Pyrus pyrifolia anti-wheel disease related protein, its coding gene and application

Abstract

This invention discloses a pear resistance to ring rot disease-related protein, its encoding gene, and its applications. The amino acid sequence of the pear resistance to ring rot disease-related protein is shown in SEQ ID NO:1. The gene encoding the pear resistance to ring rot disease-related protein, PbrTLP15, has a nucleotide sequence shown in SEQ ID NO:2. Overexpression of PbrTLP15 enhances the plant's resistance to *B. dothidea*. Virus-induced gene silencing (VIGS) revealed that silencing PbrTLP15 reduces the resistance of pear plants to *B. dothidea*. These results all indicate that PbrTLP15 possesses anti-*B. dothidea* activity. This invention provides a theoretical basis for studying the role of pear PbrTLP15 in resistance to ring rot and exploring the molecular mechanism of pear disease resistance, and also provides new insights and resources for breeding pear varieties resistant to ring rot.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to a pear resistance to ring rot disease-related protein, its encoding gene, and its applications. Background Technology

[0002] In eukaryotes, thaumatin-like proteins (TLPs) belong to the PR5 (Pathogenesis-Related Proteins 5) family. They are highly conserved and functionally diverse proteins involved in multiple processes, such as host protection, abiotic stress responses, and signal transduction. Studies have shown that TLPs are involved in various abiotic stresses, including cold, salt, drought, and pathogen infection. Kuwabara et al. (2002) found that in wheat (Triticumaestivum L.), treatment with cold stress, ABA, and Fusarium oxysporum rapidly increased the expression of TaTLPs, and transgenic lines showed improved resistance to pink snow mold (Microdochium nivale). In tobacco, TLP overexpression promoted transgenic lines to exhibit stronger cold, salt, and drought resistance. Munis et al. (2010) also found that TLP overexpression in tobacco significantly enhanced resistance to salt and drought stress, and also significantly improved resistance to Verticillium wilt. These results all indicate that TLP plays an important role in both biotic and abiotic stress processes in plants.

[0003] Pear is one of the world's oldest economically important fruit crops, cultivated on six continents, and is the third most important fruit variety. In production, pears are frequently affected by various diseases, one of which is pear ring rot. Pear ring rot is a necrotic fungal disease caused by *Botryosphaeria dothidea* (B. dothidea). Studies have shown that ring rot caused by *B. dothidea* can lead to a 25% reduction in pear yield. However, to date, little is known about TLPs in white pears. Therefore, based on genomic and transcriptomic data from white pears, this study identified the key gene *PbrTLP15*, which responds to pear ring rot, and determined its function in *B. dothidea* resistance. The results of this work will contribute to elucidating the biological functions of TLPs and the mechanisms by which they contribute to pear ring rot resistance. Summary of the Invention

[0004] The purpose of this invention is to provide a novel pear resistance to ring rot disease-related protein.

[0005] Another objective of this invention is to provide the encoding gene for the pear's resistance to ring rot-related protein and a recombinant plant expression vector.

[0006] Another object of the present invention is to provide the application of the encoding gene and the recombinant plant expression vector.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] A pear resistance to ring rot-related protein, the amino acid sequence of which is shown in SEQ ID NO:1.

[0009] The gene PbrTLP15, which encodes the pear resistance to ring rot-related protein, has the nucleotide sequence shown in SEQ ID NO:2.

[0010] A recombinant plant expression vector containing the gene PbrTLP15.

[0011] As a preferred embodiment of the present invention, the recombinant plant expression vector is obtained by inserting the gene PbrTLP15 into the XbaI and BamHI sites of the plant expression vector pCAM1300-GFP.

[0012] The application of the gene PbrTLP15 described in this invention in the construction of disease-resistant transgenic plants.

[0013] The application of the recombinant plant expression vector described in this invention in the construction of disease-resistant transgenic plants.

[0014] As a preferred embodiment of the present invention, the plant is a herbaceous plant or a woody plant.

[0015] As a further preferred embodiment of the present invention, the herbaceous plant is Arabidopsis thaliana.

[0016] As a further preferred embodiment of the present invention, the woody plant is pear callus or pear leaf.

[0017] Beneficial effects:

[0018] This invention provides a pear disease resistance-related protein, the amino acid sequence of which is shown in SEQ ID NO:1. This invention utilizes Agrobacterium-mediated genetic transformation to transform the model plants Arabidopsis thaliana and pear callus, overexpressing the PbrTLP15 gene in both Arabidopsis and pear callus. Biological functional verification of the obtained transgenic lines revealed that plants overexpressing PbrTLP15 exhibited significantly enhanced resistance to *B. dothidea*, indicating that the gene provided by this invention and its corresponding encoding gene, PbrTLP15, have the function of regulating plant disease resistance. Conversely, transient silencing of PbrTLP15 in pear seedlings using the virus-induced gene silencing (VIGS) method resulted in PbrTLP15-silenced lines exhibiting decreased disease resistance compared to the wild-type control. In conclusion, the presence of PbrTLP15 and its corresponding encoding gene has a biological function of enhancing plant disease resistance. Attached Figure Description

[0019] Figure 1 These are the symptoms of ring rot disease on pear leaves, fruits, and branches in pear orchards.

[0020] Figure 2 PbrTLPs protein sequence alignment. Blue shading indicates identical amino acids. Asterisks indicate five conserved amino acid positions.

[0021] Figure 3 PbrTLPs gene structure analysis.

[0022] Figure 4 Phylogenetic analysis and collinearity analysis of PbrTLPs. (A): Phylogenetic analysis of TLP proteins from six Rosaceae plants: white pear, apple, strawberry, plum, peach, and European pear. (B): Chromosomal localization and collinearity analysis of PbrTLPs.

[0023] Figure 5 PbrTLP15 expression pattern and subcellular localization.

[0024] (A): Expression patterns of PbrTLP15 at different time points after B. dothidea inoculation. (B): Expression patterns of PbrTLP15 in different tissue sites. (C): Subcellular localization of PbrTLP15. Tobacco epidermal cells were transiently transformed with the fusion plasmid 35S::PbrTLP15-GFP driven by the CaMV 35S promoter. AtHSBP, a cytoplasmic marker, was co-expressed with PbrTLP15 transfection in tobacco. DAPI is a nuclear marker. Bars = 50 μm. Data were analyzed using Duncan's multiple range test in SPSS (IBM SPSS22, Chicago, IL, USA) using ANOVA. Vertical bars represent standard deviation, and different letters indicate p < 0.05. Figure 6 PbrTLP15 overexpression in Arabidopsis and its response to *B. dothidea*. (A): Phenotypes of transgenic Arabidopsis (OE) and WT overexpressing PbrTLP15 after *B. dothidea* inoculation. *B. dothidea* was inoculated with rosette leaves of 30-day-old *Arabidopsis*. (BD): Expression patterns of PbrTLP15 in WT and OE *Arabidopsis*. DNA-PCR (B), M: DNA marker 2k (Sangon Biotech, B500350-0500); wild-type *Arabidopsis*; P: plasmid control; OE-1 and OE-2: PbrTLP15 overexpressing transgenic lines. The overexpression empty vector (OE) was driven by the CaMV 35S promoter. qRT-PCR assay (C) measured the transcriptional level of PbrTLP15 in transgenic *Arabidopsis*. Anti-GFP immunoassay (D). Immunoprecipitation of PbrTLP15-GFP protein using anti-GFP magnetic beads. (E): Chlorophyll content determination in WT and transgenic Arabidopsis thaliana after inoculation. (F): Callose content determination in transgenic Arabidopsis thaliana (OE) and WT after B. dothidea inoculation. Data were analyzed using Duncan's multiple range test in SPSS (IBM SPSS22, Chicago, IL, USA) ANOVA. Different lowercase letters indicate statistical significance (p<0.05, ANOVA). (GI): Leaves of transgenic Arabidopsis thaliana (OE) and WT after B. dothidea inoculation were stained with DAB (G), NBT (H), and trypan blue (I), respectively. For each treatment, leaves from three Arabidopsis thaliana plants were pooled, and then the pooled leaves were divided into three replicates. Figure 7 Overexpression of PbrTLP15 in pear callus and its response to B. dothidea.

[0025] (A): Phenotype of transgenic pear callus (OE) and wild-type (WT) callus after inoculation with *B. dothidea*. (B, C): Expression analysis of PbrTLP15 in WT and OE. DNA-PCR (B), M: DNA marker 2k (Sangon Biotech, B500350-0500); wild-type pear callus; P: plasmid control; OE-1 and OE-7: PbrTLP15 transgenic lines, overexpressing the empty vector (OE) driven by the CaMV 35S promoter; qRT-PCR (C) detection of PbrTLP15 transcription in transgenic pear callus. (D): Diameter of lesions in transgenic pear callus (OE) and WT after inoculation. (EG): Images of OE and WT after inoculation with DAB staining (E), NBT staining (F), and trypan blue staining (G), respectively. (HL): H2O2 content (H), O2- content (I), MDA content (J), POD concentration (K), and SOD activity (L) were determined in transgenic mustard (OE) and wild-type (WT) plants, respectively. Data were analyzed using Duncan's multiple range test in SPSS (IBM SPSS 22, Chicago, IL, USA) using ANOVA. Vertical bars represent standard deviations, and different letters indicate p < 0.05.

[0026] Figure 8 Silencing of PbrTLP15 reduced resistance of pear to *B. dothidea*. (A): Phenotypes of pTRV2 and pTRV2-PbrTP15 6 days after *B. dothidea* inoculation (6 dpi). (B): Relative expression levels of PbrTP15 in pTRV2 and pTRV2-PbrTP15 plants. (C): Diameter of lesions of pTRV2 and pTRV2-PbrTLP15 6 dpi after *B. dothidea* inoculation with *B. dothidea*. (DI): H2O2 content (D), O2- content (E), MDA content (F), POD activity (G), SOD activity (H), and callose content (I) of pTRV2 and pTRV2-PbrTLP15 after inoculation and without inoculation. (J): Expression of key genes SA-, JA-, and ET-. Data were analyzed using Duncan's multiple range test in SPSS (IBM SPSS 22, Chicago, IL, USA) ANOVA program. Vertical bars represent standard deviations, and different letters indicate p < 0.05. Detailed Implementation

[0027] Example 1

[0028] This invention first observed and recorded the effects of *Rhizoctonia solani* infection on pear leaves, fruits, and branches in the 'Hushu' pear resource nursery of Nanjing Agricultural University (see...). Figure 1 ).

[0029] This invention identified a class of disease-resistant proteins in the differentially expressed transcriptome data of a pear variety resistant to ring rot infected with B. dothidea (R-532). These proteins were then identified and analyzed using a whole-genome family (see...). Figure 2 Transcriptome data of PbrTLPs family members (see Table 3) were used to screen and obtain the disease resistance protein gene PbrTLP15, which showed the most significant differential gene expression in response to *Rhizoctonia solani*.

[0030] Example 2: Isolation, cloning, and construction of the overexpression vector for the PbrTLP15 gene

[0031] 0.1g of mature leaves of Dangshan pear infected with *Rhizoctonia solani* were taken and reverse transcribed using a One-step gDNA removal and cDNA synthesis kit (Transgen, China), following the manufacturer's instructions. Based on the multiple cloning site of the pCAMBIA-1300 vector and the restriction enzyme sites in the coding region of the PbrTLP15 gene, Xba I and BamHI were selected as restriction enzymes. Following general primer design principles, specific primers with restriction enzyme sites, PbrTLP15-GFP-F and PbrTLP15-GFP-R (see Table 1), were designed using DNAMAN 6.0 software. A 50μL reaction mixture included 200ng cDNA, 1× buffer (GXL Buffer), 0.2μM dNTPs, 1.25U GXL polymerase (GXL DNA Polymerase) (the aforementioned buffer and GXL DNA polymerase were purchased from TaKaRa), and 0.2μM of the aforementioned primers. The PCR reaction was performed on an Eppendorf amplification instrument according to the following program: 98°C pre-denaturation for 3 minutes, 98°C denaturation for 15 seconds, 57°C annealing for 15 seconds, 72°C extension for 90 seconds, 35 thermal cycles, 72°C extension for 10 minutes, and storage at 4°C.

[0032] After PCR products were detected by 1% agarose gel electrophoresis, DNA fragments were recovered using a gel extraction kit (purchased from Vazyme, following the instructions provided). The total volume of the pCAMBIA1300 vector double digestion system was 50 μL, containing 10 μL of pCAMBIA1300 vector plasmid obtained from plasmid extraction, 5 μL of 10×Buffer (purchased from NEB), 1 μL of Xba I, 1 μL of BamHI, and 33 μL of water. Digestion was performed at 37°C for 4 h, followed by recovery. The restriction endonuclease-digested expression vector pCAMBIA1300 and the recovered PbrTLP15 gene were ligated at 37°C for 30 min using recombinase Exnase II (purchased from Vazyme). The reaction system consisted of 4 μL of 5×CE II Buffer, 2 μL of Exnase II, 2 μL of the PCR product recovered from the PbrTLP15 gene, 6 μL of the double digestion product recovered from the pCAMBIA1300 vector, and 6 μL of water. 10 μL of the ligation product was transformed into competent DH5α cells of *E. coli* using a heat shock method. After incubation on LB agar plates containing 50 mg / L kanamycin at 37°C for 14 h, positive clones were screened. Plasmids were extracted, digested, and identified by PCR. The recombinant plasmid samples were sent to a biotechnology company for sequencing. Sequencing results showed that the full-length CDS of PbrTLP15 was 759 bp, and its nucleotide sequence is shown in SEQ ID NO.1, encoding a protein composed of 252 amino acid residues, as shown in SEQ ID NO.2. We named the recombinant vector 35S-PbrTLP15-GFP and introduced it into *Agrobacterium* GV3101 cells using a freeze-thaw method.

[0033] Example 3: Analysis of PbrTLP15 gene expression pattern and cellular localization

[0034] Samples from different tissue parts of Dangshan pear were collected from the Hushu Pear Orchard of Nanjing Agricultural University. Simultaneously, the expression pattern of the PbrTLP15 gene at different infection times after inoculation of Dangshan pear with *Rhizoctonia solani* was analyzed to verify the correlation between the obtained gene and pear resistance to *Rhizoctonia solani*. Figure 5Total RNA was extracted using the CTAB method, and the quality of the extracted samples was assessed by UV spectrophotometry and agarose gel electrophoresis. 1 μg of the extracted total RNA was reverse transcribed using a One-step gDNA removal and cDNA synthesis kit (Transgen, China), following the manufacturer's instructions. Quantitative PCR was performed on a Roche 480 PCR instrument using a quantitative PCR kit (purchased from Roche). The specific primers used were qPbrTLP15-F and qPbrTLP15-R; Tubulin was used as an internal control gene. The reaction mixture consisted of 10 μL of 2×SYBR GreenI Master Mix, 2 μL of cDNA, 4 μL of forward and reverse primers (100 μM), and 4 μL of RNase-free water. The reaction conditions were: denaturation at 95°C for 5 min; pre-denaturation at 95°C for 5 s, annealing at 60°C for 72°C, and extension for 10 s, repeated for 45 cycles.

[0035] Agrobacterium-mediated transformation was used to transform 30-day-old Nicotiana benthamiana plants. Agrobacterium containing 35S-PbrTLP15-GFP was injected into the leaves of 30-day-old Nicotiana benthamiana plants. The injected tobacco plants were first cultured in the dark for 24 hours, followed by normal culture (16 hours light / 8 hours dark) for 1-2 days. Fluorescence expression was observed using a laser scanning confocal microscope (Zeiss LSM 780). This experiment was performed in triplicate.

[0036] Example 4

[0037] Transient silencing of pear seedlings was achieved using virus-induced gene silencing (VIGS). PbrTLP15-silenced pear plants were screened and inoculated with *Rhizoctonia solani*, and the phenotypic characteristics of infected pear leaves were observed (see [link to article]). Figure 8 The specific method is as follows: 1. Construct the pTRV2-PbrTLP15 silencing vector using gene-specific primers pTRV2-PbrTLP15-F and pTRV2-PbrTLP15-R, following the steps for overexpression vectors described above; 2. Activate Agrobacterium containing the correct plasmid on solid culture medium and grow it in an incubator at 28℃ for 48 hours; 3. Add 30 mL of culture medium containing R to a 100 mL Erlenmeyer flask. + With K +1. Activated Agrobacterium was added to liquid LB medium using a pipette tip and grown in a shaker at 28℃ and 200 rpm for 12 h. 2. All bacterial cells were collected by centrifugation at 6000 rpm for 15 min using 50 mL centrifuge tubes. 3. The collected bacterial cells were resuspended in induction medium (10 mM MgCl2, 10 mM MES, 200 mM acetylsylcholine, pH 5.6), and the OD value of the bacterial solution was adjusted to 0.8-1.2. Induction was performed at room temperature for 4 h. 4. Agrobacterium transformation was performed on pear seedlings that had grown for 45 days, with at least 9 seedlings injected each time. The experiment was performed in triplicate. 5. The seedlings were first cultured in the dark for 24 h in an artificial climate chamber at 25℃, and then cultured normally (16 h light / 8 h dark) for 7-10 days. Finally, the leaves of the silenced pear plants were inoculated with Aristolochia debilis, and the relevant phenotypes were observed and recorded after 6 days.

[0038] Example 5

[0039] Agrobacterium-mediated transformation was used to transfer the constructed Agrobacterium carrying the target gene into desired plants, including Arabidopsis thaliana, pear seedlings, and pear callus. For Arabidopsis thaliana overexpressing the gene, we used the flower-dip method for introduction, as follows: 1. In a solution containing 50 mg / L K... + and 100mg / LR + 1. Streak the bacteria on double-antibiotic solid LB medium and incubate statically at 28°C for 48 hours to activate Agrobacterium; 2. Pick up a single colony from the streak using a sterilized pipette tip and place it in a container containing 50 mg / L of antibiotics. + and 100mg / LR + 1. Incubate in Erlenmeyer flasks of liquid LB medium at 28°C and 200 rpm for 12 h; 2. Collect bacterial cells by centrifugation at 5000 rpm for 2 min in 50 mL centrifuge tubes; 3. Resuspend the collected bacterial cells in an equal volume of transformation medium (1 / 2 MS; 5% sucrose (W / V); 10 μg / L 6-BA; pH adjusted to 5.7 with KOH; 0.025% surfactant (V / V)); 4. Immerse the unopened inflorescences of Arabidopsis thaliana to be transformed in the transformation medium containing bacterial cells for 2 min; 5. Incubate in a 22°C incubator in the dark for 24 h, then place under 22°C and long-day conditions (16 h light / 8 h dark). T3 generation transgenic plants were obtained after three rounds of resistance screening (750 mg / ml cephalosporin and 200 mg / ml hygromycin). The transgenic plants were then inoculated with *Rhizoctonia solani* and the related phenotypic observations were performed (see...). Figure 6Agrobacterium-mediated transformation was used to soak pear callus tissue overexpressing the gene. The resuspension of bacteria obtained in step 4 was used to transform the callus. The pear callus tissue was infected at 25°C and 120 rpm for 15 min. The infected pear callus tissue was then transferred to MS solid medium containing 200 mg / ml acetylsylcholine and cultured in the dark at 25°C for 24 h. Finally, the callus tissue was evenly spread onto MS fixation and selection medium containing 750 mg / ml cephalosporin and 100 mg / ml hygromycin. Transgenic lines were obtained through resistance screening and DNA and RNA level verification. The transgenic lines were inoculated with *Rhizoctonia solani*, and the treated materials were subjected to relevant phenotypic observations (see...). Figure 7 The results showed that plants overexpressing PbrTLP15 exhibited significantly enhanced resistance to *B. dothidea*, indicating that the gene provided in this invention and its corresponding encoding gene, PbrTLP15, have the function of regulating plant disease resistance. Conversely, transient silencing of PbrTLP15 in pear seedlings using the virus-induced gene silencing (VIGS) method resulted in PbrTLP15-silenced lines, which, upon biological function verification, showed decreased disease resistance compared to the wild-type control. This demonstrates that the presence of PbrTLP15 and its corresponding encoding gene has a biological function of enhancing plant disease resistance.

[0040] Table 1 Primers involved in this study

[0041]

[0042]

[0043] Table 2 Basic physicochemical properties of PbrTLPs

[0044]

[0045]

[0046]

[0047] Table 3. PbrTLP transcriptome data of pear leaves inoculated with *Rhizoctonia solani*

[0048]

[0049]

[0050] Notes:0:Control;1,2,3and4 indicate different times(1.5,3,4.5,and 6d,respectively)following inoculation with B.dothidea.

[0051] Note: 0: control; 1, 2, 3, and 4 represent gene expression levels after 1.5, 3, 4.5, and 6 days of inoculation with *Rhizoctonia solani*, respectively.

Claims

1. A pear resistance to ring rot-related protein, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. The gene PbrTLP15 encoding the pear resistance-to-ring rot-related protein as described in claim 1, characterized in that... The nucleotide sequence of the gene PbrTLP15 is shown in SEQ ID NO:

2.

3. A recombinant plant expression vector comprising the gene PbrTLP15 as described in claim 2.

4. The recombinant plant expression vector according to claim 3, characterized in that, The recombinant plant expression vector was obtained by inserting the gene PbrTLP15 described in claim 2 into the XbaI and BamHI sites of the plant expression vector pCAM1300-GFP.

5. The recombinant plant expression vector according to claim 3, characterized in that, The recombinant plant expression vector was obtained by inserting the gene PbrTLP15 of claim 2 between the EcoR1 and BamHI sites in the plant expression vector pTRV2.

6. The application of the gene PbrTLP15 as described in claim 2 in the construction of disease-resistant transgenic plants.

7. The use of the recombinant plant expression vector according to any one of claims 3 to 5 in the construction of disease-resistant transgenic plants.

8. The application according to claim 7, characterized in that, The plant is either a herbaceous plant or a woody plant.

9. The application according to claim 7, characterized in that, The herbaceous plant in question is Arabidopsis thaliana.

10. The application according to claim 7, characterized in that, The woody plant in question is pear callus or pear leaves.

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