Pyrus pyrifolia blast resistance gene pbhhlh030 and application thereof

Abstract

The application discloses a pear anthracnose resistance gene PpbHLH030 and application thereof, and belongs to the field of plant genetic engineering. PpbHLH030 The nucleotide sequence of the gene is shown as SEQ ID NO:1, the protein sequence encoded by the gene is shown as SEQ ID NO:2, the gene is significantly up-regulated in expression under the induction of Guignardia bidwelli and MeJA, the gene is located in a plant cell nucleus and has a conserved bHLH domain. PpbHLH030 The construction of an overexpression vector and the transformation of 'eggplant pear' calluses increase the anthracnose resistance of the calluses, and the silencing of the pear fruit PpbHLH030 gene increases the anthracnose susceptibility. The application provides an important gene resource for the cultivation of pear anthracnose resistance varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to a pear bHLH-type transcription factor. PpbHLH030 And its applications. Background Technology

[0002] In recent years, anthracnose has frequently occurred in pear-producing areas, causing premature leaf drop and fruit rot, severely impacting the pear industry. Currently, control measures for pear anthracnose mainly focus on chemical control, with limited research on the identification of resistant genes. Basic helix-loop-helix (bHLH) transcription factors are one of the largest transcription factor families in plants, widely involved in various biological processes such as plant growth and development, physiological metabolism, and stress responses. Members of this family possess a conserved bHLH domain containing approximately 60 amino acids. Based on phylogenetic analysis, plant bHLH transcription factors have been divided into 26 to 32 subfamilies, with differences in gene structure, conserved motifs, and the biological processes they regulate. For example, members of the IIIf subfamily, involved in anthocyanin and proanthocyanidin biosynthesis, also have a region at their N-terminus that interacts with R2R3-MYB transcription factors, specifically regulating downstream target genes by forming the MYB-bHLH-WDR complex. This structural diversity is the molecular basis for the functional diversity of bHLH transcription factors. Currently, research on the function of bHLH transcription factors in pear anthracnose resistance is relatively limited. Therefore, elucidating the function and mechanism of these factors in the pear's response to anthracnose can provide a theoretical basis and genetic resources for pear disease resistance breeding, and has significant scientific value. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to propose an application of bHLH transcription factors in anthracnose resistance in pears, thus making up for the lack of existing research on anthracnose resistance using bHLH transcription factors.

[0004] The present invention solves the above-mentioned technical problems through the following technical means:

[0005] This invention proposes a pear PpbHLH030 The gene, whose CDS sequence is shown in SEQ ID NO: 1.

[0006] The present invention also proposes the above. PpbHLH030 The protein encoded by the gene has the amino acid sequence shown in SEQ ID NO: 2.

[0007] The present invention also proposes the above-mentioned pear PpbHLH030 Application of genes in enhancing resistance to pear anthracnose.

[0008] Preferably, by promoting pear PpbHLH030Gene expression, which in turn promotes the synthesis of lignin and flavonoids, enhances the pear's resistance to anthracnose.

[0009] This invention also proposes a pear PpbHLH030 Application of genes in the breeding of anthracnose-resistant pear varieties.

[0010] This invention also proposes a pear PpbHLH030 Application of genes in promoting the synthesis of lignin and flavonoids in plants.

[0011] This invention also proposes a pear PpbHLH030 Gene overexpression vector, which expresses the above-mentioned pear PpbHLH030 The gene was obtained by homologous recombination into the PBI121 or pK7WG2D overexpression vector.

[0012] This invention also proposes a method for cultivating anthracnose-resistant pear varieties, which involves... PpbHLH030 Gene overexpression vectors were introduced into target plants to obtain anthracnose-resistant pear germplasm.

[0013] The preferred cultivation method specifically includes the following steps: (1) Cloning the pear described above PpbHLH030 Gene; (2) Constructing a pear PpbHLH030 Gene overexpression vectors; (3) Pear PpbHLH030 The gene overexpression vector was transformed into the target plant, and the resulting pear germplasm with improved anthracnose resistance was identified.

[0014] Preferably, the method of transformation into the target plant is either Agrobacterium-mediated transformation or gene gun transformation.

[0015] The beneficial effects of this invention are as follows: 1. This invention discloses pear bHLH-type transcription factors. PpbHLH030 The application of this gene in pear anthracnose resistance, along with its encoded amino acid sequence, is reported for the first time. RT-qPCR analysis results indicate that this gene is effective against pear anthracnose (…). Colletotrichum fructicola MeJA and other enzymes significantly upregulated expression of this gene, which is located in the plant cell nucleus and has a conserved bHLH domain; overexpression PpbHLH030 It can enhance the callus of 'eggplant-pear' and the resistance of pear fruit to anthracnose.

[0016] 2. Overexpression PpbHLH030 The gene significantly increased the activities of peroxidase (POD) and superoxide dismutase (SOD), decreased hydrogen peroxide (H2O2) content, and significantly increased lignin and flavonoid content. This invention provides valuable genetic resources for the breeding and identification of anthracnose-resistant pear varieties.

[0017] 3. This invention discovers that overexpressionPpbHLH030 Genes can enhance anthracnose resistance by increasing flavonoid and lignin content, while silencing genes can enhance resistance. PpbHLH030 This invention significantly increases the susceptibility of pear fruit to anthracnose. It provides valuable genetic resources for breeding pears with resistance to anthracnose.

[0018] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0019] Figure 1 In Embodiment 1 of the present invention PpbHLH030 Gene expression characteristics, subcellular localization, and sequence analysis diagram, where A represents inoculation with anthrax bacteria (… Colletotrichum fructicola ) 6, 12 and 24 h later, in the leaves of 'Cuiguan' pear PpbHLH030 The relative expression level; B represents the expression level in 'Cuiguan' pear leaves at 6, 12, and 24 h after MeJA treatment. PpbHLH030 The relative expression levels are shown in Figure 1; C represents the subcellular localization of PpbHLH030 protein; D represents the predicted conserved domains of PpbHLH030 protein; and E represents the phylogenetic tree of PpbHLH030 and its homologous proteins. Error bars in the figure represent the standard errors (SE) of three independent replicate experiments. Indicates the difference between the control group and the control group. p The difference was statistically significant at levels <0.01. Indicates in p The difference was significant at the level <0.0001.

[0020] Figure 2 In Embodiment 2 of the present invention PpbHLH030 Graph showing the evaluation of anthrax resistance and determination of physiological indicators in transgenic callus, where A represents... PpbHLH030 Overexpression of the healing system PpbHLH030 Relative expression level identification results; B represents transgenic callus inoculation. Colletotrichum fructicola The pathogenesis phenotypes at 3, 5, and 7 days post-inoculation; C represents the statistical analysis of the corresponding lesion diameters, with data expressed as mean ± standard error of 8 independent replicates; D, E, and F represent the determination of peroxidase (POD) activity, superoxide dismutase (SOD) activity, and hydrogen peroxide (H2O2) content at 0, 6, and 24 h post-inoculation, respectively, with data expressed as mean ± standard deviation of 3 independent replicates. Indicates the difference between the control group and the control group. p The difference was statistically significant at levels <0.05. Indicates the difference between the control group and the control group. p The difference was statistically significant at levels <0.01. Indicates the difference between the control group and the control group. p The difference was statistically significant at the level of <0.001. Indicates the difference between the control group and the control group. p The difference at the <0.0001 level was extremely significant.

[0021] Figure 3 In Embodiment 3 of the present invention PpbHLH030 The effect of transient gene silencing on anthracnose resistance in pear fruit is shown in the figure. A represents... PpbHLH030 Silent pear fruit PpbHLH030 qRT-PCR analysis of relative expression levels; B represents silent fruit inoculation. Colletotrichum fructicola The diameter of lesions was statistically analyzed at 3, 5 and 7 days after inoculation; C represents the disease phenotype at the corresponding time point; D, E and F represent the determination of peroxidase (POD) activity, superoxide dismutase (SOD) activity and hydrogen peroxide (H2O2) content at 0, 6 and 24 h after inoculation of silent fruits, respectively; data in B are expressed as mean ± standard error of 8 independent replicates; data in D~F are expressed as mean ± standard deviation of 3 independent replicates. Indicates the difference between the control group and the control group. p The difference was statistically significant at levels <0.05. Indicates the difference between the control group and the control group. p The difference was statistically significant at levels <0.01. Indicates the difference between the control group and the control group. p The difference was statistically significant at the level of <0.001. Indicates the difference between the control group and the control group. p The difference at the <0.0001 level was extremely significant.

[0022] Figure 4 In Embodiment 4 of the present invention PpbHLH030 Graph showing the analysis of JA, total flavonoids and lignin content in transgenic callus and pear fruit, where A represents overexpression. PpbHLH030 pear callus inoculation Colletotrichum fructicola JA levels at 0, 6, and 24 hours post-silencing; B represents transient silencing. PpbHLH030 pear fruit inoculation Colletotrichum fructicola JA content at 0, 6, and 24 h; C and D, flavonoid and lignin content in overexpressed callus tissue, respectively; E and F, flavonoid and lignin content in silent pear fruit, respectively. Data are expressed as mean ± standard error of three independent replicates and analyzed using one-way ANOVA. p <0.05, p <0.01, p <0.001, p <0.0001.

[0023] Figure 5 The exogenous flavonoid treatment in Example 5 of this invention Colletotrichum fructicola The effect of flavonoids on growth is shown in the figure, where A and B represent the effects of different concentrations of flavonoids on growth. C. fructicola The mycelial growth phenotype and colony diameter were statistically analyzed; C and D were the germination phenotype and germination rate of conidia under the corresponding concentration treatments, respectively. Data in B are expressed as mean ± standard deviation of more than 7 independent replicates; data in D are expressed as mean ± standard deviation of more than 200 spores. One-way ANOVA was used. p <0.05, p <0.0001. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical terms used below have the same meaning as understood by those skilled in the art.

[0025] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0026] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0027] Example 1: PpbHLH030 Gene expression, subcellular localization and amino acid sequence analysis This invention utilizes bioinformatics methods combined with PCR-specific amplification to clone... PpbHLH030 Genes. Specifically, based on predictions from the 'Crown' genome database (https: / / bigd.big.ac.cn / gwh). PpbHLH030 Amplification primers were designed based on the coding region sequence (EVM0014256). The gene amplification primer sequences are shown below: PpbHLH030 Forward primer ( PpbHLH030 -F): 5'-ATGGATTATTACAGTTTTAGCTC-3', (SEQ ID NO: 3) PpbHLH030 Reverse primer ( PpbHLH030 -R): 5'-CGAGGATGAGCTCGAGGTATCG-3' (SEQ ID NO: 4) PpbHLH030 Quantitative primers (qRT-PpbHLH030-F): 5'-AGGCATCCGTCTGCTGTG-3', (SEQ ID NO: 5) PpbHLH030 Quantitative primers (qRT-PpbHLH030-R): 5'-GGACCTGGTGAACCGAAC-3' (SEQ ID NO: 6) Actin Internal control quantitative primer (Actin-F): 5'-TGGTGTCATGGTTGGTATGG-3', (SEQ ID NO: 7) Actin Internal control quantitative primer (Actin-R): 5'-CAGGAGCAACACGAAGTTCA-3' (SEQ ID NO: 8) according to PpbHLH030 Based on the cDNA sequence, subcellular localization vector primers were designed: 1300-PpbHLH030-F: 5'-CCGTCGACCCCGGGGGTACCATGGACCCCACATTCTTCAGCAG-3'; (SEQ ID NO: 9) 1300-PpbHLH030-R: 5'-CTCACCATGAATTCGGATCCCGAGGATGAGCTCGAGGTATCG-3' (SEQ ID NO: 10) This invention uses cDNA from 'Cuiguan' tissue culture seedlings as a template for PCR amplification. The PCR amplification system is as follows: 2X Phanta Max Buffer 25 μL, cDNA 1 μL, dNTP Mix (10 mM) 1 μL, forward / reverse primers 2 μL each, Phanta Max Super-Fidelity DNA Polymerase 1 μL, and ddH2O added to a final volume of 50 μL. The amplified cDNA... PpbHLH030 The CDS fragment (SEQ ID NO: 1) is 807 bp in length and encodes 268 amino acids (SEQ ID NO: 2). The quantitative PCR reaction system consisted of: 5 μL of 2×Hieff Canace® Plus PCR Master Mix, 0.4 μL each of forward and reverse primers, 1 μL of cDNA, and water to a final volume of 10 μL. The specific PCR procedure was as follows: 95℃ pre-denaturation for 5 min, 1 cycle; 95℃ denaturation for 10 s, 55-60℃ annealing for 20 s, 72℃ extension for 20 s, 40 cycles. Three technical replicates were designed for each sample, and the quantitative results were based on 2... - CT The relative expression levels were calculated using the method described above. The results are as follows: Figure 1 As shown in Figures A and B, compared to inoculation with sterile water, PpbHLH030 In 'Cuiguan', the expression of anthracnose was significantly upregulated 24 h after induction, while the expression was significantly upregulated 6 and 12 h after exogenous MeJA treatment.

[0028] In order to investigate PpbHLH030 This study analyzes the molecular characteristics and evolutionary relationships of [a protein / organism], including its subcellular localization, conserved protein domains, and phylogenetic structure. Subcellular localization results show (e.g.) Figure 1 (as shown in C), pRI101- PpbHLH030 In tobacco leaves co-injected with the nuclear marker, the green and red fluorescent signals completely overlapped at the nucleus. Leaves co-injected with the pRI101 empty vector and the nuclear marker Agrobacterium showed green cell membranes and nuclei under green fluorescence irradiation and red nuclei under red fluorescence irradiation; the nuclei overlapped in the fused images, indicating that PpbHLH030 is localized in the nucleus. Conserved domain analysis showed (e.g.) Figure 1 (as shown in D) PpbHLH030 The protein contains typical bHLH-SF superfamily domains and ACT domains. Comparison with homologous proteins from other species indicates that it is similar to that of pear (Pyrus pyrifolia). Pyrus communis ) PcbHLH030 sand pear ( Pyrus pyrifoliaThe conserved structural domains of PybHLH030 are highly similar. Phylogenetic analysis further shows (e.g.) Figure 1 (As shown in E) PpbHLH030 It clustered with homologous proteins from the Rosaceae family and other species, with each branch exhibiting high bootstrap support values. Based on the conserved domains and evolutionary analysis results, it was named... PpbHLH030 The above results indicate that... PpbHLH030 It is located in the cell nucleus and is a typical bHLH transcription factor, which is relatively conserved in evolution.

[0029] Example 2: PpbHLH030 Evaluation of Anthracnose Resistance and Measurement of Physiological Indicators in 'Eggplant-Pear' Callus with Overexpressed Gene Wild-type callus was cultured in a dark place for 15-20 days. Callus used for infection was best when it was white, loose, and glossy. The preserved Agrobacterium bacterial suspension was shaken vigorously at a 1:100 ratio. The next day, the cells were collected by centrifugation at 5000 rpm for 10 minutes. The cells were washed with the prepared infection solution, centrifuged for 10 minutes, and the supernatant was discarded. An appropriate amount of infection solution was added, the cells were dispersed and mixed thoroughly, and the OD was adjusted. 600 = 0.6 ~ 0.8, place in a shaker at 28℃ for 30 min; then use tweezers to pick up wild-type callus and place it in the bacterial solution, gently shake to ensure even soaking, and then place in a shaker at 28℃ for 30 min; prepare an empty tissue culture bottle, line the bottle mouth with sterilized gauze, pour the callus into the gauze to filter out the bacterial solution, then place the transformed callus on filter paper and gently squeeze until semi-dry; place the callus in a pre-prepared symbiotic medium and co-culture for 2 ~ 3 days, then transfer the callus to a selection medium and incubate in the dark for one month. When green fluorescence appears, transfer to a new selection medium, and continue screening until homozygous. Select green fluorescent callus tissue, extract a portion to extract RNA, and use quantitative real-time primers to identify wild-type callus and... PpbHLH030 Gene expression status in overexpression lines. Results are as follows: Figure 2 As shown in Figure A, in the overexpression lines PpbHLH030 The expression was more than 3 times that of the control, among which PpbHLH030-1 The expression levels of strains 4 and 5 were the highest, and their anthrax resistance was evaluated.

[0030] Overexpression callus lines cultured for 15-20 days were transferred to new culture medium and spread evenly with wild-type callus, minimizing gaps between callus. The spread callus was then placed in the dark and allowed to stand for 2-3 days. Holes were punched in the culture medium using a 5 mm punch, and the mycelial side of each callus was then pressed firmly against the center of the spread callus, with the culture medium side facing up. The plates were sealed with sealing film and incubated at 25°C. Photos were taken on days 3, 5, and 7, and the diameter of the lesions was measured using IMAGEJ software.PpbHLH030 The disease resistance of transgenic callus was evaluated. Results were as follows: Figure 2 As shown in Figures B and C, the diameter of callus anthrax lesions in the overexpression line was significantly smaller than that in the control line, and the difference reached a significant level at 5 and 7 days after inoculation. These results indicate that... PpbHLH030 It can significantly enhance the resistance of pear to anthracnose, further confirming its positive regulation of pear anthracnose resistance.

[0031] Wild-type and transgenic callus, subcultured for 15-20 days, were immersed in spore suspension. After 1 minute, the callus was drained and placed in culture medium to stand. Samples were collected at 0, 6, and 24 hours for later use. Physiological parameters of wild-type callus inoculated with anthracnose spore suspension were then measured at various time points. Results are as follows: Figure 2 As shown in D~F, overexpression PpbHLH030 In the callus of the gene, POD activity was significantly higher than that in the control line (e.g., Figure 2 (As shown in D); SOD activity was significantly higher than that of the control group (e.g., D). Figure 2 (As shown in Figure E); the H2O2 content was significantly lower than that of the control group (e.g., ...). Figure 2 (As shown in F).

[0032] Example 3: PpbHLH030 Evaluation and physiological index analysis of resistance to anthracnose in pear fruit with transient gene silencing Agrobacterium-mediated transient transformation of 'Huangguan' pear fruits was performed. Agrobacterium strains containing the empty vectors pTRV1, pTRV2, and PpbHLH030-pTRV2 were suspended in 10 mM MgCl2, 10 mM MES (pH = 5.6), and 100 μM acetylsylgenone, and adjusted to OD0.05. 600 = 0.6 ~ 0.8. A 1:1 mixture of pTRV1 empty vector and pTRV2 empty vector resuspension, and a 1:1 mixture of pTRV1 empty vector and PpbHLH030-pTRV2 resuspension were placed in a 28℃ incubator for 2 h. Sixteen pear fruits of consistent size, color, and health status were selected, washed with water, and dried. The control group received 300 μL of the pTRV1 empty vector and pTRV2 empty vector mixture using a 1 mL syringe, while the experimental group received 300 μL of the pTRV1 empty vector and PpbHLH030-pTRV2 mixture using a 1 mL syringe. Four areas were injected into each fruit. The fruits were placed in 50 cm × 50 cm trays and covered with plastic wrap to maintain moisture. After 5 days of incubation at 25℃, samples were taken and inoculated with the prepared anthracnose spore suspension. The disease incidence was observed daily, and photographs were taken on days 3, 5, and 7 after inoculation. Results are as follows: Figure 3 As shown in Figure A, the silent system PpbHLH030 The expression was significantly lower than that of the unloaded treatment group.

[0033] The anthracnose resistance of transiently silenced pear lines was analyzed using an in vitro puncture inoculation method. A 10 μL pipette tip was vertically inserted into the fruit to a depth of 1 cm, creating a small pore on the fruit surface. Spore suspension was then aspirated and injected into the pore using a pipette. 5 μL of spore suspension was inoculated into each puncture site. After inoculation, the inoculated material was placed in a 28℃ incubator. The methods for photographing and measuring lesion diameter were consistent with the methods for evaluating anthracnose resistance through callus inoculation. Samples were taken at 0, 6, and 24 hours after inoculation for physiological and biochemical parameter determination. Results are as follows: Figure 3 As shown in B and C, silence. PpbHLH030 The diameter of lesions on pear fruits inoculated with the gene was larger than that of the control group at 3, 5, and 7 days after inoculation with pear anthracnose, and the difference reached a significant level at 5 and 7 days. Results of enzyme activity and other index measurements (e.g.) Figure 3 (as shown in D~F) PpbHLH030 The activities of POD and SOD in the fruit of the transiently silenced pear were significantly lower than those in the control group (e.g., Figure 3 (As shown in D and E); while the H2O2 content at 0 h was significantly higher than that of the control group (e.g., ...). Figure 3 (As shown in F).

[0034] Example 4: PpbHLH030 Analysis of JA, flavonoid and lignin content in transgenic callus and pear fruit To explore PpbHLH030 To investigate the regulatory role of jasmonic acid (JA) signaling pathway, this study measured JA content in transgenic lines treated with anthracnose for 0, 6, and 24 h. The results showed that overexpression... PpbHLH030 In the callus of the gene, the JA content was significantly lower than that of the control line at 0 h, but significantly higher than that of the control line at 24 h after inoculation (e.g., Figure 4 (As shown in A). PpbHLH030 The JA content in transiently silenced pear fruits was significantly lower than that in the control line at both 6 and 24 h after inoculation (e.g., Figure 4 (As shown in B).

[0035] To verify PpbHLH030 To further investigate the regulatory effects on the phenylpropane metabolic pathway, this study determined the contents of lignin and flavonoids in the overexpression lines. The results showed that the contents of flavonoids and lignin in the overexpression lines (OE-PpbHLH030-1, OE-PpbHLH030-4, OE-PpbHLH030-5) were significantly higher than those in the control (e.g., OE-PpbHLH030-1, OE-PpbHLH030-4, OE-PpbHLH030-5). Figure 4 As shown in C and D), the OE-PpbHLH030-1 line showed the most significant difference. In contrast, the flavonoid and lignin contents in the silent lines (pTRV-PpbHLH030-1, pTRV-PpbHLH030-2, and pTRV-PpbHLH030-3) were significantly lower than those in the control group (e.g., [reference needed]). Figure 4(As shown in E and F).

[0036] Example 5: Effects of different concentrations of flavonoids on the growth of fruit anthracnose Plate inhibition experiment: Accurately weigh 0.01 g of flavonoid powder, dissolve it in 500 μL of anhydrous ethanol to prepare a flavonoid stock solution of 20 mg / mL. Take 50 μL, 100 μL, and 200 μL of the flavonoid stock solution respectively, add them to three 50 mL bottles of PDA medium, mix well, and make the final flavonoid concentrations 20 mg / L, 40 mg / L, and 80 mg / L, respectively. Add 200 μL of anhydrous ethanol to the control group. Pour the prepared flavonoid-containing PDA medium into petri dishes and allow it to solidify. Use a 5 mm diameter punch to collect mycelial cakes from the edge of pre-cultured *Anthracnose spp.* colonies, and inoculate them in the center of PDA plates with different flavonoid concentrations (medium side facing up), with the mycelial side down. After inoculation, seal the petri dishes with sealing film and incubate at 25℃. Colony morphology was photographed and recorded on days 3 and 5 of culture, and colony diameter was measured using ImageJ software to evaluate the antibacterial activity of flavonoids against *Anthracnose citrinum*. Different concentrations (0, 20, 40, 80 mg / L) of flavonoids (e.g., *Anthracnose citrinum*) were added to PDA plates to evaluate their antibacterial activity. Figure 5 As shown in Figures A and B), the colony diameter was measured after inoculation with *Anthracnose citrinum*. It was found that the antibacterial activity gradually increased with increasing concentration. Specifically, the lesion diameter was significantly smaller than the control group (0 mg / L) at flavonoid concentrations of 20 mg / L, 40 mg / L, and 80 mg / L, indicating that flavonoids have a direct inhibitory effect on the growth of *Anthracnose citrinum*.

[0037] Spore germination experiment: First, pour 20 mL of 2% glucose onto a well-grown anthracnose fungus plate that has been cultured for about 7 days. Scrape the surface hyphae with a triangular stick, then filter the sample through gauze into a tissue culture flask. The OD of the prepared spore suspension is... 600 = 0.6, then 1 mL of spore solution was pipetted into four PCR tubes. 0 μL, 1 μL, 2 μL, and 4 μL of 20 mg / mL flavonoid stock solution were added to each of the four PCR tubes, respectively. 200 μL of spore solution was transferred to a grooved glass slide, placed in a petri dish to maintain humidity, and incubated at 25 ℃ for 12 h. The germination of conidia was observed using a stereomicroscope, and the germination rate was calculated. Results are as follows: Figure 5 As shown in Figures C and D, the conidia in the control group (0 mg / L) germinated normally and produced appressorium; the conidia in the 20 mg / L and 40 mg / L treatment groups were just beginning to germinate; while the conidia in the 80 mg / L treatment group did not germinate at all. These results further confirm that flavonoids can effectively inhibit the germination of conidia of *Anthracnose citrinum*.

[0038] PpbHLH030The gene is derived from Cuiguan Pyrus pyrifolia Cuiguan PpbHLH030 CDS sequence of the gene (SEQ ID NO: 1) ATGGATTATTACAGTTTTAGCTCAAAATCTTCATTTGGGTCGGATTTCTCTAGCTTGTTCGACCCTTTTAAGCATGACGTAAGTGGGTTCGGCGGGGCTTCGCGAAGCGGCGCTGCGGCGAATCTGCCTCACTCTCTTGTCTTGGACAGTGAGAAAGGCGAGCTTGTTAAGGCTCCGGCGAGAGTGGGGAAAAAAGAGGTGCCTGAGGCCAAGGCTTTGTTGGCTTTGAAGAATCACAGTGAGGCTGAAAGGAGGAGGAGAGAGCGAATCAATGCACATCTCTCTACACTTCGCGGTCTGGTGCCCTGCACTGAGAAGATGGACAAAGCCACATTACTTGCAGCAGTTATCAGCCAAGTGAAAGAACTGAAGATGGATGCCCTAGAATCCAGTAAGGGGTTTCTCATCCCAGTTGATGCCGATGAAGTGAAAATTGAACCCTATGTTGCGGCAGGGGATGGAACCATATCTGTGAAGGCATCCGTCTGCTGTGAATACCGGTCTGAACTTCTCTCTGACCTAAGAGAAGCCCTCGACTCCCTCCACTTAAAAATGGTGAAAGCAGAAATAGCGACTTTGGGAAACCGAGTGAAAAATGTATTCGTTCTCACCAGCTCCAAACCAGGAAGCAATGATTCTGATGCTGAAGCATACGAACTTCTTGCAAGTTCGGTTCACCAGGTCCTGAGTTCCGTCCTTGATAAAGCATCGGCCTCACCAGAATACTCGCCAAGAACAACGCTGCTGAGCAAAAGGCGAAGGCTCTCTTACTTCGATAAAATCGATACCTCGAGCTCATCCTCGTGA Cuiguan PpbHLH030 Amino acid sequence encoded by the gene (SEQ ID NO: 2) MDYYSFSSSKSSFGSDFSSLFDPFKHDVSGFGGASRSGAAANLPHSLVLDSEKGELVKAPARVGKKEVPEAKALLALKNHSEAERRRRERINAHLSTLRGLVPCTEKMDKATLLAAVISQVKELKMDALESSKGF LIPVDADEVKIEPYVAAGDGTISVKASVCCEYRSELLSDLREALDSLHLKMVKAEIATLGNRVKNVFVLTSSKPGSNDSDAEAYELLASSVHQVLSSVLDKASASPEYSPRTTLLSKRRRLSYFDKIDTSSSSS The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of pear PpbHLH030 Genes, characterized by, Its CDS sequence is shown in SEQ ID NO:

1.

2. The pear according to claim 1 PpbHLH030 Gene-encoded proteins are characterized by, Its amino acid sequence is shown in SEQ ID NO:

2.

3. The pear according to claim 1 PpbHLH030 The application of genes in enhancing resistance to pear anthracnose is characterized by, By promoting pear PpbHLH030 Gene expression, in turn, promotes the synthesis of lignin and flavonoids to increase pear resistance to anthracnose.

4. A type of pear PpbHLH030 Gene overexpression vector, characterized in that, It uses the pear described in claim 1 PpbHLH030 Genes were obtained by homologous recombination into the PBI121 vector or the pK7WG2D vector.

5. A method for cultivating anthracnose-resistant pear varieties, characterized in that, The pear as described in claim 4 PpbHLH030 Gene overexpression vectors were introduced into target plants to obtain anthracnose-resistant pear germplasm.

6. The cultivation method according to claim 5, characterized in that, Specifically, the following steps are included: (1) Cloning the pear as described in claim 1 PpbHLH030 Gene; (2) Constructing a pear PpbHLH030 Gene overexpression vectors; (3) Pear PpbHLH030 The gene overexpression vector was transformed into the target plant, and the resulting pear germplasm with improved anthracnose resistance was identified.

7. The cultivation method according to claim 5, characterized in that, The method of transformation into the target plant can be either Agrobacterium-mediated transformation or gene gun transformation.

8. The cultivation method according to claim 7, characterized in that, The method for transforming the target plant is Agrobacterium-mediated transformation.

9. The cultivation method according to claim 7, characterized in that, The method for transforming the target plant is the gene gun method.

Images