ABA-induced phaseolus vulgaris hemagglutinin promoter, and related primer pair, expression vector and application thereof

Abstract

The invention provides an ABA inducible kidney bean hemagglutinin promoter. The nucleotide sequence of the ABA inducible kidney bean hemagglutinin promoter is as shown in SEQ ID NO: 1. The invention also provides a plant recombinant expression vector containing the ABA-induced phaseolus vulgaris hemagglutinin promoter, a primer pair for amplifying the ABA-induced phaseolus vulgaris hemagglutinin promoter, and application of the ABA-induced phaseolus vulgaris hemagglutinin promoter or the plant recombinant expression vector containing the ABA-induced phaseolus vulgaris hemagglutinin promoter in ABA induced expression in plants. The ABA inducible phaseolus vulgaris hemagglutinin promoter can respond to a specific inducible factor, can effectively regulate and control expression of downstream genes in plants, and is suitable for large-scale popularization and application.

Description

Technical Field

[0001] The present invention relates to the technical field of biotechnology and plant genetic engineering, and more specifically, to the technical field of promoters for controlling the induced expression of genes in plants, and in particular to an ABA-inducible bean hemagglutinin promoter and related primer pairs, expression vectors and applications. Background Art

[0002] Promoters are important cis-acting elements for gene expression regulation, which can determine the start time of gene transcription, the intensity of expression, and the tissue specificity of expression. In plant genetic engineering, a suitable promoter is crucial for the effective expression of the target gene.

[0003] Inducible promoters are a type of promoter that can regulate gene expression in response to specific environmental signals or chemicals. Compared with constitutive promoters, inducible promoters can turn on gene expression under specific conditions, avoiding the negative impact of continued expression of target genes when not needed on plant growth and development, while also being able to more accurately regulate the response of plants to environmental changes. For example, when plants are attacked by pests and diseases, promoters that can induce the expression of related defense genes can enable plants to activate defense mechanisms in a timely manner; when plants need to adapt to adverse conditions such as drought and salinity, inducible promoters can drive the expression of stress resistance genes to improve plant tolerance. Therefore, the exploration and development of new and efficient inducible plant promoters has important scientific significance and application value.

[0004] Therefore, it is desirable to provide an ABA-inducible bean promoter that can respond to specific induction factors and effectively regulate the expression of downstream genes in plants. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, an object of the present invention is to provide an ABA-inducible bean lectin promoter, which can respond to specific induction factors, effectively regulate the expression of downstream genes in plants, and is suitable for large-scale promotion and application.

[0006] Another object of the present invention is to provide a plant recombinant expression vector, which can respond to specific induction factors, effectively regulate the expression of downstream genes in plants, and is suitable for large-scale promotion and application.

[0007] Another object of the present invention is to provide a set of primer pairs that can amplify the ABA-inducible bean hemagglutinin promoter, so that it can be used for subsequent expression vector construction to respond to specific induction factors and effectively regulate the expression of downstream genes in plants, which is suitable for large-scale promotion and application.

[0008] Another object of the present invention is to provide an application of an ABA-inducible bean hemagglutinin promoter or a plant recombinant expression vector containing the promoter, so that the expression of downstream genes in plants can be effectively regulated in response to specific induction factors, which is suitable for large-scale promotion and application.

[0009] To achieve the above objectives, in a first aspect of the present invention, an ABA-inducible bean hemagglutinin promoter is provided, wherein the nucleotide sequence of the ABA-inducible bean hemagglutinin promoter is as shown in SEQ ID NO:1.

[0010] In the second aspect of the present invention, a plant recombinant expression vector is provided, including a plant expression vector, wherein the plant recombinant expression vector also includes the above-mentioned ABA-inducible bean hemagglutinin promoter, and the plant expression vector is integrated with the ABA-inducible bean hemagglutinin promoter.

[0011] Preferably, the plant expression vector is pNC-121::GUS.

[0012] More preferably, the plant recombinant expression vector is pNC-121-Pro-PHA-L::GUS, wherein the Pro-PHA-L is the ABA-inducible Phaseolus vulgaris lectin promoter.

[0013] In the third aspect of the present invention, a set of primer pairs is provided, wherein the primer pairs are designed based on the nucleotide sequence of the above-mentioned ABA-inducible bean hemagglutinin promoter.

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

[0015] Preferably, the nucleotide sequences of the primer pair are shown in SEQ ID NO:4 and SEQ ID NO:5.

[0016] In a fourth aspect of the present invention, there is provided the use of the above-mentioned ABA-inducible bean lectin promoter or the above-mentioned plant recombinant expression vector in controlling the ABA-induced expression of genes in plants.

[0017] Preferably, the plant is Arabidopsis thaliana or Phaseolus vulgaris.

[0018] Preferably, the gene is the GUS gene.

[0019] The beneficial effects of the present invention are:

[0020] a. The nucleotide sequence of the ABA-inducible bean hemagglutinin promoter of the present invention is shown in SEQ ID NO: 1, which can respond to specific induction factors and effectively regulate the expression of downstream genes in plants, and is suitable for large-scale promotion and application.

[0021] b. The plant recombinant expression vector of the present invention comprises a plant expression vector and an ABA-inducible bean lectin promoter. The plant expression vector is integrated with an ABA-inducible bean lectin promoter, which can respond to specific induction factors and effectively regulate the expression of downstream genes in plants, and is suitable for large-scale promotion and application.

[0022] c. The primer pair of the present invention is designed according to the nucleotide sequence of the ABA-inducible bean hemagglutinin promoter, which can amplify the ABA-inducible bean hemagglutinin promoter, and thus can be used for subsequent expression vector construction to respond to specific induction factors and effectively regulate the expression of downstream genes in plants, and is suitable for large-scale promotion and application.

[0023] d. The application of the ABA-inducible bean hemagglutinin promoter or plant recombinant expression vector of the present invention in controlling ABA-induced expression of genes in plants can respond to specific induction factors and effectively regulate the expression of downstream genes in plants, which is suitable for large-scale promotion and application.

[0024] These and other objects, features and advantages of the present invention are fully reflected in the following detailed description and drawings, and can be achieved by the features, means and their combinations particularly pointed out in the description. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the PCR agarose gel electrophoresis diagram of amplifying the bean PHA-L gene promoter Pro-PHA-L, wherein lane 1 is a 2kb marker, and lane 2 is the amplification product of the primer pair specifically amplifying the bean PHA-L gene promoter Pro-PHA-L, with a size of 1500bp.

[0026] Figure 2 It is a schematic diagram of the structure of the expression vector pNC-121::GUS.

[0027] Figure 3 It is a schematic diagram of the structure of the plant recombinant expression vector pNC-121-Pro-PHA-L::GUS.

[0028] Figure 4This is the GUS staining analysis of Pro-PHA-L::GUS transgenic Arabidopsis. The blue staining indicates the location of GUS expression (as shown by the black arrow). (1) is the water treatment control group, A is the inflorescence, B is the leaf, C is the root, and D is the fruit pod; (2) is the ABA treatment group, E is the inflorescence, F is the leaf, G is the root, and H is the fruit pod.

[0029] Figure 5 This is the GUS staining analysis of Pro-PHA-L::GUS transgenic bean hairy roots. The blue staining indicates the location of GUS expression (as shown by the black arrow). (1) is the water treatment control group, and (2) is the ABA treatment group. DETAILED DESCRIPTION

[0030] In order to develop a new ABA-inducible promoter from plant sources, the present inventors have discovered an ABA-inducible Phaseolus vulgaris hemagglutinin 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 above-mentioned ABA-inducible bean hemagglutinin promoter, wherein the ABA-inducible bean hemagglutinin promoter is integrated into the plant expression vector.

[0032] The plant expression vector may be any suitable plant expression vector. Preferably, the plant expression vector is pNC-121::GUS.

[0033] The plant recombinant expression vector can be any suitable plant recombinant expression vector. More preferably, the plant recombinant expression vector is pNC-121-Pro-PHA-L::GUS, wherein the Pro-PHA-L is the ABA-inducible bean hemagglutinin promoter.

[0034] Also provided is a set of primer pairs, which are designed according to the nucleotide sequence of the above-mentioned ABA-inducible bean hemagglutinin promoter.

[0035] The primer pair may have any suitable nucleotide sequence, preferably, the nucleotide sequence of the primer pair is shown as SEQ ID NO: 2 and SEQ ID NO: 3. Alternatively, the nucleotide sequence of the primer pair is shown as SEQ ID NO: 4 and SEQ ID NO: 5.

[0036] Also provided is the use of the ABA-inducible bean hemagglutinin promoter or the plant recombinant expression vector in controlling the ABA-induced expression of genes in plants.

[0037] The plant may be any suitable plant, preferably, the plant is Arabidopsis thaliana or bean.

[0038] The gene can be any suitable gene, and can be a gene originally existing in the plant or a foreign gene from other plants. Preferably, the gene is the GUS gene.

[0039] In order to more clearly understand the technical content of the present invention, the following examples are specifically described in detail. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0040] Reagents and medicines

[0041] The antibiotics kanamycin (Kana) and rifampicin (Rif) were purchased from BBI Life Sciences Ltd.;

[0042] DNA-Marker2000 was purchased from General Biotechnology (Anhui) Co., Ltd.;

[0043] Restriction enzymes were purchased from Thermo Fisher Scientific;

[0044] KOD high-fidelity polymerase 2×Mix was purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.;

[0045] DNA fragment recovery and purification kit was purchased from Sigma;

[0046] The plasmid DNA extraction kit was purchased from Beijing Huayueyang Biotechnology Co., Ltd.;

[0047] Homologous recombination reagent Sosoo enzyme was purchased from Beijing Qingke Biotechnology Co., Ltd.

[0048] Primer synthesis and sequencing were completed by Hangzhou Youkang Biotechnology Co., Ltd.

[0049] Example 1: Promoter sequence analysis and cloning

[0050] 1. Promoter sequence analysis

[0051] The DNA sequence of bean PHA-L gene (gene number Phvul.004G158300) was downloaded from the Phytozome (https: / / phytozome-next.jgi.doe.gov / ) database, and its promoter was searched along the PHA-L gene and sequence analysis was performed to obtain the promoter of the PHA-L gene, which was named Pro-PHA-L. Its sequence is shown in SEQ ID NO: 1, and the total length of the sequence is 1500 bases.

[0052] 2. Promoter cloning

[0053] The length of 1500 bp upstream of the ATG of the PHA-L gene was selected as the promoter region of the PHA-L gene, and the primer pair PHA-L-1500-clone-F and PHA-L-1500-clone-R (see Table 2 below) was designed based on the promoter region of the PHA-L gene. In order to subsequently construct the expression vector pNC-121-Pro-PHA-L::GUS with the PHA-L gene promoter, the above primer pair was connected with a universal linker in the NC connection to obtain the primer pair PHA-L-1500-NC-F and PHA-L-1500-NC-R (see Table 2 below). The template DNA in Table 1 is the DNA of the pod of common bean variety (red flowers and white pods, Hualing High-tech Seed Breeding Research Center, Mianyang City, Sichuan Province). Common bean variety (red flowers and white pods, Hualing High-tech Seed Breeding Research Center, Mianyang City, Sichuan Province) was selected as the experimental material and cultured in a greenhouse until 20 days after flowering. The bean pod tissue was taken and quick-frozen in liquid nitrogen, and the DNA was extracted using the Huayueyang Plant DNA Extraction Kit; PCR reaction conditions: 94°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 55°C annealing for 30 s, 72°C extension for 30 s, for a total of 35 cycles.

[0054] Table 1 Promoter amplification system is as follows (total 50 μL)

[0055] Reagents Dosage KOD High-Fidelity Polymerase 2×Mix 25μL Template DNA 1μL 10 μM upstream primer 1.5μL 10 μM downstream primer 1.5μL ddH2O 21μL

[0056] Table 2 Primer sequences used for PHA-L gene promoter amplification

[0057] Primer name Primer sequence (5'-3') PHA-L-1500-clone-F As shown in SEQ ID NO: 2 PHA-L-1500-clone-R As shown in SEQ ID NO: 3 PHA-L-1500-NC-F As shown in SEQ ID NO: 4 PHA-L-1500-NC-R As shown in SEQ ID NO: 5

[0058] Experimental results:

[0059] The primers PHA-L-1500-clone-F and PHA-L-1500-clone-R were used for amplification. After 1.0% agarose gel electrophoresis, the amplified product was a 1500 bp fragment. Figure 1 As shown, the nucleotide sequence of the cloned PHA-L gene promoter is shown as SEQ ID NO: 1, with a total of 1500 bases.

[0060] Example 2: Construction of recombinant expression vector

[0061] 1. Recovery and purification

[0062] (1) Using the method of Example 1, amplify with primer pair PHA-L-1500-NC-F and PHA-L-1500-NC-R, and briefly centrifuge the PCR product. Measure its volume with a pipette and transfer it to a sterile 2 ml centrifuge tube. If the sample volume is less than 100 μL, add sterile water to 100 μL.

[0063] (2) Add 4 times the volume of XP Buffer and mix by inversion or vortexing.

[0064] (3) Place the adsorption column in the collection tube. Transfer ≤700μL of the sol solution to the adsorption column. Centrifuge at 10000rpm (8000×g) for 30-60sec. If the volume of the mixed solution is greater than 700μL, place the adsorption column in the recovery tube, transfer the remaining solution to the adsorption column, and centrifuge at 12000rpm (13400×g) for 30-60sec.

[0065] (4) Discard the filtrate and place the adsorption column in a 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.

[0066] (5) Repeat step 4.

[0067] (6) Discard the filtrate and place the adsorption column back into the collection tube. Centrifuge at 12000 rpm (13400 × g) for 2 min.

[0068] (7) Place the adsorption column in a 1.5 ml sterilized centrifuge tube, add 30 μL ddH2O (heated to 55°C) to the center of the adsorption column, and place for 5 min. Centrifuge at 12000 rpm (13400 × g) for 1 min. Discard the adsorption column and store the purified product at -20°C.

[0069] 2. Connect

[0070] This experiment uses pNC-121::GUS in the NC vector, which is called Nimble Cloning. It can clone linear DNA or DNA on a circular plasmid (entry clone) directly into the circular expression vector of the NC system through Nimble Mix, without the need for linearization of the expression vector. Its form is similar to Gateway cloning. For vectors and operation steps, refer to the literature (Yan, P.; Tuo, D.; Shen, W.; Deng, H.; Zhou, P.; Gao, XA Nimble Cloning-compatible vector system for high-throughput gene functional analysis in plants. Plant Commun 2023, 4, 100471, doi: 10.1016 / j.xplc.2022.100471).

[0071] Nimble Buffer configuration system:

[0072] 5×Nimble Buffer:

[0073] Working solution concentration Mother liquor concentration Total volume 10ml 25% PEG-8000 50％ 5ml 0.5MTris-HCLpH7.5 2M 2.5ml 50mMMgCl2 1M 0.5ml 50mM DTT 1M 0.5ml ddH2O 1.5ml

[0074] 2×Nimble Mix

[0075] 5xNimbleBuffer 20ul T5 exonuclease (1U / ul, NEB) 0.4ul SfiI (2U / ul, NEB) 4ul ddH2O 25.6ul Total volume 50ul

[0076] T5 exonuclease (1U / ul, NEB)

[0077] SfiI (2U / ul, NEB)

[0078] NC cloning reaction system

[0079] Element Dosage PCR products 10-80ng NC system expression vector 20-120ng NimbleMix 2.5μL ddH2O Make up to 5 μL

[0080] The pNC-121::GUS vector carries the GUS gene, and the full length of the vector is 14568b. Figure 2 The PCR purified product obtained above was mixed with the expression vector of the NC system (fragment, vector and sterilized water, 5 μL in total), 2.5 μL of 2xNCNimble Mix was added, pipette 10-20 times, mixed thoroughly, and reacted at 50°C (37°C is recommended for seamless cloning without the linker sequence) for 1 hour.

[0081] 3. Conversion reaction

[0082] (1) DH5α competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.) (100 μL per tube) were taken out from -80°C and quickly placed in ice. After 5 minutes, the bacterial block was thawed, and 50 μL of the competent solution was pipetted into a 1.5 ml sterile centrifuge tube. 5 μL of the recombinant product was added and the tube was placed in ice for 25 minutes.

[0083] (2) Heat shock in a 42°C water bath for 45 seconds, then quickly return to ice and let stand for 2 minutes.

[0084] (3) Add 700 μL of sterile liquid culture medium (LB) without antibiotics to the centrifuge tube, mix well, and resuscitate at 37°C, 200 rpm for 60 min.

[0085] (4) Centrifuge at 5000 rpm for 1 minute to collect the cells. Take about 100 μL of the supernatant, gently blow to resuspend the cells, and spread them on LB solid medium containing Kana resistance.

[0086] (5) Place the plate upside down in a 37°C incubator for overnight culture.

[0087] (6) Pick a single E. coli clone for colony PCR, inoculate the positive clone into LB liquid medium containing Kana, and place it in a shaker at 37°C and 200 rpm / min for expansion and culture. Send the expanded E. coli to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing analysis. After the plasmid is extracted from the clone with the correct sequencing, it is the recombinant expression vector containing Pro-PHA-L: pNC-121-Pro-PHA-L::GUS, such as Figure 3 shown.

[0088] Example 3: GUS detection of transgenic Arabidopsis

[0089] 1. Plant materials and strains

[0090] Arabidopsis thaliana common variety “Columbia” (provided by the Plant Phenotyping and Quality Safety Laboratory of China University of Jilin) ​​and Agrobacterium tumefaciens GV3101 (Shanghai Weidi Biotechnology Co., Ltd., catalog number: AE1001S).

[0091] The positive single clones with correct sequencing were shaken to extract plasmids and transformed into Agrobacterium using electroporation. The GV3101 competent cells frozen at -80℃ were thawed on ice, 1μL plasmid was added to 50μL competent cells, and the mixture was gently pipetted a few times and added to the pre-cooled electroporation cup (the electroporation cup was treated: first soaked in 75% ethanol, then soaked in anhydrous ethanol, dried at 55℃, and cooled in ice). The electroporation steps are as follows: turn on the gene transfer instrument switch, set C capacitance 25, PT; R resistance 400, PT; U voltage 2500, PT; click RD until the power rises to 99, put the electroporation cup into the groove, and click RD again, accompanied by a drip, indicating that the electroporation is complete. Then add 700μL of fresh LB culture medium (without antibiotics) to the electroporation cup, and transfer all the liquid in the electroporation cup to the original centrifuge tube after repeated pipetting, and culture at 28℃, 200rpm, and shake for 2h. Centrifuge at 5000 rpm for 1 min, discard the supernatant, concentrate to 100 μL, and spread on LB solid medium containing Kana and Rifampicin tablets (Rif). Incubate at 28°C for 36-48 hours. Verify positive clones by PCR, and store at -80°C with 50% glycerol and bacterial solution at a ratio of 1:1 (V:V) for later use.

[0092] 2. Plant transformation

[0093] The recombinant expression vector pNC-121-Pro-PHA-L::GUS was transformed into Arabidopsis thaliana by Agrobacterium-dipping method. The specific steps are as follows:

[0094] (1) Cultivation and activation of Agrobacterium

[0095] The Agrobacterium strain containing the recombinant plasmid was streaked and cultured in LB solid medium containing kanamycin and rifampicin for 48 hours. A single colony was picked and cultured overnight in 1.5 ml LB liquid medium containing kanamycin and rifampicin. The 1.5 ml overnight culture solution was transferred to a 100 ml conical flask, and LB liquid medium containing kanamycin and rifampicin was added to 15 ml and cultured overnight. After 24 hours, the cells were collected by centrifugation at 5000 rpm for 10 minutes and resuspended in MS liquid medium to an OD of 0. 600 =about 0.5.

[0096] (2) Infection of Arabidopsis thaliana

[0097] The flowers of Arabidopsis thaliana that were growing well and in full bloom were immersed in the Agrobacterium bacterial solution for 5 minutes, cultured in the dark for 48 hours in a moisturizing manner, and then cultured normally under light at 22°C.

[0098] (3) Screening of transgenic Arabidopsis

[0099] After the culture is completed, mature Arabidopsis seeds are collected and evenly spread on a screening medium containing kanamycin, and cultured in a light incubator with 80% light and 60% moderate environment. After the green positive seedlings are screened out, the positive seedlings are transferred to nutrient soil and cultured until the T1 representative seeds are harvested. Repeat the above steps until the T2 generation seeds are harvested.

[0100] (4) Transgenic Arabidopsis thaliana cultivation

[0101] The T2 generation seeds were evenly spread on the screening medium containing kanamycin, cultured in a light incubator with 80% light and 60% moderate environment for 2 weeks, and the positive seedlings were selected and transplanted to nutrient soil to grow for about 2 weeks.

[0102] 3. Observation of promoter activity

[0103] Six groups of transgenic positive plants were selected for 100μM ABA spraying and water spraying control experiments, respectively, and cultured for 24 hours. The roots of the two groups of positive seedlings were dug out, washed with clean water, dried, and immersed in Huayueyang GUS staining solution, and cultured at 37℃ for 24h to 48h. The GUS staining was observed under a microscope, and the promoter expression of the experimental group and the control group was compared. The results are shown in Figure 4 shown.

[0104] Depend on Figure 4 It can be seen that none of the organs of the transgenic Arabidopsis thaliana in the water spraying group were stained blue, while the roots and seeds in the pods of the transgenic Arabidopsis thaliana sprayed with 100 μM ABA were stained blue. These results indicate that Pro-PHA-L needs the presence of ABA to have driving ability, and its driving ability is tissue-specific and can drive gene expression in seeds and roots.

[0105] Example 4: GUS detection of transgenic bean hairy roots

[0106] 1. Plant materials and strains

[0107] Common bean varieties (red flowers and white pods, Hualing High-tech Seed Breeding Research Center, Mianyang, Sichuan Province) and Agrobacterium rhizogenes K599 (Shanghai Weidi Biotechnology Co., Ltd., catalog number: AC1080).

[0108] Transformation of recombinant expression vector into Agrobacterium rhizogenes K599:

[0109] (1) Take out the competent cells of Agrobacterium rhizogenes from the ultra-low temperature freezer and thaw them on ice until they are in an ice-water mixture.

[0110] (2) Add 1 μL of the correctly sequenced recombinant expression plasmid to the thawed competent cells, stir the bottom of the tube by hand to mix, and place on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 28°C water bath for 5 minutes, and in an ice bath for 5 minutes.

[0111] (3) Add 700 μl of TY liquid medium without antibiotics and culture at 28°C with shaking for 2 hours.

[0112] (4) Centrifuge the bacterial solution at 6000 rpm / min for 1 min, discard part of the supernatant, retain 100-150 μL, gently pipette to mix, spread on TY solid medium containing Kana, and culture inverted at 28°C for about 48 h until a single colony grows.

[0113] (5) Pick a single Agrobacterium clone and perform colony PCR.

[0114] (6) Select positive clones, shake the cells, and preserve the culture solution in 50% glycerol in an ultra-low temperature refrigerator for later use.

[0115] 2. Plant transformation

[0116] (1) Preparation of plant materials: Soak the sterilized kidney bean seeds in warm water at 28°C and 150 rpm / min in a shaking incubator overnight. The next day, spread the seeds in a square culture dish with sterile filter paper and germinate in the dark for about 36 hours. When the seeds begin to turn white, sow them in the substrate (perlite) and maintain adequate water and light conditions (day and night: 16h / 8h, 28°C / 25°C) for cultivation. After about 3 days, the kidney bean plants were obtained when the first true leaf was just beginning to unfold.

[0117] (2) Cultivation and activation of Agrobacterium

[0118] The Agrobacterium strain containing the recombinant plasmid was streaked and cultured on a solid medium containing kanamycin TY for 48 hours, and then continued to be streaked and cultured on a solid medium containing kanamycin TY for another 48 hours.

[0119] (3) Infection of bean plants

[0120] Infection process: First, use an injection needle to poke holes in the hypocotyl of kidney beans (1-2 cm below the cotyledons). While ensuring that the plant can survive, poke holes all around, about 8-10 holes. Then apply the activated bacteria to the injured part of the plant and cultivate it in a high humidity and low temperature environment (temperature: 22°C, humidity: 95%).

[0121] Culture conditions: After infection, place in an incubator at 22°C and 95% humidity for 3 days in the dark, then transfer to a light incubator and culture for 3 days in an environment of 16h / 8h day and night, 60% light, and 95% humidity. Later, adjust the light to 100%, and keep the rest unchanged. Hairy roots begin to grow after one week, and a large number of hairy roots begin to form 14 days after infection. After 21 days, the transgenic hairy roots can be identified and processed. During this period, pay attention to adding nutrient solution to the plug tray in a timely and appropriate amount.

[0122] 3. Observation of promoter activity

[0123] Six groups of transgenic positive hairy roots were selected for 100 μM ABA immersion experiment and water immersion control experiment, respectively, and cultured for 24 hours. They were immersed in Huayueyang GUS staining solution and cultured at 37℃ for 24 hours to 48 hours. The GUS staining was observed under a microscope, and the promoter expression of the experimental group and the control group was compared. The results are shown in Figure 5 shown.

[0124] Depend on Figure 5 It can be seen that the transgenic hairy roots will not be dyed blue when immersed in water, but will be dyed blue when immersed in ABA. The results show that the PHA-L gene promoter can drive gene expression in roots only in the presence of ABA, and is an ABA-inducible and tissue-specific promoter.

[0125] Therefore, the present invention discloses an inducible promoter PHA-L gene promoter Pro-PHA-L that responds to ABA. The nucleotide sequence of the promoter is shown in SEQ ID NO: 1. The sequence is 1500 bases long and is cloned from the promoter region of the kidney bean PHA-L gene (gene number Phvul.004G158300). The promoter has the following characteristics: under normal growth conditions, its transcriptional activity on downstream genes is low; when the plant is induced by abscisic acid (ABA), the promoter activity is significantly enhanced, thereby driving the downstream gene to express efficiently. A recombinant expression vector containing the above promoter can also be constructed. The recombinant expression vector can be constructed by conventional molecular cloning technology, connecting the promoter sequence to the target gene, and inserting it into a suitable plant expression vector skeleton. During the construction process, suitable multiple cloning sites, marker genes and other elements can be added as needed to facilitate subsequent transformation and screening operations.

[0126] The ABA-inducible plant promoter provided by the present invention has good induction activity and has broad application prospects in the field of plant genetic engineering. As an inducible promoter that can be expressed heterologously, it has potential application value in studying the expression and regulation of plant genes and solving food safety problems using genetic engineering methods. By constructing a recombinant expression vector containing the promoter and transforming plants, precise regulation of plant stress resistance, growth and development, quality characteristics and other aspects can be achieved, providing a powerful tool for plant genetic improvement and agricultural production.

[0127] In summary, the ABA-inducible bean lectin promoter of the present invention can respond to specific induction factors, effectively regulate the expression of downstream genes in plants, and is suitable for large-scale promotion and application.

[0128] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it is apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the specification and drawings should be regarded as illustrative rather than restrictive.

Claims

1. An ABA-inducible bean hemagglutinin promoter, characterized in that The nucleotide sequence of the ABA-inducible bean hemagglutinin promoter is shown in SEQ ID NO:

1.

2. A plant recombinant expression vector, comprising a plant expression vector, characterized in that: The plant recombinant expression vector further comprises the ABA-inducible bean hemagglutinin promoter as claimed in claim 1, and the ABA-inducible bean hemagglutinin promoter is integrated into the plant expression vector.

3. The plant recombinant expression vector according to claim 2, characterized in that: The plant expression vector is pNC-121::GUS.

4. The plant recombinant expression vector according to claim 3, characterized in that: The plant recombinant expression vector is pNC-121-Pro-PHA-L::GUS, wherein the Pro-PHA-L is the ABA-inducible bean hemagglutinin promoter.

5. A set of primer pairs, characterized in that: The primer pair is designed according to the nucleotide sequence of the ABA-inducible bean hemagglutinin promoter as claimed in claim 1.

6. The primer pair according to claim 5, characterized in that The nucleotide sequences of the primer pair are shown in SEQ ID NO:2 and SEQ ID NO:

3.

7. The primer pair according to claim 5, characterized in that The nucleotide sequences of the primer pair are shown in SEQ ID NO:4 and SEQ ID NO:

5.

8. Use of the ABA-inducible bean hemagglutinin promoter according to claim 1 or the plant recombinant expression vector according to any one of claims 2 to 4 in controlling ABA-induced expression of genes in plants.

9. The use according to claim 8, characterized in that The plant is Arabidopsis thaliana or Phaseolus vulgaris.

10. The use according to claim 8, characterized in that The gene is the GUS gene.

Images