The expression mode of the AhJ11-FAR1-5 gene under drought stress is verified by fluorescent quantitative PCR, and the result shows that the transcription water average of the gene under drought stress is obviously increased. As can be seen from FIG. 2, the relative expression level of the gene is always in an increasing trend after drought stress treatment. The results show that the AhJ11-FAR1-5 gene plays an important role in the adaptability of peanuts to drought stress. The gene is introduced into the peanut by a transgenic means, and a transgenic plant has obvious drought resistance, which shows that the AhJ11-FAR1-5 gene can obviously improve the drought resistance of the peanut.
[ description of the drawings ]
FIGS. 1a and 1b of FIG. 1 are amino acid sequences of peanut AhJ11-FAR1-5 protein compared with FAR1-5-like proteins of other peanuts;
FIG. 2 is the analysis of the expression pattern of peanut AhJ11-FAR1-5 gene under drought stress.
[ detailed description ] embodiments
The technical route of the present invention will be described in further detail with reference to specific examples, but the present invention is not limited thereto.
1.1 implementation materials:
material preparation and treatment: the material is selected from peanut variety J11, peanut seeds are cultured and germinate in Hoagland culture solution, the conditions of germination and seedling growth are 14h of light/10 h of dark, the temperature is 26 ℃, and the peanut seeds grow for 12 days for subsequent drought stress treatment. Drought stress was treated with PEG 6000. Soaking peanut root in 15% PEG6000 solution, treating for 0 hr, 6 hr, 12 hr, 18 hr, 24 hr, 36 hr and 48 hr, taking peanut root as material, and storing all materials in-80 deg.C ultra-low temperature refrigerator for use.
1.2 extraction of RNA and cDNA Synthesis
All vessels need to be treated by removing RNase. The ware was soaked with 0.1% DEPC for 12 hours and then autoclaved to remove. The solution reagent is treated with 0.1% DEPC (sterilized by autoclaving after standing at 37 deg.C for 12 hr), and the reagent not resistant to high temperature is directly treated with DEPC-H2And (O) preparation. Total RNA Extraction Single-stranded cDNA synthesis was required according to the protocol of the MiniBEST Universal RNA Extraction Kit of TAKARA, with specific reference to the SMART-RACE Kit method of Clontech. The reverse transcription product is stored in a low-temperature refrigerator at the temperature of minus 20 ℃ for later use.
1.3 AhJ11-FAR1-5 Gene clone
Cloning was performed by RACE using the kit Clontech SMART-RACE kit protocol reference kit. Primers used for RACE amplification gene full length 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-FAR 1-5: 5' -GTATAATCAGTCACTGGGAAG-3.
The RACE clone was isolated by 1% agarose gel electrophoresis and recovered and purified using UNIQ-10PCR Purification Kit (Shanghai Biotech). The purified product was linked to pGEM-T Easy vector (all Beijing is Jinzhi Biotechnology Co., Ltd.), transformed into competent E.coli (all Beijing is Jinzhi Biotechnology Co., Ltd.), cultured in LB medium, 10 positive clones were randomly selected for amplification culture, and the presence of insert was detected by PCR amplification of bacterial solution and sequenced (Sangon, Shanghai).
The sequencing results were combined to design a full-length PCR amplification product, the polymerase used for PCR amplification was TAKARA PCR MIX, and the following components 10. mu. LTAKARAPCR MIX, 1. mu.L of total cDNA, 0.5. mu.L of AhAP2ER-S1, 0.5. mu.L of AhAP2ER-S2, and 8. mu.L of sterile double distilled water were added to the 20. mu.L system. Reaction conditions are as follows: 94 ℃ 5min → (94 ℃ 1min → 57 ℃ 1min → 72 ℃ 4min)30cycles → 72 ℃ 10 min. The UNIQ-10PCR Purification Kit (Shanghai, Ltd.) was recovered and purified. The purified product was linked to pGEM-T Easy vector (all Beijing is Jinzhi Biotechnology Co., Ltd.), transformed into competent E.coli (all Beijing is Jinzhi Biotechnology Co., Ltd.), cultured in LB medium, 10 positive clones were randomly selected for amplification culture, and the presence of insert was detected by PCR amplification of bacterial solution and sequenced (Sangon, Shanghai).
An amplification primer: AhJ11-FAR 1-5-S1: 5'-CGCAGTGGTTTCCAATGGATTT-3' and AhJ11-FAR 1-5-S2: 5'-GCCACACCTGGGTTGGTGGACCCC-3' are provided.
1.4 fluorescent quantitative PCR
Functional expression of AhAP2ER gene by fluorescent quantitative PCR: the cDNA template used for the fluorescent quantitative PCR was diluted to 8 ng/. mu.L, the polymerase used was SYBR Green, the apparatus used was 7500FAST fluorescent quantitative PCR apparatus (ABI Co.), and 2. mu.L of diluted cDNA was added to each reaction system; the PCR reaction procedure was as follows: 10s at 95 ℃; 5s at 95 ℃, 30s at 60 ℃, 10s at 72 ℃ and 40 cycles; the dissolution curve was plotted with a temperature rise of 0.5 ℃ every 10 s. The relative expression was calculated as 2- Δ Δ CT. Actin is an internal reference gene of RT-PCR.
AhJ11-FAR1-5 gene fluorescent quantitative RT-PCR primer sequence is: AhJ11-FAR 1-5-R: 5'-AGGACTACTTCAAGATGTG-3' and AhJ11-FAR1-5-F: 5'-GCTGTTACTTCATTATTACGA-3'.
The primer sequence of the reference gene Actin is as follows: Actin-F: 5'-GAGGAGAAGCAGAAGCAAGTTG-3'; and Actin-R: 5'-AGACAGCATATCGGCACTCATC-3'.
2 results of the test
2.1AhJ11-FAR1-5 full-length nucleotide and coded amino acid sequence thereof
The target gene is obtained by PCR amplification and sequencing, the open reading frame of the gene is 2727bp, and 908 amino acids are coded. FIG. 1 shows that the amino acid sequence of the gene has 94% similarity to FAR1-5 protein (XP-016184350.1) of Arachis ipaensis and 68% similarity to Arachis durans related protein (XP-015950391.1) after Blast analysis on NCBI website, so the gene is named AhJ11-FAR1-5(Arachis hypaea J11 FAR1-5-like transcription factor).
FIG. 2 is the analysis of the expression pattern of peanut AhJ11-FAR1-5 gene under drought stress. The invention verifies the expression mode of AhJ11-FAR1-5 under drought stress through fluorescent quantitative PCR, and the result shows that: the transcription level of the gene under drought stress is obviously increased. As can be seen from FIG. 2, the relative expression level after drought stress treatment was always in an increasing trend. The results show that the AhJ11-FAR1-5 gene plays an important role in the adaptability of peanuts to drought stress. The gene is introduced into the peanut by a transgenic means, and a transgenic plant has obvious drought resistance compared with a contrast, which shows that the AhJ11-FAR1-5 gene can obviously improve the drought resistance of the peanut.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and those skilled in the art can make many variations and modifications of the present invention without departing from the scope of the present invention as defined in the appended claims.
Claims (4)
1. The application of a peanut transcription factor AhJ11-FAR1-5 gene in improving the drought stress resistance of peanuts is characterized in that: the AhJ11-FAR1-5 gene open reading frame is 2727bp, 908 amino acids are coded, the nucleotide SEQUENCE of the AhJ11-FAR1-5 gene is SEQUENCE1 in a SEQUENCE table, the amino acid SEQUENCE of the AhJ11-FAR1-5 gene is SEQUENCE2 in the SEQUENCE table, and the AhJ11-FAR1-5 gene is introduced into peanuts through a transgenic technology and is used for improving the drought resistance of the peanuts.
2. The use of the peanut transcription factor AhJ11-FAR1-5 gene as claimed in claim 1 for improving the drought stress resistance of peanuts, wherein the gene comprises: the transcription factor AhJ11-FAR1-5 gene expression under drought is analyzed by using fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction), and the method comprises the following steps:
diluting cDNA template used by fluorescent quantitative RT-PCR to 8 ng/muL, and adopting SYBR Green polymerase;
the PCR reaction procedure was as follows: (a)95 ℃ for 10 s; (b)95 ℃ for 5 s; (c) 30s at 60 ℃; (d)72 ℃ for 10 s; 40 cycles; and (4) drawing a dissolution curve, wherein the temperature increase gradient is 0.5 ℃ every 10s, and the internal reference gene of the RT-PCR is Actin.
3. The use of the peanut transcription factor AhJ11-FAR1-5 gene as claimed in claim 2 for improving the drought stress resistance of peanuts, wherein the gene comprises: the primer sequence for fluorescent quantitative RT-PCR is as follows: AhJ11-FAR 1-5-R: 5'-AGGACTACTTCAAGATGTG-3' and AhJ11-FAR1-5-F: 5'-GCTGTTACTTCATTATTACGA-3'.
4. The use of the peanut transcription factor AhJ11-FAR1-5 gene as claimed in claim 2 for improving the drought stress resistance of peanuts, wherein the gene comprises: the primer sequence used by the Actin is as follows: Actin-F: 5'-GAGGAGAAGCAGAAGCAAGTTG-3' and Actin-R: 5'-AGACAGCATATCGGCACTCATC-3'.
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| WO2001077161A2 (en) * | 2000-04-07 | 2001-10-18 | Basf Plant Science Gmbh | Gtp binding stress-related proteins and methods of use in plants |
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