Plant vascular tissue specific expression promoter ProAPL and application thereof

Abstract

The invention discloses a plant vascular tissue specific expression promoter ProAPL and application thereof, belongs to the technical field of plant genetic engineering, and provides a plant vascular tissue specific expression promoter ProAPL, and the nucleotide sequence of the plant vascular tissue specific expression promoter ProAPL is shown as SEQ ID NO.1. The invention further discloses a plant vascular tissue specific expression promoter ProAPL. The poplar vascular tissue specific expression promoter ProAPL provided by the invention can be effectively and specifically expressed in the poplar vascular tissue, can specifically play the function of an exogenous gene expression product in a specific tissue, and regulates and controls the continuous expression of an exogenous gene in the plant vascular tissue.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a plant vascular tissue specific expression promoter ProAPL and an application thereof. Background Art

[0002] The promoter is a sequence that provides RNA polymerase recognition and binding, and it is located upstream of the gene. Once RNA polymerase locates and binds to the promoter sequence, transcription can be initiated, so that the growth and development of the organism and the physiological process proceed in an orderly manner, thus ensuring the normal growth and development of the plant. The types of promoters can be divided into constitutive promoters, tissue-specific promoters, and inducible promoters. Constitutive promoters drive genes to be expressed continuously in all cells, tissues, and organs; tissue-specific promoters regulate genes to be expressed only in certain specific parts or organs. So far, the promoters widely used in plant expression vectors are constitutive promoters. For example, the vast majority of dicotyledonous transgenic plants use the cauliflower mosaic virus (CaMV) 35S promoter and the Agrobacterium nopaline synthase (NOS) promoter, and monocotyledonous transgenic plants mainly use the OsAct1 promoter from rice and the maize polyubiquitin gene (ZmUbi1) promoter, and these promoters have proven their effectiveness as inducible plant gene expression. However, since the genes driven by constitutive promoters are strongly expressed in different tissues and organs of plants, it has gradually been revealed that constitutive promoters can cause transgenic plants to grow slower than control plants, become shorter, have lower yields, and even die. The cambium is a key part of forest growth. It is not only responsible for the formation of wood, but also plays an important role in the radial growth and morphological construction of forests. By using cambium-specific promoters to regulate the expression of genes related to cell division, elongation, and differentiation, it can promote the division and proliferation of cambium cells and increase wood yield. The maintenance of cambium cell activity is the guarantee for the continuous thickening of woody plant stems. In the study of plant genetic engineering, the use of cambium-specific promoters to drive the specific expression of target genes in the cambium region of forest trees can effectively play the role of target genes in the cambium region, thereby purposefully improving the quality of forest trees. Therefore, it is imperative to develop promoters specific to the cambium region of forest trees and carry out targeted and precise design breeding. Summary of the invention

[0003] In order to solve the above technical problems, the present invention proposes a plant vascular tissue-specific expression promoter ProAPL and its application. The present invention provides a tissue-specific expression promoter that can effectively express specifically in poplar vascular tissue, can specifically exert the function of exogenous gene expression products in specific tissues, and regulate the continuous expression of exogenous genes in plant vascular tissues.

[0004] To achieve the above object, the present invention provides a plant vascular tissue-specific expression promoter ProAPL, the nucleotide sequence of the vascular tissue-specific expression promoter ProAPL is shown in SEQ ID NO.1.

[0005] Preferably, the vascular tissue-specific expression promoter ProAPL is specifically expressed in poplar vascular tissue.

[0006] Preferably, the vascular tissue-specific expression promoter ProAPL is specifically expressed in the poplar cambium.

[0007] The present invention also provides the application of the plant vascular tissue specific expression promoter ProAPL in regulating the specific expression of the target gene in the poplar vascular tissue.

[0008] The present invention also provides application of the plant vascular tissue specific expression promoter ProAPL in plant genetic improvement, wherein the plant is poplar.

[0009] The invention also provides an expression box containing the plant vascular tissue specific expression promoter ProAPL.

[0010] The invention also provides a recombinant expression vector containing the plant vascular tissue specific expression promoter ProAPL.

[0011] The present invention also provides an engineering bacterium containing the plant vascular tissue specific expression promoter ProAPL.

[0012] The present invention also provides a method for cultivating transgenic plants, comprising the step of transferring the vascular tissue-specific expression promoter ProAPL, the expression cassette, the recombinant expression vector or the engineering bacteria into plants; the plant is poplar.

[0013] The present invention also provides the use of the expression cassette, the recombinant expression vector or the engineering bacteria in regulating the specific expression of the target gene in the poplar vascular tissue.

[0014] Compared with the prior art, the present invention has the following advantages and technical effects: The poplar vascular tissue-specific expression promoter ProAPL of the present invention is derived from the silver gland poplar '84K' genomic DNA. The poplar vascular tissue-specific expression promoter ProAPL fragment size is 1889 bp and has the following characteristics: (1) it is located at the 5' end and upstream of the Pop_A10G069473 gene; (2) the base length is 1889 bp; (3) it has the necessary site and transcription start point for initiating transcription; (4) it is specifically expressed in the poplar vascular tissue, regulates the continuous expression of exogenous genes in specific tissues and organs of the plant, avoids energy waste and increases the effect of genetic modification, reduces plant dwarfing, gene silencing and other problems caused by constitutive expression of genes, and can not only serve the breeding of transgenic poplars, but also serve the long-term promoter transformation and design reserve resources. The present invention also discloses a recombinant expression vector containing a tissue-specific expression promoter ProAPL, which enables downstream genes to be specifically expressed in plant vascular tissues under the regulation of the tissue-specific expression promoter ProAPL of the recombinant expression vector, thereby providing a tool for studying the efficient, stable and specific expression of genes in plant vascular tissues in the field of plant genetic engineering, and has broad application prospects.

[0015] Through the study of poplar vascular tissue-specific promoter ProAPL, the regulatory mechanism of wood quality formation is revealed, which is helpful to achieve targeted improvement of wood quality and cultivate new forest varieties that meet demand. By utilizing the structural characteristics of vascular tissue to fuse functional genes with its tissue-specific promoter, a large number of special application systems are developed, which is conducive to the development of new genetic tools, the increase of vascular biomass accumulation and the enhancement of forest stress tolerance, and the application value of vascular promoter ProAPL can be fully utilized. This will provide a new scientific theoretical basis and practical application value for the molecular mechanism of vascular tissue development and wood formation in woody plants, and provide relevant theoretical and technical support for the design of molecular varieties for improving the wood properties of fast-growing forest tree species. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 Isolation and identification of the poplar vascular tissue-specific promoter ProAPL, where A is the UMAP analysis result, B is the expression position of the APL homologous gene pal-pou17833, C is the APL AspWood data analysis result, and D is the APL qPCR result; Figure 2The results of in situ expression detection of PagAPL, where A is the negative control, B is the expression position of PagAPL in the stem, the red mark in the figure represents the hybridization signal of PagAPL, and the scale bar is 200 μm; Figure 3 Schematic diagram of the construction of a vector containing the promoter ProAPL; Figure 4 For Pro PagAPL ::GUS transgenic plants, where A is the state of leaves dedifferentiating into callus after Agrobacterium transformation, B is the callus induction stage, C is the callus culture, D is the bud induction stage, and E is the root induction stage; Figure 5 are transgenic strains of APL, wherein A is strain 1, B is strain 2, C is strain 3, and D is strain 4; Figure 6 The results of the determination of GUS expression in APL transgenic plants, the red arrows in the figure represent high-expression lines; Figure 7 The results of GUS staining in APL transgenic plants, where A is strain 1, B is strain 2, and C is strain 3. The scale bar is 100 μm. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] Example 1 Populus argentea '84K' To study the material, single-cell RNA-Seq data and wood formation database analysis were used to identify poplar vascular tissue-specific genes and their expression sites, clone the corresponding promoters, and verify and evaluate the ability of specific promoters to drive gene-specific expression.

[0024] 1. Research on specific genes in the poplar gene map Poplar vascular tissue-specific genes were screened based on the poplar gene map: the candidate gene was APL (84K poplar genome Pop_A10G069473, doi:10.1093 / dnares / dsz020).

[0025] 2. Study on promoter activity in transgenic poplar plants (1) Identify homologous genes of candidate genes and analyze their tissue expression patterns using qPCR.

[0026] (2) Observe the in situ expression pattern of candidate genes using in situ PCR technology.

[0027] (3) Clone the promoter region (~2000 bp) of the APL gene that is highly expressed in the cambium.

[0028] (4) The promoter sequence obtained in (3) was constructed into the pCambia2300-Promoter-35S mini-GUS-35S-eyGFPuv vector and transformed into Agrobacterium.

[0029] (5) The vector was transformed into '84K' poplar using the leaf disc method, and transgenic plants were obtained for subsequent analysis.

[0030] 3. Study on the expression site of tissue-specific promoter (1) The transgenic plants were preliminarily identified through eyGFPuv observation and the expression level of the GUS gene was detected. Three strains with higher GUS expression multiples were selected for subsequent research.

[0031] (2) GUS histochemical staining was performed on the selected transgenic poplar plants to explore the location of their expressed genes.

[0032] (3) Verify the function of the promoter APL in specifically driving gene expression.

[0033] 4. Co-expression network analysis of vascular tissue-specific genes Data from a poplar gene mapping study were used to examine the expression of APL in different tissues. Data used for the heatmap were log2 transformed.

[0034] 5. Analysis of target gene expression level by fluorescence quantitative PCR (1) Different tissues of '84K' plants grown in soil for two months were harvested: leaves, phloem, cambium, xylem and roots. Total RNA was isolated by CTAB method and 1 μg of total RNA was reverse transcribed after quantification. The reaction was performed on a LightCycler® 480 thermal cycler (Roche, Germany) using Power 2×SYBR Real-time PCR Master Mix (TransGen Biotech, Beijing, China). UBQ was used as an internal reference (to quantify the relative transcription level of the gene in each sample). 2 -ΔΔCt Methods Analysis of gene expression levels.

[0035] (2) Real-time fluorescence quantitative PCR was used to analyze the expression of APL gene.

[0036] VI. In situ PCR localization of gene expression (1) Freshly harvested plant tissue (internode 7 of stems of '84K' tissue culture plants) was immediately fixed in formaldehyde solution, and the penetration of the fixative was enhanced by vacuum infiltration. Then, the samples were embedded in agarose, cut into blocks, and sectioned on a vibrating table. 50 µm thick sections were collected in microtubes. DNase (Takara) treatment, reverse transcription (Takara), and in situ PCR were performed in centrifuge tubes using a standard thermocycler.

[0037] (2) Genomic DNA is removed with DNase and converted to cDNA by reverse transcription. The resulting cDNA is amplified by standard gene-specific PCR incorporating digoxigenin (DIG)-labeled nucleotides. Sections are incubated with anti-DIG antibodies conjugated to alkaline phosphatase.

[0038] (3) Addition of a specific substrate for alkaline phosphatase allows colorimetric detection of DIG-labeled PCR products and compartmentalization of them into RNA synthesis sites. The sections were then transferred to microscope slides and observed using a bright-field microscope (Leica DM6B).

[0039] VII. Vector Construction and Transgenic Poplar Creation (1) The 1889 bp APL promoter (ProAPL, nucleotide sequence shown in SEQ ID NO.1) was cloned from the genomic DNA of Populus alba '84K'.

[0040]

[0041] (2) Pro was generated using the improved vector pCambia2300-Promoter-35S mini-GUS-35S-eyGFPuv PagAPL ::GUS constructs were confirmed by Sanger sequencing.

[0042] (3) The vector was then transformed into Agrobacterium GV3101 using Agrobacterium-mediated transformation. Agrobacterium cells were collected and resuspended to OD 600 =0.3~0.4. Soak the 84K poplar leaf discs in the resuspended cells for 20 min, then transfer the leaf discs to the medium for inducing callus formation and co-culture at room temperature in the dark for 2 days. -1 The plants were cultured on Kan medium for 30 days to induce the formation of stems, leaves and roots.

[0043] 8. Identification of transgenic plants (1) eyGFPuv observation Since the improved vector carries the eyGFPuv label, when the transgenic poplar seedlings are irradiated with UV light, the target plants will emit green fluorescence, thereby screening the target transgenic plants.

[0044] (2) Extract DNA from transgenic plants and detect the expression level of the GUS gene.

[0045] DNA was extracted from the APL transgenic plants screened in (1) to detect the GUS expression level, and three strains with higher expression levels were selected for subsequent experiments.

[0046] IX. Histochemical GUS staining and microscopic observation (1) The transgenic plants were incubated in 90% acetone on ice for 2 h, washed three times with washing buffer, and then placed in 1 mM X-Gluc washing buffer and incubated in the dark at 37 °C for 8 h. The plants were then washed with 75% ethanol.

[0047] (2) Slice the tissue using a Leica VT1200S, with a slice thickness of 50 μm, and observe under a Leica DM6B microscope. The washing buffer contains: 0.2 mol·L -1 NaH2PO4, 0.2 mol·L -1 Na2HPO4, 2mmol·L -1 K3[Fe(CN)6], 2mmol·L -1 K4[Fe(CN)6].

[0048] 10. Quantitative Analysis of GUS Activity The GUS quantitative detection kit (SL7161) was used to detect the GUS activity of transgenic plants. GUS reacts with 4-MUG to generate the fluorescent substance 4-MU, which produces the highest absorption peaks at 365nm and 455nm excitation wavelengths. The well-ground sample was mixed with 1mL of extraction buffer, rotated vigorously for 30s, and centrifuged at 12000rpm for 10min. Then, 150µL of supernatant was added to 150µL of 4-MUG substrate solution to generate a reaction solution. The mixture was heated at 37℃, and 100µL of the reaction solution was removed every 10min and 900µL of the stop solution was added. The final GUS activity value in the total protein was recorded as 4-MU pmol min -1 mg -1 .

[0049] 11. Results 1. Identification of Poplar Vascular Tissue-Specific Genes Using UMAP analysis (e.g. Figure 1 In Figure A, we found that the APL homologous gene PagAPL (84K poplar genome Pop_A10G069473, doi:10.1093 / dnares / dsz020) was mainly expressed in the sieve tube elements and cambium regions (e.g. Figure 1 In Figure B, the results indicate that the gene may be driven by a specific promoter. To further confirm the expression of the gene during wood development, AspWood data analysis revealed that the gene is expressed in the cambium near the phloem (e.g. Figure 1 (C), the expression pattern was the same as that of UMAP analysis. qPCR showed that the expression in stems was higher than that in leaves and roots, and PagAPL was highly expressed in the cambium (Fig. Figure 1 Middle D).

[0050] 2. In situ PCR The in situ expression of PagAPL was investigated by in situ PCR. Figure 2 Middle A and Figure 2 The results shown in B show that the PagAPL gene is mainly expressed in the cambium.

[0051] 3. Vector construction To further determine the detailed expression patterns, e.g. Figure 3 As shown, the promoter region ProAPL of PagAPL was cloned, and homologous recombination was performed using two restriction sites, EcoR I and XBa I. The ProAPL promoter was fused with the 35S mini promoter and the β-glucuronidase (GUS) reporter gene for expression.

[0052] 4. Creation of transgenic plants like Figure 4 Middle A, Figure 4 Middle B, Figure 4 Middle C, Figure 4 Medium D and Figure 4 As shown in E, Pro PagAPL ::GUS transgenic plants. The vector was modified using artificial enhanced green fluorescent protein (eyGFPuv) to make it visible, making it easier to find transgenic plants to measure GUS enzyme activity and further explore the activity characteristics of specific promoters.

[0053] 5. Identification of transgenic plants like Figure 5 Middle A, Figure 5 Middle B, Figure 5 Middle C and Figure 5 As shown in D, the vector used carries the eyGFPuv tag, and the transgenic plants can emit green fluorescence under the irradiation of a handheld fluorescent lamp, which can be quickly identified as transgenic plants. Four transgenic strains of the APL gene were obtained, namely strain 1, strain 2, strain 3 and strain 4.

[0054] To further explore the activity characteristics of specific promoters, the GUS expression level in transgenic plants was detected (e.g. Figure 6 ), the results showed that compared with the wild type, the GUS expression levels in the transgenic plants were increased, and three lines with higher GUS expression levels in the gene, namely #1, #2, and #3 in APL, were selected for subsequent experiments.

[0055] 6. GUS Histochemical Staining GUS histochemical staining was performed on transgenic poplar plants to explore its expression location. GUS staining showed that GUS signals were present in vascular tissues (e.g. Figure 7 Middle A, Figure 7 Medium B and Figure 7 (as shown in C). PagAPL :: In the GUS slices, the GUS signal is mainly in the phloem and cambium. This indicates that the promoter ProAPL of the present invention exhibits a vascular tissue-specific expression pattern in poplar and can be used in genetic engineering to start the expression of corresponding exogenous target genes.

[0056] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A plant vascular tissue-specific expression promoter ProAPL, characterized in that: The nucleotide sequence of the vascular tissue-specific expression promoter ProAPL is shown in SEQ ID NO.

1.

2. The vascular tissue-specific expression promoter ProAPL according to claim 1, characterized in that: The vascular tissue-specific expression promoter ProAPL is specifically expressed in poplar vascular tissue.

3. The vascular tissue-specific expression promoter ProAPL according to claim 1, characterized in that: The vascular tissue-specific expression promoter ProAPL is specifically expressed in the poplar cambium.

4. Use of the plant vascular tissue specific expression promoter ProAPL as claimed in claim 1 in regulating the specific expression of a target gene in poplar vascular tissue.

5. The use of the plant vascular tissue specific expression promoter ProAPL in plant genetic improvement as claimed in claim 1, characterized in that: The plant is poplar.

6. An expression cassette containing the plant vascular tissue-specific expression promoter ProAPL according to claim 1.

7. A recombinant expression vector containing the plant vascular tissue-specific expression promoter ProAPL according to claim 1.

8. An engineered bacterium containing the plant vascular tissue-specific expression promoter ProAPL according to claim 1.

9. A method for cultivating transgenic plants, characterized in that: The method comprises the steps of transferring the vascular tissue-specific expression promoter ProAPL according to claim 1, the expression cassette according to claim 6, the recombinant expression vector according to claim 7 or the engineered bacteria according to claim 8 into a plant; the plant is poplar.

10. Use of the expression cassette according to claim 6, the recombinant expression vector according to claim 7 or the engineered bacteria according to claim 8 in regulating the specific expression of a target gene in poplar vascular tissue.

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