CN115011600B - An inducible promoter GmbHLH47 promoter responding to iron deficiency stress and its application - Google Patents

Abstract

An inducible promoter GmbHLH47 promoter responding to iron deficiency stress and its application belong to the technical field of genetic engineering. In order to improve the iron stress resistance of plants, the invention provides an inducible promoter GmbHLH47 promoter in response to iron deficiency stress, which is cloned from the promoter region of soybean GmbHLH47 gene, and the nucleotide sequence is as SEQ ID NO. 1. Through the analysis of the promoter region, it was found that it contains a variety of cis-acting elements that may be involved in multiple stresses. When the transgenic plants were treated with iron-deficiency medium, it was found that the GmbHLH47 promoter could increase the expression of the exogenous gene GUS, resulting in a deeper staining of the transgenic plants, which proved that the GmbHLH47 promoter was induced by iron-deficiency stress. As a promoter induced by iron deficiency stress, its promoter region and upstream regulatory sequences play an important role in genetic engineering and improving plant resistance to iron deficiency.

Description

An inducible promoter GmbHLH47 promoter responding to iron deficiency stress and its application

technical field

The invention belongs to the technical field of genetic engineering, in particular to an inducible promoter GmbHLH47 promoter responding to iron deficiency stress and its application.

Background technique

Soybean (Glycine Max L) is one of the five major food crops in the world. It is an annual herbaceous dicotyledonous plant, and soybean seeds are rich in nutrition. Iron is one of the essential trace elements for plant growth and development, and it plays an important role in physiological metabolic processes such as photosynthesis, respiration, nitrogen fixation, protein and nucleic acid synthesis in plants. Although iron is abundant in most soils, iron is not bioavailable in the form of insoluble iron oxide. Under iron-deficiency conditions, the underground parts of soybean have poor root development, poor growth, short and thin roots, and fewer root nodules, which in turn lead to yield reduction, and in severe cases, early wilting or even death of plants.

A plant promoter is a DNA sequence that can specifically bind to RNA polymerase and its transcription factors and determine the initiation of gene transcription. The inducible promoter can accept the induction signal to induce the expression of the gene quickly and effectively, and can also relieve the stress at any time to stop the expression. For example, under abiotic stress, transcription factors (regulatory proteins) related to adversity stress specifically combine with key cis-elements in the upstream promoter of genes to enhance promoter activity, increase the expression of target genes, and improve plant stress resistance.

Contents of the invention

In order to improve the iron stress resistance of plants, the invention provides an inducible promoter GmbHLH47 promoter that starts the transcription of foreign genes under iron deficiency stress, and the GmbHLH47 promoter can drive the expression of genes under iron deficiency conditions, and the GmbHLH47 The nucleotide sequence of the promoter is shown in SEQ ID NO.1.

The present invention also provides the application of the above-mentioned GmbHLH47 promoter in improving the ability of plants to resist iron stress.

Further defined, the plant is Arabidopsis or soybean.

The present invention also provides a recombinant vector containing the above-mentioned GmbHHLH47 promoter.

The present invention also provides the application of the above-mentioned recombinant vector in improving the iron stress resistance ability of plants.

The present invention also provides a transgenic cell line or a recombinant bacterium containing the above-mentioned GmbHHLH47 promoter.

The present invention also provides the application of the above-mentioned transgenic cell line or recombinant bacteria in improving the iron stress resistance of plants.

The present invention also provides a method for improving plant resistance to iron stress, said method comprising the steps of:

(1) constructing a recombinant vector containing the GmbHLH47 promoter, the nucleotide sequence of the GmbHLH47 promoter is shown in SEQ ID NO.1;

(2) Prepare the Agrobacterium carrying the GmbHLH47 promoter: transfer the recombinant vector obtained in step (1) into Agrobacterium competent for cultivation to obtain the Agrobacterium carrying the GmbHLH47 promoter;

(3) Preparation of transgenic plants carrying the GmbHLH47 promoter: using the Agrobacterium obtained in step (2) to infect plants to obtain plants carrying the GmbHLH47 promoter.

To further define, the construction method of the recombinant vector described in step (1) is as follows: use restriction endonucleases to digest the vector pMD18T-GmbHLH47 promoter and pBI121-GUS plasmid respectively to obtain the sequence fragment of the GmbHLH47 promoter and pBI121-GUS The digested product of the plasmid, and then the sequence fragment of the GmbHLH47 promoter was ligated with the digested product of the pBI121-GUS plasmid to obtain pBI121-GmbHLH47pro::GUS.

To further define, the system of the above ligation reaction is: 5 μL of pBI121-GUS plasmid digestion product, 3 μL of the sequence fragment of the GmbHLH47 promoter, 1 μL of 10×T4 DNA ligation buffer and 1 μL of T4 DNA ligase, and the reaction temperature is 16°C, the reaction time is 12h.

Further defined, the restriction endonucleases are HindIII and BamH1.

Further defined, the plant is Arabidopsis or soybean.

It is further defined that when the plant is Arabidopsis, the timing of the infection in step (3) is that Arabidopsis reaches the flowering stage; when the plant is soybean, the timing of the infection in step (3) is soybean When cotyledons emerge.

To further define, the steps of Agrobacterium infecting Arabidopsis in the above step (3) are as follows:

(1) Take 50 μL of the preserved pBI121-GmbHLH47pro::GUS Agrobacterium liquid in the ultra-clean workbench, inoculate it into 25 mL of YEB liquid culture containing Km, Str, and Rif, and cultivate it on a shaker at 28°C for about 17 hours at 200 rpm. Place 500 μL of the activated bacterial solution in 50 mL of YEB liquid culture containing Km, Str, and Rif, and culture on a shaker at 200 rpm at 28°C for about 15 hours, and stop when the OD ₆₀₀ reaches 1.2-1.5.

(2) Put the activated Agrobacterium in a centrifuge and centrifuge at 5000rpm for 10min, discard the supernatant, collect the bacteria in the centrifuge tube, add 5% sucrose (prepared for current use) 20mL to resuspend, take 2mL to resuspend Bacteria solution, its OD value was measured at 600nm, and the OD value reached 0.9 before it could be used for transformation, and 0.04% Silwet L-77 was added to the transformation solution before transformation.

(3) Select healthy and vigorous plants. When the flower buds of Arabidopsis thaliana are about to open, the transformation effect is the best. When doing transformation, use a pipette to draw 200 μL of the newly prepared transformation solution, and add 2 drops to the flower buds of Arabidopsis thaliana. -3 drops and repeat the operation 2-3 times.

(4) After dipping the flowers, move the plants to a dark place to keep moisture for 24 hours, then move the plants to a light culture room (temperature 22°C, light 16 hours/darkness 8 hours) for normal cultivation, and prepare transformation solution again after 3 days for secondary transformation.

(5) Place T0 Arabidopsis seeds in 1/2MS medium containing Km resistance for selection.

Further definition, the step of Agrobacterium infecting soybean in step (3) is as follows:

(1) Select Heinong 53 soybean seeds, disinfect them with sterile 28% sodium hypochlorite for 5 minutes in an ultra-clean workbench, rinse them several times with sterile water, put them into MS medium with the hilum down, and culture them in dark for 6 days.

(2) The pBI121-GmbHLH47pro::GUS K599 Agrobacterium was resuspended in an ultra-clean bench to obtain a bacterial solution with an OD ₆₀₀ of 0.5-0.7, which was used to infect soybean cotyledon nodes.

(3) Take dark-cultured sterile soybeans, cut off the cotyledon at a distance of 0.3-0.5 cm with a knife, cut the cotyledon from the cotyledon node, and cut off the terminal bud. Use a tissue culture knife to make several cuts on the surface of the cotyledons and cotyledon nodes, which are called explants.

(4) Soak the explants in the bacterial solution for 30 minutes, and shake the bacterial solution from time to time. After infection, the cotyledon nodes were blotted dry with filter paper, placed on MS1 solid medium, sealed, and cultured in the dark for 5 days.

(5) After dark culture, wash the cotyledon nodes with MS3 liquid medium containing Cef and Km resistance, first rinse twice, and then soak for half an hour in order to thoroughly wash the bacteria. Cut to 0.5 cm, place the hypocotyl of the cotyledon downwards, insert it into MS3 solid medium obliquely, and culture at 25°C for 16 hours under light.

Beneficial effects of the present invention:

The GmbHLH47 promoter was cloned from the promoter region of soybean GmbHLH47 gene. Through the analysis of the promoter region, it was found that it contains a variety of cis-acting elements that may be involved in multiple stresses. When the transgenic plants were treated with iron-deficiency medium, it was found that the GmbHLH47 promoter could increase the expression of the exogenous gene GUS, resulting in a deeper staining of the transgenic plants, which proved that the GmbHLH47 promoter was induced by iron-deficiency stress. As a promoter induced by iron deficiency stress, its promoter region and upstream regulatory sequences play an important role in genetic engineering and improving plant resistance to iron deficiency.

Description of drawings

Fig. 1 is the result figure of the PCR amplification of promoter GmbHHLH47;

Figure 2 is a structural diagram of the plant expression vector pBI121-proGmbHLH47::GUS recombinant plasmid;

Fig. 3 is the graph of the GUS staining results of soybean hairy roots transformed with pBI121-proGmbHLH47::GUS; wherein, A in Fig. 3 is the cross section of iron deficiency, B in Fig. 3 is the longitudinal section of iron deficiency, and C in Fig. 3 is Iron root tip, D in Figure 3 is a normal cross section, E in Figure 3 is a normal longitudinal section, and F in Figure 3 is a normal root tip;

Figure 4 is the GUS staining results of Arabidopsis thaliana transfected with pBI121-proGmbHLH47::GUS; wherein, A in Figure 4 is the GUS staining results of wild-type Arabidopsis thaliana, and B in Figure 4 is the iron-deficiency transfected pBI121-proGmbHLH47:: GUS Arabidopsis staining results;

Fig. 5 is transproGmbHLH47 gene-positive Arabidopsis;

Fig. 6 is the hairy root of transproGmbHLH47 gene-positive soybean.

Detailed ways

The present invention will be described in further detail below in conjunction with specific examples and accompanying drawings. The following examples are convenient for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The pharmaceutical reagents used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Experimental materials: wild-type Arabidopsis thaliana seeds were preserved by our laboratory, and soybean (Glycine max) material Heinong 51 was kindly donated by the Soybean Research Laboratory of the Farming and Cultivation Institute of Heilongjiang Academy of Agricultural Sciences.

Test reagents: gel recovery kit (TIANGEN), RNA extraction kit (TIANGEN), PCR kit (TAKARA), vector ligation kit (TAKARA), plasmid extraction kit were purchased from AXYGEN Biological Company, and GUS staining kit.

Example 1: Obtaining of the GmbHHLH47 promoter

1. The method of extracting soybean DNA:

(1) Put 0.1 g of soybean leaves into a mortar precooled with liquid nitrogen, and grind to a fine powder.

(2) Transfer 50 mg of powder to a 1.5 mL sterile centrifuge tube, add 400 μL SP1 Buffer and 5 μL RNase A, and vortex at maximum speed to mix.

(3) Incubate at 65°C for 10 minutes, remove and mix twice during incubation.

(4) Add 140 μL SP2 Buffer, vortex and mix at maximum speed.

(5) Cool in ice for 5 minutes, and centrifuge at maximum speed for 10 minutes.

(6) Put the Homogenizer Comuln into a 2mL collection tube, carefully transfer the supernatant to the Homogenizer Comuln, be careful not to transfer the precipitate, and centrifuge at the maximum speed for 2 minutes.

(7) Transfer the clarified lysate to a new 1.5mL centrifuge tube, taking care not to transfer the precipitate, and measure the transferred volume.

(8) Add SP3 Buffer 1.5 times the volume of the lysate, and vortex to mix.

(9) will Insert the Mini Column into a 2mL collection tube, add 650μL of mixed lysate to In the Mini Column, centrifuge at maximum speed for 1 min and discard the filtrate.

(10) Repeat step (10).

(11) will Insert the Mini Column into a 2mL collection tube, add 650μL SPW WashBuffer to /> In the Mini Column, centrifuge at maximum speed for 1 min and discard the filtrate.

(12) Repeat step (11).

(13) will Set the Mini Column into a 2mL collection tube, and centrifuge the empty column at the maximum speed for 2 minutes.

(14) will Set the Mini Column into a new 1.5mL centrifuge tube, add 50-100μL 65℃ preheated Elution Buffer, place at room temperature for 3-5min, and centrifuge at maximum speed for 1min.

(15) Repeat step (14).

2. The method of obtaining the GmbHH47 promoter:

Find proGmbHLH47 sequence from soybean genome database, design the nucleotide sequence of 1510bp length, utilize proGmbHLH47-S and proGmbHLH47-A to carry out PCR amplification, the nucleotide sequence of proGmbHLH47-S is: 3'-AATGGAAGATGAGGAGGAT5' (as SEQ ID NO.2), the nucleotide sequence of proGmbHLH47-A is: 3'-GTGATTAAAGCCAGTTATATG-5' (shown in SEQ ID NO.3).

The reaction system was 7.2 μL of Distilled sterile water, 1 μL of 10×PCR Buffer, 0.5 μL of soybean genomic DNA, 0.8 μL of dNTP Mix (2.5 mmol/L), 0.2 μL of proGmbHLH47-S (10 μmol/L), 0.2 μL proGmbHLH47-A (10 μmol/L) and 0.1 μL rTaq DNA polymerase (5U/μL). The reaction program was 94°C for 5min, 94°C for 30s, 58.3°C for 30s, 72°C for 2min, 72°C for 10min, 4°C for 1h, and 30 cycles.

The nucleotide sequence of the GmbHLH47 promoter is shown in SEQ ID NO.1:

ATGAAGAACAATAATTCTAAGAGAAGTGGTGTAAGAAAATACCACAAATCAAAACAAATGGAAGATGAGGAGGATGATAAATTATAAAATTTGCATTATTTCAATGCTGAGATGGATTATAAAAGTTGAGTTGCTATTTGATAGAAAGGACTACATAACCAAGACGGTGATTAAGCTAGTAATCAAATTAAAATAATACGAATTCACAAAAAAAGTAATAATTA AATAATACGTGGACGAGCATGATGATGGCTGCGAGCAAGTAAAGCAATTTTAGTATCCGAACGTTTTTTGCCTTTTTGGTTTGCTTAGACTCAAAATTAAAATAAAAATTACTTTAATTTTTCGTTCTACACATTTAATAATTCTATATTAAGAACTTTTAATAAAGAATAAATAATTTTAAAAAACATAAATATTTTTAAAAATAAATTTTACGTGAATGATGTGATGA TATATACAAAAGGAAAAGAGAAAAATATGTAAACAAACGAGAAAAATAAAAAAGGGAATCATGAAAATGTACAAAAAAGAAATTAATAGAAAAAATGACTCAAATGAGTAACAATAAATACTGTATTCAAATGGATAAAAAGATAATTGTAAAAACGAGTAAAAAAGTTAGTACAAAATATAAACGGGCAAAAAAATTGTAGAAAAAAAATTAATAAGCTGCCAAAAAACAAT ACAAATGGGGAGGAAAAAAATATTCAAATAAGTAGAAAAGTAGAAAATGTGTACATGAAGGAGAAATGGATCAACAAATTATGCAAATGGATAAAAAATGGGCTGAAAAAATACAGATAGATAGAACAGAAATAATGAAAATGGATTGAAAATTGTCATTTAATTGAGAGAAAACATGTATAACCAAAATAAAATATTTCATAATAAAAAATTATATTATTATTATTACAAA AAGACCTAAAAAAAAAAAAGGATCACGTGGGGAACTCATTGGTGGTTGTTGATTGAAGCGAAGGAGCATGACCTCGCCACGCGCCTTCGCGTGACAGATGCCCTCCCGCCACACTCCATCACCACAACCCCAATCTTCTTTTCTCTCCTCTGCCCTAATTTTCCAACTTCAATTTCATTCCTATTCCAATCCTATTCCTTTCGATTCCTC TTCCCCAGTTTGGTAAGCAAAAAACCACTTTCCCTCTTTCTGCACACAATTTATCTTCCCTCTTTTTTCTATTCGGTCGTTTTCTGTTTGATCCTTGGAATTTCTCGATGCTCTGTTTTGGCTGCTATGAAGTGTGGAAATTAAAATTGGGAACTTGGTTTTGTTGTTTTTTTACCCTTTCTCTGATTGGATTGCGATAAAGGGAATTGATTTAATCA GGAAACATGGATTTTACTTTTTCATTGACCGTAAAAAAGAAATTTCCATTTCTTGGGGTGTTCGAATTACTTCTCCTTTCGTTGTAAGAATAAAAAGTACATTATTGAAGTAGTTTTGTTTTTTCTACTTTAGGAAATTTTGTTGCTGGAAGAATTGCTTAAGGGCATATAACTGGCTTTAATCAC

Using the PlantCARE online software to analyze the transcriptional regulatory elements of the promoter fragment sequence of the GmbHH47 gene, it was found that there are 26 cis-acting elements in the promoter of the GmbHH47 gene, mainly including core elements, hormone response elements, light response elements, MYB response elements, other elements and Three unnamed components.

Embodiment 2: Construction of the recombinant vector containing the GmbHLH47 promoter

1. Construct the vector pMD18T-proGmbHLH47

(1) Reaction system: 1 μL of pMD18 T vector, 3 μL of target gene fragment solution, 1 μL of ddH ₂ O, 5 μL of Solution I. The reaction temperature is 16°C, and the reaction time is more than 12h (overnight).

(2) Ligate the proGmbHLH47 plasmid fragment to the cloning vector pMD18T, and transfer the ligated product into Escherichia coli competent by the heat shock method. The specific content is as follows:

Take the competent E. coli out of the 80°C refrigerator and put it on ice quickly. After it melts, add 10 μL of the ligation product, and bathe in ice for 30 minutes; after taking it out, put it in a water bath at 42°C for 1 minute and 15 seconds, and then quickly insert it into the ice. Ice bath for 2min. Add 800 μL of LB liquid medium, place on a shaker at 200 rpm, 37°C, and shake for 1 hour; take out the centrifuge tube, centrifuge at 4000 rpm for 5 minutes, take out 800 μL of supernatant, resuspend the remaining bacteria, and dissolve the remaining about 200 μL of bacterial solution at a volume ratio of 3 Smear on two LB solid media containing ampicillin resistance at a ratio of :1; culture upside down in a 37°C incubator overnight; pick a single colony on the culture medium for identification, and pick a positive colony for amplification and culture, use The plasmid extraction kit was used to extract the pMD18T-proGmbHLH47 plasmid.

2. Construction of recombinant vector pBI121-GmbHLH47pro::GUS

(1) Digest the plasmids of pMD18T-proGmbHLH47 and pBI121:

The pMD18T-proGmbHLH47 plasmid and the pBI121-GUS plasmid were respectively digested with restriction endonucleases to obtain the sequence fragment of the GmbHLH47 promoter and the digestion product of the pBI121-GUS plasmid. Then, the sequence fragment of the GmbHLH47 promoter was ligated with the digested product of the pBI121-GUS plasmid to obtain pBI121-GmbHLH47pro::GUS. The structure of the recombinant vector is shown in FIG. 2 .

The reaction system for double digestion was: 70 μL of pMD18T-proGmbHLH47/pBI121-GUS plasmid, 10 μL of TAKARA10×KBuffer, 4 μL of Hind III, 4 μL of BamHI and 12 μL of ddH ₂ O.

The system for the ligation reaction is: 5 μL of pBI121-GUS plasmid digestion product, 3 μL of the sequence fragment of the GmbHLH47 promoter, 1 μL of 10×T4 DNA ligation buffer and 1 μL of T4 DNA ligase, the reaction temperature is 16°C, and the reaction time is for 12h.

Identification of recombinant plasmids:

Double enzyme digestion identification, extract pBI121-proGmbHLH47::GUS positive plasmid, and use Hind III and BamHI for double enzyme digestion identification, the reaction system is: 32 μL pBI121-proGmbHLH47::GUS recombinant plasmid, 5 μL T AKARA10×KBuffer, 2 μL Hind III, 2 μL of BamHI and 9 μL of ddH ₂ O yielded a target band of about 1510 bp (see Figure 1).

Embodiment 3: the preparation method of GV3101 Agrobacterium carrying proGmbHLH47

Freeze-thaw method to transform the recombinant plasmid pBI121-proGmbHLH47::GUS, the method is as follows:

(1) Thaw competent cells stored at -80°C in ice.

(2) Add 10 μL of the pBI121-GmbHLH47pro::GUS recombinant plasmid to 100 μL of Agrobacterium competent cells GV3101, and mix by hand at the bottom of the tube.

(3) Put them on ice for 5 minutes, liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes, and ice bath for 5 minutes.

(4) Add 700 μL of antibiotic-free YEB liquid medium, and shake and culture at 28° C. for 2-3 hours.

(5) Collect the bacteria by centrifuging at 6000rmp for 1min, take 100μL of the supernatant to resuspend the bacterial mass, and spread the bacterial solution on a medium containing kanamycin (50mg/mL) + streptomycin (50mg/mL) + rifampicin (50mg/mL) mL) of YEB solid medium, placed upside down and placed in a 28°C incubator for 2-3 days in the dark. Identify the transformed Agrobacterium; the identification method is: pick a single colony with relatively uniform growth, and perform colony PCR amplification with gene-specific primers; the reaction system for identification is: Template DNA colony, 1 μL of 10×Buffer, 0.8 μL dNTP Mix, 0.2 μL of proGmbHLH47-HindIII, 0.2 μL of proGmbHLH47-BamHΙ, 0.1 μL of rTaq and 7. μL of ddH ₂ O; the reaction conditions are: ①94℃, min; ②94℃, 3s; ③58.3℃, 3s; ④72°C, 1min30s; ⑤72°C, 10min; ⑥4°C, 1h; ①～⑥cycle 30 times.

Embodiment 4: the preparation method of K599 Agrobacterium carrying proGmbHLH47

Freeze-thaw method to transform the recombinant plasmid pBI121-proGmbHLH47::GUS, the method is as follows:

(1) Thaw competent cells stored at -80°C in ice.

(2) Add 1 μL of the pBI121-GmbHLH47pro::GUS recombinant plasmid to 100 μL of Agrobacterium competent cells K599, and mix by hand at the bottom of the tube.

(3) Put them on ice for 5 minutes, liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes, and ice bath for 5 minutes.

(4) Add 700 μL of TY liquid medium without antibiotics, shake and culture at 28°C for 2-3h.

(5) Collect the bacteria by centrifuging at 6000rmp for 1min, take 100μL of the supernatant to resuspend the bacterial mass, and spread the bacterial solution on a medium containing kanamycin (50mg/mL) + streptomycin (50mg/mL) + rifampicin (50mg/mL) mL) of TY solid medium, placed upside down and placed in a 28°C incubator for 2-3 days in the dark. Identify the transformed Agrobacterium; the identification method is: pick a single colony with relatively uniform growth, and perform colony PCR amplification with gene-specific primers; the reaction system for identification is: Template DNA colony, 1 μL of 10×Buffer, 0.8 μL dNTP Mix, 0.2 μL of proGmbHLH47-HindIII, 0.2 μL of proGmbHLH47-BamHΙ, 0.1 μL of rTaq and 7.7 μL of ddH ₂ O; the reaction conditions are: ①94℃, 5min; ②94℃, 30s; ③58.3℃, 30s ; ④72°C, 1min30s; ⑤72°C, 10min; ⑥4°C, 1h; ①～⑥cycle 30 times.

Embodiment 5: the method for improving soybean hairy root resistance to iron stress

① Select Heinong 53 soybean seeds, disinfect them with sterile 28% sodium hypochlorite for 5 minutes in an ultra-clean workbench, rinse them several times with sterile water, put them into MS medium with the hilum down, and culture them in dark for 6 days.

② Resuspend the pBI121-GmbHLH47 K599 Agrobacterium obtained in Example 4 in an ultra-clean workbench to obtain a bacterial solution with an _OD600 of 0.5-0.7, which is used to infect soybean cotyledon nodes.

③Take dark-cultured sterile soybeans, cut off the cotyledon 0.3-0.5cm away from the cotyledon with a knife, cut off the cotyledon from the cotyledon node, and cut off the terminal bud. Use a tissue culture knife to make several cuts on the surface of the cotyledons and cotyledon nodes, which are called explants.

④ Soak the explants in the bacterial solution for 30 minutes, and shake the bacterial solution from time to time. After infection, the cotyledon nodes were blotted dry with filter paper, placed on MS1 solid medium, sealed, and cultured in the dark for 5 days.

⑤ After dark culture, rinse the cotyledon nodes with MS3 liquid medium containing Cef and Km resistance, first rinse twice, and then soak for half an hour in order to thoroughly wash the bacteria, then dry the cotyledon nodes after washing, and cut the hypocotyls to 0.5cm, cotyledon hypocotyls facing down, inserted into MS3 solid medium obliquely, cultured at 25°C for 16h under light.

The obtained transproGmbHLH47 gene-positive soybean hairy roots are shown in FIG. 6 .

Embodiment 6: the method for improving Arabidopsis resistance to iron stress

(1) Activated Agrobacterium

Take 50 μl of the pBI121-GmbHLH47pro::GUS Agrobacterium liquid obtained in Example 3 in the ultra-clean workbench, inoculate it into 25 mL of YEB liquid culture containing Km, Str, and Rif, and culture it on a shaker at 28°C and 200 rpm for about 17 hours. Take 500 μL of the above-mentioned activated bacterial solution and place it in 50 mL of YEB liquid culture containing Km, Str, and Rif. Cultivate it on a shaker at 28°C and 200 rpm for about 15 hours, and stop when the OD ₆₀₀ is about 1.2-1.5.

(2) Put the activated Agrobacterium in a centrifuge and centrifuge at 5000rpm for 10min, discard the supernatant, collect the bacteria in the centrifuge tube, add 5% sucrose (prepared for current use) 20mL to resuspend, take 2mL to resuspend Bacteria liquid, its OD value was measured at 600nm, and the OD value reached 0.9 before it could be used for transformation, and 0.04% Silwet L-77 was added to the transformation liquid before transformation.

(3) Select healthy and vigorous plants. When the flower buds of Arabidopsis thaliana are about to open, the transformation effect is the best. When doing transformation, use a pipette to draw 200 μL of the newly prepared transformation solution, and add 2 drops to the flower buds of Arabidopsis thaliana. -3 drops and repeat the operation 2-3 times.

(4) After dipping the flowers, move the plants to a dark place to keep moisture for 24 hours, then move the plants to a light culture room (temperature 22°C, light 16 hours/darkness 8 hours) for normal cultivation, and prepare transformation solution again after 3 days for secondary transformation.

The obtained transproGmbHLH47 gene-positive Arabidopsis is shown in FIG. 5 .

Method for GUS staining of transfected pBI121-proGmbHLH47::GUS plants

The transgenic pBI121-proGmbHLH47::GUS Arabidopsis was placed in normal and iron-deficient 1/2MS medium containing Km for screening to obtain positive plants and stained. Soybean cotyledon nodes infected with pBI121-proGmbHLH47::GUS K599 Agrobacterium rhizogenes were cultured in normal MS3 medium for about a week. After a small amount of hairy roots were produced, they were replaced with iron-deficient MS3 medium until a large number of hairy roots grew. Roots are dyed.

The positive hairy root sections of normal culture and iron deficiency treatment were stained with GUS on the transverse section, longitudinal section and root tip. The staining results are shown in Fig. condition, the xylem and phloem of the hairy root had obvious blue signals, and the cortex had weak blue signals. The root tips of soybean hairy roots showed a blue signal, and under normal conditions, GUS activity was only detected in the phloem of the vascular bundles of the hairy roots, indicating that proGmbHLH47 has promoter activity and is regulated by iron deficiency.

GUS staining was performed on the transgenic pBI121-proGmbHLH47::GUS Arabidopsis and wild-type Arabidopsis. The results are shown in Figure 4. A blue signal was detected in the transgenic pBI121-proGmbHLH47::GUS Arabidopsis plants, but no GUS activity was detected in the control .

SEQUENCE LISTING

<110> Harbin Normal University

<120> An Inducible Promoter GmbHLH47 Promoter Responding to Iron Deficiency Stress and Its Application

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 1566

<212>DNA

<213> Glycine Max

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tttgttgttt ttttttaccc ctttctctga ttggattgcg ataaagggaa ttgattaat 1380

caggaaacat ggattttact ttttcattga ccgtaaaaag aaatttccat ttcttggggt 1440

gttcgaatta cttctccttt cgttgtaaga ataaaaagta cattattgaa gtagttttgt 1500

ttttctactt taggaaattt tgttgctgga agaattgctt aagggcatat aactggcttt 1560

aatcac 1566

<210> 2

<211> 19

<212>DNA

<213> Synthetic

<400> 2

aatggaagat gaggaggat 19

<210> 3

<211> 21

<212>DNA

<213> Synthetic

<400> 3

gtgattaaag ccagttatat g 21

Claims (9)

1. Inducible promoter responding to iron deficiency stressGmbHLH47A promoter, characterized in that theGmbHLH47The nucleotide sequence of the promoter is shown as SEQ ID NO. 1.

2. Claim 1 is a deviceGmbHLH47The application of the promoter in improving the iron stress resistance of plants.

3. Comprising the composition of claim 1GmbHLH47Recombinant vector of promoter.

4. Use of the recombinant vector of claim 3 for increasing the iron stress resistance of plants.

5. Comprising the composition of claim 1GmbHLH47The application of transgenic plant cell line or recombinant bacteria of promoter in raising iron stress resistance of plant.

6. A method of increasing the iron stress resistance of a plant, said method comprising the steps of:

(1) Construction of the inclusionGmbHLH47Recombinant vector of promoter, said recombinant vector comprising said promoterGmbHLH47The nucleotide sequence of the promoter is shown as SEQ ID NO. 1;

(2) Preparation of carrying bagGmbHLH47Agrobacterium of promoter: transferring the recombinant vector obtained in the step (1) into agrobacterium competence for culture to obtain the carrierGmbHLH47Agrobacterium of promoter;

(3) Preparation of carrying bagGmbHLH47Transgenic plant of promoter: infecting plants with the agrobacterium obtained in the step (2) to obtain the carrying productGmbHLH47A plant of the promoter.

7. The method of claim 6, wherein the recombinant vector of step (1) is constructed by the following steps: the vector pMD18T-GmbHLH47 promoter and pBI121-GUS plasmid are digested with restriction enzymes, respectively, to obtainGmbHLH47The sequence fragment of the promoter was digested with pBI121-GUS plasmid, and thenGmbHLH47The sequence fragment of the promoter was ligated with the cleavage product of the pBI121-GUS plasmid.

8. The method of claim 6, wherein the plant is arabidopsis thaliana or soybean.

9. The method of claim 8, wherein when the plant is arabidopsis thaliana, the timing of the infestation of step (3) is when arabidopsis thaliana reaches anthesis; when the plant is soybean, the infection time in the step (3) is when the soybean grows out of cotyledonary node.