Potato high-temperature inducible promoter pStHSP20 and application thereof

Abstract

The invention provides a potato high-temperature inducible promoter pStHSP20 and application thereof.The nucleotide sequence of the potato high-temperature inducible promoter pStHSP20 is shown as SEQ ID No.1. The potato high-temperature inducible promoter pStHSP20 is remarkably induced by high temperature, has high-temperature specific expressivity and can serve as a specific promoter to drive specific expression of a target gene under the high-temperature condition, and the specific expression of the target gene under the high-temperature condition is achieved. The method can avoid adverse effects caused by continuous expression of target genes, and has wide application value in the fields of plant genetic engineering and germplasm resource improvement.

Description

Technical Field

[0001] The invention belongs to the field of plant gene engineering technology and molecular biology, and specifically relates to the separation, identification and application of a potato high temperature inducible promoter pStHSP20. Background Art

[0002] Potato is the world's third largest food crop after rice and wheat, and is also one of the world's most important tuber crops. There are many factors that affect the growth and development of potatoes, including environmental conditions, plant hormones, and plant self-regulatory factors. Among them, temperature is one of the most important environmental factors affecting potato plant growth and tuber yield. Potato plant growth has different requirements for air and soil temperature. 20-25℃ is suitable for the growth of aboveground stems and leaves, while 15-20℃ is suitable for underground tuber formation. Potatoes prefer cool weather, and high temperature conditions can easily cause the plants to grow too tall and reduce the yield and quality of tubers. Therefore, studying the high temperature resistance mechanism of potatoes is of great significance for breeding high temperature resistant potato varieties and ensuring yield and efficiency.

[0003] The promoter is a DNA sequence in the upstream region of the 5' end of the gene that can guide the correct binding of RNA polymerase to the template and ensure the accurate and effective initiation of gene transcription. It plays an important role in the regulation of gene expression. The promoter is an important regulatory element of gene expression. Studying its structure and activity characteristics can help clarify the function of the gene and analyze the growth, development and defense mechanism of plants. According to its mode of action and function, promoters are generally divided into three categories: constitutive promoters, tissue-specific promoters, and inducible promoters. Among them, the regulation of constitutive promoters is not affected by external conditions. In plants, the expression of target genes driven by constitutive promoters can make the target genes continue to be strongly expressed in different tissues or parts. At present, some constitutive promoters, such as 35S promoter and maize ubiquitin promoter, have been widely used. However, since the target gene driven by the constitutive promoter is theoretically highly expressed in all developmental stages and all tissues of the plant, it will greatly increase the metabolic burden and energy waste of transgenic plants. Therefore, the advantages of tissue-specific promoters and inducible promoters are gradually emerging. Inducible promoters are a type of promoter that responds to special biological, physical or chemical signals formed in the long-term process of plant adaptation to the environment. In the absence of inducing factors, the target gene driven by them is not expressed or expressed at the background level. Once the inducing factor appears in the environment, the expression of the target gene will increase rapidly. Compared with the adverse effects that constitutive promoters may bring, the advantage of inducible promoters is that they can enable plants to adjust the transcription level of specific genes according to different types of special signal molecules such as biological, physical, and chemical signals, reasonably reduce energy consumption in plants, and avoid the negative effects of excessive gene expression on plant growth and development. Therefore, the isolation and identification of stress-induced promoters in plants has become one of the research hotspots of plant genetic engineering in recent years.

[0004] In the study of the molecular mechanism of potato's resistance to high temperature, a high temperature inducible promoter is needed to upregulate the expression of the target gene, and then analyze the function of the target gene in potato's response to high temperature stress, so as to minimize the adverse effects of the target gene on other tissues and organs. Although there have been reports on potato inducible promoters, there are still few isolated and identified high temperature inducible promoters. Therefore, isolating and identifying more high temperature inducible promoters and studying their expression response to high temperature stress will not only help to analyze the molecular mechanism of potato's resistance to high temperature stress, but also provide applicable promoter reference sequences for the study of plant resistance to high temperature stress, and provide a theoretical basis for the creation of new high temperature resistant materials.

[0005] The inventors found through research that the potato StHSP20 gene is significantly induced by high temperature and may play an important role in potato's response to high temperature stress. In the process of gene expression, the promoter is one of the key factors that determine whether the gene is transcribed and how efficient the transcription is. Therefore, cloning and functional analysis of the StHSP20 promoter has important theoretical significance and application value for analyzing the molecular mechanism of plant resistance to high temperature stress and breeding improvement. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a potato high-temperature inducible promoter pStHSP20 and its application. The potato high-temperature inducible promoter pStHSP20 is significantly induced by high temperature, has high-temperature specific expression, can be used as a specific promoter to drive the specific expression of the target gene under high temperature conditions, can avoid the adverse effects caused by the continuous expression of the target gene, and has wide application value in the fields of plant genetic engineering and germplasm resource improvement.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a potato high temperature inducible promoter pStHSP20, whose nucleotide sequence is one of the following:

[0009] a) having the nucleotide sequence as described in SEQ ID No.1;

[0010] b) having a sequence complementary to the nucleotide sequence defined in a);

[0011] c) a nucleotide sequence having more than 90% homology with the nucleotide sequence defined in a) and having the same high temperature inducible function.

[0012] The present invention also provides the use of the potato high temperature inducible promoter pStHSP20 to induce the expression of a target gene at high temperature. In one embodiment of the present invention, the pStHSP20::pRC18 vector was transferred into the Agrobacterium strain GV3101, and the transformed Agrobacterium strain was transferred into the model plant Arabidopsis thaliana, proving that the promoter pStHSP20 can drive the expression of the target gene at a high temperature of 38°C.

[0013] The present invention also provides a method for cloning or isolating the potato high temperature inducible promoter pStHSP20, which comprises using the primer pair SEQ ID No. 2 and SEQ ID No. 3 to obtain the nucleotide sequence of the promoter pStHSP20 from potato DM total genomic DNA by PCR amplification.

[0014] The present invention also provides primers for extending the potato high temperature inducible promoter pStHSP20, including a forward primer sequence with a nucleotide sequence as shown in SEQ ID No.2 and a reverse primer sequence with a nucleotide sequence as shown in SEQ ID No.3.

[0015] The present invention also provides a gene expression cassette, a recombinant expression vector, a recombinant bacterium and a transgenic plant cell line containing the potato high temperature inducible promoter pStHSP20, specifically:

[0016] 1) The gene expression cassette is composed of a potato high temperature inducible promoter pStHSP20, an exogenous target gene and a transcription termination sequence connected in a specific direction. In one embodiment of the present invention, the exogenous target gene is specifically a β-glucuronidase gene (GUS).

[0017] 2) The recombinant expression vector is a plant recombinant expression vector comprising the gene expression cassette described in 1). In one embodiment of the present invention, the expression vector is specifically pRC18.

[0018] 3) The recombinant bacteria are recombinant bacteria comprising the gene expression cassette described in 1).

[0019] 4) The transgenic plant cell line is a transgenic plant cell line comprising the gene expression cassette described in 1).

[0020] The present invention also provides the use of the above-mentioned potato high temperature inducible promoter pStHSP20, a gene expression cassette containing the potato high temperature inducible promoter pStHSP20, a recombinant expression vector, a recombinant bacterium, and a transgenic plant cell line in preparing transgenic plants. The transgenic plants of the present invention can be obtained by any known transgenic technology capable of introducing exogenous genes into plant cells or plant tissues; in one embodiment of the present invention, the transgenic technology is Agrobacterium-mediated transformation.

[0021] The present invention also provides the use of the potato high temperature inducible promoter pStHSP20, gene expression cassette, recombinant expression vector, recombinant bacteria and transgenic plant cell line in cultivating high temperature resistant plant varieties.

[0022] Preferably, the plant is a dicotyledonous plant or a monocotyledonous plant, more preferably a potato.

[0023] Beneficial effects of the present invention: (1) The present invention provides a potato high temperature inducible promoter pStHSP20, whose nucleotide sequence is shown in SEQ ID No. 1. The potato high temperature inducible promoter pStHSP20 is significantly induced by high temperature and has high temperature specific expression. It can be used as a specific promoter to drive the specific expression of the target gene under high temperature conditions, which can avoid the adverse effects caused by the continuous expression of the target gene and has a wide range of application value in the fields of plant genetic engineering and germplasm resource improvement. (2) The potato high temperature inducible promoter pStHSP20 provided by the present invention can also be used for functional analysis and identification of plant heat resistance related genes, which helps to enrich the molecular mechanism of regulating potato resistance to high temperature stress and provide an effective gene reserve for the subsequent use of plant genetic engineering methods to improve plant stress resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the construction of the recombinant expression vector pStHSP20-GUS of the high temperature inducible promoter pStHSP20.

[0025] Figure 2 This is the electrophoresis diagram of the cloned promoter pStHSP20.

[0026] Figure 3 These are the results of GUS staining of pStHSP20-GUS transgenic Arabidopsis seedlings under normal growth conditions and high temperature stress.

[0027] Figure 4 This is the result graph of GUS gene expression level in pStHSP20-GUS transgenic Arabidopsis seedlings under normal growth conditions and high temperature stress treatment.

[0028] Figure 5 This is a quantitative detection diagram of GUS activity in pStHSP20-GUS transgenic Arabidopsis seedlings under normal growth conditions and high temperature stress. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and should not be construed as limiting the present invention; the experimental methods in the following examples, unless otherwise specified, are all conventional methods; the materials, reagents and instruments used in the following examples, unless otherwise specified, are all commercially available products; in the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA. Without departing from the spirit and essence of the present invention, modifications or substitutions made by those skilled in the art to the methods, steps or conditions of the present invention are all within the scope of the present invention.

[0030] The main strain used in the present invention is Escherichia coli Trelief TM 5α, Agrobacterium strain GV3101, the expression vector used was pRC18 (kanamycin resistance), the vector came from the Chongqing Key Laboratory of Tuber Biology and Genetic Breeding. Primer synthesis and sequencing were completed by Sangon Biotech Co., Ltd.

[0031] Example 1: Obtaining potato high temperature inducible promoter pStHSP20

[0032] 1) Extraction of total genomic DNA from potato DM

[0033] Take about 0.1g of young potato leaves and place them in a 2mL sterile centrifuge tube with two steel balls; freeze them in liquid nitrogen for about 5 minutes and grind them in a 40Hz grinder. The sample was quickly ground into powder for 20 seconds; 600 μL of 2×CTAB extract preheated at 65°C (the extract and the reducing agent were mixed in a volume ratio of 500:1) was added; the centrifuge tube was placed in a 65°C water bath for 0.5 h, and the centrifuge tube was gently shaken every 10 minutes or so; the sample was taken out and cooled at room temperature for use, 500 μL of chloroform-isoamyl alcohol mixture (24:1) was added, the mixture was vigorously shaken and mixed, centrifuged at 12000 rpm for 10 minutes, and the supernatant was transferred to a new centrifuge tube; an equal volume of isopropanol was added to the supernatant, the mixture was mixed by inversion, and the mixture was placed at -20°C for precipitation for more than 30 minutes, centrifuged at 12000 rpm for 10 minutes, and the supernatant was discarded; the precipitate was washed once with 75% ethanol, centrifuged at 12000 rpm for 5 minutes, and the supernatant was discarded; the precipitate was naturally dried and dissolved in 50 μL of deionized water; it was stored at -20°C.

[0034] 2) Cloning of potato high temperature inducible promoter pStHSP20 and construction of expression vector

[0035] The promoter reference sequence 2Kb upstream of the transcription start site of StHSP20 was obtained from http: / / spuddb.uga.edu / index.shtml, and the primers required for cloning pStHSP20 were further designed using snap gene software (the forward primer and reverse primer sequences were GCCAGTGCCAAGCTTATATAGAATTCCGATATCATG, SEQ ID No. 2 and TCCGGTACCTCTAGATGCTAACAAAATACTCTGATG, SEQ ID No. 3, respectively, wherein in the forward primer, the sequence GCCAGTGCC is the upstream sequence of the fusion site of the vector pRC18, and the sequence AAGCTT is the HindIII restriction site; in the reverse primer, the sequence TCCGGTACC is the downstream sequence of the fusion site of the vector pRC18, and the sequence TCTAGA is the XbaI restriction site). The potato DM genomic DNA obtained above was used as a template and PCR amplification was performed using the high-fidelity polymerase 2×KeyPo Master Mix (Dye Plus) of Nanjing Novozymes Biotech Co., Ltd.

[0036] a) PCR reaction system (50 μL):

[0037]

[0038] b) PCR reaction procedure:

[0039]

[0040] After amplification, 1% agarose gel electrophoresis was used to detect the target band ( Figure 2 ). The target fragment with a sequence length of about 2000 bp was purified and recovered using the TIANGEN Universal DNA Purification and Recovery Kit.

[0041] The pRC18 vector was double-digested with HindIII and XbaI, and the linear vector was obtained by gel recovery.

[0042] c) Enzyme digestion reaction system:

[0043]

[0044] The purified PCR target fragment and the restriction-cut linear vector were recombined using the ClonExpress MultiS One Step Cloning Kit of Nanjing Novozymes Biotechnology Co., Ltd.

[0045] d) Homologous recombination reaction system:

[0046]

[0047] Next, the E. coli transformation experiment was carried out using TreliefTM 5α E. coli competent cells, and single clones were picked for colony PCR positive clone detection. The positive clones screened were sequenced for verification, and the vector with correct sequencing was named pStHSP20-GUS ( Figure 1 ), and further used the Plasmid Miniprep Kit (ZP101) from Beijing Zhuangmeng International Biogene Technology Co., Ltd. to extract the plasmid.

[0048] Example 2: Transformation of Arabidopsis thaliana with the recombinant expression vector pStHSP20-GUS

[0049] 1) Transform the recombinant expression vector pStHSP20-GUS into Agrobacterium

[0050] Agrobacterium competent cells GV3101 were used for Agrobacterium transformation experiments. The specific steps are as follows: GV3101 competent cells were quickly inserted into ice, and after melting, 1 μg of plasmid was added; ice bath for 5 minutes, liquid nitrogen cold shock for 5 minutes, 37°C metal bath heat shock for 5 minutes, and ice bath for 5 minutes; 200 μL of YEB medium without antibiotics was added to the centrifuge tube in the clean bench, and after pipetting and mixing, it was revived at 28°C and 200 rpm for 2-3 hours; 200 μL of bacterial solution was aspirated and applied to YEB solid medium containing rifampicin (25 μg / mL) and kanamycin (50 μg / mL), and after inverted culture in a constant temperature incubator at 28°C for 2 days, positive clones were screened.

[0051] 2) Agrobacterium-mediated genetic transformation of Arabidopsis

[0052] Wild-type Arabidopsis thaliana was transformed by the inflorescence infiltration method, specifically: Agrobacterium containing the pStHSP20-GUS plasmid was activated in YEB liquid medium, cultured overnight at 28°C, 200-220rpm; the above bacterial solution was added to 50mL YEB liquid medium containing the corresponding antibiotics until the OD 600 =0.1, 28°C, 200-220rpm, culture until OD 600 = about 0.7; 5000rpm, 6min centrifugation to collect bacteria, discard the supernatant and resuspend Agrobacterium in the infection solution (containing 5g / 100mL sucrose and 50μL / 100mL SilwetL-77) according to the proportion; the pruned wild-type Arabidopsis inflorescence is completely immersed in the above Agrobacterium suspension for 10-20s, and after the infection is completed, it is cultured in the dark for 2d, and then transferred to normal light for continued cultivation until harvest.

[0053] Example 3: Screening and identification of pStHSP20-GUS transgenic Arabidopsis

[0054] Take the transgenic seeds obtained in Example 2 and stored by low temperature drying. Under a sterile environment, place an appropriate amount of seeds in a sterile 1.5mL centrifuge tube, add an appropriate volume of 75% alcohol for disinfection for 8min, pour out the alcohol, and wash with sterile water for 3 times. The sterilized seeds are evenly sown on a 1 / 2MS medium containing hygromycin (25μg / mL), and placed in 4℃ dark vernalization for 2d, and then cultured for 10-15d under the conditions of temperature 22℃, humidity 60%, light and dark alternation (lighting time 16h, dark time 8h). At this time, dark green, hypocotyl elongation, and normal true leaf growth of resistant seedlings can be seen, while yellow or white seedlings are non-resistant wild-type seedlings. The selected resistant seedlings are transplanted into a nutrient matrix (vermiculite: nutrient soil = 1:2, V / V) for continued cultivation. Young tissues of the screened T1 generation positive transgenic plants were taken, and genomic DNA was extracted for PCR detection. The forward primer sequence and reverse primer used for detection were TCGAATAATTCACATATTC, SEQ ID No. 4 and CGCGATCCAGACTGAATG, SEQID No. 5, respectively. The results showed that all positive transgenic plants contained the target bands. After repeated screening, T2 generation plants and seeds were obtained, and the T2 generation seeds were further screened to finally obtain T3 generation homozygous plants for subsequent experiments.

[0055] Example 4: Identification of high temperature inducible activity of pStHSP20

[0056] 1) High temperature stress treatment

[0057] In order to verify the response of pStHSP20 to high temperature stress, two strains pStHSP20-GUS#2 and pStHSP20-GUS#11 were selected from the obtained positive plants for high temperature stress induction activity analysis. The sterilized T3 generation homozygous transgenic Arabidopsis and wild-type Arabidopsis seeds were sown on 1 / 2MS culture medium using the Arabidopsis disinfection method in Example 3, and after 2 days of vernalization at 4°C, they were cultured normally for 10 days. The treatment group was then treated at 38°C for 3 hours, and the control group was kept in a 22°C incubator for growth.

[0058] 2) GUS histochemical staining analysis

[0059] The Arabidopsis seedlings of the treatment group and the control group were immersed in the substrate X-Gluc staining reaction solution, vacuumed for 5 minutes, and incubated in the dark at 37°C for 12 hours. The staining solution was removed, and then decolorized with 75% ethanol 2-3 times, and observed and photographed under a stereoscope. The results are shown in the figure. Under normal and high temperature induction conditions, the wild-type Arabidopsis could not be stained (no GUS gene expression). Under normal conditions, only the stem tip of the pStHSP20-GUS transgenic Arabidopsis was slightly colored, and the rest of the tissues were not stained. After being treated at 38°C for 3 hours, the pStHSP20-GUS transgenic Arabidopsis showed dark blue, indicating that the promoter pStHSP20 was significantly induced by high temperature ( Figure 3 ).

[0060] 3) Analysis of GUS gene expression level

[0061] The RNA of Arabidopsis seedlings in the above-mentioned treatment group and control group was extracted using the FastPureUniversal Plant Total RNA Isolation Kit RC411-01 from Nanjing Novazon Biotechnology Co., Ltd., and then the reverse transcription kit of Yisheng Biotechnology (Shanghai) Co., Ltd. was used. Ⅲ1St Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) was used for reverse transcription. The cDNA synthesized by the above reverse transcription was used as a template for specific PCR amplification of the GUS gene. The forward primer sequence and the reverse primer sequence were CGGTCAGTGGCAGTGAAGGG, SEQ ID No.6 and CGAGGTACGGTACGGTAGGAGTTGG, SEQID No.7, respectively. Three replicates were set for each sample, and the Ct value of the sample was counted. The data was calculated using the 2-ΔΔCT formula to analyze the relative expression of the target gene. The results showed that under high temperature stress, the expression level of the GUS gene regulated by the promoter pStHSP20 was significantly upregulated, indicating that the promoter pStHSP20 was significantly induced by high temperature ( Figure 4 ).

[0062] 4) Quantitative analysis of GUS enzyme activity

[0063] The sample to be tested was ground into powder in a pre-cooled mortar with liquid nitrogen, and its weight was accurately weighed; 1 mL of extract solution was added to every 100 mg of powder, and vortexed to mix it thoroughly; centrifuged at 12,000×g, 4°C for 10 min, aspirated the supernatant into a new centrifuge tube, and placed on ice for use; the protein concentration of each supernatant was determined according to the Bradford protein assay; 150 μL of supernatant was taken, 150 μL of preheated 4-MUG substrate solution was added, mixed, and incubated at 37°C for reaction; 100 μL of reaction solution was taken and added to 900 μL of reaction stop solution at 10 min and 20 min respectively; the fluorescence intensity was measured using a fluorescence microplate reader under the conditions of excitation wavelength 365 nm and emission wavelength 456 nm, and the concentration of 4-MU produced by each sample reaction was obtained through the 4-MU standard curve, and the GUS enzyme activity was calculated; each sample was repeated 3 times. The results showed that after 38℃ high temperature treatment for 3h, GUS activity increased significantly, indicating that the activity of pStHSP20 was significantly induced by high temperature stress treatment ( Figure 5 ).

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A potato high temperature inducible promoter pStHSP20, whose nucleotide sequence is one of the following: a) having the nucleotide sequence as described in SEQ ID No.1; b) having a sequence complementary to the nucleotide sequence defined in a); c) a nucleotide sequence having more than 90% homology with the nucleotide sequence defined in a) and having the same high temperature inducible function.

2. Use of the potato high temperature inducible promoter pStHSP20 according to claim 1 to induce the expression of a target gene under high temperature.

3. A method for cloning or isolating the potato high temperature inducible promoter pStHSP20 according to claim 1, characterized in that: The method comprises the following steps: using the primer pair of SEQ ID No. 2 and SEQ ID No. 3 to obtain the nucleotide sequence of the promoter pStHSP20 from the total genomic DNA of potato DM by PCR amplification.

4. Primers for extending the potato high temperature inducible promoter pStHSP20 according to claim 1, characterized in that: It includes a forward primer sequence with a nucleotide sequence as shown in SEQ ID No.2 and a reverse primer sequence with a nucleotide sequence as shown in SEQ ID No.

3.

5. A gene expression cassette, a recombinant expression vector, a recombinant bacterium and a transgenic plant cell line containing the potato high temperature inducible promoter pStHSP20 according to claim 1, specifically: 1) The gene expression cassette is composed of a potato high temperature inducible promoter pStHSP20, an exogenous target gene and a transcription termination sequence connected in a specific direction; 2) the recombinant expression vector is a plant recombinant expression vector comprising the gene expression cassette described in 1); 3) The recombinant bacteria is a recombinant bacteria comprising the gene expression cassette described in 1); 4) The transgenic plant cell line is a transgenic plant cell line comprising the gene expression cassette described in 1).

6. The recombinant expression vector according to claim 5, characterized in that The expression vector of the recombinant expression vector is pRC18.

7. Use of the potato high temperature inducible promoter pStHSP20 according to claim 1, the gene expression cassette, the recombinant expression vector, the recombinant bacteria, the transgenic plant cell line according to claim 5, and the recombinant expression vector according to claim 6 in preparing transgenic plants.

8. Use of the potato high temperature inducible promoter pStHSP20 according to claim 1, the gene expression cassette according to claim 5, the recombinant expression vector according to claim 5, the recombinant bacteria according to claim 5, the transgenic plant cell line according to claim 5, and the recombinant expression vector according to claim 6 in cultivating high temperature resistant plant varieties.

9. The use according to claim 8, characterized in that: The plant is a dicotyledonous plant or a monocotyledonous plant.

10. The use according to claim 9, characterized in that: The plant is potato.

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