Barley hvmbf1c gene promoter and use thereof

Abstract

The application discloses a barley HvMBF1c gene full-length promoter sequence and application thereof. A 2000 bp or so nucleotide sequence at the 5' end upstream of a coding region of the HvMBF1c gene is used as a full-length promoter, specific upstream and downstream primers are designed, and a target promoter fragment is amplified through PCR, wherein the sequence has not only a core promoter element, but also functional elements responding to adversity stress induction and plant hormone induction. The sequence is fused with a GUS reporter gene, and the recombined expression vector is transformed into wild-type Arabidopsis thaliana, and GUS staining detection is carried out on positive clone plant tissues, so that it is determined that the HvMBF1c gene promoter can exercise normal functions in the transgenic plants and has a constitutive promoter characteristic. The promoter of the application provides a suitable regulatory element for plant expression vector construction, and has important application value for barley stress resistance transgenic breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to an HvMBF1c gene promoter sequence and its application. Background Technology

[0002] The promoter, located upstream of the 5' end of the corresponding regulatory gene, is the central regulator of gene transcription. It recognizes and binds to RNA polymerase, initiating the transcription process. Containing various cis-acting elements that bind to transcription factors, it participates in regulating the expression of downstream genes, enabling plants to effectively regulate growth and development and respond to changes in the external environment. Plant promoters are mainly classified into three categories: constitutive promoters, tissue-specific promoters, and inducible promoters. The gene expression characteristics initiated by each type of promoter exhibit diversity and significant species-specific differences. Some plant genes do not have promoters belonging to only one category and sometimes exhibit characteristics of other types of promoters.

[0003] Currently, with the rapid development of molecular biology techniques and the gradual improvement of transgenic technology, it has become a reality to improve plant (crop) yield, quality, and adaptability to abiotic stress through transgenic methods. While the acquisition of superior exogenous genes has greatly promoted the development of transgenic breeding, improving the expression level and tissue specificity of exogenous genes in transgenic plants severely restricts the application value and scope of transgenic technology. The main reason for this is the lack of superior gene promoters; for example, in current transgenic technology, constitutive promoters are still mainly CaMV35S. Therefore, discovering new promoters in plants and studying their functions has significant economic and application value for the development of plant transgenic technology.

[0004] Multiprotein bridging factor 1 (MBF1), a ubiquitous gene in eukaryotes, is considered a highly conserved transcriptional coactivator. Previous studies have shown that MBF1 participates in regulating plant growth, development, and responses to abiotic stress. In our previous research on genes beneficial to salt-tolerant germination in barley seeds, we found that the expression levels of HvMBF1a and HvMBF1b did not change significantly under salt stress, while the expression of HvMBF1c was significantly upregulated at both the transcriptional and protein levels. We speculate that HvMBF1c may be involved in regulating salt tolerance during barley seed germination and could serve as an important candidate gene for molecular breeding of salt-tolerant barley (Feng Juling, Wang Juncheng, Yao Lirong, et al. Cloning of barley HvMBF1c and its expression pattern in response to salt stress [J / OL]. Journal of Plant Genetic Resources: 1-15). Therefore, cloning the promoter sequence of the HvMBF1c gene and performing functional analysis can provide important promoter sequence resources for molecular breeding practices in barley salt tolerance. Summary of the Invention

[0005] The key technical problem this invention aims to solve lies in the HvMBF1c gene promoter sequence and its application. To solve the above technical problem, this invention adopts the following technical solution: 1. The promoter of the barley HvMBF1c gene, the sequence of which is shown in SEQ ID No.1 of the sequence listing, and contains 2000 bp nucleotides.

[0006] 2. The method for cloning the HvMBF1c gene promoter sequence includes: (1) extracting DNA, (2) PCR amplification, and (3) ligating the cloning vector and sequencing.

[0007] 3. The method for constructing an expression vector for the HvMBF1c gene promoter sequence includes: (1) vector selection and promoter cloning, (2) vector ligation, and (3) double enzyme digestion verification.

[0008] 4. The method for transforming and culturing Arabidopsis thaliana with the HvMBF1c promoter of Agrobacterium, including: (1) transforming Agrobacterium, (2) transforming Arabidopsis thaliana, and (3) screening and verification of positive plants.

[0009] 5. Application of the HvMBF1c gene promoter, wherein the application is constitutive expression of the GUS gene in Arabidopsis leaves and roots.

[0010] 6. Application of the HvMBF1c gene promoter, wherein the application is constitutive expression of heteronucleotide sequences.

[0011] Beneficial Effects: This invention provides a method for preparing the promoter sequence of the barley HvMBF1c gene and performs bioinformatics analysis, enabling rapid and accurate acquisition of the promoter sequence while clarifying its sequence characteristics. It also provides an application of the barley HvMBF1c gene promoter sequence in the field of plant stress resistance gene technology. Furthermore, it confirms that the barley HvMBF1c gene promoter belongs to the constitutive promoter type, providing a basis for its application in plant transgenic breeding practices. The 2000 bp upstream nucleotide sequence of the 5' end of the barley HvMBF1c gene was used as the full-length promoter sequence. Using this as a template, upstream primer HvMBF1c-F1 and downstream primer HvMBF1c-R1 were designed. The 2000 bp upstream promoter fragment of the HvMBF1c gene was obtained by PCR amplification, and cis-acting element analysis of sequence characteristics was performed. Then, it was fused with the GUS reporter gene and transformed into wild-type Arabidopsis thaliana. GUS staining was performed on different tissues of positive clones to confirm that the HvMBF1c gene promoter performs normal function in transgenic plants and has constitutive promoter characteristics.

[0012] In summary, this invention is the first to discover the promoter sequence of the salt stress response gene HvMBF1c in barley and confirm that the promoter is constitutively expressed, providing a suitable regulatory element for the construction of plant genetic engineering expression vectors and providing a new promoter for the efficient expression of heterologous nucleotide sequences in plant tissues. Attached Figure Description

[0013] Figure 1 DNA was extracted from barley leaves. M: DL15000+2000 DNA Marker; Lanes 1-3: Leaf DNA.

[0014] Figure 2 PCR amplification of the HvMBF1c promoter and identification of positive clones. A: PCR amplification of the HvMBF1c promoter; B: identification of promoter-positive clones; M: DL2000 DNA Marker; Lanes 1-2: promoter amplification products; Lanes 3-8: bacterial culture of promoter-positive clones.

[0015] Figure 3 This is a sequence diagram of the HvMBF1c promoter.

[0016] Figure 4 In the image, A represents the amplification and purification of the HvMBF1c promoter; M represents the DL2000 DNA Marker; lanes 1-2 contain PCR amplification products; and lanes 3-4 contain gel recovery products. Figure 4 B represents the identification of HvMBF1c promoter-positive recombination clones; M: DL2000 DNA Marker; lanes 1-6: bacterial suspensions of fusion-expressing recombination-positive clones. Figure 4 In the middle lane, C represents the double digestion verification of the HvMBF1c promoter fusion expression recombinant plasmid. M: DL15000+2000 DNA Marker; Lanes 1-2: Fusion expression double digestion plasmid; Lane 3: pBI121-GUS plasmid.

[0017] Figure 5 PCR validation of Agrobacterium fusion expression of recombinant plasmid in bacterial culture. M: DL2000 DNA Marker; Lanes 1-6: Agrobacterium fusion expression bacterial culture; Lane 7: ddH2O.

[0018] Figure 6 This is for resistance screening of positive transgenic Arabidopsis thaliana. The part circled in red in the figure is the positive transgenic Arabidopsis thaliana.

[0019] Figure 7 PCR identification of HvMBF1c fusion expression positive transgenic plants. M: DL2000 DNA Marker; lanes 1-8: positive transgenic plants; lane WT: wild-type plants; lane +: fusion expression recombinant plasmid; lane -: ddH2O.

[0020] Figure 8 The distribution of cis-acting elements in the HvMBF1c promoter region.

[0021] Figure 9 GUS staining for Arabidopsis thaliana seedlings transfected with the HvMBF1c promoter. A: Arabidopsis thaliana transfected with the HvMBF1c promoter; B: Root of wild-type Arabidopsis thaliana; C: Whole plant of wild-type Arabidopsis thaliana. Specific implementation methods Unless otherwise specified, the methods and apparatus used in the following embodiments of this invention are conventional methods and apparatus; the equipment and reagents used are all conventional equipment and reagents purchased from reagent companies. To make the objectives, technical solutions, and advantages of this invention clearer, the specific implementation methods of this invention are described in detail below with reference to specific embodiments. Examples of these preferred embodiments are illustrated in the specific embodiments. It should also be noted that, in order to avoid obscuring the technical solution of this invention due to unnecessary details, only technical solutions and / or processing steps closely related to the solution according to this invention are shown in the embodiments, while other details that are not closely related are omitted.

[0022] Example 1 This embodiment provides the HvMBF1c gene promoter sequence, wherein the barley HvMBF1c gene promoter sequence is shown in SEQ ID No.1 of the sequence listing and contains 2000 bp nucleotides.

[0023] Example 2 This embodiment provides a method for cloning the HvMBF1c gene promoter sequence, including: 1. DNA Extraction. Select a number of plump, uniformly sized barley seeds, disinfect and clean them, and evenly place them in a germination box lined with two layers of moist germination paper. Incubate at room temperature until the three-leaf stage. Extract total DNA from barley leaves using a plant genomic DNA extraction kit. Refer to the kit instructions for specific extraction procedures. Detect DNA integrity using 1% agarose gel electrophoresis, and determine the concentration and purity of DNA using a micro spectrophotometer (DNA / Proteins Analyzer P100+, USA). Successfully extracted DNA (…) Figure 1 Store at -20 ℃ for later use.

[0024] 2. PCR amplification. Based on... HvMBF1c Design upstream primers based on the 2000 bp promoter sequence of the gene. HvMBF1c -F1 (5'-TGCTTTCTTTCAACATGTATAC-3') and downstream primer HvMBF1c-R1 (5'-TGCTCCCTTCGCTTCTGTTC-3'), primers were synthesized by a professional company, and the target region was amplified using barley leaf DNA as a template. The amplification system (25 μL) consisted of: 12.5 μL of 2×Taq PCR MasterMix, 1.0 μL of upstream primer (10 μmol / L), 1.0 μL of downstream primer (10 μmol / L), 1 μL of DNA (100 ng / μL), and 9.5 μL of sterile ddH2O. The landing PCR amplification program was as follows: 94 ℃ pre-denaturation for 5 min; 15 cycles of 94 ℃ for 30 s, 70 ℃ for 30 s, and 72 ℃ for 3 min; 30 cycles of 94 ℃ for 30 s, 55 ℃ for 30 s, and 72 ℃ for 3 min; and a 10-min extension at 72 ℃. The amplified products were detected by 1% agarose gel electrophoresis, yielding a band that met the expected target size of 2000 bp. Figure 2 -A).

[0025] 3. Ligate the cloning vector and sequence. Recover the amplified product using an enhanced agarose gel DNA recovery kit (refer to the instruction manual for recovery procedures). Then, combine the purified gel product with the cloning vector pMD. TM 19-T (Dalian Solarbio Biotechnology Co., Ltd.) was ligated at 16 ℃ for approximately 12 h. The reaction system consisted of: purified product - 4 μL, Solution Ⅰ - 5 μL, pMD TM 19-T Vector -1 μL. The ligation product was transformed into DH5α using the heat shock method. After incubation at 37 ℃ in the dark for 12-16 h, large, round, white single colonies were picked and incubated at 37 ℃ and 220 rpm for about 12 h with shaking. The turbid E. coli culture was then subjected to colony PCR identification, which yielded a 2000 bp target band. Figure 2 -B). Positive clone bacterial cultures with appropriate band sizes were sent to Lanzhou Tianqi Gene Biotechnology Co., Ltd. for sequencing. The sequenced sequences showed 100% homology with the target sequence. Figure 3 This indicates that the clone has been successfully created. HvMBF1c The full length of the gene promoter.

[0026] Example 3 This embodiment provides a method for constructing an expression vector for the HvMBF1c gene promoter sequence, including: 1. Vector selection and promoter cloning. Using pBI121-GUS as the expression vector, and based on the requirements for constructing the recombinant vector, the above embodiments were performed. HvMBF1c Design upstream primers by adding HindIII and BamHI restriction enzyme sites to the upstream and downstream primers of the gene promoter. HvMBF1c -F2 (5'-CCCAAGCTTTGCTTTCTTTCAACATGTATAC-3') and downstream primer HvMBF1c -R2(5'-CGGGATCCTGCTCCCTTCGCTTCTGTTC-3'). The target fragment was re-amplified using the above method and validated by gel extraction and purification. The results consistently yielded bands consistent with the expected target, with a size of 2000 bp. Figure 4 -A), followed by the purification product and pMD TM Sequencing was performed on the recombinant DH5α-positive clonal bacterial culture containing the 19-T vector.

[0027] 2. Vector Ligation. Extract the appropriate DH5α bacterial culture plasmid and pBI121-GUS empty vector bacterial culture plasmid using the kit (extraction method as per the instruction manual). Perform double enzyme digestion on both plasmids at 37 ℃. The digestion system (60 μL) consisted of: sterile ddH2O - 12 μL, 10×T Buffer - 6 μL, BSA - 6 μL, HindIII - 3 μL, BamHI - 3 μL, and plasmid - 30 μL. After 1% agarose gel electrophoresis, the promoter fragment and vector fragment were purified separately using gel electrophoresis. The purified products were ligated using T4 DNA ligase at 16 ℃. The ligation system (10 μL) consisted of: HvMBF1c Promoter fragment - 6 μL, pBI121-GUS vector fragment - 1 μL, sterile ddH2O - 1 μL, 10×T4 DNA ligase buffer - 1 μL, T4 DNA ligase - 1 μL (Note: HvMBF1c After adding the promoter fragment, pBI121-GFP vector fragment, sterile ddH2O, and 10×T4 DNA ligase buffer, the mixture was briefly centrifuged for a few seconds, incubated at 65 ℃ for 3 min, and then placed on ice for a few seconds before adding T4 DNA ligase. The ligation product was then transformed again into DH5α using the heat shock method. PCR verification of the recombinant positive clone of the promoter and pBI121-GUS yielded a target band of 2000 bp. Figure 4 -B).

[0028] 3. Double enzyme digestion verification. Positive monoclonal bacterial cultures with suitable band sizes were sent to the company for sequencing. The plasmid from the sequencing-suitable DH5α bacterial culture was then extracted again and subjected to double enzyme digestion. The digestion system (20 μL) consisted of: sterile ddH2O - 4 μL, 10×T Buffer - 2 μL, BSA - 2 μL, HindIII - 1 μL, BamHI - 1 μL, and plasmid - 10 μL. The digestion products were detected by 1% agarose gel electrophoresis, revealing a vector band of approximately 15000 bp and a promoter band of approximately 2000 bp. Figure 4 -C), indicating HvMBF1c The promoter has been ligated to the pBI121-GUS expression vector, and pBI121- has been successfully constructed. HvMBF1cpro ::GUS fusion expression vector.

[0029] Example 4 This embodiment provides a method for transforming and culturing Agrobacterium thaliana with the HvMBF1c promoter, including: 1. Transformation with Agrobacterium. The fusion expression recombinant plasmid pBI121- was transformed using a freeze-thaw method. HvMBF1cpro ::GUS was used to transform Agrobacterium competent cells GV3101. The specific transformation steps were performed according to the manufacturer's instructions. After transformation, single colonies were selected and cultured in LB liquid medium containing Rif and Kans, and incubated at 28°C and 220 rpm for 1-2 days. PCR verification of the Agrobacterium culture containing the recombinant plasmid was performed, and the results showed a band size of 2000 bp (…). Figure 5 (This is consistent with the expected result.)

[0030] 2. Transformation of Arabidopsis thaliana. The Agrobacterium tumefaciens PCR products with appropriate band sizes were finally sequenced. The Agrobacterium tumefaciens with appropriate sequencing results were used to prepare the infection solution and transformed into wild-type Arabidopsis thaliana by inflorescence infection method. After the Arabidopsis thaliana matured, T0 generation transgenic seeds were harvested from individual plants.

[0031] 3. Screening and verification of positive plants. Harvested Arabidopsis seeds were treated with low temperature and sterilized before being sown on 1 / 2 MS solid medium containing the antibiotic Kan (50 ug / ml). After incubation at 26 ℃ for approximately 15 days, positive seedlings with good growth and relatively green cotyledons were selected. Figure 6 The seedlings were carefully transplanted into sterilized Arabidopsis thaliana culture medium. Once they reached the seedling stage, DNA was extracted from the leaves of both the positive seedlings and wild-type Arabidopsis thaliana. HvMBF1c -F2 and HvMBF1c -R2 primer pair HvMBF1c The promoter fragment was used for PCR detection, and transgenic positive seedlings were finally obtained. Figure 7 ).

[0032] Example 5 This embodiment provides the application of the HvMBF1c gene promoter sequence, including: Functional element analysis of the promoter sequence using Plant CARE software revealed that, in addition to the core promoter elements TATA-box, CAAT-box, and Unnamed_4, the sequence region also contained several hormone-response and stress-response elements. For example, the MYC, TGACG-motif, and CGTCA-motif elements were associated with methyl jasmonate (MeJA); the TCA-element element was associated with salicylic acid; the ABRE, GARE-motif, and TGA-element elements were involved in abscisic acid, gibberellin, and auxin responses, respectively; the MYB element was associated with various stresses and metabolic regulation; the MYB recognition site element was associated with drought stress; and the TC-rich repeats element was involved in regulating abiotic stress defense and stress response. Furthermore, the region also contained light-response-related regulatory elements such as G-Box, GT1-motif, I-box, TCT-motif, Box 4, and GATA-motif; and elements related to plant root characteristics, such as motif I. Figure 8 ).

[0033] Arabidopsis seedlings or tissues transfected with the HvMBF1c gene promoter were completely immersed in GUS staining solution and incubated overnight at 37°C. After destaining in 75% ethanol, the GUS staining was observed and photographed. Results showed that leaves and roots of Arabidopsis seedlings transfected with the HvMBF1c gene promoter fragment were stained, and the staining was quite deep. However, leaves and roots of wild-type Arabidopsis without any vector were not stained blue, indicating the absence of GUS reporter gene expression. Figure 9 This indicates that the cloned HvMBF1c gene promoter has strong constitutive promoter activity, providing a suitable regulatory element for the efficient expression of heterologous nucleotide sequences in plant tissues.

[0034] The above description is only a specific embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. The application of the HvMBF1c gene promoter, characterized in that... The application is to constitutively express heteronucleotides in Arabidopsis leaves and roots; the HvMBF1c gene promoter sequence is shown in SEQ ID No.

1.

2. The application of the HvMBF1c gene promoter according to claim 1, characterized in that... The heteronucleotide is the GUS gene.

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