Application of PbrATL18 in drought and anthracnose resistance genetic improvement of pear

Abstract

The application discloses application of a Pyrus betulaefolia E3 ubiquitin ligase gene PbrATL18 in genetic improvement of plant drought resistance and anthracnose resistance and belongs to the technical field of genetic engineering. The nucleotide sequence of the PbrATL18 gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2. The plant drought resistance and / or anthracnose resistance performance is improved by overexpressing the PbrATL18 gene in the plant or improving the protein activity. The application constructs a gene overexpression Arabidopsis transformation vector and a gene silencing P. betulaefolia seedling transformation vector respectively, obtains positive plant seedlings, and carries out drought and anthracnose treatment. The results show that overexpression of the E3 ubiquitin ligase gene PbrATL18 can effectively maintain the balance of active oxygen in cells, keep the cell osmotic potential stable, and significantly improve the plant drought resistance and anthracnose resistance performance, thereby providing a new idea for high drought tolerance and disease resistance breeding of pear trees.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the PbrATL18 gene, an E3 ubiquitin ligase gene from Pyrus pyrifolia, in the genetic improvement of plant drought resistance and anthracnose. Background Technology

[0002] Pears are widely cultivated globally, and in China, they are the third largest fruit crop after apples and citrus. The planting area is vast, forming a "three-region, four-point" production area layout pattern from Northeast China to Guangxi, Yunnan, and Shandong. Although the pear planting area is extensive and most pear orchards are rationally planned, due to differences in environmental factors across regions, the impact of various natural disasters, and long-term irrational development and utilization by humans, my country's land problems are becoming increasingly serious. This poses a significant threat to agricultural production, food security, and the cultivation of various fruits, vegetables, and ornamental plants, and also exposes the pear industry to the impacts of salinity, drought, frost damage, and floods. Stressful environments are one of the main factors limiting plant growth. Stress is divided into abiotic stress and biotic stress. Among abiotic stresses, drought stress is one of the most harmful and widespread. With continuous population growth and excessive mining activities, soil erosion and desertification are becoming increasingly serious, leading to more and more arable land being abandoned due to drought. Among biotic stresses, fungal infection is a highly harmful one. Fungal hyphae can infect plant roots, stems, leaves, and fruits, causing huge economic losses. For pear crops, anthracnose, caused by Golgiformis, is one of the most widespread fungal diseases. In the Yangtze River basin of China, anthracnose outbreaks occur every summer during periods of high temperature and heavy rainfall, causing significant damage to pear yields. Therefore, there is an urgent need to obtain drought-resistant and disease-resistant pear varieties through breeding.

[0003] In recent years, there has been an increasing number of reports on the use of genetic engineering to enhance resistance to biotic and abiotic stresses. For example, overexpression of NbATL60 and StRFP1 can improve the resistance of potatoes and tobacco to late blight. The Arabidopsis ATL9 protein can be induced by chitin and participates in basal resistance to the biotic fungal pathogen, Arabidopsis powdery mildew. VpRH2 can interact with VpGRP2A (a glycine-rich RNA-binding protein) and plays a positive role in the resistance of grapes to powdery mildew. Arabidopsis ATL78 improves drought resistance by mediating ABA-dependent stomatal closure and regulating ROS homeostasis. IbATL38 is strongly induced by NaCl and ABA; Arabidopsis plants overexpressing IbATL38 can improve salt tolerance by inducing the expression of a series of stress-related genes and reducing H2O2 content. Overexpression of PtXERICO in Arabidopsis improves the plant's ability to resist high temperatures and drought by regulating transpiration.

[0004] However, to date, there have been no reports of obtaining drought-resistant and disease-resistant pear varieties through breeding. Summary of the Invention

[0005] The purpose of this invention is to provide an application of the PbrATL18 E3 ubiquitin ligase gene in *Pyrus pyrifolia* in the genetic improvement of plant drought resistance and anthracnose, in order to solve the problems existing in the prior art. This invention overexpresses the PbrATL18 E3 ubiquitin ligase gene in plants, which can significantly improve the drought resistance and anthracnose resistance of plants, providing a new approach for breeding highly drought-resistant and disease-resistant pear trees.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the application of the Pyrus pyrifolia E3 ubiquitin ligase gene PbrATL18 in improving the drought resistance and / or anthracnose resistance of plants, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] Furthermore, the amino acid sequence of the protein encoded by the PbrATL18 gene of the pear E3 ubiquitin ligase is shown in SEQ ID NO.2.

[0009] Furthermore, by overexpressing the PbrATL18 gene or increasing the activity of the protein in plants, the drought resistance and / or anthracnose resistance of the plants can be improved.

[0010] The present invention also provides a recombinant vector of the Pyrus pyrifolia E3 ubiquitin ligase gene PbrATL18, which is obtained by amplifying the Pyrus pyrifolia E3 ubiquitin ligase gene PbrATL18 with amplification primers and then ligating it into an overexpression vector.

[0011] Furthermore, the nucleotide sequences of the amplification primers are shown in SEQ ID NO.3-4.

[0012] The present invention also provides the application of the recombinant vector as described above in improving the resistance and / or drought tolerance of plants to anthracnose.

[0013] Furthermore, the plant includes Arabidopsis thaliana or Pyrus pyrifolia.

[0014] Furthermore, the anthrax pathogen includes *Colletotrichum fructicola*.

[0015] The present invention discloses the following technical effects:

[0016] This invention utilizes gene-overexpressing Arabidopsis thaliana transformation vectors and gene-silencing *Pyrus pyrifolia* seedling transformation vectors to obtain positive seedlings. These seedlings were then subjected to drought and anthracnose treatments. Results showed that the transgenic Arabidopsis thaliana positive seedlings exhibited significantly lower electrical conductivity, significantly higher chlorophyll content, and a 2-fold increase in seed germination rate and root growth compared to the wild type, indicating that the positive seedlings possess stronger drought tolerance than the wild type. Virus-silenced *Pyrus pyrifolia* seedlings showed higher MDA levels and lower chlorophyll content and electrical conductivity than the wild type. These results indicate that silencing the E3 ubiquitin ligase gene PbrATL18 weakens the plant's drought and anthracnose resistance, while overexpression of the E3 ubiquitin ligase gene PbrATL18 effectively maintains the balance of intracellular reactive oxygen species and stabilizes the cell's osmotic potential, thus enabling the plant to better adapt to drought and anthracnose stress. Therefore, overexpression of the E3 ubiquitin ligase gene PbrATL18 in Pyrus pyrifolia can significantly improve the plant's drought and anthracnose resistance, providing a new approach for breeding highly drought-resistant and disease-resistant pear trees. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the technical process of the present invention;

[0019] Figure 2 The expression patterns of PbrATL18 under different stress treatments: A represents high salt stress; B represents drought stress; and C represents anthracnose stress.

[0020] Figure 3 Identification of Arabidopsis thaliana overexpressing PbrATL18; A is a T0 generation transgenic plant; B is a T1 generation transgenic plant; C is the identification result of the internal control gene Actin;

[0021] Figure 4 A is for the identification of PbrATL18 silenced plants; A shows the detection results of gene-specific primers and internal control primer Tubulin; B is a statistical graph of the expression level of the target gene in wild-type and PbrATL18 silenced plants.

[0022] Figure 5Phenotypic and physiological data of Arabidopsis thaliana overexpressing PbrATL18 under drought treatment; (a) seed germination rate; (b) comparison of root length; (c) growth status; (d) chlorophyll fluorescence imaging; (e)-(f) histochemical staining results of DAB and NBT enzymes; (g) statistical results of seed germination rate; (h) statistical results of root length; (i) statistical results of electrical conductivity; (j) statistical results of Fv / Fm; (k)-(p) MDA, H2O2, and anti O2, respectively. - Statistical charts of POD, CAT and SOD content;

[0023] Figure 6 Phenotypic and physiological data of plants overexpressing PbrATL18 treated with anthracnose; (a) Anthracnose mycelial spread; (b)-(e) Statistical graphs of CAT, PAL, CHI, and PPO content;

[0024] Figure 7 Phenotypic and physiological data of PbrATL18 silent plants under drought treatment; (a)-(b) growth status before and after drought; (c)-(d) chlorophyll fluorescence imaging before and after drought; (e)-(f) histochemical staining results of DAB and NBT enzymes; (g) conductivity statistics; (h)-(n) MDA, Fv / Fm, H2O2, and anti O2, respectively. - Statistical charts of POD, CAT and SOD content;

[0025] Figure 8 Phenotypic and physiological data of anthracnose treatment in PbrATL18 silent plants; (a)-(b) and (e) show the comparison of leaf lesion diameter between the control group and gene-silenced plants; (c)-(d) and (f)-(g) show the comparison of PAL, CHI, CAT and PPO contents, respectively. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] As a widely used rootstock in the pear industry, *Pyrus pyrifolia* exhibits extremely high stress and disease resistance, making it an ideal material for researching drought tolerance and anthracnose resistance in woody plants and for cloning related resistance genes. Therefore, the inventors chose to clone stress and disease resistance-related genes from *Pyrus pyrifolia* for their research on resistance genetic engineering.

[0032] Example 1: Cloning of the full-length cDNA of the PbrATL18 gene, an E3 ubiquitin ligase gene from *Pyrus pyrifolia*.

[0033] An E3 ubiquitin ligase gene, PbrATL18, was screened from *Pyrus pyrifolia*. Primers were designed based on the sequence of the PbrATL18 gene and Primer Premier 5.0. The forward primer sequence was 5'-ATGGGCCGCCCCCTGGAG-3' (SEQ ID NO.3); the reverse primer sequence was 5'-TTACTCCCTCCTAGTGCACTCGG-3' (SEQ ID NO.4). The full-length primer was amplified from *Pyrus pyrifolia* using RT-PCR, and its nucleotide sequence is shown in SEQ ID NO.1.

[0034] ATGGGCCGCCCCCTGGAGCCCGAGCAGATCGAGCAGCCCCACACCGTCCGCCCGCCGCTCGAGCAGTACGGCCCCGCCCCCCAGCAGGGATACTCCCTCAGCGGCAAGATCATGCTCAGCGCCATCGTCATCCTCTTTTTCGTCGTCATCCTCATGGTCTGCCTCCACCTCTACGCCCGCTGGTACCTTCTTCGCGCCCGCCGCCGCCGTCTCCGCCGCAACCACAGAAACCGCCGCACCCACATCGTCTTTCACGAGGACGCCGCTGCCCTGTCCGCCGCCCCCACCCGCGGCCTCGACGCCTCCGTCCTCAACTCCCTCCCCGTATTTGTTTATTCGTCGAAATTGGATCAGATGGCGTTGTTTCAGCAGCAGCCGATTTTGGAGTGCGCCGTCTGCTTGTCGGAATTCGAGGACGACGAAACGGGGCGTTTGCTTCCGAAGTGTAAGCACAGCTTCCACATCGAGTGTATTGATATGTGGTTCCATTCTCACTCCACGTGTCCCCTTTGCCGGGCCCCCGTGGAACTGAGCCCGGAGTCTGAAACCCGGCCCGACGTGCTTCTTAGTGTCTGCGAACCTGACGGAGGCGAACCGGGTCCGCGCTCCGATTTGTGCTCGGAGTGTTGCAATTCCGAGGCGACGTCGTCAGGGGCGTGGAGAAAGCCGTCGAACATCGTGGTTCCGAGAAGGAACGAGAGTTTCGGGAGGGGGGAGGACTCCGGACGCGGCGAATCACCGGCAGGTCAGTCTTTTAGGTCGCCGATGAGTCGGATGTTGTCATTTAGGAGAATACTAAGCCGGGAAAGACGAAACGGCGGCGTTTCACCTTCGGGAGTAAACGCGGGGAGTTGCAGTTCGGTGGCCGAGTCGGATATCGAACTCGGTGGCCGACAAGGGACCACCGAGTGCACTAGGAGGGAGTAA;

[0035] The amino acid sequence of the encoded protein is shown in SEQ ID NO.2:

[0036] MGRPLEPEQIEQPHTVRPPLEQYGPAPQQGYSLSGKIMLSAIVILFFVVILMVCLHLYARWYLLRARRRRLRRNHRNRRTHIVFHEDAAALSAAPTRGLDASVLNSLPVFVYSSKLDQMALFQQQPILECAVCLSEFEDDETGRLLPKCKHSFHI ECIDMWFHSHSTCPLCRAPVELSPESETRPDVLLSVCEPDGGEPGPRSDLCSECCNSEATSSGAWRKPSNIVVPRRNESFGRGEDSGRGESPAGQSFRSPMSRMLSFRRILSRERRNGGVSPSGVNAGSCSSVAESDIELGGRQGTTECTRRE*.

[0037] The detailed steps are as follows: The synthesis of the first strand of cDNA was performed according to the instructions of the TIANGEN reverse transcription kit. The obtained first-strand cDNA was used for amplification of the E3 ubiquitin ligase gene PbrATL18. The total PCR reaction volume was 50 μl, including 1 μl of *Pyrus pyrifolia* cDNA, 2.5 μl each of forward and reverse primers, 1 μl of enzyme, 25 μl of buffer, and 18 μl of sterile dd water. PCR was performed according to the following program: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 58℃ annealing for 90 s, 72℃ extension for 90 s, 35 cycles, followed by a 72℃ extension for 10 min after each cycle. After amplification, a single-band PCR product was generated. After electrophoresis on a 1% agarose gel, the target band was excised and recovered according to the instructions of the gel recovery kit.

[0038] The purified product was ligated into the pEASY-BluntZero vector at a gene-to-vector molar ratio of 3:1. The total reaction volume was 5 μl, consisting of 4.5 μl of the PCR-purified product and 0.5 μl of the vector. Ligation was performed at 25°C for 30 min, followed by transformation into competent E. coli DH5α cells using the heat shock method. PCR verification and sequencing were performed using primers for the target gene sequence (performed by Shanghai Sangon Biotech Co., Ltd.).

[0039] Example 2

[0040] qRT-PCR analysis of the E3 ubiquitin ligase gene PbrATL18 under different abiotic conditions

[0041] To analyze the response pattern of the E3 ubiquitin ligase gene PbrATL18 in *Pyrus pyrifolia* to high salt, drought, and anthracnose treatments, the expression pattern of the E3 ubiquitin ligase gene PbrATL18 was analyzed using Real-time PCR. RNA was extracted using the Plant Total RNA Isolation Kit Plus from Chengdu Fuji Biotechnology Co., Ltd., and the synthesis of the first strand of DNA was performed according to the TANGEN reverse transcription kit manual. The forward primer sequence used for qRT-PCR was 5'-TTTAGGTCGCCGATGAGTCG-3' (SEQ ID NO. 5); the reverse primer sequence was 5'-TACTCCCGAAGGTGAAACGC-3' (SEQ ID NO. 6). The 10 μl reaction mixture contained: 5 μl 2×SYBR PremixExTaq, 0.1 μl cDNA, 0.4 μl primers (with Tubulin as internal control primers (SEQ ID NO. 7 and SEQ ID NO. 8)), and 4.5 μl water. The real-time quantitative PCR program is as follows: pre-denaturation at 95℃ for 3 min, followed by 40 cycles of 95℃ for 3 s, 60℃ for 10 s, 72℃ for 30 s, 95℃ for 5 s, and 65℃ for 1 min.

[0042] See results Figure 2 ,in, Figure 2 A represents the relative expression level of the gene encoding the present invention, which was analyzed by real-time quantitative PCR at corresponding time points in *Pyrus pyrifolia* seedlings (non-transgenic) treated with 200 mM NaCl. Figure 2 B represents the expression pattern of *Pyrus pyrifolia* seedlings under room temperature drought dehydration at different time points; Figure 2 C represents the relative expression levels of the coding gene of *Pyrus pyrifolia* seedlings at corresponding time points after treatment with anthracnose spore suspension. This was analyzed using real-time quantitative PCR. Figure 2 As can be seen from B and 2C, the E3 ubiquitin ligase gene PbrATL18 has a very strong response to drought and anthrax, but a moderate response to salt stress. Figure 1 This is a schematic diagram of the technical process for the E3 ubiquitin ligase gene PbrATL18 encoding gene of the present invention under high salt, drought and anthrax stress.

[0043] Example 3

[0044] Genetic transformation of Arabidopsis thaliana

[0045] 1. Construction of plant transformation vectors

[0046] Based on the multiple cloning site of the PCMBIA1300 vector and the coding region sequence of the PbrATL18 gene, restriction enzyme sites XbaI and BamHI were added. Following general primer design principles, upstream and downstream PCR primers were designed using Primer Primer 5.0 software: upstream primer: 5'-gagaacacgggggactctagaATGGGCCGCCCCCTGGAG-3' (SEQ ID NO. 9) and downstream primer: 5'-gcccttgctcaccatggatccCTCCCTCCTAGTGCACTCGG-3' (SEQ ID NO. 10). PCR amplification was performed using the PbrATL18 gene clone as a template. The annealing temperature for PCR amplification was 58℃, and the PCR reaction system and amplification program were the same as those for the PbrATL18 gene clone. After amplification, gel purification and recovery were performed. The PCMBIA1300 vector double digestion reaction volume was 40 μl, containing: 10 μl of the PCMBIA1300 vector plasmid, 4 μl of 10×M buffer, 1 μl each of XbaI and BamHI, and 24 μl of double-distilled water. After digestion at 37℃ for 3-4 h, the product was purified and recovered. In the ligation reaction system, the molar ratio of PbrATL18 gene to PCMBIA1300 vector was 2:1, with a total reaction volume of 10 μl. This system contained: 1 μl of 10× buffer, 1 μl of DNA recombinase, 4 μl of the double-digested and recovered PbrATL18 gene, 2 μl of the double-digested and recovered PCMBIA1300 vector product, and 2 μl of double-distilled water. The reaction was carried out at 37℃ for 30 min to obtain the ligation product. The ligation product was transformed into *E. coli* DH5α and cultured on LB agar plates containing 50 mg / L kanamycin for 16 h. After selecting positive clones, the cells were shaken to extract plasmids for PCR identification. Sequencing confirmed the absence of coding frame mutations, and a recombinant clone containing the inserted target fragment was obtained. This clone was named p1300-PbrATL18 recombinant vector, and the recombinant vector p1300-PbrATL18 was introduced into Agrobacterium GV3101 using the freeze-thaw method.

[0047] 2. The steps of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana are as follows:

[0048] (1) Agrobacterium culture: Take Agrobacterium tumefaciens bacterial culture stored in an ultra-low temperature freezer, streak it on LB agar plates containing 50 mg / L kanamycin and 50 mg / L rifampin, and incubate at 28℃ for 36-48 hours. Scrape off the streaks and add them to liquid MS medium (2.37 g / L MS + 50 g / L sucrose + 0.1 mg / L IBA, pH = 5.8). Incubate at 28℃ for 30 min with shaking. When the bacterial concentration reaches OD = 0.8-1.0, add 200 μl / L of surfactant sweet77 for inoculation.

[0049] (2) Inoculation: Take wild-type Arabidopsis plants with stems about 10cm tall, remove all seed pods, then place them upside down in a glass bottle containing the prepared Agrobacterium tumefaciens bacterial solution, vacuum the bottle, maintain a pressure of 0.05Mpa for 5min, and place them on their side away from light for 24 hours.

[0050] (3) Cultivation: Cultivate the plants according to conventional methods until they bear fruit and harvest mature seeds (T0 generation).

[0051] 3. Screening of transgenic positive seedlings

[0052] The above method was used to obtain T0 generation Arabidopsis seeds transgenic with the PbrATL18 gene. The surface of the T0 generation seeds was sterilized and they were evenly distributed on MS selective medium containing 50 mg / L hygromycin and 50 mg / L termethin. They were cultured at 22℃ under light for 16 h / d. After one week of growth, plants with fast growth and long root length were selected and transplanted into sterilized nutrient soil for a period of time.

[0053] 3.1 DNA extraction from transgenic Arabidopsis thaliana

[0054] Following the above method, transgenic Arabidopsis thaliana with the PbrATL18 gene was obtained. DNA was extracted from each Arabidopsis thaliana plant, and primers were designed for PCR amplification to identify positive seedlings.

[0055] (1) Take an appropriate amount of Arabidopsis thaliana leaves and grind them into powder with liquid nitrogen. Then add 500 μl of CTAB (100 mmol / L Tris-HCl pH 8.0, 1.5 mmol / L NaCl, 50 mmol / L EDTA pH 8.0, 2% w / v CTAB, fully dissolved in a 65℃ water bath) and 10 μl of β-mercaptoethanol, and mix well.

[0056] (2) Heat in a 65℃ water bath for 30 min, and remove and gently invert every 10 min to mix; centrifuge at room temperature for 10 min at 10000g; take the supernatant, add 500μl of chloroform-isoamyl alcohol (chloroform:isoamyl alcohol volume ratio is 24:1), and invert to mix;

[0057] (3) Centrifuge at 10000g for 10 min, take 450 μl of the supernatant into a new 1.5 ml centrifuge tube, add 450 μl of isopropanol, and mix by inverting the tube.

[0058] (4) Centrifuge at 10000g for 10 min, discard the supernatant, rinse twice with 1 mL of 75% ethanol, centrifuge at 10000g for 10 min to completely remove the ethanol, and place in a clean bench to air dry until the DNA becomes colorless and transparent.

[0059] (5) Add 50 μl of ultrapure water, place in a 65℃ incubator to dissolve for 40 min, and then perform gel detection.

[0060] 3.2 Detection of positive transgenic plants

[0061] PCR amplification was performed using gene-specific primers. The reaction procedures and systems are shown in Tables 1 and 2, respectively. The upstream primer 5'-ATGGGCCGCCCCCTGGAG-3' (SEQ ID NO.11) and the downstream primer 5'-CTCCCTCCTAGTGCACTCGG-3' (SEQ ID NO.12) were used for PCR identification. Lines that amplified fragments of the expected size were considered positive transgenic lines.

[0062] Table 1 PCR reaction procedure

[0063]

[0064]

[0065] Table 2 PCR reaction system

[0066] Reaction components Dosage (μl) Template DNA 1 PCR Buffer 2 dNTD Mix (2.5 mmol / L) 1.6 Right-side forward primer 1 Left-side reverse primer 1 Taq DNA polymerase (5U) 0.2 Nuclease-free water 13.2

[0067] PbrATL18 was expressed in Arabidopsis thaliana via Agrobacterium-mediated transformation. Molecular genetic analysis identified transgenic Arabidopsis with a single-copy homozygous insertion, stably expressing PbrATL18 from generations T1 to T2, thus demonstrating stable inheritance of the phenotypic trait. Insertion site analysis confirmed that the phenotypic changes in the PbrATL18 transgenic materials were not caused by the transgenic manipulation affecting other genes. Therefore, this transgenic material provides material support for the research of this invention. PCR was used to identify T0 generation transgenic plants such as… Figure 3 A, Using semi-quantitative identification of T1 generation transgenic plants such as Figure 3 B and C, OE1 and 7 are two overexpression lines.

[0068] Example 4

[0069] Instantaneous transformation of wild pear seedlings

[0070] 1. Construction of virus-induced gene silencing vector

[0071] The viral silencing vector was constructed according to the method in Example 4. The viral silencing vector pTRV2 had two restriction enzyme sites: XbaI and SmaI. Primers were designed using Primer Primer 5.0 software according to general primer design principles. The upstream primer was 5'-CAAGATCATGCTCAGCGCCA-3' (SEQ ID NO.13); the downstream primer was 5'-GACAAGCAGACGGCGCAC-3' (SEQ ID NO.14). The PbrATL18 gene was amplified and inserted between the two restriction enzyme sites on the vector to obtain the recombinant vector pTRV2-PbrATL18, which was then transformed into Agrobacterium GV3101 competent cells.

[0072] 2. Virus-induced gene silencing in pear seedlings

[0073] (1) Agrobacterium culture: Agrobacterium tumefaciens culture stored in an ultra-low temperature freezer was cultured in LB liquid medium supplemented with kanamycin 50 mg / L and rifampin 50 mg / L at 28°C and 220 rpm for 12 h. The cultured bacterial culture was centrifuged at 6000g for 10 min to collect the bacterial cells. The precipitate was resuspended in infection solution (10 mM MgCl2, 10 mM MES, 200 mM acetylsyl syringone, pH 5.6) until the concentration reached OD = 0.8-1.0;

[0074] (2) Induction of bacterial culture: Place the bacterial culture with adjusted OD value in the dark and induce at room temperature for 4 hours at 100 rpm;

[0075] (3) Pear seedling injection: pTRV1 and pTRV2 bacterial solutions were mixed in a 1:1 ratio as the control group, and pTRV1 and pTRV2-PbrATL18 bacterial solutions were mixed in a 1:1 ratio as the experimental group. The seedlings were injected with pear seedlings that were 45 days old, had the same growth status and good health.

[0076] 3. Identification of virus-induced gene silencing suppression positive vaccines

[0077] After injection, pear seedlings were treated in the dark at room temperature for 12 hours, followed by 5 days of normal culture. RNA was extracted from seedlings of each line from both the control and experimental groups. The RNA structure was verified by gel electrophoresis, and its concentration was determined using Nanodrop (200-1000 ng / µl). The total RNA volume was adjusted to 3 µg before reverse transcription into cDNA. Tubulin from pear was then used as an internal control for amplification. The nucleotide sequence of the Tubulin primers is as follows:

[0078] Tubulin forward primer: 5'-TGGGCTTTGCTCCTCTTAC-3' (SEQ ID NO.7);

[0079] Tubulin reverse primer: 5'-CCTTCGTGCTCATCTTACC-3' (SEQ ID NO.8).

[0080] like Figure 4 The bands amplified by Tubulin were all of uniform brightness, indicating that the concentration of reverse-transcribed cDNA was the same. Then, qRT-PCR was performed using PbrATL18 specific primers and pear internal reference primer Tubulin to analyze the expression level of the test lines. Based on the expression level of the PbrATL18 gene, three plants with lower expression levels were selected as virus silencing positive lines.

[0081] See results Figure 4 As shown, qRT-PCR was used to detect gene expression levels in gene-silenced positive plants using gene-specific primers and the internal reference primer Tubulin. Figure 4 This indicates that the PbrATL18 gene in the positive strains of *Pyrus pyrifolia* seedlings was silenced by the virus.

[0082] Example 5

[0083] 1. Identification of resistance in PbrATL18 transgenic plants

[0084] To determine whether the PbrATL18 transgenic Arabidopsis thaliana is associated with drought stress resistance, both the control and transgenic lines were subjected to short-term and long-term drought stress treatments. Seeds from the same batch of PbrATL18 transgenic lines (OE1 and OE7) and wild-type (WT) Arabidopsis thaliana were sterilized and sown on MS medium. Approximately 3 days after germination, they were transplanted into soil for further cultivation. Transgenic plants of different ages were subjected to drought treatment, and their phenotypes after treatment were observed. Germination rates were recorded, and electrical conductivity, chlorophyll content, and root length were measured.

[0085] When transgenic Arabidopsis seeds were sown on MS medium containing 10% PEG, the germination rate of transgenic Arabidopsis seeds was significantly higher than that of WT (Wheat Flood Control and Sterile Ingredients). Figure 5 a,g). Additionally, transplanting 7-day-old Arabidopsis seedlings onto MS medium containing 10% PEG and culturing them under light for 7 days showed that WT root length was shorter and the plants experienced greater stress. Figure 5 b, h). 14-day-old transgenic Arabidopsis thaliana and WT potted seedlings were subjected to drought treatment. After 21 days, compared to the transgenic line, WT showed weaker growth and more wilted leaves. Figure 5 c). Electrical conductivity and MDA are important indicators for measuring the degree of cell damage. Before drought treatment, there was no significant difference in electrical conductivity and MDA content between WT and transgenic lines. After drought treatment, the electrical conductivity of WT was significantly lower than that of transgenic lines, and the MDA content was significantly higher than that of transgenic lines. Figure 5The values ​​of i,k indicate that drought stress caused more severe damage to WT plants. Chlorophyll fluorescence imaging showed that WT plants suffered greater chlorophyll damage compared to transgenic plants. Figure 5 d). Fv / Fm (optimal / maximum photochemical efficiency) is an important indicator of plant light energy conversion efficiency, and this parameter generally decreases significantly under stress conditions. After drought stress treatment, the Fv / Fm of WT plants was significantly lower than that of transgenic plants ( Figure 5 j) indicates that the light energy conversion efficiency of WT plants was lower after stress. Since ROS homeostasis in plants is an important indicator of plant stress resistance, we performed DAB and NBT enzyme histochemical staining on the leaves of Arabidopsis thaliana potted seedlings after drought treatment. The results are as follows: Figure 5 Figures e and f show that the staining of WT leaves was deeper after treatment. We also measured H2O2 and anti-O2 in the leaves of WT and transgenic plants before and after drought treatment. - The content of (antioxidant anions) is shown in the figure. Figure 5 As can be seen from the data (l,m), under drought treatment, WT leaves accumulated more H2O2 compared to transgenic plant leaves, while the content of antioxidant anions decreased, resulting in reduced anti-O2 content. - With O2 - The negative correlation indicates that more O2 also accumulated in the WT blades. - Compared to the transgenic lines, the WT and transgenic lines suffered greater ROS damage. Considering the important role of antioxidant enzymes in ROS scavenging, we used a kit to detect the POD, CAT, and SOD contents of WT and transgenic plants before and after drought treatment. The results showed that the antioxidant enzyme activities in transgenic plants after drought treatment were significantly higher than those in WT plants. Figure 5 The n, o, p) values ​​indicate that the transgenic plants have a stronger reactive oxygen species (ROS) scavenging capacity compared to WT plants. In conclusion, PbrATL18 can enhance the ROS scavenging capacity of transgenic plants by increasing the activity of antioxidant enzymes, thus protecting the plants from stress damage and improving their drought tolerance.

[0086] 2. Analysis of anthrax resistance in PbrATL18 transgenic pear callus tissue

[0087] To identify the effect of PbrATL18 overexpression on plant resistance to anthracnose, PbrATL18 was overexpressed in pear callus tissue, followed by inoculation with anthracnose fungal cakes. The mycelial spread rate and various physiological indicators were observed to determine the effect of PbrATL18 overexpression on plant resistance to anthracnose. Figure 6As shown in (a), 15 days after inoculation with mycelium, the hyphae of wild-type WT callus culture dish spread throughout the entire dish, while the spread area of ​​pear callus overexpressing PbrATL18 was significantly smaller than that of wild-type WT. Catalase (CAT), chitinase (CHI), phenylalanine ammonia-lyase (PAL), and polyphenol oxidase (PPO) are all physiological indicators that can measure the plant's resistance to pathogens. The results of this study indicate that after anthracnose infection, the contents of CAT, CHI, PAL, and PPO in PbrATL18 overexpressing pear callus were significantly higher than those in the control. Figure 6 (b, c, d, e) , while before infection, the content of each enzyme activity was not significantly different. In conclusion, pear callus overexpressing PbrATL18 is more resistant to anthracnose infection than wild-type WT.

[0088] Example 6

[0089] Identification of resistance in PbrATL18 virus-silenced plants

[0090] 1. Drought resistance analysis of virus-silenced Pyrus pyrifolia seedlings

[0091] To determine whether the gene encoded by PbrATL18 is closely related to drought tolerance in plants, wild-type and virus-silenced positive *Pyrus pyrifolia* seedlings were subjected to drought treatment for a certain period. PbrATL18-silenced plants and controls were treated with drought for 14 days together; the PbrATL18-silenced plants exhibited more severe wilting compared to the control. Figure 7 a, b). The results of conductivity and MDA content tests indicated that PbrATL18-silenced plant cells suffered greater cell damage. Figure 7 g, h). Under the chlorophyll fluorescence imaging system, it was clearly observed that the chlorophyll in the PbrATL18 silent plants was significantly damaged ( Figure 7 c, d), correspondingly, the Fv / Fm of PbrATL18 silent plants was significantly lower than that of the control ( Figure 7 i). Histochemical staining and physiological data testing showed that the H2O2 content and O2 content in PbrATL18 silenced plants were significantly reduced. - The content was significantly higher than the control after drought treatment. Figure 7 e, f, j, k). Further analysis of the activities of antioxidant enzymes POD, CAT, and SOD in PbrATL18-silenced plants and control plants showed that the contents of these enzymes in PbrATL18-silenced plants were significantly lower than those in the control. Figure 7 In summary, *Pyrus pyrifolia* silencing PbrATL18 via VIGS showed greater sensitivity to drought stress and lower activity of antioxidant enzymes compared to the control.

[0092] 2. Analysis of anthracnose resistance in virus-silenced pear seedlings

[0093] To investigate the function of PbrATL18 in combating anthracnose, we used sterile needles to puncture detached leaves from PbrATL18-silenced plants and controls, followed by spraying with anthracnose spore suspension. The leaves were then incubated in the dark at 25°C for 5 days. The results showed that the diameter of lesions on the leaves of PbrATL18-silenced plants was significantly larger than that of the control. Figure 8 a, b, e). Catalase (CAT), chitinase (CHI), phenylalanine ammonia-lyase (PAL), and polyphenol oxidase (PPO) are all physiological indicators that can measure the plant's resistance to pathogens. The results of this study indicate that after anthracnose infection, the contents of CAT, CHI, PAL, and PPO in the leaves of PbrATL18 silent plants were significantly lower than those in the control. Figure 8 (c, d, f, g) Before infection, the contents of PAL and CHI were not significantly different. The contents of CAT and PPO in the control group were significantly higher than those in the PbrATL18 silenced plants before treatment, but the total contents were lower. In conclusion, Pear trees silenced with VIGS were more susceptible to anthracnose infection than the control.

[0094] Analysis of the above results shows that the PbrATL18 gene is closely related to plant drought resistance and anthracnose. Overexpression of the PbrATL18 gene can effectively enhance the reactive oxygen species scavenging capacity of transgenic plants, maintain intracellular ion balance and osmotic potential homeostasis, thereby improving the plant's drought resistance and anthracnose resistance.

[0095] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A use of a pear E3 ubiquitin ligase gene PbrATL18 in improving drought and / or anthracnose resistance of a plant, characterized in that, The nucleotide sequence of the PbrATL18 E3 ubiquitin ligase gene of Pyrus betulaefolia is shown as SEQ ID NO.

1. The plant is Arabidopsis thaliana or Pyrus betulaefolia.

2. Use according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the PbrATL18 E3 ubiquitin ligase gene of Pyrus betulaefolia is shown as SEQ ID NO.

2.

3. Use according to claim 1, characterized in that, The drought resistance and / or anthracnose resistance of the plant is improved by overexpressing the PbrATL18 E3 ubiquitin ligase gene of Pyrus betulaefolia in the plant.

4. The use of a recombinant vector of Pyrus betulaefolia E3 ubiquitin ligase gene PbrATL18 in improving the resistance of plants to anthracnose bacteria and / or drought tolerance, characterized in that, The recombinant vector is obtained by amplifying the PbrATL18 E3 ubiquitin ligase gene of Pyrus betulaefolia in claim 1 by using the amplification primer, and then inserting it into an overexpression vector. The plant is Arabidopsis thaliana or Pyrus betulaefolia.

5. Use according to claim 4, characterized in that, The nucleotide sequence of the amplification primer is shown as SEQ ID NO. 3-4.

6. Use according to claim 4, characterized in that, The anthracnose pathogen is Colletotrichum gloeosporioides.

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