Gibberella-induced promoter, expression vector comprising the same, and use thereof

Abstract

The present application discloses a Gibberella-induced promoter, an expression vector containing the promoter and application, and the nucleotide sequence of the promoter is shown as SEQ ID NO. 1. The Gibberella-induced promoter of the present application can drive the rice blast disease-resistant gene PigmR The wheat transgenic material obtained by the method has the characteristics of obtaining Gibberella resistance while the agronomic traits are not affected. The present application overcomes the limitation of insufficient wheat self-resistant Gibberella gene and has strong application value.

Description

Technical Field

[0001] This invention relates to wheat genetics and breeding, and more specifically, to a Fusarium graminearum inducible promoter, an expression vector containing the promoter, and its applications. Background Technology

[0002] Currently, wheat scab control employs a comprehensive prevention and control system, including cultivation management, precision pesticide application, and the breeding and application of resistant varieties. Cultivation management measures include returning straw to the field for full decomposition to reduce the initial inoculum population; optimizing sowing time and density to avoid overlap between the heading and flowering stages and the hot, humid rainy season; scientific fertilization to enhance plant resistance; and improving drainage to reduce field humidity and disrupt pathogen infection conditions. Pesticide control involves preventative spraying with highly effective, low-toxicity agents such as cyazofamid and tebuconazole during the susceptible window from wheat heading to early flowering, rotating and mixing pesticides to delay the development of resistance; and using modern spraying equipment to improve droplet uniformity and operational efficiency. Breeding and planting resistant varieties is the most economical and effective fundamental approach, with the cloning of resistance genes and the selection of novel resistance molecular modules being key breeding techniques. Currently, the bottleneck restricting the breeding of resistant varieties is the limited number of wheat's own scab resistance genes, and the fact that most of them have weak effects.

[0003] Cross-species gene utilization is an emerging technology for crop pest and disease control. This strategy involves introducing functional genes from animals, microorganisms, or other plants into target crops, endowing them with disease resistance without affecting other agronomic traits. The most successful example of this strategy is the breeding of insect-resistant cotton. Insecticidal protein genes from Bacillus thuringiensis were introduced into the cotton genome, successfully breeding new cotton varieties specifically resistant to lepidopteran pests (such as bollworm and pink bollworm) and promoting their cultivation. This has played a supporting role in ensuring my country's cotton self-sufficiency rate and is a landmark achievement in using biotechnology to solve practical problems in agricultural production. However, there are few reports on the application of this technology in the control of wheat scab. Summary of the Invention

[0004] To address the lack of reports on the application of cross-species gene technology in the control of wheat scab, this invention provides an inducible promoter for Fusarium graminearum, an expression vector containing this promoter, and its application. This Fusarium graminearum inducible promoter is linked to a rice blast resistance gene. PigmR By linking the two materials together to a suitable vector and transferring them into wheat cells, wheat materials with significantly enhanced resistance to Fusarium head blight can be obtained without altering other agronomic traits.

[0005] The key to cross-species gene utilization lies in the selection of target genes and their expression in the target species, ultimately conferring specific traits to the target species without affecting other traits. Considering the current level of public acceptance of genetically modified crops, the selection of target genes from closely related species is preferable. For wheat scab, genes derived from its close relatives (such as long-spike wheatgrass and tufted wheatgrass) or gramineous crops (such as rice and maize) are the optimal choice.

[0006] Fusarium head blight is a facultative nutritional fungus. In the early stages of infection, it is in a vivotrophic form, obtaining nutrients from the host's living cells; in the later stages, it becomes a catastrophe-like form, obtaining nutrients from the host's dead cells. This nutritional mode is similar to that of rice blast fungus. The rice genome carries a large number of blast resistance genes, suggesting a theoretical basis for utilizing these genes for the control of wheat Fusarium head blight.

[0007] The present invention aims to screen suitable rice blast resistance genes and promoters in wheat induced by Fusarium head blight, and use these promoters to drive the expression of rice blast resistance genes in susceptible wheat to verify whether they are resistant to Fusarium head blight without affecting other agronomic traits, thus providing theoretical and technical support for the prevention and control of wheat Fusarium head blight.

[0008] To achieve the above objectives, the present invention provides an inducible promoter for Fusarium graminearum, the nucleotide sequence of which is shown in SEQ ID NO.1:

[0009] A second aspect of the present invention provides an expression vector comprising the promoter described above. Further, it also includes... PigmR Gene.

[0010] A third aspect of the present invention provides primers for amplifying the promoter described above, comprising primers 224200-proF1 and 224200-proR1.

[0011] The fourth aspect of this invention provides the application of the above-described promoter in improving wheat scab resistance.

[0012] Specifically, the promoter drives PigmR Gene overexpression can enhance wheat resistance to Fusarium head blight.

[0013] Through the above technical solution, the present invention achieves the following beneficial effects: 1. The wheat scab inducible promoter of the present invention can drive the rice blast resistance gene. PigmR This method expresses the gene in wheat to enhance wheat resistance to Fusarium head blight. The resulting transgenic wheat materials acquire resistance to Fusarium head blight without affecting agronomic traits.

[0014] 2. This invention provides a method for improving wheat resistance to Fusarium head blight by utilizing disease-resistant genes across species. This method overcomes the limitation of insufficient Fusarium head blight resistance genes in wheat itself and has strong application value. Attached Figure Description

[0015] Figure 1 This invention utilizes pathogen-inducible promoters and PigmR Flowchart illustrating gene-based enhancement of wheat scab resistance; Figure 2 yes PigmR Phenotypic (A), statistical data (B), and gene expression analysis (C) of overexpression rice materials inoculated with Fusarium graminearum 21 days later; Figure 3 yes PigmR Overexpression in wheat materials PigmR Gene expression analysis; double asterisks (**) indicate that, compared with Fielder, overexpressing plants showed higher expression levels. PigmR Gene expression levels were significantly higher ( P When the value increases to <0.01, the black dots in the box plot represent data points; Figure 4 yes PigmR The percentage of diseased spikelets in wheat 21 days after inoculation with Fusarium graminearum; double asterisks (**) indicate that the percentage of diseased spikelets in overexpressing plants was significantly higher than that in Fielder plants. PThe value decreases to <0.01. In the box plot, the black dots represent data points, i.e., the diseased spikelet rate of each ear. Figure 5 yes PigmR The agronomic traits of wheat overexpressing the gene were investigated, including plant height (A), tiller number (B), spike length (C), thousand-grain weight (D), grain length (E), and grain width (F). Double asterisks (**) indicate that the corresponding agronomic trait values ​​of the overexpressing plants were significantly different from those of Fielder plants. P <0.01) increases or decreases; black dots in the box plot represent data points; Figure 6 This is an analysis of the expression levels of genes selected from transcriptome screening after Fielder inoculation with Fusarium graminearum. Double asterisks (**) indicate significant differences in gene expression levels compared to before inoculation (0 h). P When the value is <0.01, the black dots in the box plot represent data points. Figure 7 pGreenII 0800 is used to verify promoter activity. LUC vector map; Figure 8 It is driven by pathogen-inducible promoters. PigmR Wheat plants expressing the effect of inoculation with Fusarium head blight PigmR Expression level analysis; double asterisks (**) indicate significantly higher gene expression levels compared to Fielder. P When the value is <0.01, the black dots in the box plot represent data points. Figure 9 It is driven by pathogen-inducible promoters. PigmR The expression represents the statistical percentage of diseased spikelets in wheat plants after inoculation with Fusarium graminearum; double asterisks (**) indicate a significantly higher percentage of diseased spikelets compared to Fielder. P When the value is less than 0.01, the black dots in the box plot represent data points. Figure 10 It is driven by pathogen-inducible promoters. PigmR The agronomic traits of wheat plants were assessed, including plant height (A), number of tillers (B), spike length (C), thousand-grain weight (D), grain length (E), and grain width (F). The black dots in the box plot represent data points. Detailed Implementation

[0016] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0017] like Figure 1The following embodiments mainly include two parts: First, screening suitable rice blast resistance genes: summarizing published rice blast resistance genes and selecting genes with broad-spectrum resistance and clear resistance mechanisms; second, screening suitable Fusarium head blight inducible promoters: using transcriptome sequencing analysis to select genes induced by Fusarium head blight and extracting their promoter sequences. These promoters are then used to drive the expression of the aforementioned resistance genes in wheat, and Fusarium head blight resistance is analyzed to verify the effectiveness of the selected promoters and resistance genes.

[0018] Example 1. Screening and Validation of Rice Blast Resistance Genes (1) Screening for rice blast resistance genes Published literature has summarized and analyzed cloned rice blast resistance genes (Mao et al., 2021, Science in China Life Sciences). The vast majority of these blast resistance genes exhibit pathogen race-specific resistance, meaning they are resistant only to specific blast fungus strains. This is because the encoded products of these genes can only recognize specific effector factors secreted by the pathogen. Only a very small number of genes exhibit broad-spectrum resistance, meaning they are resistant to most blast fungus strains. Considering that Fusarium head blight and rice blast fungus secrete different effector factors, those resistance genes with race-specific resistance cannot successfully recognize Fusarium head blight and cannot initiate a resistance response. Therefore, selecting genes with broad-spectrum resistance is necessary to potentially elicit a response to Fusarium head blight.

[0019] Among the few broad-spectrum resistance genes for rice blast, those cloned from local varieties... PigmR The gene possesses strong resistance and a broad spectrum of resistance. The disease resistance signal transduction network mediated by this gene contains both basic resistance components and specific resistance triggered by pathogen effector proteins, suggesting that when this gene is transferred into wheat, the basic resistance component can confer resistance to Fusarium head blight.

[0020] (2) Overexpression PigmR Analysis of genetically modified rice materials inoculated with Fusarium graminearum Fusarium graminearum, which causes wheat scab, can also infect rice. Further analysis is needed. PigmR Whether genes can provide resistance to Fusarium graminearum, and whether... PigmR Inoculation analysis was performed on rice materials that overexpressed the gene (a gift from Professor Deng Yiwen of Zhejiang University).

[0021] When rice reached the flowering stage, following a method reported in the literature, rice panicles were sprayed with Fusarium graminearum spore solution and covered with plastic bags to maintain moisture for 3 days. After removing the plastic bags, disease incidence data were collected 21 days later. Results showed that, compared to the transgenic receptor Nipponbare, overexpression of [a specific strain / product]... PigmR The disease resistance of rice materials was significantly enhanced. Figure 2 ),show PigmR The gene can enhance rice's resistance to Fusarium graminearum.

[0022] (3) Overexpression PigmR Analysis of genetically modified wheat materials inoculated with Fusarium head blight For further analysis PigmR Whether the gene can provide resistance to Fusarium graminearum in wheat, PigmR The gene is overexpressed in wheat.

[0023] From the above overexpression PigmR The open reading frame of the gene was obtained from rice materials. The specific method was as follows: Leaf samples were taken from the rice materials, and total RNA was extracted using the TransZol RNA Extraction Kit (Beijing TransGen Biotech Co., Ltd.) according to the instructions provided with the kit. 5 μg of total RNA was added to DNase I (Invitrogen, USA) to remove genomic DNA, and then used as a template for oligo(dT) amplification. 18 Total cDNA was obtained by reverse transcription using oligomeric primers and M-MLV reverse transcriptase (Invitrogen, USA). A pair of PCR primers, PigmR-F (5′-CAGGTCGACTCTAGA), was designed. GGATCC ATGGCGGAGACGGTGCTG-3′ (SEQ ID NO.2, underlined) Bam HI restriction site) and PigmR-R (5′-CTCGAGGGTACCCGG GGATCC TCAGCCAGCTTGAGCTGTGC-3′ (SEQ ID NO.3, underlined) Bam (HI restriction site), and amplified using the obtained cDNA as a template. PigmR The open reading frame of the gene (GenBank accession number APF29096) was ligated into the wheat overexpression vector pUbiGW, which carries a maize ubiquitin promoter with constitutive and overexpression characteristics (He et al., 2024). The successfully constructed vector was introduced into the Fusarium head blight-susceptible wheat variety Fielder using Agrobacterium-mediated genetic transformation (Zhou et al., 2024).

[0024] In this embodiment, a total of 5 independently transformed plants were obtained. Spikelets were collected from the transformed plants at the flowering stage, total RNA was extracted and reverse transcribed, and the RNA was detected by qPCR. PigmR Gene expression levels were determined using primer pairs PigmR-RT-F1 (5′-TTCCTTTCTCTTTATCATAATT-3′, SEQ ID NO.4) and PigmR-RT-R1 (5′-AGATTCCAACCTGCACTTGCCT-3′, SEQ ID NO.5). Results showed ( Figure 3), among these transformed plants PigmR Gene expression levels were significantly increased.

[0025] Inoculation analysis of genetically transformed plants in the T2 generation was conducted against Fusarium head blight. During the wheat flowering stage, a single-flower drip inoculation method was used, injecting pathogenic spores into the spikelets. Disease incidence was assessed three weeks later. Results showed ( Figure 4 Compared to the infected control Fielder, PigmR Overexpression of the gene significantly enhances disease resistance in plants. P <0.01).

[0026] (4) Overexpression PigmR Agronomic trait analysis of wheat materials based on genes Published literature indicates that overexpression in rice PigmR Genes that cause decreased seed setting rate and other unfavorable agronomic traits, therefore, the overexpression of the above-mentioned genes... PigmR Agronomic traits of wheat materials were analyzed using genetic methods. The results showed that ( Figure 5 Compared to Fielder, overexpression PigmR The wheat plant height, ear length, thousand-grain weight, grain length, and grain width were all reduced, while the number of tillers increased. These results suggest that rational utilization... PigmR The prerequisite for improving wheat resistance to Fusarium head blight is to make PigmR The expression level is kept at an appropriate level so that it can improve wheat resistance to Fusarium head blight without affecting agronomic traits.

[0027] Example 2. Screening and Validation of Inducible Promoters for Fusarium Head Bollworm (1) Identification of the inducible gene of Fusarium graminearum The wheat variety Fielder is susceptible to Fusarium head blight. Based on published literature, Fielder spikelets were inoculated with spores of Fusarium head blight strain PH1 during the flowering stage using the single-flower drip inoculation method. Spikelets were collected before inoculation and 2 days after inoculation for RNA extraction.

[0028] Total RNA was extracted using the method described in Example 1 and subjected to transcriptome sequencing analysis (Shenzhen BGI Genomics Co., Ltd.).

[0029] Genes that were not expressed before vaccination (FPKM = 0) and whose expression levels increased significantly after vaccination were selected for further analysis (parameters were set as baseMean ≥ 50, log2FoldChange ≥ 5, and FPKM ≥ 10 after vaccination) (Table 1).

[0030] Table 1. Information on the inducible genes of the screened Fusarium wilt pathogens

[0031] qPCR primers were designed for the above genes to verify whether their expression was induced by Fusarium graminearum. The primer sequences are shown in Table 2.

[0032] Table 2. qPCR primer information for the screened Fusarium graminearum inducible genes

[0033] Fielder spikelets at the flowering stage were inoculated using the same method described above. RNA was extracted from spikelets before inoculation and at 24 h and 48 h after inoculation. Total cDNA from the spikelets was obtained by reverse transcription using the method described in Example 1. qPCR analysis was performed using the primers in Table 2. The results are as follows: Figure 6 As shown in Table 1, the expression levels of the selected genes increased significantly after inoculation with Fusarium graminearum, but the increase was not the same. Among them, the expression level of TraesCS6A02G224200 increased the most, so this gene was selected for further analysis.

[0034] (2) Identification of the inducible promoter of Fusarium graminearum DNA was extracted from Fielder leaves using a high-efficiency plant genomic DNA extraction kit (Beijing Qingke Biotechnology Co., Ltd.) and used as a template for PCR amplification. The genome sequence of TraesCS6A02G224200 was downloaded from the WheatOmics website (https: / / wheatomics.sdau.edu.cn / ), and the sequence 2000 bp upstream of the start codon ATG of TraesCS6A02G224200 was extracted as the promoter. A pair of PCR primers, 224200-proF1 (5′-GTAACAATATGAAATTTTGTTCAAATCAA-3′, SEQ ID NO.6) and 224200-proR1 (5′-TGCTAGCTTTGCTCACTCCTATGC-3′, SEQ ID NO.7), were designed. Using Fielder leaf DNA as a template, the promoter sequence of TraesCS6A02G224200 was amplified using the high-fidelity DNA polymerase Phanta Max Master Mix (Nanjing Novizan Biotechnology Co., Ltd.). This sequence was then ligated into the pGreenII-0800-Luc vector using a single-fragment one-step cloning kit (Nanjing Novizan Biotechnology Co., Ltd.). Figure 7The pGreenII-0800-Luc vector, purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd., was sequenced and analyzed by Suzhou Genewise Biotechnology Co., Ltd. The vector carries two important elements: a Renilla luciferase (RLuc) gene driven by a CaMV 35S constitutive promoter as an internal reference; and a Firefly luciferase (FLuc) gene driven by the test promoter. Sequencing results showed that the PCR-amplified TraesCS6A02G224200 promoter sequence was identical to the sequence downloaded from the website.

[0035] The pGreenII-0800-Luc vector, containing the TraesCS6A02G224200 promoter sequence, was transformed into Agrobacterium GV3101 (pSoup-p19) competent cells and used to infect tobacco leaves. Simultaneously, tobacco cells were infected with the empty pGreenII-0800-Luc vector as a negative control. After incubation in the dark for 48 h, the infected tobacco leaves were quickly cut and placed in liquid nitrogen, ground into powder, and the protein was extracted using the Dual Luciferase Reporter Assay Kit (Nanjing Novizan Biotechnology Co., Ltd.) according to the accompanying instructions. The fluorescence signals produced by FLuc and RLuc catalysis were detected using a microplate reader (Tecan, Austria), and the FLuc / RLuc ratio was used to eliminate differences in infection efficiency. The results (Table 3) showed that there was no significant difference in the FLuc and RLuc ratios between the empty vector control group and the TraesCS6A02G224200 promoter experimental group, indicating that the TraesCS6A02G224200 promoter is a weak promoter and cannot drive the expression of the target gene under normal conditions.

[0036] Table 3. Promoter activity analysis of TraesCS6A02G224200

[0037] (1) Each experimental group was infected with 6 tobacco leaves. The mean value refers to the mean ± standard deviation of the 6 tobacco leaves. t -test calculation P value.

[0038] The above treatment was repeated. After incubating tobacco leaves infected with Agrobacterium in the dark for 24 h, wounds were made on the infected leaves using a syringe needle. The spores of Fusarium head blight strain PH1 were injected into the infected leaves using a syringe with the needle removed, and the leaves were incubated in the dark for another 24 h to detect promoter induction by Fusarium head blight. Tobacco leaf proteins were extracted using the same method, and the fluorescence signals catalyzed by FLuc and RLuc were detected using a microplate reader. The results (Table 4) showed that the FLuc to RLuc ratio in the TraesCS6A02G224200 promoter experimental group was significantly higher than that in the empty vector control, indicating that the TraesCS6A02G224200 promoter was induced by Fusarium head blight.

[0039] Table 4. Induction activity analysis of TraesCS6A02G224200 promoter

[0040] (1) Each experimental group was infected with 6 tobacco leaves. The mean value refers to the mean ± standard deviation of the 6 tobacco leaves. t -test calculation P value.

[0041] Example 3. Driving with Fusarium graminearum inducible promoter PigmR Genes that enhance wheat resistance to Fusarium head blight Using restriction endonucleases Pst I. The maize ubiquitin promoter carried by the wheat overexpression vector pUbiGW was removed. Primer pair 224200-pro-F2 (5′-GCTTAATTAAACTAGT was used. CTGCAG GTAACAATATGAAATTTTGTTCAAATCAA-3′)(SEQ ID NO.8, underlined Pst I restriction site) and 224200-pro-R2 (5′-TAGTAGGCCTAGGG CTGCAG TGCTAGCTTTGCTCACTCCTATGC-3′) (SEQ ID NO.9, underlined) Pst Using the pGreenII-0800-Luc plasmid containing the TraesCS6A02G224200 promoter sequence from Example 2 as a template, the TraesCS6A02G224200 promoter sequence was amplified and ligated into the pUbiGW plasmid that had the maize ubiquitin promoter removed, using a single-fragment one-step cloning kit (Nanjing Novizan Biotechnology Co., Ltd.).

[0042] Using the method in Example 1 above, PigmRThe open reading frame was linked to the modified pUbiGW plasmid, and it was introduced into the Fusarium head blight-susceptible wheat variety Fielder using Agrobacterium-mediated genetic transformation.

[0043] In this embodiment, a total of 7 independently transformed plants were obtained. During the flowering stage, a single-flower drip inoculation method was used. One spike was selected from each plant, and three florets were inoculated. Two days later, the inoculated florets were harvested, total RNA was extracted and reverse transcribed, and the qPCR method described in Example 1 was used to detect RNA in the transformed plants. PigmR Gene expression levels. The results showed ( Figure 8 After inoculation, these transformed plants PigmR The significantly increased gene expression level indicates that the inducible promoter of Fusarium graminearum was induced and driven after inoculation. PigmR Gene overexpression.

[0044] The genetically transformed plants of generation T2 were inoculated with Fusarium head blight, and the disease incidence was analyzed three weeks later. The results showed that ( Figure 9 Compared to the infected control Fielder, the inducible promoter of Fusarium graminearum drives... PigmR The gene significantly enhances the disease resistance of plants. P <0.01).

[0045] Further analysis of the agronomic traits of the genetically transformed plants showed no significant changes in plant height, tiller number, panicle length, thousand-grain weight, grain length, and grain width. Figure 10 This indicates that the TraesCS6A02G224200 is used to start the sub-driver. PigmR Gene expression in wheat can improve wheat resistance to Fusarium head blight without affecting agronomic traits.

[0046] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0047] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0048] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A Fusarium graminearum inducible promoter, characterized in that, The nucleotide sequence of the promoter is shown in SEQ ID NO.

1.

2. An expression vector, characterized by, Includes the promoter described in claim 1.

3. The expression vector according to claim 2, characterized in that, Also includes PigmR Gene.

4. Primers for amplifying the promoter of claim 1, characterized in that, This includes primers 224200-proF1 and 224200-proR1.

5. The application of the promoter according to claim 1 in improving wheat scab resistance.

6. The application according to claim 5, characterized in that, The promoter drives PigmR Gene overexpression can enhance wheat resistance to Fusarium head blight.

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