CN102643851A - Prokaryotic expression vector of Arabidopsis transcription factor Dof1 (DNA-binding with one finger) and application of prokaryotic expression vector - Google Patents

Abstract

The invention discloses a prokaryotic expression vector pET32a- Dof1 for highly expressing transcription factors. This vector is a dedicated vector for prokaryotic expression containing the Arabidopsis transcription factor gene ( Dof1 ). The present invention uses RT-PCR to clone the Dof1 gene from the model plant Arabidopsis thaliana, controls its expression in Escherichia coli with the T7 promoter, overexpresses it in Ecoli BL21 when the lactose operon is induced by IPTG, and expresses the recombinant protein It is secreted into the cytoplasm and does not form inclusion bodies. The isolated recombinant protein can be used as an antigen for the preparation of Dof1 antibody.

Description

Prokaryotic expression vector of Arabidopsis transcription factor Dof1 gene and its application

technical field

The invention belongs to the field of genetic engineering, in particular to a prokaryotic expression carrier pET32a- Dof1 of transcription factor gene Dof1 and its application in preparing Dof1 recombinant protein.

Background technique

Dof transcription factor is a plant-specific transcription factor, since the cDNA sequence of the first Dof protein was obtained in maize (Yanagisawa S, Izui K. 1993, J Biol Chem, 268:16028-16036), Dof protein genes in many monocotyledonous and dicotyledonous plants were isolated, such as Arabidopsis, tobacco, pumpkin, potato, barley, wheat, rice, etc. (Yanagisawa S. 2002, Trends Plant Sci, 12:555-560). Studies have shown that Dof transcription factors participate in a variety of biological processes during plant growth and development, including participating in multiple pathways such as light response and carbon and nitrogen metabolism, seed development and germination, plant hormone response, and microtubule system development.

Studies have found that maize Dof1 is in Arabidopsis (Yanagisawa S, Sheen J. Plant Cell, 1998, 10:75-89) and in potato (Yanagisawa S, Izui K. J Biol Chem, 1993, 268:16028-16036; Kumar R, Taware R. Mol Biol Rep, 2009, DOI 10.1007/s 11033- 008-9436-8) overexpression can increase the expression of enzymes related to carbon skeleton synthesis, and then participate in the expression regulation of light-responsive genes. In addition, a kind of protein of corn Dof family-PBF ( Prolamin box Binding Factor) can also be used as a trans-acting factor of storage protein gene to regulate the transcription of seed storage protein gene (Mena M, et al. Plant J, 1998, 16: 53-62).

The Dof gene in wheat plays an important role in regulating light-responsive genes, mainly related to photosynthesis and sucrose transport (Tanaka MY, Takahata Y, Nakayama H, et al. Planta, 2009, DOI 10.1007/s00425-009 -0979-2). The PpDof5 transcription factor of pine trees regulates the expression of GS genes essential for ammonia assimilation during tree development (Rueda-Lopez M, Crespillo R, Canovas FM, et al. Plant J, 2008, 56(1):73-85). The Dof protein gene SRF1 of sweet potato negatively regulates the expression of a vacuolar reverse transcriptase gene Ibßfruct2 to affect the carbon metabolism of roots (Isabel-LaMoneda I, Díaz I, Martínez M, et al. Plant J, 2003, 33:329- 340). The Dof family of transcription factors in rice includes 30 Dof genes, among which the known DNA-binding domain of Dof3 can combine with GARE (Gibberellic Acid Response Element) to participate in gibberellin regulation of the expression of aleurone hydrolase genes in grain germination (Park DH, Lim PO, Kim JS, et al. Plant J, 2003, 34:161~171), OsDof may also participate in the antioxidant process caused by bacteria (Mou ZL, Fan WH, Dong XN, Cell, 2003, 113: 935 -944) and related to photoperiodic flowering regulation (Li DJ, Yang CH, Li XB, et al. Planta, 2009, 229: 1159~1169).

Studies have shown that the Arabidopsis Dof transcription factor family includes 36 genes (Lijavetzky D, Carbonero P, Vicente-Carbajosa J. BMC Evol Biol, 2003, 3:17), Yanagisawa et al. found that the overexpression of maize Dof1 in Arabidopsis can promote the increase of the expression of enzymes related to carbon skeleton synthesis, and the PEPC and pyruvate kinase (PK) genes of Arabidopsis The expressions were all activated, and the enzymatic activities of PEPC and PK were significantly increased. Along with the reduction of glucose content, the content of some free amino acids, especially glutamic acid, was increased, and the nitrogen content of Arabidopsis plants transfected with Dof1 gene was increased by 30%, and the growth of Arabidopsis thaliana was also improved under low nitrogen conditions. The Arabidopsis COG1 gene encodes a Dof transcription factor, which acts as a negative regulator of the phyA and phyB signaling pathways during the transfer of phytochrome signals. Overexpression of COG1 reduces the response of transgenic Arabidopsis plants to red and far-red light. Sensitivity (Ward JM et al. Plant Cell, 2005, 17:475-485). The Dof transcription factor OBP3 is also involved in the regulation of Arabidopsis phytochrome and cryptochrome signal transduction (Iwamoto M, Higo K, Takano M. Plant Cell Environ, 2009, 32(5): 592-603). Kang HG et al found that Dof protein can stimulate auxin (Kang HG, et al. Proc Natl Acad Sci USA, 1997, 94: 7685-7690) and salicylic acid (Kang HG. Plant J, 2000, 21:329-339) generate a response. In addition, Dof transcription factors are also involved in the formation of microtubule organization in plants (Pineau C, et al. Plant J, 2005, 44: 271-289; Konishi, et al. Plant and Cell Physiol, 2007, 48: S197-S197; Yong Guo, et al. The Plant Cell, 2009, 21: 3518–3534), seed germination (Gualberti G, Papi M, Bellucci L, et al. Plant Cell, 2002, 14: 1253~263) and plant flowering process (Fabio Fornara, et al. Developmental Cell, 2009, 17: 75–86).

Because Dof protein has multiple characteristics, people have strengthened the in-depth research on Dof protein, and the overexpression of this transcription factor can be used to improve the genetic manipulation of crop metabolism. Dof1 protein-specific antibody is an effective tool for detecting the expression level of Dof1 protein in plants. So far, there are no reports on prokaryotic expression of Arabidopsis transcription factor Dof1 gene and preparation of Dof1 antibody.

Contents of the invention

The purpose of the present invention is to provide a prokaryotic expression vector (pET32a- Dof1 ) of Arabidopsis transcription factor Dof1, which contains T7 promoter and terminator, bacterial ribosome binding site and a group consisting of 6 amino acids Amino acid tag His-Tag and the full-length sequence of Dof1 gene cDNA.

In the present invention, there is a T7 promoter and a bacterial ribosome binding site RBS upstream of the Dof1 gene, an operator sequence that can be induced by IPTG is located downstream of the T7 promoter, and there is a 6 amino acid sequence immediately upstream of the start codon of the Dof1 gene Composed of histidine tags His-Tag.

The Dof1 gene in the vector of the present invention is derived from the cDNA of Arabidopsis thaliana (ecotype Col), and its accession number in GenBank is NM_104048.3.

Another object of the present invention is to apply the prokaryotic expression vector of the specific cDNA fragment of Arabidopsis Dof1 gene in the preparation of Dof1 recombinant protein and specific peptide. Using the vector provided by the invention to transform Escherichia coli (BL21) can realize the high-level expression of Dof1 protein, and recover the Dof1 protein secreted into the cytoplasm, which can be used for the preparation of Dof1-specific antibody.

In order to realize the above-mentioned purpose of the present invention, the present invention provides following technical scheme:

1. Amplification of the cDNA fragment of the Dof1 gene

Find the gene sequence of the coding region of Arabidopsis thaliana pigment regulatory protein gene Dof1 from GenBank, and design a pair of primers with the following sequence:

Dof15: 5'-CA CCATGG ATCTGACGTCAGC-3', the underline is the enzyme cutting site Nco I,

Dof13: 5'- GTCGAC TCAATTCTTTCTCCATTCTGTTC-3', the underline is the enzyme cutting site Sal I,

RT-PCR was performed on the total RNA of Arabidopsis thaliana ( Ecotype, Columbia ) to obtain the full length of the coding region of Dof1 .

2. Construction of prokaryotic expression vector pET32a- Dof1

(1) Recover and purify the gene fragment of the coding region of Dof1 , connect it to the pMD18-T vector, extract the plasmid DNA by alkaline lysis, and obtain the recombinant vector pMD18- Dof 1 by PCR detection and enzyme digestion detection.

(2) The plasmids pMD18- Dof 1 and pET32a were digested with Nco I and Sal I to obtain the Dof1 gene and pET32a vector fragments respectively, which were recovered after electrophoresis, ligated and transformed to obtain the recombinant plasmid pET32a- Dof1 .

3. Optimization of recombinant protein induction conditions of Dof1 gene

The prokaryotic expression vector pET32a- Dof1 was introduced into E. coli protein expression special receptor strain BL21 by heat stimulation method, and transformed colonies were obtained. IPTG was used to induce the expression of Dof1 recombinant protein, and the expression time, IPTG concentration and temperature conditions were optimized. Optimal expression conditions for recombinant proteins. The experimental results showed that the optimal expression condition of the protein was induced by 1mM IPTG at 28°C for 6h.

Technical advantage and effect of the present invention:

A series of Dof transcription factor genes have been identified from plants, which are involved in regulating the expression of related genes in multiple physiological and biochemical processes of plants. The genetic manipulation of them can improve the quality and resistance of crops. The present invention uses Arabidopsis thaliana cDNA as a template to clone the full length of the Dof1 transcription factor gene and construct its prokaryotic expression vector pET32a- Dof1 , which contains a T7 promoter, a bacterial ribosome binding site and a histidine tag. Transformation of Escherichia coli BL21 with the vector can achieve high-level expression of Dof1 protein, recover the Dof1 protein secreted into the cytoplasm, and prepare antiserum. It lays the foundation for the study of function and molecular function; on the other hand, it provides a technical means for the detection of expression levels of other transcription factors. The method disclosed for the first time the prokaryotic expression of Arabidopsis transcription factor Dof1 gene and the preparation of Dof1 protein antibody.

Description of drawings

Figure 1 is a schematic diagram of electrophoresis detection of Arabidopsis total RNA in the present invention, wherein lanes 1-2 are the proposed total RNA of Arabidopsis thaliana.

Fig. 2 is a schematic diagram of the TA cloning strategy of the Dof1 gene in the present invention.

Figure 3 is a schematic diagram of the amplification and TA clone electrophoresis detection of the Dof1 gene of the present invention, wherein A is reverse-transcribed into cDNA using Arabidopsis RNA as a substrate for RT-PCR, M: λDNA/ Hind III; 1-3: Dof1 RT-PCR amplification product; B is the electrophoresis detection of recombinant plasmid pMD18- Dof1 , M: positive control (plasmid with a molecular weight of 3.3 kb); 1-2: recombinant plasmid pMD18- Dof1 ; C is the expression of recombinant plasmid pMD18- Dof1 PCR detection, M: DL2000; 1: Positive control (PCR product using Arabidopsis cDNA as template); 2-3: PCR product amplified with Dof15 and Dof13 primers using pMD18- Dof1 plasmid as template; D is recombinant plasmid Enzyme digestion detection of pMD18- Dof1 , M: DL2000; 1-2: Nco I and Sal I double digestion product.

Fig. 4 is a schematic diagram of the construction strategy of the prokaryotic expression vector pET32a- Dof1 of the present invention.

Figure 5 is a schematic diagram of electrophoretic detection of pMD18- Dof1 and pET-32a plasmids of the present invention using Nco I and Sal I digestion results, where M: Trans 2K plus; 1-3: pMD18- Dof1 plasmid digestion results; 4-5: pMD18 - Dof1 plasmid control; 6-8: pET-32a plasmid digestion results; 9: pET-32a plasmid control.

Figure 6 is the electrophoresis detection diagram of the gel recovery results of the pMD18- Dof1 and pET-32a plasmids of the present invention after digestion with Nco I and Sal I, where M: Trans 2K plus; 1: the recovered Dof1 fragment; 2: the recovered pET -32a fragment.

Fig. 7 is a schematic diagram of electrophoresis detection of the pET32a- Dof1 plasmid of the present invention, wherein 1-5: pET32a- Dof1 plasmid. 6: pET-32a plasmid control.

Figure 8 is the PCR detection electropherogram of pET32a- Dof1 of the present invention (primers use Dof15 and Dof13), where M: Marker; 1-2: negative control (PCR products amplified with ddH2O and pET32a plasmids as templates respectively); 3- 7: Electrophoresis detection image of PCR amplification product using pET32a- Dof1 plasmid as template; 8: Positive control (PCR product amplified using pMD18- Dof1 plasmid as template).

Fig. 9 is an enzyme-digested electrophoresis detection diagram of pET32a- Dof1 of the present invention, wherein M: Trans 2K plus; 1: double-digested product using Nco I and Sal I.

Figure 10 is the PCR detection electrophoresis of BL21 in the present invention (primers use Dof15 and Dof13), where M: Trans 2K plus; 1: Positive control (PCR product amplified using the pMD18- Dof1 plasmid as a template); 2-3 : PCR product amplified using BL21 bacterial solution transformed into pET32a- Dof1 as template; 4-5: Negative control (PCR product amplified using BL21 bacterial solution transformed into pET-32a as template); 6: Negative control ( PCR products were amplified using the pET-32a plasmid as a template).

Figure 11 is the SDS-PAG electrophoresis detection diagram of the total protein of the present invention, wherein M: Protein Marker-0431; 1: BL21 transformed with pET32a- Dof1 (not induced by IPTG); 2: BL21 transformed with pET-32a (induced without IPTG) ); 3-5: BL21 transfected with pET32a- Dof1 (37 ℃, 1 mM IPTG induced for 2, 4, 6 h); 6: BL21 transformed with pET-32a (37 ℃, 1 mM IPTG induced for 4 h); 7- 9: BL21 transfected with pET32a- Dof1 (28°C, induced with 1 mM IPTG for 2, 4, and 6 h); 6: BL21 transformed with pET-32a (28°C, induced with 1 mM IPTG for 4 h).

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention is not limited to the content described.

The reagents used in this example are mainly molecular biology experiment reagents, various restriction endonucleases, Taq DNA polymerase, dNTP, etc. Co., Ltd. (Dalian), the plasmid extraction kit was purchased from Biotech Biotechnology Co., Ltd., and the rest of the reagents were of domestic analytical grade, and the instruments were commonly used in molecular biology and genetic engineering laboratories.

The primer sequences used were all synthesized in Shanghai Sangong, and the methods used in the examples of the present invention were conventional methods unless otherwise specified.

Example 1: Preparation and detection of Arabidopsis total RNA

Arabidopsis total RNA was extracted using TRIzoL Reagent (RNA extraction reagent, Invitrogen) with slight modifications to the instructions. Take 0.1 g of young Arabidopsis thaliana, add 1 ml of TRIzoL RNA extract, mix well and let stand at room temperature for 5 min, add 0.2 ml chloroform, shake and mix, 4 ℃, 12000 Centrifuge at rpm/min for 15 min. Transfer the supernatant and add 0.5 ml isopropanol, mix well, place at room temperature for 10 min, and then centrifuge at 12000 rpm/min for 10 min. Discard the supernatant, wash the precipitate with 1 ml of 75% ethanol, centrifuge at 7500 rpm/min at 4 °C for 5 min, dry the precipitate in vacuum, and dissolve the RNA in water treated with 20 μl diethyl pyrocarbonate (DEPC), at -20 °C. Take 1ul RNA and use 1.2% agarose gel for electrophoresis detection, the result (Figure 1) shows that the quality of the extracted RNA meets the requirements.

Example 2: Synthesis of Arabidopsis cDNA

Using Arabidopsis total RNA as a template, cDNA was synthesized using RevertAid ^TM -MuLV Reverse Transcriptase Kit (reverse transcription kit, Fermentas). Take 0.1-0.5 μg of plant total RNA, Oligo(dT) 50ng, 10 mM dNTP mix 1 μl, make up to 9 μl with DEPC-treated water, mix well, collect it at the bottom of the tube by brief centrifugation, and place it at a constant temperature of 65 ℃ to dry Heat in a hot heater for 5 min, ice-bath for 10 min, add 11 μl of reaction mixture (10×RT Buffer 4 μl, 25 mM MgCl ₂ 4 μl, 0.1 M DTT 2 μl, RNase inhibitor 1 μl), mix well, briefly Collect it at the bottom of the tube by centrifugation, incubate at 25°C for 2 minutes, add 1 μl RevertAid ^TM -MuLV reverse transcriptase, mix well, incubate at 25°C for 20 minutes, then incubate at 42°C for 70 minutes, keep in an ice bath for 10 minutes, and store at -20°C spare.

Example 3: Amplification and TA cloning of Dof1 gene

The strategy of amplification of Dof1 gene and TA cloning is shown in Figure 2. The first choice is to search for the full-length sequence of the coding region of Dof1 from GenBank, and design a pair of primers. The sequence is as follows:

Dof15: 5'-CA CCATGG ATCTGACGTCAGC-3',

Dof13: 5'- GTCGAC TCAATTCTTTCTCCATTCTGTTC-3',

Primer Dof15 at the 5' end added an Nco I site at the end; primer Dof13 at the 3' end added a Sal I site at the end.

Use Dof1 gene upstream and downstream specific primers Dof15 and Dof13 for RT-PCR, add 3 μl of cDNA as a template to the RT-PCR reaction mixture, add 50ng of specific primers Dof15 and Dof13, 2.5 μl dNTP (2.5 mM,) Add 0.25 μl of Ex taq DNA polymerase (5 U/μl) and 2.5 μl of 10×Ex taq enzyme reaction buffer (Nippon Takabio), and add double distilled water to make the final volume of the reaction 25 μl. Heat at 94°C for 2 minutes on the PCR instrument, then perform 30 cycles of reaction according to the program of 94°C, 30 seconds, 55°C, 30 seconds, 72°C, 45 seconds, and finally extend the reaction at 72°C for 10 minutes. AKT1 gene was amplified by PCR reaction. After the reaction was completed, the PCR amplification product of Dof1 was separated by agarose gel electrophoresis (Figure 3A), the full-length fragment (600 bp) of the Dof1 coding region was recovered and purified, and then cloned using the TA cloning kit of TaKaRa. Connected to the pMD18-T (Dalian Bao Biological Company) carrier, the experimental operation was carried out according to the instructions of the kit, and after overnight reaction, the reaction mixture was used to transform Escherichia coli competent DH5α (purchased from Tiangen Biochemical Technology Company), and the alkaline lysis method Plasmid DNA was extracted, and subjected to 1% agarose gel electrophoresis (Figure 3B), and the recombinant plasmid pMD18 - Dof1 whose size was consistent with the theoretical value was selected for further PCR detection. A DNA fragment of about 600 bp in Dof1 gene could be amplified from the successfully cloned recombinant plasmids (Fig. 3C). According to the multiple cloning sites at both ends of the positive recombinant plasmid pMD18- Dof1 , the recombinant plasmid was digested with Nco I and Sal I, and the digested product was detected by 1% agarose gel electrophoresis, and the recombinant plasmid pMD18- Dof1 was successfully connected to produce 2 One of the bands was about 600 bp of the Dof1 gene DNA insert, and the other was a 2.7 kb vector fragment (Figure 3D). Sequence analysis proved that the insert in the vector was the full-length fragment of the coding region of Dof1 . After reconfirming that the plasmid was successfully connected, re-transform Escherichia coli DH5α, pick a single colony for liquid culture, and purify the plasmid pMD18- Dof1 with the kit.

Example 4: Construction of prokaryotic expression vector pET32a- Dof1

The construction strategy of pET32a- Dof1 is shown in Figure 4. Cut the purified plasmid vectors pET32a (Novagen) and pMD18- Dof1 with Nco I (Fermentas) and Sal I (Fermentas), and separate the cut vectors by agarose gel electrophoresis. Vector and insert fragments (Figure 5), the vector fragment (5.9kb) produced by cutting pET32a and the DNA fragment of Dof1 gene (600 bp) produced by cutting pMD18- Dof1 were recovered from the gel (Figure 6), and then used TaKaRa's ligase kit ligates the pET32a fragment and the DNA fragment of the Dof1 gene to produce prokaryotic expression pET32a- Dof1 . Use the ligation reaction mixture to transform high-efficiency (10 ⁸ ) Escherichia coli competent (DH5α, Tiangen Biochemical Technology), spread the transformed Escherichia coli on the LB plate added with ampicillin (Amp, 100 μg/ml), Cultivate overnight at 37°C, screen Amp-resistant recombinant colonies, extract plasmids from Amp-resistant recombinant colonies (Figure 7), perform PCR amplification detection with upstream and downstream primers of the Dof1 gene (Figure 8), and successfully connect the plasmid vector pET32a- Dof1 can amplify a Dof1 fragment with a size of 600bp. Nco I and Sal I were used to digest the recombinant plasmid for detection, and the bands generated by the successfully ligated plasmid on the agarose gel electrophoresis pattern were consistent with the expected results (Figure 9). After reconfirming the successful connection of the plasmid by sequencing, re-transform Escherichia coli DH5α, pick a single colony and culture it with liquid LB, and purify the plasmid pET32a- Dof1 with the kit.

Example 5: Prokaryotic expression of pET32a- Dof1

Select the correct pET32a- Dof1 plasmid and transform it into Escherichia coli BL21. The specific steps are as follows: add 2 ul of the purified plasmid to 100 μl of competent E. coli BL21, after ice bathing for 20 min, heat stimulation at 42°C for 45 s, and ice bathing for 10 min ; Add 900 μl SOC liquid medium, incubate on a shaker at 37°C and 200 rpm for 60 min; centrifuge at 10,000 rpm for 1 min; absorb the supernatant on the ultra-clean table, and when there is about 100 μl left, use a gun to mix well and transfer to the On the LB plate with Amp resistance, spread evenly with a sterile triangular glass rod; cultivate overnight at 37°C; pick a single clone, inoculate it in LB liquid medium supplemented with Amp, and culture overnight at 37°C on a shaker at 180 rpm. Use the upstream and downstream primers of the Dof1 gene for colony PCR amplification detection, and a 600bp Dof1 band can be found in the positive clones that have been successfully transformed (Figure 10).

Pick the BL21 Escherichia coli strain containing pET32a- Dof1 that was confirmed to be successfully transformed and inoculate it in the LB liquid medium containing Amp. Cultivate on a shaker at 180 rpm for about 2 hours, and measure the OD value of the bacterial solution to 0.6-0.8. After that, after taking out 2 ml of the bacterial liquid, add IPTG (final concentration of 1 mM) to the remaining bacterial liquid for induction, and cultivate them on a shaker at 180 rpm at 37°C and 28°C respectively, and take samples every two hours. The samples were induced with 1 mM IPTG for 2, 4, and 6 h, respectively. Centrifuge the obtained bacterial solution at 4°C and 12000 rpm for 2 min, discard the supernatant, wash twice with 1 ml Washing Buffer (10% glycerol, 100 mM Tris-HCl (pH7.4)), add 100 ul ddH ₂ O , sonicate until the solution is clear, centrifuge at 12000 rpm for 2 min at 4 °C, add 25 ul of 5×Loading Buffer, boil in a boiling water bath for 10 min, and store at -20 °C for protein analysis.

Example 6: SDS-PAGE electrophoresis detection of prokaryotic expression vector pET32a- Dof1

Separating gel (8 ml ddH ₂ O; 10 ml Solution A: 30% acrylamide stock solution; 5.3 ml Solution B: 1.5 M Tris (pH8.8), 0.4% SDS; 0.25 ml 10% ammonium persulfate; 0.013 ml TEMED solution) and stacking gel (6.3 ml ddH ₂ O; 1.6 ml Solution A: 30% acrylamide stock solution; 2.1 ml Solution C: 0.5 M Tris (pH6.8), 0.4% SDS; 0.13 ml 10% ammonium persulfate ; 0.018 ml TEMED solution), 10 ul of each sample was loaded, the voltage was 100 V, the current was 70-90 mA, after electrophoresis, the separation gel was taken out, and the staining solution (0.25g Coomassie brilliant blue dissolved in 90ml methanol: water ( 1:1) and 10mL glacial acetic acid solution to filter out particulate matter) for 30 min, then use decolorization solution (90% methanol, 2% glacial acetic acid) to decolorize for 60 min, remove the staining solution, add distilled water, and shake at 45 rpm to decolorize to the bar with clear. It can be seen that the specific band of Dof1 appears, but there is no such band in the pET32a- Dof1 bacterial solution induced by adding IPTG, and the BL21 bacterial solution transformed by the pET32a empty vector cultured with IPTG (Figure 11). The expression of Dof1 protein was the highest after 1 mM IPTG induction for 6 hours.

SEQUENCE LISTING

<110> Kunming University of Science and Technology

<120> Prokaryotic expression vector of Arabidopsis transcription factor Dof1 gene and its application

<160> 2

<170> Patent In version 3.5

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<211> twenty one

<212> dna

<213> artificial sequence

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caccatggat ctgacgtcag c twenty one

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gtcgactcaa ttcttctcca ttctgttc 28

Claims (4)

1. Arabidopis thaliana Dof1The segmental prokaryotic expression carrier of gene specific cDNA is characterized in that: contain T7 promotor and terminator, bacterial ribosome binding site, one by form histidine-tagged of 6 amino acid and Dof1Special cDNA fragment.

2. Arabidopis thaliana according to claim 1 Dof1The segmental prokaryotic expression carrier of gene specific cDNA is characterized in that: Dof1There are T7 promotor and bacterial ribosome binding site RBS in the upper reaches of gene, and the downstream of T7 promotor have can be by IPTG inductive operator sequence, near Dof1The upstream from start codon of gene fragment is a histidine-tagged sequence His-Tag who is made up of 6 amino acid.

3. according to the Arabidopis thaliana of claim 1 Dof1The segmental prokaryotic expression carrier of gene specific cDNA is characterized in that: transcription factor gene Dof1CDNA derive from Arabidopis thaliana, it is NM_104048.3 in the GenBank accession number.

4. the Arabidopis thaliana of claim 1 Dof1The application of the segmental prokaryotic expression carrier of gene specific cDNA in preparation Dof1 recombinant protein and special peptide section.

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