Plant vascular tissue specific expression promoter ProSMXL5 and application thereof

Abstract

The invention discloses a plant vascular tissue specific expression promoter ProSMXL5 and application thereof, belongs to the technical field of plant genetic engineering, and provides a plant vascular tissue specific expression promoter ProSMXL5, and the nucleotide sequence of the plant vascular tissue specific expression promoter ProSMXL5 is as shown in SEQ ID NO.1. The invention also discloses a plant vascular tissue specific expression promoter ProSMXL5 and application thereof, belongs to the technical field of plant genetic engineering, and provides a plant vascular tissue specific expression promoter ProSMXL5. The poplar vascular tissue specific expression promoter ProSMXL5 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 ProSMXL5 and an application thereof. Background Art

[0002] In order to make exogenous genes play an efficient role in plants while reducing adverse effects on plants, people are paying more and more attention to the research and application of specific promoters. Under the regulation of tissue-specific promoters, namely organ-specific promoters, gene expression only occurs in certain specific organs or tissues, showing developmental regulation. Specific promoters can overcome the waste caused by constitutive promoters to initiate non-specific, continuous and efficient expression of exogenous genes in recipient plants, and can also overcome the phenomenon of gene silencing or co-suppression caused by repeated use of the same promoter to drive two or more exogenous genes. In addition, plant genetic engineering research also requires promoters with tissue-specific high expression, so that exogenous target genes can be efficiently expressed in specific tissues. Phloem is a complex tissue in plants that has the functions of transporting assimilated products, storing and supporting, and plays an important role in the growth and development of plants. Phloem-specific promoters are of great significance for a deep understanding of the growth and development regulation mechanism of forest trees. By studying specific promoters related to phloem, we can further reveal the regulatory rules of phloem-related gene expression in the growth, development and response to environmental changes of forest trees, and provide a solid theoretical basis for genetic improvement and directional breeding of forest trees. The growth cycle of forest trees is long and the growth regulation mechanism is complex. In order to effectively play the role of phloem development-related genes and achieve the goal of creating high-quality and high-yield wood, it is urgent to develop phloem-specific promoters for forest trees. Summary of the invention

[0003] In order to solve the above technical problems, the present invention proposes a plant vascular tissue-specific expression promoter ProSMXL5 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 ProSMXL5, the nucleotide sequence of the vascular tissue-specific expression promoter ProSMXL5 is shown in SEQ ID NO.1.

[0005] Preferably, the vascular tissue-specific expression promoter ProSMXL5 is specifically expressed in poplar vascular tissue.

[0006] Preferably, the vascular tissue-specific expression promoter ProSMXL5 is specifically expressed in poplar phloem.

[0007] The present invention also provides the application of the plant vascular tissue specific expression promoter ProSMXL5 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 ProSMXL5 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 ProSMXL5.

[0010] The invention also provides a recombinant expression vector containing the plant vascular tissue specific expression promoter ProSMXL5.

[0011] The present invention also provides an engineering bacterium containing the plant vascular tissue-specific expression promoter ProSMXL5.

[0012] The present invention also provides a method for cultivating transgenic plants, comprising the step of transferring the vascular tissue-specific expression promoter ProSMXL5, 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 ProSMXL5 of the present invention is derived from the silver gland poplar '84K' genomic DNA. The poplar vascular tissue-specific expression promoter ProSMXL5 fragment size is 1996bp and has the following characteristics: (1) it is located at the 5' end and upstream of the Pop_A18G035979 gene; (2) the base length is 1996bp; (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 ProSMXL5, which enables downstream genes to be specifically expressed in plant vascular tissues under the regulation of the tissue-specific expression promoter ProSMXL5 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] By utilizing the structural characteristics of the phloem to fuse functional genes with its tissue-specific promoter ProSMXL5, a large number of special application systems have been developed, which is conducive to the development of new genetic tools, the increase of wood biomass accumulation and the research on enhancing forest stress tolerance. For example, in the genetic engineering of pest resistance, placing genes with insect resistance or antibacterial activity under the regulation of phloem-specific promoters can make these genes highly expressed in the phloem, thereby making the forest phloem resistant to pests and diseases without causing unnecessary effects on the normal physiological functions of other tissues. Through the study of poplar phloem-specific promoter ProSMXL5, the regulatory mechanism of wood quality formation is revealed, which is helpful to achieve the directional improvement of wood quality and cultivate new high-yield and high-resistant forest varieties. 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 ProSMXL5, where A is the UMAP analysis result, B is the expression position of the SMXL5 homologous gene pal-pou28037, C is the SMXL5 AspWood data analysis result, and D is the SMXL5 qPCR result; Figure 2 The results of in situ expression detection of PagSMXL5, where A is the negative control, B is the expression position of PagSMXL5 in the stem, the red mark in the figure represents the hybridization signal of PagSMXL5, and the scale bar is 200 μm; Figure 3 Schematic diagram of constructing a vector containing the promoter ProSMXL5; Figure 4 For Pro PagSMXL5 ::GUS transgenic plants, where A is the dedifferentiated callus of leaves infected by the leaf disc method, B is the callus induced buds, and C is the transgenic regenerated buds under UV light irradiation; Figure 5 are transgenic strains of SMXL5, wherein A is strain 3, B is strain 4, C is strain 7, D is strain 8, E is strain 15, F is strain 17, G is strain 19, and H is strain 21; Figure 6 The results of GUS expression determination in SMXL5 transgenic plants, the red arrows in the figure represent high-expression lines; Figure 7 These are the results of GUS staining in SMXL5 transgenic plants, where A is strain 3, B is strain 8, and C is strain 21. 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 co-expression network analysis of the wood formation process 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 SMXL5 (84K poplar genome Pop_A18G035979, 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 SMXL5 gene that is highly expressed in the phloem.

[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 SMXL5 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 SMXL5 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 using the 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). Methods Analysis of gene expression levels.

[0035] (2) Real-time fluorescence quantitative PCR was used to analyze the expression of SMXL5 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 1996 bp SMXL5 promoter (ProSMXL5, 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 PagSMXL5 ::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 SMXL5 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 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 SMXL5 homologous gene pal-pou28037 (poplar single-cell database scRNAPal) is mainly expressed in phloem parenchyma cells and phloem cells (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 was expressed in 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 PagSMXL5 was highly expressed in phloem (Fig. Figure 1 Middle D).

[0050] 2. In situ PCR The in situ expression of PagSMXL5 was investigated by in situ PCR. Figure 2 Middle A and Figure 2 The results shown in B show that the PagSMXL5 gene is mainly expressed in the phloem.

[0051] 3. Vector construction To further determine the detailed expression patterns, e.g. Figure 3 As shown, the promoter region of PagSMXL5, ProSMXL5, was cloned, and homologous recombination was performed using two restriction sites, EcoR I and XBa I. The ProSMXL5 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 Medium B and Figure 4As shown in C, Pro PagSMXL5 ::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, Figure 5 Middle D, Figure 5 Middle E, Figure 5 Medium F, Figure 5 Medium G and Figure 5 As shown in H, 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. Eight transgenic strains of the SMXL5 gene were obtained, namely strain 3, strain 4, strain 7, strain 8, strain 15, strain 17, strain 19 and strain 21.

[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 #3, #8, and #21 in SMXL5, 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). PagSMXL5 :: In the GUS slices, the GUS signal is mainly in the developing phloem. This indicates that the promoter ProSMXL5 of the present invention exhibits a vascular tissue-specific expression pattern in poplar and can be used in genetic engineering to initiate 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 ProSMXL5, characterized in that: The nucleotide sequence of the vascular tissue-specific expression promoter ProSMXL5 is shown in SEQ ID NO.

1.

2. The vascular tissue-specific expression promoter ProSMXL5 according to claim 1, characterized in that: The vascular tissue-specific expression promoter ProSMXL5 is specifically expressed in poplar vascular tissue.

3. The vascular tissue-specific expression promoter ProSMXL5 according to claim 1, characterized in that: The vascular tissue-specific expression promoter ProSMXL5 is specifically expressed in poplar phloem.

4. Use of the plant vascular tissue specific expression promoter ProSMXL5 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 ProSMXL5 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 ProSMXL5 according to claim 1.

7. A recombinant expression vector containing the plant vascular tissue-specific expression promoter ProSMXL5 according to claim 1.

8. An engineered bacterium containing the plant vascular tissue-specific expression promoter ProSMXL5 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 ProSMXL5 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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