Wheat TaWRKY104 gene for promoting plant drought resistance and its application

Abstract

The present invention relates to the field of plant biotechnology, and in particular to a wheat TaWRKY104 gene for promoting plant drought resistance and an application thereof. The nucleotide sequence of the TaWRKY104 gene is shown in SEQ ID NO: 1. In the present invention, the TaWRKY104 gene is overexpressed and transformed into Arabidopsis for functional verification. The result shows that the drought resistance of Arabidopsis overexpressing the TaWRKY104 gene is significantly improved compared with that of the wild-type Arabidopsis. Therefore, the TaWRKY104 gene plays a positive regulatory role in the drought resistance process of wheat, provides a theoretical basis and gene resources for wheat molecular breeding, and is of great significance for obtaining drought-resistant and high-yield strains in the future and improving the drought resistance of wheat.

Description

Technical Field

[0001] The invention relates to the field of plant biotechnology, and in particular to a wheat TaWRKY104 gene for promoting plant drought resistance and an application thereof. Background Art

[0002] Drought has a great impact on wheat production and has become one of the important factors affecting wheat yield. WRKY proteins are involved in plant defense responses to pathogens, abiotic stresses such as drought tolerance, salt tolerance, low temperature tolerance, growth and development, and other physiological and biochemical reactions, and are characterized by rapidity and instantaneity.

[0003] Both wheat TaWRKY1 and TaWRKY33 genes are induced by drought. Overexpression of TaWRKY1 and TaWRKY33 genes in Arabidopsis increased the germination rate and survival rate of Arabidopsis under drought stress conditions and reduced the leaf water loss rate. The drought resistance of Arabidopsis plants overexpressing TaWRKY33 gene was stronger than that of plants overexpressing TaWRKY1 gene.

[0004] However, there are currently few known wheat genes that have the function of regulating plant drought resistance, and it is necessary to discover more wheat genes with drought resistance functions. Summary of the invention

[0005] In order to solve the above technical problems, the present invention aims to clarify the application of TaWRKY104 gene in promoting plant drought resistance.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A first aspect of the present invention provides a wheat TaWRKY104 gene for promoting plant drought resistance, wherein the nucleotide sequence of the TaWRKY104 gene is shown in SEQ ID NO: 1, and the drought resistance of transgenic plants is improved by overexpressing the TaWRKY104 gene in plants; the plants include Arabidopsis thaliana.

[0008] The present invention found in the study that compared with wild-type Arabidopsis, the survival rate of Arabidopsis overexpressing TaWRKY104 gene is much higher than that of wild-type Arabidopsis. Therefore, it is speculated that the TaWRKY104 gene has a certain resistance to drought stress, providing a theoretical basis for using TaWRKY104 to improve wheat drought resistance.

[0009] The second aspect of the present invention provides a recombinant expression vector, which comprises the TaWRKY104 gene.

[0010] In another preferred embodiment, the recombinant expression vector is obtained by inserting the TaWRKY104 gene between the Pst I and Kpn I sites of the pSuper1300 overexpression vector plasmid.

[0011] The third aspect of the present invention provides a recombinant engineered bacterium comprising the recombinant expression vector.

[0012] In another preferred embodiment, the recombinant expression vector is obtained by transferring the recombinant expression vector into Agrobacterium competent cells.

[0013] The engineered bacteria can be understood as the engineered bacteria used by those skilled in the art in the transgenic process, such as Agrobacterium competent cells EHA105. However, with the development of science and technology, the selection of the engineered bacteria may change, or the application fields for non-transgenic purposes also involve the use of vectors and engineered bacteria, but only those containing the genes or vectors of the present invention are within the scope of protection of the present invention.

[0014] The fourth aspect of the present invention provides the use of the recombinant expression vector in cultivating drought-resistant transgenic plants. Compared with the wild type, the drought resistance of the transgenic plants is improved.

[0015] In another preferred embodiment, the plant comprises Arabidopsis thaliana.

[0016] The fifth aspect of the present invention provides the use of the recombinant expression vector in promoting drought resistance in plants, wherein the plants include Arabidopsis thaliana.

[0017] The sixth aspect of the present invention provides the use of the recombinant engineered bacteria in promoting drought resistance in plants, wherein the plants include Arabidopsis thaliana.

[0018] The method for improving drought resistance of Arabidopsis thaliana provided by the present invention specifically comprises target gene cloning, target gene overexpression vector construction, target gene transformation into Agrobacterium, target gene transformation into Arabidopsis thaliana, positive strain identification, phenotype observation, and determination of drought-resistant materials.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention first discovered the role of overexpressed TaWRKY104 gene in regulating drought resistance in Arabidopsis. The present invention overexpressed TaWRKY104 gene and transformed it into Arabidopsis for functional verification. The results showed that Arabidopsis overexpressing TaWRKY104 gene had significantly improved drought resistance compared with wild-type Arabidopsis. Therefore, TaWRKY104 gene plays a positive regulatory role in the drought resistance of wheat, providing a theoretical basis and gene resources for molecular breeding of wheat, which is of great significance for obtaining drought-resistant and high-yield strains in the future and improving wheat drought resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Phenotypic diagram of drought stress test for overexpressing Arabidopsis thaliana.

[0022] Figure 2 Survival statistics of drought stress test for overexpressing Arabidopsis thaliana.

[0023] Figure 3 These are the physiological indexes of Arabidopsis leaves before and after drought treatment, where (a) is the soluble sugar content, (b) is the proline content, (c) is the protein content, (d) is the SOD content, (e) is the CAT content, and (f) is the POD content. DETAILED DESCRIPTION

[0024] The technical solutions in the present invention will be described clearly and completely below in conjunction with the specific implementation of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The present invention provides a new use of the TaWRKY104 gene, wherein the nucleotide sequence of the TaWRKY104 gene is shown in SEQ ID NO.1:

[0026] ATGCATATATGCATGGATCAAGGGAGCCAATTGGGGATGGCTTACTGTCTTCCTAACCTCAGTGTGCCAGATCACTACTACACCACACCTGTTCCTCTTTCTCCACTTCAACTACCCTTCCATCCCAAGCCTCTCCAGATGCCATTCGACCAGGAAGAAGCCTTGATGCTCTCTTCTGACCATTGTGGGCTATACCCGCTGCCGGCGCTTCCGCTCGGCAGCGGCCACTCCGCCCGCGCGCCTGCCATCGTCTGCGAGAAGCCCACGGTCGGTTTCATGCCTAATATTGGGGCTGAGGAGGTGGGCACGTCGGTGACGGCAAGAGTTGGCTATGAGGGTGCCACTGCCTGTAATGGTTATTCTAGTAATACATGGTGGAGGGGCTCGACGATGTTGGCGGGGGAGAAGGGGAAGATGAAGGTGAGGAGGAAGATGAGGGAGCCGAGGTTTTGCTTCCAGACCAGAAGCGACGTGGATGTACTTGACGATGGCTACAAGTGGAGGAAATACGGGCAGAAGGTTGTCAAGAACAGTCTGCATCCCAGGAGCTACTTCAGGTGCACGCACAGCAACTGCCGCGTGAAGAAGCGCGTGGAGCGGCTGTCGACGGACTGCCGCATGGTGATCACCACGTACGAGGGCCGCCACACGCACCCCCCCTGCGACGACAACTCCTCCTCCTCCGGCGACAACACCACCACCTGCTTCTGA。

[0027] The amino acid sequence of the TaWRKY104 protein is shown in SEQ ID NO.2:

[0028] MHICMDQGSQLGMAYCLPNLSVPDHYYTTPVPLSPLQLPFHPKPLQMPFDQEEALMLSSDHCGLYPLPALPLGSGHSARAPAIVCEKPTVGFMPNIGAEEVGTSVTARVGYEGATACN GYSSNTWWRGSTMLAGEKGKMKVRRKMREPRFCFQTRSDVDVLDDGYKWRKYGQKVVKNSLHPRSYFRCTHSNCRVKKRVERLSTDCRMVITTYEGRHTHPPCDDNSSSSGDNTTTCF.

[0029] Example 1: Cloning of TaWRKY104 gene.

[0030] Using Qingmai No. 6 as the material, when the wheat grows to the stage of two leaves and one heart, 1 g of wheat leaves are removed with tweezers, and RNA is extracted using a plant RNA extraction kit, wherein the plant RNA extraction kit is purchased from Aikerui Biological Company. The specific method is as follows.

[0031] 1. Lysis of plant tissue.

[0032] (1) Transfer the fresh plant tissue sample to a mortar pre-cooled with liquid nitrogen, and grind the plant tissue in the mortar until it becomes powder, while continuously adding liquid nitrogen during the grinding process.

[0033] (2) Transfer 100 mg of the powdered sample into a 1.5 mL sterile, enzyme-free centrifuge tube containing 500 μL of lysis buffer, and ensure that there are no visible particles. Before use, confirm that 50× reducing agent has been added to the lysis buffer.

[0034] (3) The lysate was allowed to stand at room temperature for 2 minutes and then centrifuged at 12,000 rpm and 4°C for 5 minutes.

[0035] (4) Carefully pipette the supernatant into a new 1.5 mL sterile, enzyme-free centrifuge tube.

[0036] 2. Purification.

[0037] (1) Add 250 μL of anhydrous ethanol to the above lysate and mix well with a pipette. If obvious sticky matter or precipitation appears, pipette several times to break up the precipitate.

[0038] (2) Transfer the above mixed solution and precipitate to the plant RNA filter column, centrifuge at 12000 rpm for 2 minutes at room temperature, and discard the filtrate.

[0039] (3) Add 600 μL of RWA buffer to the plant RNA filter column, centrifuge at 12,000 rpm for 1 minute at room temperature, and discard the filtrate.

[0040] (4) Add 750 μL of buffer RWB to the plant RNA filter column, centrifuge at 12,000 rpm for 1 minute at room temperature, and discard the filtrate.

[0041] (5) DNase I digestion: Prepare DNase I, i.e., DNase 1 reaction solution according to Table 1 and mix well. Add 50 μL of DNase 1 reaction solution to the center of the Plant RNA Mini Column membrane, let stand at room temperature for 15 minutes, add 350 μL of buffer RWB to the center of the plant RNA filter column membrane, centrifuge at 12,000 rpm at room temperature for 1 minute, and discard the filtrate.

[0042] Table 1 Reaction system

[0043]

[0044] (6) Add 750 μL of buffer RWB to the plant RNA filter column, centrifuge at 12,000 rpm for 1 minute at room temperature, and discard the filtrate.

[0045] (7) Place the adsorption column of the plant RNA filter column in a new 2.0 mL collection tube and centrifuge at 12,000 rpm for 2 minutes at room temperature.

[0046] (8) Place the adsorption column of the plant RNA filter column on a new filter column, add 70 μL of sterile water to the center of the adsorption column membrane, let it stand at room temperature for 5 minutes, and then centrifuge at 12000 rpm for 2 minutes at room temperature to elute the RNA, which can be used for subsequent experiments. If subsequent experiments are not performed immediately, the dissolved RNA can be stored at -80°C.

[0047] 3. Reverse transcribe RNA into cDNA using a cDNA synthesis kit, where the cDNA synthesis kit was purchased from Acre Biotech. The specific steps are as follows.

[0048] (1) Prepare RNA template solution on ice according to Table 2 and place in PCR instrument for denaturation and annealing reaction at 65°C for 5 min; store at 4°C.

[0049] Table 2 RNA template solution

[0050]

[0051] (2) Prepare the reverse transcription reaction system according to Table 3 and synthesize cDNA.

[0052] Table 3 Reverse transcription reaction system

[0053]

[0054] The reaction conditions for cDNA synthesis are shown in Table 4.

[0055] Table 4 cDNA synthesis reaction conditions

[0056]

[0057] Gene-specific primers were designed as shown in Table 5 below, and the full-length coding frame of TaWRKY104 was amplified using cDNA as a template and connected into the intermediate vector p-EASY. The specific method is as follows.

[0058] Table 5 Gene-specific primers

[0059]

[0060] The cloning reaction system is shown in Table 6.

[0061] Table 6 Cloning reaction system

[0062]

[0063] Mix gently and react at 37°C for 15 minutes. After the reaction is completed, place the centrifuge tube on ice to obtain a ligation product.

[0064] (3) Transformation.

[0065] S1. Add the ligation product to 100 μL of E. coli competent cells. Add the ligation product when the E. coli competent cells have just been thawed, flick to mix, and place on ice for 30 minutes.

[0066] S2. Heat shock in a 42°C water bath for 45 seconds and immediately place on ice for 3 minutes.

[0067] S3. Add 700 mL of LB liquid culture medium and culture at 200 rpm and 37°C for 1 hour.

[0068] S4, centrifuge at 5000rpm for 3 minutes, discard 700mL supernatant, take 100μL bacterial solution to spread on the plate, and place it in a 37℃ incubator for 16h; pick 10 single clones on the plate and add them to 700mL LB liquid culture medium, culture at 200rpm and 37℃ for 6 hours. After successful sequencing, extract the plasmid for use.

[0069] Example 2: Construction of TaWRKY104 gene recombinant expression vector.

[0070] The Super1300-TaWRKY104 expression vector was constructed, and the homology arm primers were designed according to the restriction sites selected in the multiple cloning site of the pSuper1300 vector, as shown in Table 7. The TaWRKY104 gene was amplified using a high-fidelity enzyme, and the pSuper1300 vector was double-digested to construct the Super1300-TaWRKY104 expression vector. The specific steps are as follows.

[0071] Table 7 Gene-specific primers

[0072]

[0073] (1) The pSuper1300 restriction enzyme digestion system is shown in Table 8, and the TaWRKY104 amplification system is shown in Table 9.

[0074] Table 8 pSuper1300 restriction enzyme digestion system

[0075]

[0076] Table 9 TaWRKY104 amplification system

[0077]

[0078] The linear vector fragment and target fragment after enzyme digestion were recovered by gel, and the recovery accuracy was tested.

[0079] (2) Use seamless cloning to connect the amplified target gene with the linearized vector. The specific steps refer to the integrated ultra-fast cloning kit of Nanjing Novozymes Biotechnology Co., Ltd. The specific steps are as follows: The cloning reaction system is shown in Table 10.

[0080] Table 10 Cloning reaction system

[0081]

[0082] Mix gently and react at 50°C for 15 minutes. After the reaction is complete, place the centrifuge tube on ice.

[0083] Transformation: S1. Add the ligation product to 100 μL of E. coli competent medium. When the competent cells are just thawed, add the ligation product, flick to mix, and place on ice for 30 minutes. The E. coli competent medium was purchased from Qingke Biotechnology Co., Ltd.

[0084] S2. Heat shock in a 42°C water bath for 45 seconds and immediately place on ice for 3 minutes.

[0085] S3. Add 700 mL of LB liquid culture medium and culture at 200 rpm and 37°C for 1 hour.

[0086] S4. Centrifuge at 5000 rpm for 3 minutes, discard 700 ml of supernatant, take 100 μL of bacterial solution to spread on the plate, and place it in a 37°C incubator for 16 hours.

[0087] After PCR identification, the positive colonies were sent for testing, and the bacterial solution with correct sequencing was amplified and the plasmid DNA was extracted for later use.

[0088] Example 3: Construction of transgenic lines overexpressing TaWRKY104.

[0089] 1. Arabidopsis thaliana cultivation.

[0090] (1) Seed disinfection: Take an appropriate amount of Arabidopsis seeds and place them in 75% alcohol for disinfection for 2 minutes, then wash them with sterile water 5 times, each washing lasting 2 minutes.

[0091] (2) Cultivation process: Thoroughly sterilized wild-type Arabidopsis seeds are recorded as WT and evenly sown on the surface of 1 / 2MS solid culture medium to absorb surface moisture. To complete the vernalization process, the culture dish is placed in a refrigerator at 4°C for 3 days. Then it is transferred to a plant incubator in the laboratory, where the incubator conditions are 21°C, 16 / 8h, light / dark. When Arabidopsis grows four true leaves, healthy seedlings are selected and transferred to soil, and the plants are continued to be maintained in the plant incubator until they begin to enter the bolting stage. At this time, the stems that have grown seeds are cut off, and those flower buds that have not yet borne fruit are retained for subsequent Agrobacterium infection experiments.

[0092] 2. Transformation of Agrobacterium.

[0093] The successfully constructed pSuper1300-TaWRKY104 recombinant plasmid was introduced into Agrobacterium as follows: (1) Thaw the competent Agrobacterium cells stored at -80°C on ice. Under sterile conditions, add 10 μL of plasmid DNA to the competent cells, mix gently, and place on ice for 5 min.

[0094] (2) Quickly freeze the competent cells in liquid nitrogen for 5 min, then quickly place them in a 37°C water bath for 5 min without shaking the water surface, and then place the centrifuge tube in an ice water bath for 5 min.

[0095] (3) Under sterile conditions, add 800 mL of LB liquid medium and culture at 28°C with shaking for 3 h to allow the bacteria to revive.

[0096] (4) Centrifuge at 5000 rpm for 1 min to collect the bacteria, retain about 100 μL of the supernatant and gently pipette to mix, then spread on an LB solid plate containing Kan and Rif resistance antibiotics and culture for 48 h.

[0097] (5) After PCR identification of positive colonies, the correct bacterial solution was amplified and set aside.

[0098] 3. Infection of inflorescence by Agrobacterium.

[0099] (1) Preparation of bacteria: Inoculate the above Agrobacterium bacterial culture into 10 mL of LB liquid culture medium containing Kan and Rif resistance antibiotics, and culture in a shaker at 220 rpm at 28°C for 12 h.

[0100] (2) Select 1 mL of bacterial liquid and inoculate it into 100 mL of LB liquid medium containing Rif and Kan resistance antibiotics, culture for 18 hours, and proliferate to OD600 of 1.2 under the same culture conditions. Then, centrifuge in a 5000 rpm centrifuge for 10 minutes, remove the supernatant, collect the Agrobacterium that has settled, use the Arabidopsis transformation infiltration solution as shown in Table 11, resuspend the Agrobacterium cells that have settled, and then adjust to OD600 of 1.0.

[0101] Table 11 Arabidopsis infiltration solution formula

[0102]

[0103] The flower buds of Arabidopsis thaliana were briefly immersed in the infiltration solution for 12 seconds. During the process, the Agrobacterium infiltration solution was prevented from contacting the soil to cause adverse effects on the tender roots of Arabidopsis thaliana. After the infection was completed, the Arabidopsis thaliana was placed in a dark environment for 48 hours and then placed in a light incubator for further cultivation. The incubator was maintained at 23°C, 16 / 8h, light / darkness. After the Arabidopsis thaliana matured, T0 generation seeds were collected from individual plants.

[0104] 4. Homologous breeding of transgenic Arabidopsis thaliana.

[0105] After the T0 generation seeds are disinfected, they are evenly sown with a pipette tip on a 1 / 2MS medium containing 25 mg / L hygromycin to culture T1 generation Arabidopsis. Transgenic plants that grow well under the hygromycin selection pressure are selected and transplanted into the soil for single plant culture. After the Arabidopsis matures, T1 generation seeds are collected from individual plants.

[0106] The T1 generation seeds were cultured in the same way as the T2 generation Arabidopsis, and the transgenic plants that grew well under the hygromycin selection pressure were selected and transplanted into the soil for single plant culture. DNA and RNA were extracted from each Arabidopsis plant when it was cultured to the bolting stage, and the obtained DNA was used as a template to perform PCR amplification using the cross-vector specific primers of TaWRKY104. The insertion of the TaWRKY104 gene into Arabidopsis was confirmed by 1% agarose gel electrophoresis. The strains with the correct bands were detected by RNA fluorescence quantitative detection, and the three strains with the highest expression of TaWRKY104 were selected for further cultivation. After the Arabidopsis matured, the T2 generation seeds were collected.

[0107] Using the T2 seeds of the three selected strains, 100 seeds were taken out for disinfection, and evenly sown with a toothpick on a 1 / 2MS medium containing 25 mg / L hygromycin, and cultured according to the above method. When they grew to 4 leaves, positive seedlings with consistent growth were selected, and after they could take root normally on the culture medium, they were transplanted into the soil and placed in a light incubator for further culture, wherein the incubator conditions were 23°C, 16 / 8h, light / darkness. After the Arabidopsis matured, T3 seeds were collected from individual plants to obtain homozygous transgenic Arabidopsis seeds.

[0108] Example 4: Drought stress test.

[0109] Natural drought treatment: The sterilized WT and three transgenic seeds, the three strains are called OE1, OE2 and OE3, are sown on 1 / 2MS medium, and placed in a 22℃ constant temperature greenhouse after 3 days of vernalization, where the light cycle is 16h light / 8h dark. After 10 days, they are moved into pots filled with nutrient soil, and 6 Arabidopsis thaliana are moved into each pot for natural drought treatment, that is, continuous no watering treatment. During this period, pay attention to observe the phenotypic differences between the transgenic strains and the wild type. After 10 days, the phenotypes are recorded and photographed, and then rewatering treatment is carried out, with three biological replicates. After 3 days of rewatering, the phenotypic changes are observed and the survival rates of the transgenic strains and the wild type are counted respectively. The results are as follows Figure 1 and Figure 2 As shown, from Figure 1 It can be seen that Arabidopsis thaliana overexpressing OE1, OE2 and OE3 showed a certain degree of wilting after 10 days of natural drought. Figure 2 It can be seen that after 3 days of rehydration, the survival rate of overexpression Arabidopsis was higher than that of wild-type Arabidopsis.

[0110] Before and 10 days after drought treatment, Arabidopsis leaves were taken to measure the following physiological indices: soluble sugar content, proline content, protein content, superoxide dismutase content, catalase content, and peroxidase content. The results are as follows: Figure 3 As shown. Among them, superoxide dismutase is recorded as POD, catalase content is recorded as MDA, and peroxidase is recorded as CAT. Figure 3 It can be seen that after drought treatment, the soluble sugar content, proline content, protein content, superoxide dismutase content, catalase content, and peroxidase content of OE1, OE2, and OE3 were all higher than those of the WT group, which shows that the TaWRKY104 gene has a certain resistance to drought stress.

[0111] Example 5: Application of TaWRKY104 gene in genetic improvement and breeding of wheat drought resistance.

[0112] In production practice, the above genes can be transformed into wheat explant cells, and then the transformed callus tissue can be cultivated into plants. Through transgenic methods, plant expression vectors can be used to transform explant cells to cultivate drought-resistant wheat.

[0113] In production practice, the above genes can also be used to enhance the efficiency and accuracy of breeding targets through molecular marker-assisted selection breeding methods. For example, molecular markers are used to associate the target gene with the drought resistance trait of wheat, and the presence of the target gene is detected to achieve the purpose of selecting the target trait. The present invention discovered the function and specific application of the TaWRKY104 gene in regulating plant drought resistance, which provides a new gene resource for wheat drought resistance breeding and also lays the foundation for further analysis of the molecular mechanism of TaWRKY104 regulating plant drought resistance.

[0114] Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Obviously, various changes and modifications may be made to the present invention by those skilled in the art without departing from the spirit and scope of the present invention. Thus, the present invention is intended to include such changes and modifications if they fall within the scope of the technical equivalents of the present invention.

Claims

1. An application of wheat TaWRKY104 gene in promoting drought resistance in Arabidopsis thaliana, characterized in that: The nucleotide sequence of the wheat TaWRKY104 gene is shown in SEQ ID NO:

1. By overexpressing the wheat TaWRKY104 gene in Arabidopsis thaliana, the Arabidopsis thaliana is made drought-resistant.

2. The use of the wheat TaWRKY104 gene in promoting drought resistance in Arabidopsis according to claim 1, characterized in that: Overexpression of the wheat TaWRKY104 gene in Arabidopsis is achieved by transforming Arabidopsis thaliana with a recombinant engineered bacterium containing the wheat TaWRKY104 gene.

3. The use of the wheat TaWRKY104 gene in promoting drought resistance in Arabidopsis according to claim 2, characterized in that: The recombinant engineering bacteria are obtained by transferring the recombinant expression vector containing the wheat TaWRKY104 gene into Agrobacterium competent cells.

4. The use of the wheat TaWRKY104 gene in promoting drought resistance in Arabidopsis according to claim 3, characterized in that: The recombinant expression vector is obtained by inserting the wheat TaWRKY104 gene between the PstI and KpnI sites of the pSuper1300 overexpression vector plasmid.

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