Heavy metal-inducible promoters, related primer pairs, expression vectors, and applications

Abstract

This invention provides a heavy metal-inducible promoter, the nucleotide sequence of which is shown in SEQ ID NO:1. It also provides a plant recombinant expression vector containing the heavy metal-inducible promoter, primer pairs for amplifying the heavy metal-inducible promoter, and the application of the heavy metal-inducible promoter or plant recombinant expression vector in controlling the heavy metal-induced expression of genes in plants. The heavy metal-inducible promoter of this invention can control the heavy metal-induced expression of genes in plants, providing a useful core component for studying plant gene expression, regulation, and using genetic engineering methods to alter plant tolerance and accumulation patterns of heavy metals, as well as developing probes for detecting heavy metals. It is suitable for large-scale application.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology and plant genetic engineering technology, more particularly, to the field of promoters for controlling gene expression in plants, and particularly refers to a heavy metal inducible promoter and related primer pair, expression vector and application. BACKGROUND

[0002] In plants, some metals such as manganese (Mn) and copper (Cu) are essential cofactors for growth and development, which maintain protein structure or catalyze enzymatic reactions. Some non-essential metal elements, such as cadmium (Cd), nickel (Ni), and chromium (Cr), can cause great toxicity to plants even at low concentrations. Whether essential or non-essential, when the concentration of metals exceeds a certain threshold, it can cause irreversible damage to plant growth and reduce yield. These heavy metals not only accumulate in primary producers in the food chain, but also can be enriched through the food chain to affect human health. Therefore, studying the response of heavy metals in plants has practical significance for solving food safety problems.

[0003] Several heavy metal inducible genes have been found in plants, such as the MTP gene encoding a zinc finger protein, which is mainly involved in the absorption and transport of various heavy metal ions including Cd, Mn, and Ni, and has been reported to be induced by heavy metals in many plants such as Arabidopsis, alfalfa, and poplar. The Phaseolus vulgaris PvSR2 gene specifically responds to heavy metal stress, but not to other environmental stresses such as heat shock, ultraviolet radiation, and viral infection. The gene promoter is a DNA sequence located upstream of the gene coding region, which contains multiple cis-acting elements that can be recognized by RNA polymerase and regulate transcription. Promoters and their contained cis-acting elements control gene expression to a large extent by activating or inhibiting transcription. Unlike constitutive promoters, inducible promoters only initiate transcription of foreign genes under specific conditions stimulated by certain signals. The promoter sequences of OsGSTU5, OsGSTU37, and OsHSP18.6 isolated from rice are Cd inducible promoters, and the GUSplus reporter gene connected behind these promoters has a very low background under normal conditions, but is strongly induced by Cd treatment. The promoter of the PvSR2 gene cloned from Phaseolus vulgaris heterologously expressed in tobacco was found to exhibit a metal-responsive inducible promoter.

[0004] Promoters are important elements of expression vectors for genetic engineering, and the study of metal inducible promoters is of great significance for clarifying the expression regulation mechanism of metal inducible genes and improving crop tolerance to heavy metals. The development of new heavy metal inducible promoters from plants can provide useful core elements for studying plant gene expression, regulation, and using genetic engineering methods to change plant tolerance to heavy metals and enrichment methods, and for developing probes for detecting heavy metals. SUMMARY

[0005] In order to overcome the above-mentioned defects in the prior art, one object of the present application is to provide a heavy metal inducible promoter, which can control the expression of genes in plants induced by heavy metals, can provide useful core elements for the research of plant gene expression, regulation, and the change of plant tolerance and enrichment of heavy metals by using genetic engineering methods, and the development of probes for detecting heavy metals, and is suitable for large-scale popularization and application.

[0006] Another object of the present application is to provide a plant recombinant expression vector, which can control the expression of genes in plants induced by heavy metals, can provide useful core elements for the research of plant gene expression, regulation, and the change of plant tolerance and enrichment of heavy metals by using genetic engineering methods, and the development of probes for detecting heavy metals, and is suitable for large-scale popularization and application.

[0007] Another object of the present application is to provide a set of primers, which can amplify the heavy metal inducible promoter, so as to be used for subsequent expression vector construction, so as to control the expression of genes in plants induced by heavy metals, can provide useful core elements for the research of plant gene expression, regulation, and the change of plant tolerance and enrichment of heavy metals by using genetic engineering methods, and the development of probes for detecting heavy metals, and is suitable for large-scale popularization and application.

[0008] Another object of the present application is to provide the application of a heavy metal inducible promoter or a plant recombinant expression vector containing the promoter, so as to control the expression of genes in plants induced by heavy metals, can provide useful core elements for the research of plant gene expression, regulation, and the change of plant tolerance and enrichment of heavy metals by using genetic engineering methods, and the development of probes for detecting heavy metals, and is suitable for large-scale popularization and application.

[0009] In order to achieve the above objects, in the first aspect of the present application, a heavy metal inducible promoter is provided, characterized in that the nucleotide sequence of the heavy metal inducible promoter is shown in SEQ ID NO: 1.

[0010] In the second aspect of the present application, a plant recombinant expression vector is provided, comprising a plant expression vector, characterized in that the plant recombinant expression vector further comprises the above-mentioned heavy metal inducible promoter, and the heavy metal inducible promoter is integrated into the plant expression vector.

[0011] Preferably, the plant expression vector is pNC-35S::GFP.

[0012] More preferably, the plant recombinant expression vector is pNC-35S::GFP-PvGIP1Pro::GUS, wherein the PvGIP1Pro is the heavy metal inducible promoter.

[0013] In a third aspect of the invention, a set of primer pairs is provided, characterized in that the primer pairs are designed according to the nucleotide sequence of the heavy metal inducible promoter described above.

[0014] Preferably, the nucleotide sequences of the primer pair are shown in SEQ ID NO:2 and SEQ ID NO:3.

[0015] In a fourth aspect of the invention, the application of the above-described heavy metal-inducible promoter or the above-described plant recombinant expression vector in controlling the heavy metal-induced expression of genes in plants is provided.

[0016] Preferably, the plant is a common bean.

[0017] Preferably, the plant is a common bean hairy-rooted plant.

[0018] Preferably, the gene is the GUS gene, or the heavy metal is Cd, Cr, Mn, Ni or Cu.

[0019] The beneficial effects of this invention are as follows:

[0020] a. The nucleotide sequence of the heavy metal inducible promoter of the present invention is shown in SEQ ID NO:1. It can control the heavy metal-induced expression of genes in plants and can provide a useful core component for studying plant gene expression, regulation and using genetic engineering methods to change the tolerance and accumulation of heavy metals in plants, and developing probes for detecting heavy metals. It is suitable for large-scale application.

[0021] b. The plant recombinant expression vector of the present invention includes a plant expression vector and a heavy metal inducible promoter. The plant expression vector integrates a heavy metal inducible promoter, which can control the heavy metal-induced expression of genes in plants. It can provide a useful core component for studying plant gene expression, regulation and using genetic engineering methods to change the tolerance and accumulation mode of plants to heavy metals, and developing probes for detecting heavy metals. It is suitable for large-scale promotion and application.

[0022] c. The primer pairs of the present invention are designed based on the nucleotide sequences of heavy metal-inducible promoters, which can amplify heavy metal-inducible promoters and thus be used for subsequent expression vector construction to control the heavy metal-induced expression of genes in plants. They can provide useful core components for studying plant gene expression, regulation and using genetic engineering methods to change the tolerance and accumulation of heavy metals in plants, and developing probes for detecting heavy metals, and are suitable for large-scale application.

[0023] d. The application of the heavy metal-inducible promoter or plant recombinant expression vector of the present invention in controlling the heavy metal-induced expression of genes in plants can control the heavy metal-induced expression of genes in plants. It can provide useful core components for studying plant gene expression, regulation and using genetic engineering methods to change the tolerance and accumulation mode of plants to heavy metals, and developing probes for detecting heavy metals. It is suitable for large-scale promotion and application.

[0024] These and other objects, features and advantages of the present invention will be fully apparent from the following detailed description and drawings, and can be achieved by the features, means and combinations thereof specifically pointed out in the specification. Attached Figure Description

[0025] Figure 1 This is a PCR electrophoresis image of the PvGIP1 promoter. Lane 1 is a 2kb marker, lane 2 is a negative control, and lane 3 is the amplification product of the primer pair specifically amplifying the PvGIP1 promoter. The amplified product contains the PvGIP1 promoter PvGIP1Pro and part of the expression vector sequence, with a size of 2211bp.

[0026] Figure 2 This is a schematic diagram of the structure of the expression vector pNC-35S::GFP.

[0027] Figure 3 This is a schematic diagram of the structure of the plant recombinant expression vector pNC-35S::GFP-PvGIP1Pro::GUS.

[0028] Figure 4 These are positive roots of 35S::GFP-PvGIP1Pro::GUS transgenic common bean hairy root plants, among which the roots showing green fluorescence are positive roots (see hollow arrows).

[0029] Figure 5 This is a GUS staining analysis of 35S::GFP-PvGIP1Pro::GUS transgenic bean hairy root plants after heavy metal treatment. The roots showing blue fluorescence are GUS-expressing roots (see solid arrows), and NaCl is the control treatment group. Detailed Implementation

[0030] In order to develop new heavy metal inducible promoters from plants, the inventors have discovered a heavy metal inducible promoter through extensive research, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0031] A plant recombinant expression vector is also provided, comprising a plant expression vector and the aforementioned heavy metal inducible promoter, wherein the heavy metal inducible promoter is integrated into the plant expression vector.

[0032] The plant expression vector can be any suitable plant expression vector, preferably pNC-35S::GFP.

[0033] The plant recombinant expression vector can be any suitable plant recombinant expression vector, more preferably, the plant recombinant expression vector is pNC-35S::GFP-PvGIP1Pro::GUS, wherein PvGIP1Pro is the heavy metal inducible promoter.

[0034] A set of primer pairs is also provided, which are designed based on the nucleotide sequences of the aforementioned heavy metal-inducible promoters.

[0035] The primer pair may have any suitable nucleotide sequence, preferably as shown in SEQ ID NO:2 and SEQ ID NO:3.

[0036] The application of the aforementioned heavy metal-inducible promoters or plant recombinant expression vectors in controlling the heavy metal-inducible expression of genes in plants is also provided.

[0037] The plant can be any suitable plant, but preferably, it is a common bean. More preferably, it is a common bean hairy-rooted plant.

[0038] The gene can be any suitable gene, either a gene that is naturally present in the plant or a foreign gene from other plants. Preferably, the gene is the GUS gene.

[0039] The heavy metal can be any suitable heavy metal, preferably Cd, Cr, Mn, Ni or Cu.

[0040] To provide a clearer understanding of the technical content of this invention, the following embodiments are provided for detailed description. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: ColdSpring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0041] Example 1:

[0042] 1. Promoter sequence analysis

[0043] The DNA sequence of the common bean PvGIP1 gene (gene number Phvul.001G120800) was downloaded from the Phytozome database (https: / / phytozome-next.jgi.doe.gov / ). The promoter of the PvGIP1 gene was located and sequenced, thus obtaining the promoter of the PvGIP1 gene, which was named the common bean heavy metal inducible promoter PvGIP1Pro. Its sequence is shown in SEQ ID NO: 1, with a full length of 2167 bases.

[0044] 2. Promoter cloning

[0045] The 2167 bp upstream of the ATG region of the PvGIP1 gene was selected as the PvGIP1 promoter region. To construct the expression vector pNC-35S::GFP-PvGIP1Pro::GUS containing the PvGIP1 promoter, primer pairs were designed based on the PvGIP1 promoter region. The restriction enzyme sites were SbfI and SpeI. Homologous sequences near the SbfI and SpeI restriction sites of the vector pNC-35S::GFP were added to the primer pairs (see Table 2 below). In Table 1, the template DNA was a DNA sample from common bean roots, provided by the Plant Phenotyping and Quality Safety Laboratory of China Jiliang University; the negative control was a reaction system without template DNA; KOD high-fidelity polymerase 2×Mix was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.

[0046] Table 1 shows the promoter amplification system (total 50 μL).

[0047] Reagent Amount KOD high fidelity polymerase 2 x Mix 25 μL Template DNA 1 μL 10 μM Upstream primer 1.5 μL 10 μM Downstream primer 1.5 μL dd H2O 21 μL

[0048] Table 2 Primer sequences used for PvGIP1 promoter amplification

[0049]

[0050]

[0051] The restriction endonuclease recognition site in Pv-2304-0800-F is cctgcagg, and the restriction endonuclease recognition site in Pv-2304-0800-R is actagt. The sequences preceding the restriction endonuclease recognition sites are homologous sequences near the SbfI and SpeI restriction sites of the vector pNC-35S::GFP.

[0052] Experimental results:

[0053] After 1.0% agarose gel electrophoresis, the amplified product was a 2211bp fragment, see [link to electrophoresis]. Figure 1As shown in the figure, the nucleotide sequence of the cloned PvGIP1 promoter is shown in SEQ ID NO:1, which contains 2167 bases.

[0054] 3. Construction of binary expression vectors containing PvGIP1Pro::GUS

[0055] a. Strains and plasmids

[0056] The *E. coli* strain used in this invention is DH5α (purchased from Shanghai Weidi Biotechnology Co., Ltd.); the plant expression vector pNC-35S::GFP (see [link to relevant documentation]). Figure 2 As shown in the figure, the GFP gene was replaced by an NC universal adapter at the NC frame of the pNC-Cam2304-MCS35S vector (purchased from Hainan Nixing Biotechnology Co., Ltd.).

[0057] b. Reagents and Chemicals

[0058] Antibiotics kanamycin (Kana) and rifampin (Rif) were purchased from BBI Life Sciences Co., Ltd.; DNA-Marker 2000 was purchased from General Biotechnology (Anhui) Co., Ltd.; restriction endonucleases were purchased from Thermo Fisher Scientific; KOD high-fidelity polymerase 2×Mix was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.; DNA fragment recovery and purification kits, plasmid DNA extraction kits, and homologous recombination reagent ClonExpress II were purchased from Nanjing Novizan Biotechnology Co., Ltd. Primer synthesis and sequencing were performed by Hangzhou Youkang Biotechnology Co., Ltd.

[0059] c. Recovery and purification

[0060] (1) Briefly centrifuge the PCR products, enzyme reaction solution, or crude DNA (including genomic DNA). Measure the volume with a pipette and transfer it to a sterile 1.5 ml or 2 ml centrifuge tube. If the sample volume is less than 100 μL, add sterile water to bring it up to 100 μL.

[0061] (2) Add 5 volumes of Buffer GDP and mix by inverting or vortexing. If you need to recover DNA fragments smaller than 100 bp, add 1.5 volumes of anhydrous ethanol (sample + Buffer GDP).

[0062] (3) Place the adsorption column into the collection tube. Transfer ≤700 μL of sol solution into the adsorption column. Centrifuge at 10000 rpm (8000 × g) for 30-60 seconds. If the volume of the mixed solution is greater than 700 μL, place the adsorption column into the recovery tube, transfer the remaining solution into the adsorption column, and centrifuge at 12000 rpm (13400 × g) for 30-60 seconds.

[0063] (4) Discard the filtrate and place the adsorption column in the collection tube. Add 700 μL of Buffer GW (with anhydrous ethanol added) to the adsorption column. Centrifuge at 12000 rpm (13400 × g) for 30–60 seconds.

[0064] (5) Repeat step 4.

[0065] (6) Discard the filtrate and place the adsorption column back into the collection tube. Centrifuge at 12000 rpm (13400 × g) for 2 min.

[0066] (7) Place the adsorption column in a 1.5 ml sterile centrifuge tube, add 20-30 μL of Elution Buffer to the center of the adsorption column, and incubate for 2 min. Centrifuge at 12000 rpm (13400 × g) for 1 min. Discard the adsorption column and store the DNA at -20℃.

[0067] d. Construction of expression carriers

[0068] (1) The pNC-35S::GFP plasmid was double-digested with restriction endonucleases SbfI and SpeI. The large fragment of the digested product was recovered to obtain the linearized vector. The amplification product obtained in step 2 was then double-digested with restriction endonucleases SbfI and SpeI. The large fragment of the digested product was recovered to obtain the insert fragment. Homologous recombination was performed using the ClonExpress II recombination reaction system, as shown below. This reaction must be prepared on ice.

[0069] Component Amount Linearized vector 180 ng Insert fragment 90 ng 5 x CE II Buffer 1 μL Exnase II 0.5 μL ddH2O Make up to 5 μL

[0070] (2) Use a pipette to gently aspirate and mix (do not shake to mix), and briefly centrifuge to collect the reaction volume at the bottom of the tube.

[0071] (3) React at 37℃ for 30 min; then cool to 4℃ or immediately place on ice to cool.

[0072] (4) Take out DH5α competent cells (50 μL per tube) from -80℃, quickly insert them into ice, wait for the bacterial block to melt after 5 minutes, add 5 μL of recombinant product, and let stand in ice for 25 minutes.

[0073] (5) Heat shock in a 42℃ water bath for 45 seconds, then quickly put it back into ice and let it stand for 2 minutes.

[0074] (6) Add 700 μL of antibiotic-free sterile liquid culture medium (LB) to the centrifuge tube, mix well, and thaw at 37°C and 200 rpm for 60 minutes.

[0075] (7) Centrifuge at 5000 rpm for 1 minute to collect the bacterial cells, and take about 100 μL of supernatant. Gently resuspend the bacterial block by blowing and spread it on LB solid medium containing Kana resistance.

[0076] (8) Invert the plate and incubate it overnight in a 37°C incubator.

[0077] (9) Select single clones of *E. coli* for colony PCR. Inoculate positive clones into LB broth containing Kana and amplify them in a shaker at 37°C and 200 rpm. Send the amplified *E. coli* culture to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing analysis. After extracting plasmids from correctly sequenced clones, the resulting binary expression vector containing PvGIP1Pro::GUS is obtained: pNC-35S::GFP-PvGIP1Pro::GUS. The recombinant vector map is shown below. Figure 3 As shown.

[0078] 4. Transgenic plants with hairy roots from common beans

[0079] a. Plant materials and strains

[0080] The seeds of the common bean cultivar LP098 and Agrobacterium rhizogenes R1000 were provided by the Plant Phenotyping and Quality Safety Laboratory of China Jiliang University.

[0081] b. Transformation of Agrobacterium rhizogenes R1000 with recombinant expression vector

[0082] (1) Remove the competent cells of Agrobacterium rhizogenes from the ultra-low temperature freezer and place them on ice to thaw.

[0083] (2) Add 1 μL of the correctly sequenced recombinant expression plasmid to the thawed competent cells and mix carefully.

[0084] (3) Transfer the liquid mixture from (2) to an empty and dry electrostatic cup, place the electrostatic cup into the instrument slot, and perform electrostatic rotation.

[0085] (4) After successful electroporation, add 800 μL of sterile LB liquid medium without antibiotics to the electroporation cup, and continuously pipette the medium to transfer it to the original centrifuge tube containing competent cells. Incubate in a shaker at 28°C and 200 rpm / min for 2 hours.

[0086] (5) Centrifuge the bacterial culture at 5000g / min for 1 minute, discard part of the supernatant, keep 100-150μL, gently mix with a pipette, spread it on LB solid medium containing Kana and Rif, and incubate at 28℃ upside down for about 48h until single colonies grow.

[0087] (6) Select Agrobacterium single clones for colony PCR.

[0088] (7) Select positive clones and shake them, and store the bacterial solution in an ultra-low temperature freezer with 50% glycerol for later use.

[0089] b. Genetically modified common bean mediated by Agrobacterium rhizogenes R1000

[0090] (1) Preparation of plant material: Sterilized seeds were soaked overnight in warm water at 28℃ and 150 rpm / min in a shaker. The next day, the seeds were spread evenly in a square petri dish with sterile filter paper and germinated in the dark for about 36 hours. When the seeds first showed white sprouts, they were sown into the substrate (perlite) and cultured under sufficient moisture and light conditions (day / night: 16h / 8h, 28℃ / 25℃). After about 3 days, bean plants with the first true leaf emerging were obtained.

[0091] (2) Infection process: First, use an injection needle to make holes in the hypocotyl of the bean (1-2 cm below the cotyledon). Ensure that the plant can survive, then make about 8-10 holes around the perimeter. Then, apply the activated bacteria to the injured part of the plant and culture it in a high humidity and low temperature environment (temperature: 22℃, humidity: 95%).

[0092] (3) Culture conditions: After infection, the plants were placed in an incubator at 22℃ and 95% humidity for 3 days in the dark. Then, they were transferred to a light incubator and cultured continuously for 3 days at 16h / 8h day / night, 60% light, and 95% humidity. Later, the light was adjusted to 100%, while other conditions remained unchanged. Hairy roots began to grow after one week. A large number of hairy roots began to form 14 days after infection. The transgenic hairy roots could be identified after 21 days. During this period, nutrient solution should be added to the seedling trays in a timely and appropriate manner.

[0093] 5. Heavy metal-induced experiments

[0094] (1) Positive roots were identified by observing green fluorescence. Positive common bean plants with positive hairy roots, namely 35S::GFP-PvGIP1Pro::GUS transgenic common bean hairy root plants (see...) Figure 4 Transfer the plants from perlite to a water-filled culture bottle and culture them for 3 days to allow them to adapt to the hydroponic environment in advance.

[0095] (2) Positive plants were randomly assigned to ensure that the number of replicates for each treatment was greater than or equal to 3, for a total of 6 treatments: CdCl2, CuCl2, CrCl2, MnCl2, NiCl2, and NaCl, with a final concentration of 50 μM for each solution. NaCl was used as the control treatment. After 8 h of treatment, positive roots were collected, washed, and stained with β-glucosidase (GUS). The positive roots after the above treatment were immersed in GUS staining solution (ready-to-use GUS staining solution, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) and incubated at 37 °C for 18 h.

[0096] Results Analysis: The positive roots of the 35S::GFP-PvGIP1Pro::GUS transgenic bean hairy-root plants exhibited green fluorescence. The positive roots in the control NaCl-treated group showed no obvious blue color in GUS staining, but the positive roots in the CdCl2, CuCl2, CrCl2, MnCl2, and NiCl2-treated groups showed localized blue staining, indicating that the PvGIP1 promoter PvGIP1Pro has promoter function and can initiate the expression of exogenous genes under heavy metal stress. The PvGIP1 promoter PvGIP1Pro is a heavy metal-inducible promoter. GUS staining results are shown below. Figure 5 As shown.

[0097] Therefore, this invention discloses a heavy metal-responsive inducible promoter, PvGIP1Pro, whose nucleotide sequence is shown in SEQ ID NO: 1, with a full length of 2167 bases. It was cloned from the promoter region of the common bean gene PvGIP1 (gene number Phvul.001G120800). When transgenic common bean hairy-root plants were treated with NaCl as a control, it was found that the PvGIP1 promoter did not significantly drive the expression of the exogenous gene GUS in the roots. However, when the transgenic common bean hairy-root plants were treated with heavy metals Cd, Cr, Mn, Ni, or Cu, the PvGIP1 promoter induced the expression of the exogenous gene GUS in the roots, resulting in blue staining of the transgenic positive roots, demonstrating that the promoter was significantly induced by heavy metal stress. As an inducible promoter capable of heterologous expression, it has potential application value in studying plant gene expression and regulation, and in using genetic engineering methods to solve food safety problems.

[0098] In summary, the heavy metal-inducible promoter of the present invention can control the heavy metal-induced expression of genes in plants. It can provide a useful core component for studying plant gene expression, regulation, and using genetic engineering methods to change the tolerance and accumulation patterns of plants to heavy metals, as well as for developing probes for detecting heavy metals. It is suitable for large-scale application.

[0099] In this specification, the invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations can be made without departing from the spirit and scope of the invention. Therefore, the specification and drawings should be considered illustrative rather than restrictive. sequence list <110> China Jiliang University <120> Heavy metal-inducible promoters, related primer pairs, expression vectors, and applications <160> 3 <210> 1 <211> 2167 <212> DNA <213> Phaseolus vulgaris Linn. <220> <221> Promoter <222> (1)...(2167) <223> Nucleotide sequence of the promoter PvGIP1Pro of the Phaseolus vulgaris PvGIP1 gene <400> 1 caagtgaata gatgacctcc ttatacatgg gaaaattttc aacaacacaa taactttgaa 60 gggacaggat gaacacttaa tgacacatgc aaaagttgtg tcatgctagt ataacttatt 120 catataaagt catgtcatgc atacacaact tactttctac gtaaattttt taaaacttta 180 ttatttggat aactccttaa aattacatta taatgataaa tttgtaacat ttattctcta 240 aaataaacct aatgcgtaga aagttaaaaa cactaaaaat acaaagagta tatttttata 300 aaattacaag tttttatagt aaaaaaaaga aataaatgac aaatatagta ttagattaat 360 gtagttagat aagattccaa ttttaaaatt taaataataa ataataaata atattttaag 420 aaaaaaacca ttaacaaaaa taataattga agaataataa taaattttat tttgtattcc 480 ctgtttattt ccaattaaac aactaataac caatctcatt gtacaaaact ggaaattttt 540 actctagatg aaaaccaatg attgagccag tgactaaaag aaaaaatatt ttaattttta 600 atgtttgata taaaattgta acataaacaa attaaaagtt gttatgaact atttgttgga 660 gatcttattg ttaagaatgt gggctttaag cctaactcaa cctcattaaa ctggcttatg 720 gggtgaggtt tgtacccagt tatatacaat atcctaatct ctactcgatg tgaaatctcc 780 aacactattt tatatttttt tatgaaatat aattttgtct tttaaatcaa tttgtttatt 840 ttcttataat ttgatattat attaattttt cggtttatta tccaattttt ctattattaa 900 tggatataat gtcaatatat taattagtat aattattatg atatgtttta ttttgaatat 960 gataaatcaa atacaatttt ttttactata taaaattatg gtattattta tttaatataa 1020 tatatttatt aaacaagttc aatcgcagtt taatatttat ccagtttaat aactaatcca 1080 atttcaaagg ttacaaaac attgttattt tgaccttat ataccaaaga accattatagtt 1140 ttctcatatg aattttttct gttgattttc gggattcatt catatttcag acagcacatg 1200 catggaccgt agccggatcc gttagtcggc ccaattttaa aaaaataaca tataaagtta 1260 tttcttccag caccccgtca ttttctcagc atccattaat atcttaaaag acgattttac 1320 cccttttaa atgacttttg gattaatttt ctgaaatctt ccagacacct atttcggaat 1380 ttttataatt gtattccgga aaacgttttt ttggaattttt caaaaaaaaa ttcataaatt 1440 tcttttggaa cgtgaatagt actttctgga atctgttttt gcaataaatt ttctgaaaaa 1500 acatttagaa agaactttct ggaataagag cagattagaa gatatatttt aaacattttc 1560 cgtatttgac agatgcatg aagaagtttg aggggggtga aagaagaaac acccaatctt 1620 ttatttgatg gtccaaggat gaggcccaaa cgtatttcat ctgcgggtaa atcgcgtcgc 1680 tccgaaatcg cgttcactta accctaagaa aaagccccaa atcgaacttc attcactgaa 1740 atcgcgccgt ttgtgtctc gccgcgcgcg cctgtcgttc tctcgccgt cagtggcgcc 1800 gcttctgttc tcctgctcg catttggcac tgttgcgccg ttgttcaaga tcgcgcgcac 1860 cgcttgccaa agcagtcgcc gtcgtgctcg ccgctgtccc tctctattct tctgctgccg 1920 tggttctgca cctcccctct ctgtaatctt caatttttctg attgtgctgt cgcgtgctcc 1980 acttttctat tttaattgtt ttaattattt tttgatttca gtcgcggaac tgttcctctc 2040 ttttgtttca gttttttgata tagatcctgg agttacgatt tttaattcca ttttgttcct 2100 ttttttgttt caggaatttt aatttttgtg atactaatga attgaacatt ctctgcccat 2160 ttatcag 2167 <210> 2 <211> 52 <212> DNA <213> Artificial sequence <220> <221> misc_feature <222> (1)...(52) <223> Forward primers for amplifying the promoter PvGIP1Pro of the common bean PvGIP1 gene <400> 2 aggctaatct ggggacctgc aggcaagtga atagatgacc tccttataca tg 52 <210> 3 <211> 45 <212> DNA <213> Artificial sequence <220> <221> misc_feature <222> (1)...(45) <223> Reverse primer for amplifying the promoter PvGIP1Pro of the common bean PvGIP1 gene <400> 3 aagttcttct cctttactag tctgataaat gggcagagaa tgttc 45

Claims

1. A heavy metal inducible promoter, characterized in that, The nucleotide sequence of the heavy metal inducible promoter is shown as SEQ ID NO:

1.

2. A plant recombinant expression vector comprising a plant expression vector, characterized by, The plant recombinant expression vector further comprises the heavy metal inducible promoter according to claim 1, and the heavy metal inducible promoter is integrated into the plant expression vector.

3. Use of the heavy metal inducible promoter according to claim 1 or the plant recombinant expression vector according to claim 2 in heavy metal induced gene expression in plants, wherein the heavy metal is Cd, Cr, Mn, Ni or Cu.

4. Use according to claim 3, wherein the compound is ###0002### The plant is Phaseolus vulgaris.

5. The use according to claim 3, wherein the compound is ###0002### The gene is GUS Gene.

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