Soybean high temperature inducible promoter and its application

Abstract

A soybean high-temperature inducible promoter and its application. The present invention relates to a soybean inositol galactosidase synthase gene promoter sequence that can be used as a promoter to regulate gene expression under high-temperature stress. The promoter of the present invention is derived from the promoter sequence of the soybean inositol galactosidase synthase gene GmGolS, named GmGolSP, and its base sequence is shown in SEQ ID NO: 1. The sequence contains a variety of stress-related cis-acting elements. The cis-acting elements are MBS, ARE, TC-rich repeats, ABRE, CGTCA-motif and GC-motif. The soybean high-temperature inducible promoter of the present invention is used to induce heat-resistant gene expression under high-temperature stress, and can initiate the efficient expression of heat-resistant related genes under high-temperature conditions, thereby cultivating practical and effective heat-resistant plant varieties. The present invention is applied to the field of plant genetic engineering.

Description

Technical Field

[0001] The invention relates to a soybean high-temperature inducible promoter and application thereof. Background Art

[0002] Soybean is an important economic crop, cultivated worldwide. It is not only a major source of protein and lipids for humans, but also a vital livestock feed and industrial raw material, and holds great medical value. However, adverse environmental conditions such as low temperatures, drought, and high salinity can affect soybean growth and development, leading to reduced soybean yields and severe economic losses. In addition to conventional breeding techniques, recent advances in molecular biology and the maturity of plant genetic engineering breeding technologies have made it possible to improve soybean stress resistance through genetic engineering.

[0003] A promoter is a DNA sequence upstream of a gene that binds RNA polymerase and transcription factors. It contains multiple cis-acting elements and can regulate gene expression at the transcriptional level. Promoters can be divided into three types based on their mode of action: constitutive, specific, and inducible. In genetic engineering, constitutive promoters such as CaMV35S are commonly used to control target gene expression. While these promoters can overexpress target genes, their constitutive expression is not specific to the specific organism, environment, or time of day. While using constitutive promoters to control the expression of stress-related genes can improve a plant's resilience to adverse conditions, overexpression of the stress-related proteins occurs even under normal plant growth conditions, resulting in a wasteful metabolic process. Furthermore, prolonged over-accumulation of stress-related proteins in plants under normal conditions can negatively impact normal plant growth, leading to morphological abnormalities, growth retardation, and even death in transgenic plants. Inducible promoters, upon stimulation by external signals, can rapidly and precisely regulate the expression of exogenous genes. Compared to constitutive promoters, inducible promoters can minimize the negative effects of gene overexpression on plants. Therefore, cloning and applying stress-inducible promoters can achieve efficient, controllable and specific (site-specific, timing-specific, quantitative) regulation of stress-resistant gene expression, which has great advantages in improving plant stress resistance and has become a hot topic in current research.

[0004] There are several reports of high-temperature-inducible promoters both domestically and internationally. The Arabidopsis AtHSP70b promoter exhibits high-temperature-induced expression in tobacco; the alfalfa MsMBF1c promoter is significantly induced by heat stress; the OsHsfB2c and PM19 promoters cloned from rice are both highly heat-inducible, while the Hsfp promoter is only heat-inducible in young panicles; and the creeping bentgrass HSP26.7 promoter is strongly induced by heat stress, with its activity being higher in reproductive organs. However, overall, the number of high-temperature-inducible promoters currently available for use in genetic engineering is relatively small compared to other types of inducible promoters. Therefore, the cloning and identification of new and effective high-temperature-inducible promoters remains a key research priority. Summary of the Invention

[0005] The purpose of the present invention is to provide a soybean high temperature inducible promoter and its application

[0006] The promoter of the present invention is derived from the promoter sequence of soybean galactinol synthase gene GmGolS and is named GmGolSP. The base sequence thereof is shown in SEQ ID NO: 1.

[0007] The sequence contains multiple stress-related cis-acting elements.

[0008] The invention relates to a recombinant expression vector comprising a soybean high temperature inducible promoter, wherein the original vector of the recombinant expression vector is pCAMBIA1301.

[0009] The soybean high-temperature inducible promoter of the present invention is used in inducing the expression of heat-resistant genes under high-temperature stress.

[0010] The present invention uses real-time fluorescence quantitative PCR to detect the expression of the GmGolS gene in soybean leaves under 42°C heat treatment. GmGolS expression significantly increased, indicating that the GmGolS gene has heat-induced expression properties. Therefore, a soybean genome database was searched for sequences approximately 2000 base pairs upstream of the GmGolS gene. Primers were designed and a 1739-base pair of GmGolS promoter sequences were cloned from the soybean leaf genome, designated GmGolSP. GmGolSP was constructed into the plant expression vector pCAMBIA1301 and transformed into tobacco plants using the leaf disc method. Hygromycin selection and genomic DNA PCR were used to identify GmGolSPT1-generation transgenic tobacco plants. The GmGolSP transgenic tobacco plants were heat-treated at 42°C for 2 hours. GUS histochemical staining and real-time fluorescence quantitative PCR were performed on untreated and heat-treated tobacco leaves to detect GUS reporter gene expression. This confirmed that GmGolSP exhibits heat-induced activation properties under heat stress, indicating that it is a heat-inducible promoter.

[0011] The advantages of the present invention over the prior art are:

[0012] Although the use of constitutive promoters to control the expression of stress-related genes can improve the stress resistance of plants under adverse conditions, the related stress-resistant proteins will be over-expressed even under normal plant growth conditions, which is a waste for plant metabolism. Moreover, the long-term excessive accumulation of stress-resistant proteins in plants under normal conditions will also have a negative impact on the normal growth of plants, causing abnormal morphology, growth retardation or even death of transgenic plants. Adversity-induced promoters only start the expression of downstream genes in large quantities when plants are subjected to adverse stress, which can avoid plant growth defects caused by the continuous expression of exogenous genes in transgenic plants. The present invention cloned a soybean high-temperature inducible promoter. The acquisition of this promoter provides a favorable tool for plant heat-resistant genetic engineering research, which can start the efficient expression of heat-resistant related genes under high temperature conditions, thereby cultivating practical and effective heat-resistant plant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Real-time fluorescence quantitative PCR detection results of GmGolS gene expression levels under untreated 0h and high temperature treatment;

[0014] Figure 2 PCR amplification results of the GmGolS gene promoter sequence, where M is DL2000 Marker and 1 is the amplified band;

[0015] Figure 3 Prediction and analysis of cis-acting elements in GmGolSP sequences;

[0016] Figure 4 The results of double enzyme digestion of the recombinant vector pCAMBIA1301-GmGolSP, where M is DL2000 Marker and 1 is the double enzyme digestion band;

[0017] Figure 5 PCR amplification results of Agrobacterium tumefaciens containing the pCAMBIA1301-GmGolSP recombinant vector, where M is the DL2000 marker and 1 is the PCR amplification band;

[0018] Figure 6 PCR identification results of GmGolSPT0 generation positive transgenic tobacco, where M is DL2000 Marker; 1 is water control; 2 is wild-type tobacco negative control; 3 is pCAMBIA1301-GmGolSP recombinant vector positive control; 4-6 are GmGolSPT0 generation positive transgenic tobacco amplification bands;

[0019] Figure 7 GUS histochemical staining results of untreated wild-type tobacco leaves;

[0020] Figure 8 GUS histochemical staining results of wild-type tobacco leaves treated with high temperature for 2 h;

[0021] Figure 9 GUS histochemical staining results of untreated GmGolSPT1 generation transgenic tobacco leaves;

[0022] Figure 10 The results of GUS histochemical staining of GmGolSPT1 transgenic tobacco leaves after high temperature treatment for 2h;

[0023] Figure 11 GUS histochemical staining results of positive control pCAMBIA1301 transgenic tobacco leaves;

[0024] Figure 12 Real-time fluorescence quantification of GUS gene expression in GmGolSP transgenic tobacco treated with high temperature for 2 h. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is not limited to the specific implementation methods listed below, but also includes any reasonable combination of the specific implementation methods.

[0026] Specific embodiment 1: The sequence of the soybean high temperature inducible promoter in this embodiment is shown as SEQ ID NO: 1 in the sequence listing.

[0027] Specific embodiment 2: This embodiment differs from specific embodiment 1 in that the sequence contains a cis-acting element related to stress. Other steps and parameters are the same as those of specific embodiment 1.

[0028] Specific embodiment 3: This embodiment differs from specific embodiment 1 or 2 in that the cis-acting elements are MBS, ARE, TC-rich repeats, ABRE, CGTCA-motif and GC-motif, respectively. Other steps and parameters are the same as those of specific embodiment 1 or 2.

[0029] In this embodiment, MBS is the drought-induced MYB transcription factor binding site, ARE is the anaerobic inducible element, TC-richrepeats is the defense and stress response element, ABRE is the abscisic acid response element, CGTCA-motif is the jasmonic acid response element, and GC-motif is the hypoxia-inducible element.

[0030] Specific embodiment 4: This embodiment comprises a recombinant expression vector of a soybean high temperature inducible promoter.

[0031] Specific embodiment 5: This embodiment differs from specific embodiment 4 in that the original vector of the recombinant expression vector is pCAMBIA1301. Other steps and parameters are the same as those of specific embodiment 4.

[0032] Specific embodiment 6: This embodiment uses a soybean high-temperature inducible promoter to induce the expression of heat-resistant genes under high temperature.

[0033] Example 1: This example will verify the sequence of the soybean high temperature inducible promoter of the present invention through the following experiments:

[0034] (1) Real-time fluorescence quantitative PCR detection of GmGolSP gene expression under high temperature treatment

[0035] In a 25°C incubator, Hoagland nutrient solution was prepared for hydroculture of soybean seedlings, and the lighting conditions were 16h light / 8h dark. When the first three-leaf compound leaf of the soybean seedlings was fully expanded, high temperature stress treatment was carried out: the soybean seedlings were placed in a 42°C incubator, and 0.1g of the first three-leaf compound leaf of the soybean seedlings was weighed at 0h (untreated) and 1h, 2h, 5h and 10h after high temperature treatment, and quickly placed in liquid nitrogen and stored at -80°C for later use. The total RNA of each of the above samples was extracted using RNAisoPlus reagent (purchased from Takara) and analyzed by agarose gel electrophoresis and OD 260 / 280 The quality of the extracted RNA was tested by RT-PCR. The first-strand cDNA was synthesized using a cDNA reverse transcription kit (purchased from Novoprotein).

[0036] Real-time quantitative PCR primers were designed based on the ORF sequence of the GmGolS gene (GenBank accession number: NM001251098). The upstream primer was 5′-TCTAAGCCTTGGAGGTACACTGG-3′, and the downstream primer was 5′-GGCACGGACGAACTTGACTTC-3′. The soybean constitutively expressed gene β-Tubuin (GenBank accession number: GMU12286) was used as an internal reference gene (F: 5′-GGAAGGCTTTCTTGCATTGGTA-3′; R: 5′-AGTGGCATCCTGGTACTGC-3′) and soybean leaf cDNA was used as a template on a BIO-RADCFX96 Real-Time PCR instrument. The real-time quantitative PCR reaction system was as follows: 10 μL of 2×TB Green Premix Ex Taq II (purchased from Takara), 2 μL of cDNA, and 0.8 μL of each of the upstream and downstream primers, all added to 20 μL with water. The reaction procedure was as follows: pre-denaturation at 95°C for 30 s; 95°C for 5 s, 58°C for 30 s, 40 cycles. Each treatment was repeated 3 times. －△△CtThe relative expression of genes was calculated by the method shown in Table 1 and Figure 1 As shown in Figure 3, the expression level of GmGolS gene was significantly increased under high temperature treatment.

[0037] Table 1 Relative expression of GmGolS gene under high temperature treatment (values ​​are the average of three replicates)

[0038]

[0039] (II) Cloning of the GmGolS gene promoter sequence and analysis of cis-acting elements

[0040] Based on the soybean GmGolS gene cDNA sequence, the soybean genome database GmGDB (http: / / www.plantgdb.org / GmGDB / ) was searched to obtain the promoter sequence of about 2000 bp upstream. Primers were designed using the primer design software Primer5. The sequences are as follows: F: 5'- GTCGAC AGGATTTAGTAACGTAGGGCC-3' (the underline represents the SalⅠ restriction site); R: 5'- CCATGG GATCTCAGTGATGATGAGTGAGTAG-3' (the underline represents the NcoⅠ restriction site). Genomic DNA from soybean leaves was extracted using a plant genomic DNA extraction kit (purchased from TaKaRa). PCR amplification was performed using genomic DNA as a template. The PCR program was as follows: 94°C for 8 minutes; 94°C for 40 seconds, 56°C for 40 seconds, and 72°C for 1 minute for 30 cycles; and extension at 72°C for 8 minutes. The amplified bands obtained were as follows: Figure 2 The amplified fragment was recovered and ligated into the cloning vector pMD18-T (purchased from TaKaRa). The positive plasmid obtained from the screening was named pMD18-T-GmGolSP and sent to Shanghai Bioengineering for sequencing to verify the correctness of the sequence. The PlantCARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) database was used for the prediction and analysis of promoter cis-acting elements.

[0041] The promoter sequence of GmGolS gene GmGolSP was obtained by PCR amplification, which is 1739 bp in length (as shown in SEQ ID NO: 1 in the sequence list). It is predicted that the GmGolSP sequence contains a variety of cis-acting elements related to adversity. Figure 3Cis-acting elements associated with stress include a drought-inducible MYB transcription factor binding site (MBS), two anaerobic-inducible elements (AREs), a defense and stress-responsive element (TC-richrepeats), an abscisic acid-responsive element (ABRE), a jasmonic acid-responsive element (CGTCA-motif), and a hypoxia-inducible element (GC-motif). Homology blasting of the GmGolSP sequence in the NCBI database revealed no promoter sequences with high homology, indicating that GmGolSP is a novel promoter sequence.

[0042] (III) Genetic transformation of tobacco with GmGolSP and identification of transgenic tobacco

[0043] Plant expression vector construction method:

[0044] The pMD18-T-GmGolSP plasmid was double-digested with SalⅠ and NcoⅠ restriction enzymes (purchased from TaKaRa). The digestion product was recovered by agarose gel electrophoresis and then ligated with the pCAMBIA1301 vector (which was also double-digested with SalⅠ and NcoⅠ) (the purpose was to remove the CaMV35S promoter on the pCAMBIA1301 vector). The ligation product was transformed into Escherichia coli DH5α and verified by double-digestion of the plasmid (the results are shown in Figure 2). Figure 4 The recombinant vector was transformed into Agrobacterium EHA105 and detected by PCR (the results are shown in Figure 5 As shown), a transgenic engineering bacterium containing the pCAMBIA1301-GmGolSP recombinant vector was obtained.

[0045] Tobacco NC89 (purchased from China Tobacco Seed Co., Ltd.) was transformed with pCAMBIA1301-GmGolSP using the Agrobacterium infection tobacco leaf disc method. The pCAMBIA1301 empty vector was also used as a positive control. The specific method is as follows:

[0046] 1. Place tobacco seeds in a 1.5 mL centrifuge tube, soak and disinfect with 10% sodium hypochlorite for 3 minutes, and rinse with sterile water 4-5 times;

[0047] 2. Spread tobacco seeds on MS medium, culture conditions: 16h (28℃) light / 8h (22℃) dark;

[0048] 3. Take sterile tobacco leaves that have been grown for 1-2 months after germination and cut them into 0.5cm 2 Small pieces of the culture medium (with the main vein removed) were inoculated into MS differentiation medium (MS supplemented with 3 mg / L 6-BA and 0.2 mg / L NAA) and pre-cultured for 2 days;

[0049] 4. Pick a single clone of pCAMBIA1301-GmGolSP and incubate it in 5 mL of YEP (10 g / L peptone, 10 g / L yeast extract, 5 g / L NaCl) supplemented with 50 μg / mL rifampicin and 50 mg / L kanamycin. Cultivate at 28°C with shaking at 120 rpm for 24 h.

[0050] 5. Transfer the above culture into 50 mL of YEP supplemented with 50 μg / mL rifampicin and 50 μg / mL kanamycin at a ratio of 1:100, and culture at 28°C with shaking at 120 rpm until the OD 600 =0.4-0.5;

[0051] Centrifuge at 65000rpm for 15min at room temperature, remove the supernatant, and resuspend the cells in MS liquid culture concentrate until OD 600 = about 0.5;

[0052] 7. Infect the pre-cultured tobacco leaves in the resuspension for 20 minutes, dry the bacterial liquid on the leaf surface, place them on the co-culture medium (pH adjusted to about 5.4), and co-culture for 3 days;

[0053] 8. Wash the co-cultivated tobacco leaves with MS liquid medium containing 500 mg / L carbenicillin, air dry, and transfer to screening medium (MS supplemented with 3 mg / L 6-BA, 0.2 mg / L NAA, 500 mg / L carbenicillin, and 8 mg / L hygromycin). Subculture once every 15 days.

[0054] 9. When the resistant buds grow to 1 cm, transfer them to rooting culture concentrate (MS supplemented with 200 mg / L carbenicillin and 5 mg / L hygromycin) to promote root growth;

[0055] 10 After the roots of the tobacco seedlings are well developed, move them into the soil and manage them normally.

[0056] Screening and identification methods for transgenic tobacco:

[0057] 1. Take T0 generation tobacco leaves and extract total leaf DNA according to the instructions of the Plant Genomic DNA Extraction Kit (purchased from TaKaRa Company);

[0058] 2. Dilute the extracted DNA 10-fold and take 1 μL as a template. Perform conventional PCR verification using primers (F: 5'-AGGATTTAGTAACGTAGGGCC-3'; R: 5'-GATCTCAGTGATGATGAGTGAGTAG-3'). Untransformed wild-type tobacco was used as a negative control, and plasmid pCAMBIA1301-GmGolSP was used as a positive control.

[0059] 3. Harvesting seeds of positive transgenic tobacco plants, i.e., T0 generation seeds;

[0060] 4. Plant the T0 generation seeds in the soil to obtain T1 generation transgenic tobacco seedlings, and continue to extract DNA for PCR testing. Take the T1 generation positive transgenic tobacco plants for subsequent experiments.

[0061] Figure 6 This is the PCR identification result of GmGolSP transgenic tobacco genomic DNA, indicating that GmGolSP has been successfully integrated into the tobacco genome.

[0062] (IV) GUS histochemical staining of leaves of GmGolSP transgenic tobacco under high temperature treatment

[0063] Eight-week-old GmGolSP T1 transgenic tobacco plants grown in pots were placed in a 42°C incubator for high-temperature treatment. Leaves from untreated T1 transgenic tobacco and leaves from T1 transgenic tobacco that had been high-temperature treated for 2 hours were excised. Leaves from wild-type tobacco that had been untreated and high-temperature treated for 2 hours served as negative controls, and leaves from untreated pCAMBIA1301 transgenic tobacco served as positive controls. GUS histochemical staining was performed by adding GUS staining solution (Cloning and Expression Activity Analysis of the Soybean Stearate-ACP Desaturase Gene Promoter, Zhang Qinglin et al., 2011), incubating overnight at 37°C, and decolorizing with 75% ethanol until the background color disappeared.

[0064] GUS histochemical staining results Figure 7-11 As shown, wild-type tobacco leaves that were not treated or treated with high temperature for 2 h were not stained blue ( Figure 7 and Figure 8 ); Untreated GmGolSP T1 transgenic tobacco leaves were stained blue ( Figure 9 ), indicating that GmGolSP can initiate the expression of GUS reporter gene; the leaves of GmGolSP T1 transgenic tobacco treated with high temperature for 2 h were stained blue ( Figure 10 ), and Figure 9 Darker than Figure 11 Compared with the positive control, the color is similar, indicating that the activation activity of GmGolSP is enhanced after high temperature treatment, which increases the expression of the downstream reporter gene GUS.

[0065] (V) Real-time fluorescence quantitative PCR detection of GUS gene expression under high temperature treatment of GmGolSP transgenic tobacco

[0066] Eight-week-old GmGolSP T1 transgenic tobacco plants grown in pots were placed in a 42°C incubator for 2 hours. 0.1 g of leaves from the untreated (0 h) and the T1 transgenic tobacco plants that were treated for 2 hours were excised and immediately placed in liquid nitrogen and stored at -80°C until further use. Total RNA from the tobacco leaves was extracted using RNAiso Plus reagent (purchased from Takara), and first-strand cDNA was synthesized using a cDNA reverse transcription kit (purchased from Novoprotein) according to the manufacturer's instructions.

[0067] A BIO-RAD CFX96 Real-Time PCR instrument was used, using the constitutively expressed tobacco gene α-tubulin (GenBank accession number: AB052822) as an internal reference gene (F: 5′-ATGAGAGAGTGCATATCGAT-3′; R: 5′-TTCACTGAAGAAGGTGTTGAA-3′). Real-time PCR primers for the GUS gene were as follows: F: 5′-GATCGCGAAAACTGTGGAAT-3′; R: 5′-TAATGAGTGACCGCATCGAA-3′. Tobacco leaf cDNA was used as a template. The real-time PCR reaction system and procedure were the same as above, except that the annealing temperature was changed to 55°C.

[0068] Table 2 Relative expression of GUS gene in GmGolSP transgenic tobacco under high temperature treatment (values ​​are the average of three replicates)

[0069]

[0070] As shown in Table 2 and Figure 12 As shown in the figure, the real-time fluorescence quantitative PCR results showed that GmGolSP could significantly increase the expression of downstream GUS reporter gene when treated with high temperature for 2h. Sequence Listing <110> Qiqihar University <120> Soybean high temperature inducible promoter and its application <160> 13 <210> 1 <211> 1793 <212> DNA <213> Soybean (Glycine max (L.) Merr) <220> <223> Soybean high temperature inducible promoter GmGolSP. <400> 1 aggatttagt aacgtagggc ccagtcggtg gtgatagtat acactattgg gggcggtggc 60 aggtaacctg aaaacggata ttctattctt tctttttgt cgtttagta ccaactgagg 120 tagctttcc acatggaat attaacttgg agagaggttg attagtgtca agaaggatca 180 taaatatgtg attagttagt tatctgtag catcacttct taattcat gtcagcttac 240 attttacttt tcccttattt tggaagttg gtttcgaat tactttgtt tttttttttt 300 aactctccaa attacataa acatagaggt ttaaacttaa aacattatt tgttctgcca 360 aaaaacctat ggtcaatgtt gagaggtttt tattaaggtc tctcctc tggtctcttg 420 taggaaca aagctttatt gcaatttct cgattcttca tatgtccact taagtggtat 480 ttttctatta tttctcctaa gtgtaagaat aaaggggatc acaccgaac ttcagagtt 540 gcagtgagaa aaaaataatc taatgcaatt attgacta catatttgaa taaatattag 600 taagaaaatg cgttagaact tactaggga acgtgttttct ctaaatgtttt caatgataa 660 aaaattgaa gttaatata gttaattaaaaaaaaaaaaaaaaaaataaaaa 720 780 aaaaataaat tatctataa aatataaaca tcatgtcaaa tgaaaaataat tctcgttatt 840 aatagtgaca agtaacgcat aataacattg gttcttttaa aactatctct tttccttgag 900 tatcataaaa cgaaatgatg catccgtcaa tcactatgat tcatatgttt tcttttaaaa 960 aaagagatt atatttttt tggcaaaaaa tacttttatg tatttatata aaagaggaaag 1020 attctgattc atgttcccaa acgttaatta ttttgattca ggtcttttta ttatattttc 1080 catttaagat gaaataaaaa ataactgcaa aataattatt ttgatataag aatgtggttg 1140 ccaattgttt actatatta tatttatgtg aagattttat tgttcagtta acaaaactta 1200 aatgctacag gatttttata agttataact atatcttgca tgtctatcgc tatcaagatt 1260 1320 1380 agtgaaagac tatatctaca accgcgtgag tggaaacgaa ttaactaatc aaggaaagat 1440 tatccattat ccctcaccgt gggattccaa agccatctcc ttaaccgtta gattacttga 1500 gagtgcaatg gcgacaatac ccccccgaag ctctatataa gcaaccacag catttccccg 1560 accatcacaa caacaaacca acaaagatct tccttacaaa agtttgtttt caaagtgtgt 1620 tttgtttccc aaatcctact cttgtgacca caacccttcc tcctctttct tttgaaacct 1680 ctttttttct attccccaac caaacaagca aacgctactc actcatcatc actgagatc 1739 <210> 2 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Upstream primers for real-time fluorescence quantitative PCR of GmGolSP gene. <400> 2 tctaagcctt ggaggtacac tgg 23 <210> 3 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Downstream primers of GmGolSP gene real-time fluorescence quantitative PCR. <400> 3 ggcacggacg aacttgactt c 21 <210> 4 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> β-Tubuin PCR upstream primers for the internal reference gene. <400> 4 ggaaggcttt cttgcattgg ta 22 <210> 5 <211> 19 <212> DNA <213> Artificial sequence <220> <223> PCR downstream primers for the internal reference gene β-Tubuin. <400> 5 agtggcatcc tggtactgc 19 <210> 6 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Upstream primers for soybean GmGolSP amplification. <400> 6 gtcgacagga tttagtaacg tagggcc 27 <210> 7 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Soybean GmGolSP amplification downstream primers. <400> 7 ccatgggatc tcagtgatga tgagtgagta g 31 <210> 8 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Upstream primers for PCR amplification of transgenic tobacco. <400> 8 aggatttagt aacgtagggc c 21 <210> 9 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Downstream primers for PCR amplification of transgenic tobacco. <400> 9 gatctcagtg atgatgagtg agtag 25 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Upstream primers for PCR amplification of the internal reference gene α-tubulin. <400> 10 atgagagagt gcatatcgat 20 <210> 11 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Downstream primers for PCR amplification of the internal reference gene α-tubulin. <400> 11 ttcactgaag aaggtgttga a 21 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Upstream primers for real-time fluorescence quantitative PCR of GUS gene. <400> 12 gatcgcgaaa actgtggaat 20 <210> 13 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Downstream primers for real-time fluorescence quantitative PCR of GUS gene. <400> 13 taatgagtga ccgcatcgaa 20

Claims

1. Application of a soybean high temperature inducible promoter in inducing heat-resistant gene expression at high temperatures, characterized in that: The nucleotide sequence of the promoter is shown in SEQ ID NO: 1 in the sequence listing.

2. The use according to claim 1, characterized in that The sequence contains a cis-acting element related to stress.

3. The use according to claim 2, characterized in that The cis-acting elements are MBS, ARE, TC-rich repeats, ABRE, CGTCA-motif and GC-motif.

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