Promoter responsive to pathogenic stress and use thereof

Abstract

The application discloses a pathogen stress-responsive promoter and application thereof, wherein the promoter is derived from an upstream regulatory sequence of a Litchi chinensis Sonn. LcMYB1 gene, and the nucleotide sequence is shown as SEQ ID NO. 5. The LcMYB1 promoter can be specifically induced and activated by Peronophythora litchii stress; when not infected by pathogenic bacteria, the background transcriptional activity of a downstream gene driven by the LcMYB1 promoter is very low, and the expression is mainly located in leaf veins; after infection by Peronophythora litchii, the LcMYB1 promoter can quickly start high-efficiency expression of the downstream gene, and the expression position is diffused from the leaf veins to surrounding tissues. The application further provides a recombinant expression vector, a recombinant bacterium and a transgenic plant containing the promoter, and application of the promoter in regulation of expression of a reporter gene, screening of disease-resistant materials and cultivation of disease-resistant plants. The provided promoter has pathogen induction specificity and tissue expression specificity, provides a novel inducible regulatory element for plant disease-resistant molecular breeding, and has important application value.

Description

Technical Field

[0001] This application belongs to the field of plant genetic engineering technology, specifically relating to a promoter that responds to pathogen stress and its application. Background Technology

[0002] Lychee is a highly distinctive subtropical economic fruit tree in southern my country, with a long history of cultivation and a wide planting area. It possesses extremely high economic and market value and is a crucial pillar crop for rural revitalization and the development of specialty forestry and fruit industries in South China. During large-scale lychee cultivation, disease stress severely restricts the stable development of the lychee industry. Among these, downy mildew is the most frequent and damaging fungal disease. This disease can infect lychee leaves, flower spikes, and fruits, and is highly prone to outbreaks in rainy and humid environments, causing flower and fruit drop, fruit rot, and continued spread during post-harvest storage and transportation. This not only significantly reduces lychee yield but also severely degrades the appearance and quality of the fruit, resulting in substantial economic losses to the lychee industry.

[0003] Traditional production relies primarily on chemical fungicides to control downy mildew. However, long-term, excessive application of pesticides easily leads to pesticide residues, environmental pollution, and increased pathogen resistance, which is inconsistent with the requirements of green agriculture and high-quality fruit production. In plant genetic engineering breeding, promoters, as core cis-acting elements for gene expression regulation, can precisely regulate the spatiotemporal and intensity of target gene expression. Among them, pathogen-inducible promoters can specifically initiate the expression of downstream disease-resistant genes under pathogen infection stress, while expressing them at low levels or not at all under normal growth conditions. This enhances plant disease resistance while avoiding the growth depletion and developmental defects caused by constitutive overexpression, making them ideal regulatory elements for creating disease-resistant germplasm.

[0004] Currently, most existing inducible promoters are derived from model plants, exhibiting weak expression activity and insensitive induction response in the litchi genetic context. Furthermore, there is a lack of visual inducible promoter resources that can clearly trace the pathogen infection process and are suitable for in vivo litchi detection. Existing promoters generally suffer from poor tissue specificity, low induction folds, and high background expression, severely limiting the progress of molecular breeding research for litchi disease resistance. Summary of the Invention

[0005] The technical problem this application aims to solve is to overcome the deficiencies in existing technologies, such as the lack of inducible promoters with clear tissue expression patterns and efficient responses to downy mildew stress in litchi, and to provide a promoter that responds to pathogen stress. The promoter provided in this application is expressed at a low level only in leaf veins when not infected by the pathogen, but its expression is significantly enhanced after induction by downy mildew and spreads to surrounding tissues. It can precisely drive the expression of downstream genes, solving the problem of insufficient regulatory elements in molecular breeding for litchi disease resistance.

[0006] This application adopts the following technical solution to solve the above-mentioned technical problems: This application provides an LcMYB1 promoter that responds to pathogen stress, characterized in that the nucleotide sequence of the LcMYB1 promoter is shown in SEQ ID NO.5.

[0007] In some embodiments, when the LcMYB1 promoter responding to pathogen stress is not infected by pathogens, the LcMYB1 transcriptional activity is extremely low, and under normal conditions, the basal transcriptional level of the downstream GUS gene it drives is less than one-sixth of the peak transcriptional level induced by pathogen infection.

[0008] In some embodiments, the expression sites of the LcMYB1 promoter include: leaf veins and parts of the root; In some embodiments, the LcMYB1 promoter is significantly induced and activated after infection with Phytophthora litchii, driving efficient expression of downstream genes, and the expression region spreads from leaf veins to surrounding tissues.

[0009] This application also provides a method for cloning the LcMYB1 promoter in response to pathogen stress. Genomic DNA is extracted from *Litsea cubeba* leaves and used as a template. PCR amplification is performed using primers SEQ ID NO.1 and SEQ ID NO.2 under the following conditions: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 25 s, 60℃ annealing for 25 s, 72℃ extension for 50 s, for a total of 35 cycles; final extension at 72℃ for 2 min, and storage at 4℃. The LcMYB1 promoter is then obtained.

[0010] This application also provides a recombinant expression vector comprising the LcMYB1 promoter prepared above; The initial vector for the recombinant expression vector was pGreen-GUS.

[0011] In some embodiments, the method for constructing the recombinant expression vector includes: using GUS as a reporter gene, ligating the LcMYB1 promoter to the GUS reporter gene; specifically, the LcMYB1 promoter fragment is tandemly fused with the GUS reporter gene; the vector containing the GUS reporter gene is digested with the restriction endonuclease BcuI to obtain a linearized vector; after purification, the LcMYB1 promoter and the linearized vector are seamlessly ligated via homologous recombination using the Accurate Biotechnology OK Clon DNA Ligation Kit II, achieving effective fusion of the LcMYB1 promoter and the downstream GUS reporter gene. The recombinant expression vector is pLcMYB1:GUS; the primers used to construct the recombinant expression vector are shown in SEQ ID NO. 3 and SEQ ID NO. 4.

[0012] This application also provides a transgenic cell line or recombinant bacteria comprising the LcMYB1 promoter prepared above or the recombinant expression vector prepared above. The recombinant bacteria include Escherichia coli for cloning and Agrobacterium for plant transformation.

[0013] This application also provides a transgenic plant comprising the recombinant expression vector prepared above; the transgenic plant is obtained by transforming the recombinant expression vector into Agrobacterium GV3101 and then transforming it by Agrobacterium-mediated transformation, including Arabidopsis thaliana, tobacco or litchi.

[0014] This application also provides an application of the LcMYB1 promoter prepared above, the application including: the application in regulating the specific expression of GUS reporter gene in response to litchi downy mildew stress; the application in visually tracing the pathogen infection process and the early disease resistance response of plants; and the application in breeding litchi downy mildew resistant plant varieties.

[0015] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this application.

[0016] All reagents and raw materials used in this application are commercially available.

[0017] The positive and progressive effects of this application are as follows: The promoter provided by this application has typical pathogen-inducible characteristics. Its background expression level is low when it is not infected by Phytophthora litica, and it can be rapidly activated after infection, realizing precise spatiotemporal regulation of downstream genes and avoiding the negative effects of continuous expression of constitutive promoters. The promoter has a clear tissue expression pattern. Under healthy conditions, it is mainly expressed in trace amounts in the leaf veins. After pathogen induction, the expression signal diffuses from the leaf veins to the surrounding tissues, which can intuitively reflect the pathogen infection path and the early disease resistance response sites of plants, providing convenience for the study of pathogen-plant interaction. This application constructs a recombinant expression vector based on the pGreen-GUS vector, using GUS as a reporter gene. Promoter activity can be rapidly and intuitively determined through GUS staining. The operation is simple and the results are reliable, making it suitable for promoter function verification and high-throughput screening. This promoter is derived from litchi itself and is adapted to the genetic background of litchi. It can be directly used for molecular breeding of litchi to resist downy mildew. It can also be applied to model plants such as Arabidopsis thaliana and tobacco, with a wide range of applications and important theoretical and applied value. Attached Figure Description

[0018] This application can be better understood by referring to the description given below in conjunction with the accompanying drawings. These drawings, together with the detailed description below, are included in and form part of this specification, and are used to further illustrate preferred embodiments of the application and explain the principles and advantages of the application. Wherein: Figure 1Clustering heatmap of differentially expressed genes in the transcriptome of disease-resistant litchi variety 'Shangfanzhi' (SFZ) and disease-susceptible litchi variety 'Guiwei' (GW) in response to downy mildew infection.

[0019] Figure 2 The image shows the agarose gel electrophoresis results of the PCR amplification products of the LcMYB1 promoter.

[0020] Figure 3 for pLcMYB1:GUS Image showing the Basta resistance screening results of transgenic Arabidopsis thaliana positive plants.

[0021] Figure 4 The image shows the PCR identification results of the LcMYB1:GUS transgenic Arabidopsis positive line.

[0022] Figure 5 This image shows the inoculation treatment of Phytophthora litica spores into the T3 generation pLcMYB1:GUS transgenic Arabidopsis thaliana line.

[0023] Figure 6 Image showing GUS histochemical staining analysis of pLcMYB1:GUS transgenic Arabidopsis thaliana before and after downy mildew infection.

[0024] Figure 7 This is a graph showing the quantitative analysis of GUS enzyme activity in pLcMYB1:GUS transgenic Arabidopsis thaliana before and after infection with Phytophthora lichei. Detailed Implementation

[0025] The present application is further illustrated below by way of embodiments, but these embodiments are not intended to limit the scope of the present application. Experimental methods not specifically described in the following embodiments are performed according to conventional methods and conditions, or as selected in accordance with the product instructions.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0027] All raw materials used in the following examples are commercially available.

[0028] Example 1. Obtaining the LcMYB1 gene Transcriptional analysis was performed on litchi samples from the disease-resistant variety 'Shangfanzhi' (SFZ) and the susceptible variety 'Guiwei' (GW) after 24 hours of treatment with litchi downy mildew (P) and a mock treatment, respectively. The LcMYB1 gene was identified. Figure 1 ). Figure 1In the heatmap, the horizontal axis represents different treatment groups, including the uninoculated control (GW_Mock) and pathogen-inoculated treatment (GW_P) of the susceptible material 'Guiwei', and the uninoculated control (SFZ_Mock) and pathogen-inoculated treatment (SFZ_P) of the resistant material 'Shangfanzhi'; the vertical axis represents differentially expressed genes, and the left side shows a hierarchical clustering tree constructed based on expression profile similarity. The colors in the heatmap, from blue to red, represent the relative levels of gene expression after Z-score normalization. The LcMYB1 gene, which is marked, showed significant induced expression after inoculation of the resistant material 'Shangfanzhi' with Phytophthora downyensis (SFZ_P). Example 2. Cloning of the LcMYB1 promoter 1. Experimental materials: Healthy leaves of the 'Feizixiao' lychee were selected, flash-frozen in liquid nitrogen, and stored at -80℃ for later use.

[0029] 2. Genomic DNA Extraction: Genomic DNA was extracted from the leaves of *Litsea cubeba* using the high-efficiency plant genomic DNA extraction kit (DP350) from Tiangen Biotech (Beijing) Co., Ltd. DNA purity and integrity were assessed by agarose gel electrophoresis, and DNA concentration was detected by Nanodrop to ensure that the DNA quality met the requirements for PCR amplification.

[0030] 3. Primer design and synthesis: Specific primers were designed based on the upstream sequence of the litchi LcMYB1 gene. The primer sequences are SEQ ID NO.1 and SEQ ID NO.2, respectively. The primers were synthesized by a conventional biotechnology company.

[0031] 4. PCR amplification: Using extracted litchi genomic DNA as a template, PCR amplification was performed. The amplification system and program were set up according to the conventional PCR reaction. The target fragment of the LcMYB1 promoter was obtained. Figure 2 ).

[0032] 5. Sequence verification: After the PCR amplification product was recovered by agarose gel electrophoresis, it was ligated into the pMD19-T cloning vector, transformed into E. coli DH5α competent cells, and positive clones were selected for sequencing to obtain the nucleotide sequence of the LcMYB1 promoter (as shown in SEQ ID NO.5).

[0033] Figure 2 The DL2000 lane is used as the DNA molecular weight standard, and the Promoter lane is used as the LcMYB1 promoter PCR amplification product. The band size is consistent with the expected 2000bp. The band is single and clear, with no obvious non-specific amplification products, indicating that the LcMYB1 promoter fragment was successfully amplified and can be used for subsequent vector construction. Example 3. Construction of GUS reporter gene vector 1. Enzyme Digestion Reaction: The LcMYB1 promoter fragment, verified by sequencing, and the pGreen-GUS vector were selected and subjected to double enzyme digestion. HindIII and BamHI were used as restriction enzymes, both purchased from Thermo Fisher Scientific. The enzyme digestion system was prepared strictly according to the restriction enzyme reagent instructions. Both samples were placed in a 37°C water bath for enzyme digestion. After the reaction, the digestion products were purified using conventional nucleic acid recovery methods for later use.

[0034] 2. Ligation Reaction: The purified LcMYB1 promoter fragment was ligated to the pGreen-GUS vector digested fragment using the OK ClonClon DNA Ligation Kit from Aikerui Biotechnology Co., Ltd., to construct the recombinant expression vector pLcMYB1:GUS. The ligation reaction was strictly performed according to the kit instructions to ensure efficient ligation of the promoter and vector fragment.

[0035] 3. Transformation and Validation: The above ligation product was transformed into E. coli DH5α competent cells, and single colonies were selected as positive clone candidates. The recombinant plasmids of each candidate clone were extracted and verified by double enzyme digestion and DNA sequencing. Only after confirming that the LcMYB1 promoter fragment has been correctly inserted into the vector and that there are no mutations or deletions in the sequence can the recombinant vector be used for subsequent experiments.

[0036] Example 4. Construction of recombinant bacteria The recombinant expression vector pLcMYB1:GUS was transformed into Escherichia coli DH5α (for vector cloning and amplification) and Agrobacterium GV3101 (for plant genetic transformation), respectively. Positive clones were selected and identified by PCR to obtain recombinant Escherichia coli and recombinant Agrobacterium.

[0037] Example 5. Obtaining and Identifying Transgenic Plants 1. Agrobacterium-mediated transformation of Arabidopsis thaliana: Recombinant Agrobacterium GV3101 (containing pLcMYB1:GUS vector) was used to transform Arabidopsis thaliana Col-0 ecological strains using the flower immersion method, and T0 generation seeds were harvested.

[0038] 2. Positive selection: T0 generation seeds were sown on MS medium containing basta resistance selection agent to obtain positive transgenic Arabidopsis seedlings. PCR identification confirmed that the promoter fragment had been integrated into the Arabidopsis genome. Figure 3 and Figure 4 ).

[0039] 3. Obtaining homozygous lines: Through continuous screening up to the T3 generation, transgenic Arabidopsis homozygous lines were obtained for subsequent promoter function verification.

[0040] Figure 3 Arabidopsis seeds were screened for Basta herbicide and cultured on Basta-containing 1 / 2 MS medium for 7-10 days. The left-hand area is... pLcMYB1:GUS The transgenic lines are shown in red circles, indicating surviving and normally growing positive seedlings. The control (Col-0) on the right is a non-transgenic wild-type line, whose seedlings turned yellow and died due to Basta sensitivity. This demonstrates that the exogenous fragment has been successfully integrated into the Arabidopsis genome and can stably express the Basta resistance gene. Scale bar = 5cm; Figure 4 Using genomic DNA from different lines (#1~#8) as templates, PCR amplification was performed using specific primers; the marker was the DL2000 DNA molecular weight standard, with the arrow indicating the 1000bp position; all tested lines amplified clear bands of the expected size, with no non-specific amplification products, indicating that the exogenous pLcMYB1:GUS fragment had been successfully integrated into the Arabidopsis genome, and all eight lines were transgenic positive plants; Example 6. Functional Verification of the Promoter 1. Experimental grouping: T3 generation was selected. pLcMYB1:GUS Transgenic Arabidopsis homozygous lines and wild-type Col-0 seedlings were sown on 1 / 2 MS medium containing basta and cultured for 7-10 days. Two independent T3 generation transgenic lines and wild-type Col-0 control seedlings were inoculated with *Phytophthora lichee* spore suspension and cultured at 22℃ under 16h light / 8h dark conditions for 24h to provide stress treatment material for subsequent GUS histochemical staining and promoter activity analysis. Figure 5 ).

[0041] 2. GUS staining detection: Arabidopsis seedlings were collected 24 h after inoculation, and GUS staining solution was added. The seedlings were stained at 37°C in the dark for 8 h. After decolorization with 75% ethanol gradient, the distribution and intensity of GUS signals were observed.

[0042] 3. Results Analysis: In the control group (Mock, uninoculated with pathogen), only extremely weak GUS signals were detected in the leaf veins of Arabidopsis thaliana seedlings, indicating that the promoter's basal expression level was extremely low and mainly expressed in the leaf veins. In the treatment group (inoculated with Phytophthora litchifolia spores), the GUS signal of Arabidopsis thaliana seedlings was significantly enhanced, and the expression area gradually spread from the leaf veins to the surrounding tissues. With the extension of inoculation time, the signal intensity continued to increase, indicating that this promoter can be significantly induced and activated by Phytophthora litchifolia stress and has a specific spatiotemporal expression pattern. Figure 6 and Figure 7 ).

[0043] Figure 5On Basta 1 / 2 MS medium, two independent T3 generation transgenic lines (pLcMYB1:GUS#3, #4) and wild-type Col-0 control seedlings were inoculated with Phytophthora lichee spore suspension and cultured at 22℃ under 16h light / 8h dark conditions for 24h to provide stress treatment material for subsequent GUS histochemical staining and promoter activity analysis; scale bar = 5cm.

[0044] Figure 6 The paper demonstrates two independent T3 generation transgenic lines (#3 and #4) in the uninoculated control (Mock) and the litchi downy mildew inoculation treatment (…). P. litchii GUS staining results under [specific conditions]: The Mock group showed only a weak background blue signal in the leaf veins, while after inoculation with *Phytophthora inerme*, significantly enhanced GUS staining appeared in the leaves and some roots, with the blue signal spreading from the leaf veins to the surrounding tissues. This result indicates that the LcMYB1 promoter has extremely low background activity under normal conditions and is only specifically induced and activated under pathogen stress, driving efficient expression of downstream genes. This provides direct histochemical evidence for the pathogen stress response function of the promoter described in this patent.

[0045] Figure 7 Two independent T3 generation transgenic lines (#3 and #4) were inoculated with *Phytophthora litchii* as an uninoculated control (Mock) and as *Phytophthora litchii*. GUS enzyme activity was measured using a fluorescence method, and the fold induction was calculated based on the Mock group. The results showed that the baseline level of GUS activity in both lines was extremely low under Mock conditions, while after inoculation with the pathogen, GUS activity was increased by approximately 5-6 times, with significant differences. This quantitative result is consistent with the histochemical staining results, directly demonstrating that the LcMYB1 promoter has pathogen-stress-specific inducing activity, providing quantitative evidence for the function of the promoter described in this patent.

[0046] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof in this application are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0047] Although this application has been disclosed above through the description of specific embodiments, it should be understood that those skilled in the art can devise various modifications, improvements, or equivalents to this application within the spirit and scope of the appended solutions. Such modifications, improvements, or equivalents should also be considered to be included within the scope of protection claimed in this application.

Claims

1. A response to pathogen stress LcMYB1 The promoter is characterized in that, The nucleotide sequence of the LcMYB1 promoter is shown in SEQ ID NO.

5.

2. The response to pathogen stress as described in claim 1 LcMYB1 The promoter is characterized in that, When not infected by pathogens, the transcriptional activity of LcMYB1 is extremely low, and the basal transcriptional level of the downstream GUS gene driven by it is less than one-sixth of the peak transcriptional level induced by pathogen infection.

3. The promoter for responding to pathogen stress as described in claim 1 or 2, characterized in that, The expression sites of the LcMYB1 promoter include: leaf veins and part of the root.

4. The LcMYB1 promoter in response to pathogen stress as described in claim 3, characterized in that, The LcMYB1 promoter was significantly induced and activated after infection with Phytophthora downy mildew on litchi, driving the efficient expression of downstream genes, and the expression area spread from the leaf veins to the surrounding tissues.

5. A method for cloning the LcMYB1 promoter in response to pathogen stress as described in any one of claims 1 to 4, characterized in that, Genomic DNA was extracted from the leaves of the lychee 'Feizixiao' variety and used as a template for PCR amplification with primers SEQ ID NO.1 and SEQ ID NO.

2. Preferably, the amplification conditions are: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 25 s, 60℃ annealing for 25 s, 72℃ extension for 50 s, for a total of 35 cycles; and finally 72℃ final extension for 2 min, and storage at 4℃ to obtain the LcMYB1 promoter.

6. A recombinant expression vector, characterized in that, It includes the LcMYB1 promoter as described in any one of claims 1 to 4; The initial vector for the recombinant expression vector was pGreen-GUS.

7. The method for constructing the recombinant expression vector as described in claim 6, characterized in that, include: Using GUS as a reporter gene, the LcMYB1 promoter was operatively linked to the GUS reporter gene; Preferably, the specific method for the ligation operation is as follows: the LcMYB1 promoter fragment is tandemly fused with the GUS reporter gene to construct a linearized vector. The vector containing the GUS reporter gene is then digested with the restriction endonuclease BcuI to obtain a linearized vector. After purification, the LcMYB1 promoter and the linearized vector are seamlessly ligated via homologous recombination using the Accurate Biotechnology OK Clon DNA Ligation Kit II, achieving effective fusion of the LcMYB1 promoter and the downstream GUS reporter gene. The recombinant expression vector is pLcMYB1:GUS. The primers used to construct the recombinant expression vector are shown in SEQ ID NO.3 and SEQ ID NO.

4.

8. A transgenic cell line or recombinant bacteria, characterized in that, It comprises the LcMYB1 promoter as described in any one of claims 1 to 4 or the recombinant expression vector as described in claim 6; The recombinant bacteria include Escherichia coli for cloning and Agrobacterium for plant transformation.

9. A transgenic plant, characterized in that, The transgenic plant includes the recombinant expression vector as described in claim 3; the transgenic plant is obtained by transforming the recombinant expression vector into Agrobacterium GV3101 and then transforming it by Agrobacterium-mediated transformation, including Arabidopsis thaliana, tobacco, or litchi.

10. An application of the LcMYB1 promoter as described in any one of claims 1 to 4, characterized in that, The applications include: regulating the expression of GUS reporter genes in response to downy mildew stress in litchi; visualizing and tracing the pathogen infection process and early plant resistance responses; and breeding plant varieties resistant to downy mildew in litchi.

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