Plant vascular tissue-specific expression promoter ProSMXL5 and its application

Abstract

The present invention discloses a plant vascular tissue-specific expression promoter ProSMXL5 and its application, belonging to the technical field of plant genetic engineering. A plant vascular tissue-specific expression promoter ProSMXL5 is provided, and the nucleotide sequence of the vascular tissue-specific expression promoter ProSMXL5 is shown as SEQ ID NO.1. The present invention provides the poplar vascular tissue-specific expression promoter ProSMXL5, which can effectively and specifically express in the poplar vascular tissue, can specifically play the function of the exogenous gene expression product in a specific tissue, and regulate the continuous expression of the exogenous gene in the plant vascular tissue.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to a plant vascular tissue-specific expression promoter ProSMXL5 and its application. Background Art

[0002] In order to enable foreign genes to play an efficient role in plants while reducing adverse effects on plants, people currently pay more and more attention to the research and application of specific promoters. Under the regulation of tissue-specific promoters, that is, organ-specific promoters, the expression of genes only occurs in certain specific organs or tissues, showing developmental regulation. Specific promoters can overcome the waste caused by the non-specific continuous and efficient expression of foreign genes driven by constitutive promoters in recipient plants, and can also overcome the phenomenon of gene silencing or co-suppression that may be caused by repeatedly using the same promoter to drive two or more foreign genes. In addition, in plant genetic engineering research, promoters with high tissue-specific expression are also needed to enable foreign target genes to be highly expressed in specific tissues. The phloem is a complex tissue in plants that has functions of transporting assimilates, storing, and supporting, and plays an important role in the growth and development of plants. Phloem-specific promoters are of great significance for deeply understanding the growth and development regulation mechanism of forest trees. By studying phloem-related specific promoters, the regulation rules of phloem-related gene expression during the growth, development, and response to environmental changes of forest trees can be further revealed, providing a solid theoretical basis for the genetic improvement and directional cultivation of forest trees. Forest trees have a long growth cycle and a complex growth regulation mechanism. 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] To solve the above technical problems, the present invention provides a plant vascular tissue-specific expression promoter ProSMXL5 and its application. The tissue-specific expression promoter provided by the present invention can be effectively and specifically expressed in the vascular tissue of poplar, can specifically play the function of the expression product of the foreign gene in a specific tissue, and regulate the continuous expression of the foreign gene in the plant vascular tissue.

[0004] To achieve the above object, the present invention provides a plant vascular tissue-specific expression promoter ProSMXL5, and the nucleotide sequence of the vascular tissue-specific expression promoter ProSMXL5 is as shown in SEQ ID NO.1.

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

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

[0007] The present invention also provides the use of the plant vascular tissue-specific expression promoter ProSMXL5 in regulating the specific expression of a target gene in the vascular tissue of poplar trees.

[0008] The present invention also provides the use of the plant vascular tissue-specific expression promoter ProSMXL5 in plant genetic improvement, wherein the plant is a poplar tree.

[0009] The present invention also provides an expression cassette containing the plant vascular tissue-specific expression promoter ProSMXL5.

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

[0011] The present invention also provides an engineered 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 engineered bacterium into a plant; the plant is a poplar tree.

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

[0014] Compared with the prior art, the present invention has the following advantages and technical effects:

[0015] The Poplar Vascular Tissue-Specific Expression Promoter ProSMXL5 of the present invention is derived from the genomic DNA of Populus alba × Populus glandulosa '84K'. The size of the Poplar Vascular Tissue-Specific Expression Promoter ProSMXL5 fragment is 1996bp, and it 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 sites for initiating transcription and the transcription start point; (4) It is specifically expressed in the poplar vascular tissue, regulating the continuous expression of foreign genes in specific plant tissues and organs, avoiding energy waste and increasing the effect of transgenes, reducing problems such as plant dwarfing and gene silencing caused by constitutive expression of genes. It can not only serve for transgenic poplar breeding, but also reserve resources for long-term promoter modification and design. The present invention also discloses a recombinant expression vector containing the tissue-specific expression promoter ProSMXL5, which enables the downstream gene to be specifically expressed in the plant vascular tissue under the regulation of the tissue-specific expression promoter ProSMXL5 of the recombinant expression vector, providing a tool for the research on the efficient, stable and specific expression of genes in the plant vascular tissue in the field of plant genetic engineering, and having broad application prospects.

[0016] Utilize the structural characteristics of the phloem to fuse its tissue-specific promoter ProSMXL5 with functional genes, develop a large number of special application systems, which is conducive to the research on developing new genetic tools, increasing the accumulation of wood biomass and enhancing the stress tolerance of forest trees. For example, in the genetic engineering of pest and disease resistance, placing genes with insecticidal or antibacterial activity under the regulation of the phloem-specific promoter can enable these genes to be highly expressed in the phloem, so that the phloem of forest trees can resist pests and diseases without causing unnecessary impacts on the normal physiological functions of other tissues. Through the research on the poplar phloem-specific promoter ProSMXL5, revealing the regulatory mechanism of wood quality formation helps to achieve the directional improvement of wood quality and cultivate new high-yield and highly resistant forest tree varieties. Brief Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following-described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0018] 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 AspWood data analysis result of SMXL5, and D is the SMXL5 qPCR result;

[0019] Figure 2 In-situ expression detection results of PagSMXL5. Among them, A is the negative control, B is the expression position of PagSMXL5 in the stem. The red marks in the figure represent the hybridization signals of PagSMXL5, and the scale bar is 200 μm;

[0020] Figure 3 Schematic diagram of the vector construction containing the promoter ProSMXL5;

[0021] Figure 4 For Pro PagSMXL5 ::GUS transgenic plants. Among them, A is the callus formed by the dedifferentiation of the leaves infected by the leaf disc method, B is the callus inducing buds, and C is the transgenic regenerated buds under the irradiation of the uv lamp;

[0022] Figure 5 Transgenic lines of SMXL5. Among them, A is line 3, B is line 4, C is line 7, D is line 8, E is line 15, F is line 17, G is line 19, and H is line 21;

[0023] Figure 6 Results of the determination of GUS expression levels in transgenic plants of SMXL5. The red arrows in the figure represent the high-expression lines;

[0024] Figure 7 GUS staining results of transgenic plants of SMXL5. Among them, A is line 3, B is line 8, and C is line 21, and the scale bar is 100 μm. Detailed implementation manners

[0025] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to those documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0028] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the description of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of this invention are obvious to those skilled in the art. The description and examples of this invention are merely exemplary.

[0029] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0030] Example 1

[0031] Taking Populus alba×P. glandulosa‘84K’ as the research material, using single-cell RNA-Seq data and co-expression network analysis during wood formation, to identify Populus 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.

[0032] I. Research on specific genes in the Populus gene map

[0033] Screen Populus vascular tissue-specific genes according to the Populus gene map: The candidate gene is SMXL5 (Pop_A18G035979 in the 84K Populus genome, doi: 10.1093 / dnares / dsz020).

[0034] II. Research on promoter activity of Populus transgenic plants

[0035] (1) Identify the homologous genes of the candidate gene and analyze its tissue expression pattern using qPCR.

[0036] (2) Observe the in-situ expression pattern of the candidate gene by in-situ PCR technology.

[0037] (3) Clone the promoter region (~2000bp) of the SMXL5 gene highly expressed in the phloem.

[0038] (4) The promoter sequence obtained in (3) was constructed into the pCambia2300 - Promoter - 35S mini - GUS - 35S - eyGFPuv vector and transformed into Agrobacterium tumefaciens.

[0039] (5) The vector was used to transform '84K' poplar by the leaf disc method, and transgenic plants were obtained for subsequent analysis.

[0040] III. Study on the expression sites of tissue - specific promoters

[0041] (1) Transgenic plants were preliminarily identified by observing eyGFPuv, and the expression level of the GUS gene was detected. Three lines with higher GUS expression multiples were selected for subsequent studies.

[0042] (2) GUS histochemical staining was performed on the selected transgenic poplar plants to explore the location of the expressed gene.

[0043] (3) Verify the function of the promoter SMXL5 in specifically driving gene expression.

[0044] IV. Co - expression network analysis of vascular tissue - specific genes

[0045] Data from poplar gene mapping studies were used to detect the expression of SMXL5 in different tissues. The data for the heatmap were log2 - transformed.

[0046] V. Analysis of the expression level of the target gene by fluorescence quantitative PCR

[0047] (1) Different tissues were harvested from '84K' plants grown in soil for two months: 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 carried out on a LightCycler® 480 thermal cycler (Roche, Germany) using the Power 2×SYBR Real - time PCR Premix (TransGen Biotech, Beijing, China). UBQ was used as an internal reference (to quantify the relative transcriptional level of this gene in each sample). The method was used to analyze the gene expression level.

[0048] (2) The expression of the SMXL5 gene was analyzed by real - time fluorescence quantitative PCR.

[0049] VI. In - situ PCR localization of gene expression

[0050] (1) Freshly harvested plant tissues (the 7th internode of the stem of the '84K' tissue-cultured plant) were immediately fixed in formaldehyde solution, and the penetration of the fixative was enhanced by vacuum infiltration. Then, the samples were embedded in agarose, trimmed, and sectioned on a vibratome. 50-μm-thick sections were collected into microtubes. DNase (Takara) treatment, reverse transcription (Takara), and in situ PCR were performed in centrifuge tubes using a standard thermal cycler.

[0051] (2) Genomic DNA was removed by DNase and converted into cDNA by reverse transcription. The cDNA was amplified by standard gene-specific PCR incorporating digoxigenin (DIG)-labeled nucleotides. The sections were incubated with an anti-DIG antibody conjugated to alkaline phosphatase.

[0052] (3) The addition of a specific substrate for alkaline phosphatase allowed for colorimetric detection of the DIG-labeled PCR products and their localization as RNA synthesis sites. Then, the sections were transferred to microscope slides and observed using bright-field microscopy (Leica DM6B).

[0053] VII. Vector construction and creation of transgenic poplars

[0054] (1) A 1996-bp SMXL5 promoter (ProSMXL5, nucleotide sequence shown in SEQ ID NO.1) was cloned from the genomic DNA of Populus alba × Populus glandulosa '84K'.

[0055]

[0056] (2) The Pro PagSMXL5 ::GUS construct was generated using the modified vector pCambia2300 - Promoter - 35S mini - GUS - 35S - eyGFPuv and verified by Sanger sequencing.

[0057] (3) Subsequently, the vector was transformed into Agrobacterium tumefaciens GV3101 using Agrobacterium - mediated transformation. The Agrobacterium cells were collected and resuspended to an OD 600 value between 0.3 and 0.4. The leaf discs of Populus alba×P. glandulosa tissue - cultured were soaked in the resuspended cells for 20 min, and then the leaf discs were transferred to the medium for inducing callus formation and co - cultured in the dark at room temperature for 2 d. At 25 °C, they were cultured on 40 mg·L -1 Kan medium for 30 d to induce the formation of stems, leaves and roots.

[0058] VIII. Identification of transgenic plants

[0059] (1) Observation of eyGFPuv

[0060] Since the modified vector carries the eyGFPuv tag, after irradiating the transgenic poplar seedlings with a uv lamp, the target plants will emit green fluorescence, and thus the target transgenic plants can be screened.

[0061] (2) Extract the DNA of transgenic plants and detect the expression level of the GUS gene.

[0062] Extract DNA from the SMXL5 transgenic plants screened in (1) to detect the GUS expression level, and select 3 lines with higher expression levels for subsequent experiments.

[0063] IX. Histochemical GUS staining and microscopic observation

[0064] (1) Incubate the transgenic plants in 90% acetone - ice for 2 h, wash them 3 times with washing buffer, then place them in 1 mM X - Gluc washing buffer and incubate in the dark at 37 °C for 8 h, and wash the plants with 75% ethanol.

[0065] (2) Section with a Leica VT1200S, with a section thickness of 50 μm, and observe under a Leica DM6B microscope. The washing buffer contains: 0.2 mol·L -1 NaH 2 PO 4 、0.2 mol·L -1 Na 2 HPO 4 、2 mmol·L -1 K 3 [Fe(CN) 6, 2 mmol·L -1 K 4 [Fe(CN) 6 .

[0066] X. Quantitative Analysis of GUS Activity

[0067] The GUS activity of transgenic plants was detected using a GUS quantitative detection kit (SL7161). GUS reacts with 4-MUG to produce the fluorescent substance 4-MU, which has the highest absorption peak at the excitation wavelengths of 365 nm and 455 nm. Well-ground samples were mixed with 1 mL of extraction buffer, vortexed vigorously for 30 s, and centrifuged at 12,000 rpm for 10 min. Then, 150 μL of the supernatant was added to 150 μL of the 4-MUG substrate solution to form a reaction solution. The mixture was heated at 37 °C, and 100 μL of the reaction solution was taken out every 10 min and added to 900 μL of the termination solution. The GUS activity value in the final total protein was recorded as 4-MU pmol min -1 ·mg -1 .

[0068] XI. Results

[0069] 1. Identification of Poplar Vascular Tissue-Specific Genes

[0070] Using UMAP analysis (as shown in Figure 1 A), it was found that the SMXL5 homologous gene pal-pou28037 (scRNAPal of the poplar single-cell database) was mainly expressed in phloem parenchyma cells and phloem cells (as shown in Figure 1 B), indicating that the gene might be driven by a specific promoter. To further confirm the expression of the gene during wood development, AspWood data analysis showed that this gene was expressed in the phloem (as shown in Figure 1 C), and the expression pattern was the same as that of UMAP analysis. qPCR showed that the expression in the stem was higher than that in the leaves and roots, and PagSMXL5 was highly expressed in the phloem (as shown in Figure 1 D).

[0071] 2. In Situ PCR

[0072] The in situ expression of PagSMXL5 was investigated by in situ PCR technology, and as shown in the results of Figure 2 A and Figure 2 B, the PagSMXL5 gene was mainly expressed in the phloem.

[0073] 3. Vector Construction

[0074] To further determine the detailed expression pattern, as shown in Figure 3As shown, the promoter region ProSMXL5 of PagSMXL5 was cloned, and homologous recombination was carried out using two restriction enzyme 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.

[0075] 4. Creation of transgenic plants

[0076] As Figure 4 shown in A of Figure 4 and B of Figure 4 and C of PagSMXL5 as shown, transgenic Pro PagSMXL5 ::GUS plants were obtained by transforming Populus alba×Populus glandulosa '84K' leaves using Agrobacterium-mediated transformation. The vector was modified using enhanced yellow fluorescent protein (eyGFPuv) to make it visible, which made it easier to find transgenic plants for measuring GUS enzyme activity and further explore the activity characteristics of specific promoters.

[0077] 5. Identification of transgenic plants

[0078] As Figure 5 shown in A of Figure 5 and B of Figure 5 and C of Figure 5 and D of Figure 5 and E of Figure 5 and F of Figure 5 and G of Figure 5 and H of Figure 5 as shown, the vector used carried the eyGFPuv tag, and transgenic plants could emit green fluorescence under the irradiation of a hand-held fluorescent lamp, enabling rapid identification of transgenic plants. Eight transgenic lines of the SMXL5 gene were obtained, namely line 3, line 4, line 7, line 8, line 15, line 17, line 19, and line 21.

[0079] To further explore the activity characteristics of specific promoters, the GUS expression levels in transgenic plants were detected (as Figure 6 shown), and the results showed that the GUS expression levels in transgenic plants were all increased compared with those of the wild type. Three lines with relatively high GUS expression levels in the gene, namely #3, #8, and #21 in SMXL5, were selected for subsequent experiments.

[0080] 6. GUS histochemical staining

[0081] GUS histochemical staining was performed on transgenic poplar plants to explore the expression location. GUS staining showed that GUS signals were present in the vascular tissue (as Figure 7 shown in A of Figure 7 and B of Figure 7 and C of PagSMXL5In the GUS sections, the GUS signal is mainly in the developing phloem. It shows that the promoter ProSMXL5 of the present invention exhibits a vascular tissue-specific expression pattern in poplar and can be applied to initiate the expression of corresponding foreign target genes in genetic engineering.

[0082] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A poplar phloem-specific expression promoter ProSMXL5, characterized in that: The nucleotide sequence of the poplar phloem-specific expression promoter ProSMXL5 is shown in SEQ ID NO.

1.

2. Use of the poplar phloem-specific expression promoter ProSMXL5 as described in claim 1 in regulating the specific expression of a target gene in the poplar phloem.

3. The use of the poplar phloem-specific expression promoter ProSMXL5 in plant genetic improvement as claimed in claim 1, characterized in that: The plant is poplar.

4. An expression cassette containing the poplar phloem-specific expression promoter ProSMXL5 according to claim 1.

5. A recombinant expression vector containing the poplar phloem-specific expression promoter ProSMXL5 according to claim 1.

6. An engineered bacterium containing the poplar phloem-specific expression promoter ProSMXL5 according to claim 1.

7. A method for cultivating transgenic plants, characterized in that: The method comprises the steps of transferring the poplar phloem-specific expression promoter ProSMXL5 described in claim 1, the expression cassette described in claim 4, the recombinant expression vector described in claim 5 or the engineered bacteria described in claim 6 into a plant; the plant is poplar.

8. Use of the expression cassette according to claim 4, the recombinant expression vector according to claim 5 or the engineered bacteria according to claim 6 in regulating the specific expression of a target gene in poplar phloem.

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