A lotus leaf-specific promoter pNnCYP80G and its application

Abstract

This invention belongs to the field of plant genetic engineering and molecular biology, specifically disclosing a lotus leaf-specific promoter pNnCYP80G. Using a recombinant plant expression vector with the pNnCYP80G promoter, transgenic Arabidopsis plants were constructed, achieving… GUS The gene was specifically and highly expressed in Arabidopsis leaves, and the promoter pNnCYP80G responded to methyl jasmonate and mechanical damage treatments, further enhancing its expression. GUS Gene expression in leaves. The promoter pNnCYP80G can specifically express exogenous genes in plant leaf tissues, thereby improving leaf quality traits and effectively reducing the impact of exogenous gene expression on other organs and tissues.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and molecular biology technology, specifically relating to a lotus leaf tissue-specific promoter pNnCYP80G and its applications. Background Technology

[0002] Promoters play a crucial role in transgenic engineering, responsible for initiating and finely regulating the transcription initiation process of downstream genes. They also control the site, timing, and intensity of gene expression.

[0003] A promoter is a DNA sequence located upstream of the coding region of a gene. Based on expression patterns, they can be classified into three categories: constitutive promoters, inducible promoters, and tissue-specific promoters. Tissue-specific promoters function only in specific tissues or organs. In transgenic engineering, the rational use of tissue-specific promoters can accurately control gene expression in specific tissues, safely improve plant traits, and effectively reduce the impact of exogenous genes on normal plant growth and development. Therefore, exploring the function of tissue-specific promoters has become a research hotspot in the field of plant genetic engineering and has significant scientific importance.

[0004] Promoters typically contain cis-acting elements that control gene expression. Hormone and abiotic stress response elements are among the main components of promoters. Some tissue-specific promoters are regulated by hormones and abiotic signals. For example, the soybean root-specific promoter Glyma12g02240 enhances downstream gene expression and improves plant salt tolerance in response to salt stress. The rose RhPIP promoter enhances GUS gene expression activity in plant leaves in response to GA hormone treatment. The lily DXR promoter regulates the expression of the target gene only in flower tissues, and its expression is induced by injury and methyl jasmonate.

[0005] Lotus is an important aquatic economic crop in my country, with a wide cultivation area and abundant germplasm resources. Lotus leaves are crucial nutrient tissues and organs, rich in various secondary metabolites, and have been developed into a popular weight-loss and health food. The development of lotus leaf tissue-specific promoters plays a vital role in the production and utilization of leaf secondary metabolites. Furthermore, it holds broad application prospects in research on improving the quality traits of plant leaf organs. Summary of the Invention

[0006] The purpose of this invention is to provide a leaf-specific promoter pNnCYP80G, which enables the target gene to be specifically and efficiently expressed in leaf tissues.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The lotus leaf-specific promoter pNnCYP80G has the sequence shown in SEQ ID NO.1. Primer sequences used for amplifying the pNnCYP80G promoter are shown in SEQ ID NO.2 and 3.

[0009] A recombinant expression vector comprising the promoter pNnCYP80G. Preferably, the recombinant expression vector is constructed using the plant expression vector pBI101.

[0010] Transgenic plants containing the promoter pNnCYP80G.

[0011] The application of the pNnCYP80G promoter or a recombinant expression vector containing the pNnCYP80G promoter in driving the specific expression of the target gene in plant leaves. Preferably, the plant is lotus and Arabidopsis thaliana.

[0012] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0013] 1. The promoter described in this invention is the specific promoter pNnCYP80G from lotus leaves, which was screened by transcriptome sequencing technology. This method has high accuracy and strong reliability.

[0014] 2. The promoter pNnCYP80G described in this invention drives the target gene to be specifically and highly expressed in leaves. Therefore, the expression of the target gene will not affect the plant's vegetative growth and development. In the future, its tissue-specific expression characteristics can be used to directionally modify plant leaf traits, such as improving photosynthetic efficiency, resisting pests and diseases, or altering metabolic pathways in leaves. Attached Figure Description

[0015] Figure 1 The results of the analysis of the expression of the NnCYP80G gene regulated by the promoter pNnCYP80G in different tissues of lotus.

[0016] Figure 2 This is a plasmid map of the plant expression vector pBI101.

[0017] Figure 3 A schematic diagram of the restriction enzyme sites for constructing a recombinant expression vector by fusing the GUS gene with the promoter pNnCYP80G.

[0018] Figure 4 The results show the GUS staining and quantitative fluorescence analysis of the GUS gene in different parts of Arabidopsis thaliana.

[0019] Figure 5 GUS staining results for Arabidopsis thaliana leaf damage and methyl jasmate treatment. Detailed Implementation

[0020] This invention screened a lotus leaf-specific promoter based on transcriptome analysis of different tissues of lotus, cloned the promoter using PCR technology, constructed a fusion vector of the promoter and GUS reporter gene using homologous recombination, obtained transgenic Arabidopsis positive plants using Agrobacterium, and analyzed the promoter expression site and promoter activity using GUS histochemical staining and quantitative PCR technology for different plant tissues.

[0021] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. These specific embodiments are merely illustrative and do not limit the scope of protection of the invention. Any modifications or substitutions made to the invention using the embodiments as a guide without departing from the spirit and essence of the invention are within the scope of the invention.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials, reagents, and instruments used in the following examples are commercially available. The primers and sequencing used in the following examples were performed by Wuhan Qingke Co., Ltd.; the experimental material, the lotus variety 'Qiuxing', was obtained from the Lotus Germplasm Resource Conservation Platform of Wuhan Botanical Garden, Chinese Academy of Sciences; the wild-type Arabidopsis thaliana Col-0, the competent Escherichia coli strain TOP10, the competent Agrobacterium tumefaciens strain GV3101, and the modified plant genetic expression vector pBI101 were all preserved in the inventor's laboratory.

[0023] Example 1

[0024] Obtaining the full-length sequence of the lotus pNnCYP80G promoter:

[0025] Analysis of transcriptome data from various tissues of the lotus variety 'Qiuxing' revealed that the NnCYP80G gene is specifically expressed in lotus leaf tissues. Figure 1 Based on the predicted NnCYP80G sequence from the lotus whole genome sequence, a 1418 bp upstream fragment was selected as the promoter sequence, named pNnCYP80G, whose nucleotide sequence is shown in SEQ ID NO.1. Amplification primers were designed as shown in SEQ ID NO.2 and 3. In the upstream primer (SEQ ID NO.2), the first 10 bases are the homologous recombination arm of the plant genetic expression vector pBI101, followed by the SalI restriction site, and the remaining sequence is the upstream amplification primer sequence. In the downstream primer (SEQ ID NO.3), the first 8 bases are the homologous recombination arm of the plant genetic expression vector pBI101, followed by the XbaI restriction site, and the remaining sequence is the downstream amplification primer series.

[0026] Genomic DNA was extracted from the lotus variety 'Qiuxing' using the Tiangen reagent kit. Using this DNA as a template, the promoter pNnCYP80G sequence was amplified using the primers described above. The PCR amplification reaction system is as follows:

[0027]

[0028] The PCR amplification program is as follows: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min, 12℃ ∞.

[0029] Take 5 μl of PCR product and verify the amplified sequence size by 1% agarose gel electrophoresis. The PCR amplification product showed only one band of about 1500 bp after electrophoresis. The DNA product was recovered and purified using a PCR product recovery kit.

[0030] Example 2

[0031] pNnCYP80G promoter-GUS gene fusion vector transformed into Arabidopsis thaliana:

[0032] (1) Construction of a plant expression vector fused with the pNnCYP80G GUS gene

[0033] The plant expression vector pBI101 was digested with SalI and XbaI. The double digestion system is as follows:

[0034]

[0035] The enzyme digestion reaction was carried out in a 37°C water bath for 2 hours. The digestion products were separated by 1% agarose gel electrophoresis, and the purified plant expression vector pBI101 was recovered using an agarose gel electrophoresis recovery kit. The purified PCR product fragment was ligated to the recovered pBI101 linear vector via homologous recombination to construct the plant expression vector pBI101 with the pNnCYP80G promoter fused to the GUS gene. The ligation system is as follows:

[0036]

[0037] Add 5 μl of the ligation product to an EP tube containing 50 μl of competent TOP10 cells, gently aspirate to mix, incubate on ice for 30 min, then heat shock in a 42°C water bath for 1 min, and incubate on ice for 2 min. In a clean bench, add 200 μl of antibiotic-free LB liquid medium to the EP tube. Incubate the bacterial suspension at 37°C on a shaker at 220 rpm for 1 h, then evenly spread it onto a solid LB agar plate containing 50 μg / L kanamycin. Incubate at 37°C inverted for 12–16 h. In a clean bench, add 500 μl of LB liquid medium containing 50 μg / L kanamycin to the EP tube. Using a sterile 10 μl pipette tip, pick a healthy single colony from the solid LB agar plate, gently mix it into the LB liquid medium, and incubate the bacterial suspension at 37°C on a shaker at 220 rpm for 24 h. Perform PCR verification on the bacterial suspension. The amplification primers are shown in SEQ ID NO. 2 and 3. The reaction system is as follows:

[0038]

[0039]

[0040] Single colonies that test positive are sent to a sequencing company for sequencing. Plasmids are extracted from single colonies that are correctly sequenced, and the recombinant vector is named pBI101-pNnCYP80G-GUS based on the promoter it contains.

[0041] (2) Transform the recombinant vector into Agrobacterium.

[0042] Add 100 ng of recombinant vector to an EP tube containing 30 μl of Agrobacterium tumefaciens competent cells GV3101, gently aspirate to mix, and transfer all the liquid to a clean, sterile electroporation cuvette. Cap the cuvette and place it in an electroporator. Electroporate once at 2500 V / 6 ms. After electroporation, add 500 μl of antibiotic-free LB broth medium to the cuvette, gently aspirate to mix, and transfer all the liquid to a clean, sterile EP tube. Incubate at 28°C and 220 rpm for 3 h on a shaker. Spread the mixture evenly onto solid LB agar plates containing 50 μg / L kanamycin and 50 μg / L rifampin, and incubate upside down at 28°C for 36–48 h. Select single colonies for colony PCR verification using the same reaction system as in Example 2.

[0043] (3) Transformation of Arabidopsis thaliana by flower dipping method

[0044] Select Col-0 type Arabidopsis thaliana plants in full bloom, water them thoroughly, remove the siliques, and inoculate them with a resuspended Agrobacterium tumefaciens infection solution containing the recombinant vector (OD). 600 =0.8) Soak Arabidopsis inflorescences for 1 min 30 s, infect Arabidopsis and culture in the dark for 24 h, then transfer to normal photoperiod culture until T0 generation seeds mature.

[0045] The harvested T0 generation seeds were screened on 1 / 2 MS seed germination medium containing 50 μg / L kanamycin. Normally growing resistant seedlings were selected and planted to harvest T1 generation seeds. These seeds were then screened again on 1 / 2 MS seed germination medium containing 50 μg / L kanamycin. The ratio of normally growing seedlings to abnormally growing seedlings was 3:1, indicating single-copy insert plants. Normally growing resistant seedlings were then planted, and after the plants matured, genomic DNA was extracted from wild-type plants and the pBI101-pNnCYP80G-GUS transgenic line. PCR was performed using promoter amplification primers for positive identification. Arabidopsis plants that tested positive by PCR were further cultured to harvest T2 generation seeds. The same germination medium method was used to screen for normally growing T2 generation plants, which were the homozygous single-copy insert transgenic lines.

[0046] Example 3

[0047] GUS identification of genetically modified plants:

[0048] GUS staining analysis was performed using a GUS staining kit (Zhongke Ruitai, catalog number: RTU4032). Leaves, inflorescences, and pods of wild-type Col-0 and T2 generation transgenic Arabidopsis thaliana lines were subjected to GUS histochemical staining. Results showed that no blue staining was observed in any tissue of wild-type Arabidopsis thaliana. Tissue staining results of the pBI101-pNnCYP80G-GUS line showed that the GUS gene was mainly expressed in the leaf tissue of Arabidopsis thaliana. Figure 4 A). Quantitative detection of the GUS gene was performed on different tissues of Arabidopsis thaliana. The quantitative primers are shown in SEQ ID NO. 4 and 5. The specific procedure was as follows: RNA was extracted from roots, stems, leaves, inflorescences, and seeds of Arabidopsis thaliana transgenic with the pBI101-pNnCYP80G-GUS gene, and then reverse transcribed into cDNA. The cDNA was used as a template for quantitative real-time PCR analysis of the GUS gene, with three biological replicates. The results showed that the GUS gene expression in leaves of the pBI101-pNnCYP80G-GUS transgenic line was significantly higher than that in other tissues. Figure 4 B).

[0049] Example 4

[0050] Damage to genetically modified plants and methyl jasmonate treatment:

[0051] Leaves of pBI101-pNnCYP80G-GUS transgenic plants were damaged using sterilized needles, with Col-0 Arabidopsis thaliana as a control. GUS staining was performed 24 hours later. Col-0 and pBI101-pNnCYP80G-GUS T2 generation Arabidopsis thaliana seeds were germinated on 1 / 2 MS medium. When the seeds had two true leaves, they were sprayed with 100 μM methyl jasmonate (MeJA), and GUS staining was performed three days later. GUS staining results showed that after damage treatment, the blue color of the leaves in the damaged area was significantly deeper than that in the undamaged area. Figure 5 A). Spraying with MeJA after seed germination promotes the expression of GUS protein in transgenic Arabidopsis leaves, resulting in deeper leaf staining. Figure 5 B). This indicates that the activity of the pNnCYP80G promoter in the leaves is impaired and regulated by the methyl jasmonate signaling pathway.

[0052] In summary, this invention provides a lotus leaf-specific promoter pNnCYP80G, the nucleotide sequence of which is shown in SEQ ID NO.1; this promoter drives the high expression of the target gene in lotus leaves. Linking the promoter pNnCYP80G to the GUS gene and transforming Arabidopsis thaliana allows for the high expression of GUS in Arabidopsis leaves via this promoter. The lotus leaf-specific promoter pNnCYP80G has significant application value in functional studies of genes related to plant leaf development and metabolic synthesis, as well as in plant metabolic engineering regulation research.

[0053] It should be understood that the above specific implementation methods are only used to explain the basic principles of the present invention and are not intended to limit the present invention in any way. All changes and improvements made based on the present invention shall still fall within the protection scope of the present invention.

Claims

1. Use of the promoter pNnCYP80G for driving the specific expression of a gene of interest in the leaves of a plant, characterized in that, The sequence of the promoter is shown as SEQ ID NO.

1.

2. Use of a recombinant expression vector containing the promoter pNnCYP80G for driving the specific expression of a gene of interest in the leaves of a plant, characterized in that, The sequence of the promoter is shown as SEQ ID NO.

1.

3. Use according to claim 1 or 2, characterized in that, The plants are lotus and Arabidopsis.

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