Application of compound in improving resistance of lily to gray mold

Abstract

The invention discloses application of a compound to improvement of resistance of lily to gray mold. The compound is any one of the following: (1) a sequence for silencing the LhNCED3 gene in lily; and (2) a sequence for knocking out the LhNCED3 gene in the lily. According to the invention, a gene for negatively regulating and controlling the gray mold resistance of lilies is found through research, the LhNCED3 gene is obtained by cloning from 'Sobang' lily leaves, the function of the LhNCED3 gene is analyzed through a virus-induced gene silencing technology, the lily gray mold can be effectively regulated and controlled by applying the LhNCED3 gene, and an important candidate gene is provided for molecular breeding of the gray mold resistance of the lilies.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to the application of a compound in improving the resistance of lily to gray mold. Background Art

[0002] lily( Lilium spp.) are bulbous flowers. However, the pathogenic fungus Botrytis cinerea ( Botrytis elliptica ) is the most common disease in lily cultivation, which seriously affects cut flower production and bulb breeding, and restricts the promotion and application of varieties.

[0003] The pathogenic fungus, Botrytis cinerea, is a typical saprophytic pathogen with a relatively narrow host range, limited to plants of the genus Lilium in the Liliaceae family. It primarily harms lily leaves, stems, and floral organs. It can also infect underground organs, causing bulb rot. The occurrence of gray mold is closely related to environmental temperature and humidity. Botrytis cinerea is most active when temperatures are between 20 and 23°C and relative humidity is above 90%, favoring spore production and disease spread. Waterlogging, excessive planting density, and poor ventilation can also accelerate disease outbreaks. Botrytis cinerea rapidly reproduces and spreads by feeding on dead host tissue. When the host plant's nutrients are depleted, hyphae aggregate to form hard, black sclerotia, which serve as its primary source of infection. Under suitable conditions, sclerotia in the soil or on diseased plant debris produce large numbers of conidia, which are then spread by wind, rain, and other external forces, leading to further infection.

[0004] Currently, production mainly relies on chemical agents to control lily gray mold. However, these agents cannot completely inhibit the pathogenic fungus, and the pathogenic fungus is prone to developing disease resistance.

[0005] In terms of biological control, green control technology is of great significance for the efficient, high-quality and safe production of lilies, but these measures have not been widely used in actual production. For example, the invention application with publication number CN1926995A discloses a microbial anti-lily gray mold agent and its preparation method and application, which discloses a Streptomyces luteus (Gamboge) that can be used to control gray mold in lilies. Streptomyces luteogriseus )ECO 00001.

[0006] Production practice has proved that the application of disease-resistant varieties is the most economical, effective and environmentally friendly disease prevention and control measure, and the screening of highly resistant germplasm resources is an important way to breed disease-resistant lily varieties.

[0007] In recent years, with the widespread application of high-throughput sequencing technology, the discovery of genes related to the gray mold resistance signaling pathway in lily has achieved fruitful results. Many studies are of great significance for the storage of disease-resistant genes and the cultivation of highly resistant germplasm. Research has not yet touched upon the precise positioning and cloning of disease-resistant genes and the functional verification, which are the basis for analyzing the molecular mechanism of plant disease resistance. This part of work needs to be carried out in depth. The mechanism of lily resistance to gray mold is still unknown. In-depth analysis of the molecular mechanism of disease resistance and the discovery of disease-resistant genes are important breakthroughs in the cultivation of disease-resistant germplasm (Li Xinran, Wu Yun, Ren Ziming, et al. Research progress on gray mold in lily [J]. China Plant Protection Guide, 2024, 44(04): 20-27.). Summary of the Invention

[0008] In view of the above-mentioned deficiencies in the prior art, the present invention provides an application of a compound in improving the resistance of lily to gray mold.

[0009] The present invention first provides the use of a compound in improving the resistance of lily to gray mold, wherein the compound is any one of the following: (1) Used to separate lily LhNCED3 gene silencing sequences; (2) Used to separate lily LhNCED3 Gene knockout sequence.

[0010] Preferably, lily LhNCED3 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0011] More preferably, lily LhNCED3 The promoter sequence of the gene is shown in SEQ ID NO.2.

[0012] Preferably, the compound is used for LhNCED3 The sequence of gene silencing, the sequence of the specific fragment used for gene silencing is shown in SEQ ID NO.3.

[0013] During gene silencing, the specific fragment of gene silencing is cloned into a plant expression vector and transferred into lily using Agrobacterium to make the lily LhNCED3 Gene silencing.

[0014] The present invention also provides a method for improving the resistance of lily to gray mold, wherein LhNCED3 Gene silencing or knockout.

[0015] Preferably, lily LhNCED3 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0016] The present invention also provides a method for screening lilies with high resistance to gray mold, by detecting LhNCED3 The lower the expression amount of the gene is, the higher the resistance of the lily plant to botrytis blight is.

[0017] The application finds a gene for negatively regulating the resistance of lily to botrytis blight, and the gene is cloned from leaves of 'Sorbonne' lily. LhNCED3 The application analyzes the function of the gene through virus-induced gene silencing (VIGS) technology, and the gene can be used for effectively regulating the botrytis blight of lily, thereby providing an important candidate gene for molecular breeding of lily resistance to botrytis blight. BRIEF DESCRIPTION OF DRAWINGS

[0018] 图1 For LhNCED3 Expression analysis of the gene in different tissues of 'Sorbonne' lily, and significant difference comparison is compared with the expression level in the scale, **P<0.01.

[0019] 图2 For LhNCED3 Expression analysis of the gene in different resistance lilies inoculated with botrytis elliptica, wherein, B. elliptica inoculated (‘Sorbonne’) indicates that the high resistance lily variety 'Sorbonne' is inoculated with botrytis elliptica; Mock (‘Sorbonne’) indicates that the high resistance lily variety 'Sorbonne' is not inoculated with botrytis elliptica as a control; B. elliptica inoculated (‘Tresor’) indicates that the high sensitive lily variety 'Tresor' is inoculated with botrytis elliptica; Mock (‘Tresor’) indicates that the high sensitive lily variety 'Tresor' is not inoculated with botrytis elliptica as a control; *P<0.05, **P<0.01.

[0020] 图3 For LhNCED3 Expression analysis of the gene treated with exogenous hormones ABA or JA for different times, *P<0.05, **P<0.01.

[0021] 图4 For silencing LhNCED3 Disease phenotype observation and lesion area statistics of 'Sorbonne' petals after silencing the gene, wherein, 图4 A: lily petals LhNCED3 Phenotype change after silencing the gene; 图4 B: lily petals LhNCED3 Lesion area statistics after silencing the gene: 图4 C: LhNCED3 Silencing efficiency of the gene, *P<0.05.

[0022] 图5 For silencing LhNCED3 Endogenous ABA content in 'Sorbonne' petals after silencing the gene, *P<0.05. DETAILED DESCRIPTION

[0023] Example 1

[0024] Cloning of the lily LhNCED3 gene and promoter and analysis of cis-acting elements.

[0025] The experimental lilies were grown in the school greenhouse. Lilium Total RNA from the tissue of the oriental hybrid 'Sorbonne') was reverse transcribed into cDNA, subjected to RT-PCR, and the amplified products were cloned into T-Vector pMDTM19 and sequenced. Seamless cloning was used to insert each product into the designated vector. After transformation into competent Escherichia coli, 100 μL of the transformation product was plated on LB + kanamycin (Kan) solid medium, and positive single clones were randomly selected and sent to a biotechnology company for sequencing. NCEDs The complete protein coding region (CDS) sequence. The CDS sequence is 1725 bp long and encodes 574 amino acids. The gene sequence is shown in SEQ ID NO.1. Three rounds of amplification were performed by Genome Walking, and the third round of RT-PCR products were selectively sequenced to obtain the gene. LhNCEDS3 The upstream promoter sequence is shown in SEQ ID NO.2.

[0026] Online analysis using the Plant Care database LhNCED3 Promoters contain key cis-acting elements.

[0027] PlantCare analysis results show LhNCED3 The promoter contains cis-acting elements related to plant hormone response, growth and development, and participation in adversity. LhNCED3 There are 62 cis-acting elements in the gene, such as abscisic acid (ABA) and multiple cis-acting elements directly involved in jasmonic acid (JA) response.

[0028] Example 2

[0029] lily LhNCED3 Gene expression patterns in different tissue locations.

[0030] Healthy, disease-free 'Sorbonne' lilies were selected as test materials: roots, stems, leaves, petals, and scales. Total RNA was extracted from 'Sorbonne' lilies using an RNA extraction kit (RC411, Novozymes, Nanjing) using the following procedure: (1) Select Buffer EL lysis buffer, take an appropriate amount of the sample that has been quick-frozen in liquid nitrogen and grind it into powder and immediately add 600 μL, vortex vigorously for 30 seconds, and then centrifuge at 12000 rpm at -4℃ for 5 minutes.

[0031] (2) Transfer 500 μL of the supernatant to FastPure gDNA-Filter Columns III, centrifuge at 1200 rpm, -4°C for 30 sec, discard FastPure gDNA-Filter Columns III, and collect the filtrate.

[0032] (3) Add 0.5 times the volume of the filtrate (about 250 μL) of anhydrous ethanol to the collection tube and shake to mix for 15 seconds.

[0033] (4) Transfer the mixed solution to FastPure RNA Columns V, centrifuge at 12,000 rpm at -4°C for 30 seconds, and discard the filtrate.

[0034] (5) Add 700 μL of Buffer RWA to FastPure RNA Columns V, centrifuge at 12,000 rpm at -4°C for 30 seconds, and discard the filtrate.

[0035] (6) Add 500 μL of Buffer RWB to FastPure RNA Columns V, centrifuge at 12,000 rpm at -4°C for 30 seconds, discard the filtrate, and repeat this step.

[0036] (7) Place FastPure RNA Columns V back into the collection tube and centrifuge at 12,000 rpm at -4°C for 2 min.

[0037] (8) Transfer FastPure RNA Columns V to new RNase-free Collection Tubes 1.5 mL centrifuge tubes, add 50 μL of RNase-free ddH2O to the center of the adsorption column membrane, and centrifuge at 12,000 rpm at -4°C for 1 min.

[0038] The extracted RNA was used in the following experiments.

[0039] RNA concentration and purity were confirmed by NanoDrop 2000 spectrophotometer and 1.0% agarose gel electrophoresis. Qualified RNA was reverse transcribed and synthesized into cDNA using the reverse transcription kit (R323-01, Novozymes, Nanjing) according to the instructions. The specific operation method is as follows: (1) The reaction system for genomic DNA removal is shown in Table 1.

[0040] Table 1

[0041] Mix gently by pipetting. PCR reaction conditions: 42°C, 2 min.

[0042] (2) Prepare the reverse transcription reaction system as shown in Table 2.

[0043] Table 2

[0044] Mix gently by pipetting. Perform reverse transcription reaction: 37°C for 15 min; 85°C for 5 sec.

[0045] (3) After the reversal is completed, qRT-PCR is used to detect lilies LhNCED3 Gene expression in different tissues of lily. The specific steps of the fluorescence quantitative PCR (qRT-PCR) experiment are as follows: The primers and internal control LhGADPH sequence for qRT-PCR are shown in Table 3.

[0046] Table 3

[0047] The reaction system is shown in Table 4.

[0048] Table 4

[0049] Reaction conditions: 95°C, 5 min; 95°C, 10 sec, 60°C, 20 s, 72°C, 20 s, 40 cycles.

[0050] The results are as follows 图1 As shown, the results show that: LhNCED3 The gene expression levels are relatively high in petals and leaves, and the order from high to low is petals, leaves, stems, roots, and scales.

[0051] Example 3

[0052] Comparison of lilies with different resistance after inoculation with Botrytis cinerea LhNCED3 Gene expression.

[0053] Place healthy and disease-free leaves in a culture dish with two layers of moist gauze on the bottom, use a sterile punch to cut out fungus blocks of uniform size from the culture medium (mycelium facing down) and inoculate them on the back of the leaves. For the control, use a culture medium of uniform size that does not contain Botrytis cinerea. Place the inoculated leaves in a plastic tray, spray a small amount of sterile water, and seal with plastic wrap to keep it moist. Incubate in the dark in an incubator at 25°C.

[0054] The leaves were collected before inoculation (0 h), 12 h, 24 h, 36 h, and 48 h after inoculation, and the results were detected by qRT-PCR. LhNCED3 The steps for expressing the gene in the highly resistant lily variety 'Sorbonne' and the highly susceptible lily variety 'Tresor' were the same as those in Example 2.

[0055] The results are as follows 图2 As shown, the results showed that after inoculation with Botrytis cinerea, LhNCED3 The overall expression level of the gene in the two lilies showed an upward trend compared with the control group. 48 hours after inoculation, the expression level of the gene in 'Chuanshuo' increased by 7.9 times compared with that in 'Suobang'.

[0056] Example 4

[0057] Study on the effect of external hormone on lily LhNCED3 Influence of gene expression.

[0058] To explore LhNCED3 Whether the gene can respond to exogenous hormones, 100 μM·L -1 ABA (abscisic acid) and 1mM L -1 JA (jasmonic acid) solution was sprayed on the leaves of 'Sorbonne' lily, while the control group (CK group) was sprayed with sterile water. Sampling time was 1 hour, 12 hours, 24 hours, 36 hours, and 48 hours. Three samples were collected at the same time point for all treatments, and each time point was repeated three times. After quick freezing in liquid nitrogen, the samples were stored in a -80℃ freezer. RNA was then extracted and reverse transcribed, and detected by qRT-PCR. LhNCED3 The expression level of the gene was determined by the same steps as in Example 2.

[0059] The results are as follows 图3 The results showed that within 24 h of ABA and JA treatment, LhNCED3 The gene expression level showed a trend of increasing first and then decreasing, but it still showed an upward trend. After 36 h of treatment, the gene expression level increased significantly. Specifically, after 36 h and 48 h of ABA treatment, LhNCED3 The gene expression levels changed by 11.3 and 21.3 times that of the control group. LhNCED3 The expression levels of the genes changed by 4.4 times and 5.1 times that of the control group.

[0060] Example 5

[0061] lily LhNCED3 Analysis of disease resistance in 'Sorbonne' petals after gene silencing.

[0062] The specific steps for constructing the gene silencing (VIGS) vector and transiently transforming the petals of 'Sorbonne' are as follows: A specific fragment of 350 bp in length was selected as the VIGS silencing fragment, and the target fragment was amplified to construct pTRV2- LhNCED3 The sequence of the recombinant plasmid and the specific fragment used for gene silencing is shown in SEQ ID NO. 3, and the primer sequences used are shown in Table 5.

[0063] Table 5

[0064] EHA105 bacterial cultures containing the pTRV2 empty plasmid and the pTRV2 recombinant plasmid were mixed in equal proportions with EHA105 bacterial cultures containing pTRV1 and incubated under a vacuum of -0.75 MPa for 10 minutes. After releasing the vacuum, the petals were washed with deionized water and stored in 0.4% agar at 8°C. After 3 days of culture, the induction rate of lily petals was statistically analyzed by fluorescence quantitative analysis.

[0065] The results are as follows 图4 As shown. By constructing a gene silencing vector and transiently transforming lily petals with vacuum-assisted Agrobacterium infiltration. VIGS induction LhNCED3 The results of silencing showed that the gene expression level after VIGS silencing was significantly reduced to 50.2% of that in the empty load treatment (TRV2-00 group). LhNCED3 After the treatment (group), the area of ​​petal lesions was 35.7% of that of the empty load treatment.

[0066] Example 6

[0067] lily LhNCED3 Analysis of ABA content in petals of 'Sorbonne' after gene silencing.

[0068] The endogenous ABA content of the petals after silencing the gene in Example 5 was determined. The specific operation was as follows: The sample was quickly ground into powder in liquid nitrogen, 50 mg of the ground sample was weighed, and 10 μL of 100 ng·mL -1 Vortex for 10 min at 4°C, 12000 r·min -1The supernatant was collected and concentrated in a new centrifuge tube under the following conditions. The concentrated solution was reconstituted with 100 μL of 80% methanol / water solution, filtered through a 0.22 μm filter membrane, and placed in an injection vial for LC-MS / MS analysis.

[0069] The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) and tandem mass spectrometry (MS / MS). An MWDB database is constructed based on standard samples to perform qualitative analysis of mass spectrometry data.

[0070] The results are as follows 图5 As shown, the results show that: through transient silencing LhNCED3 The gene significantly inhibited the susceptibility of 'Sorbonne' lily petals to Botrytis cinerea. Further endogenous ABA content detection revealed that the endogenous ABA content of lily petals in the empty load treatment (TRV2-00 group) was 9.94 ng·g -1 , and silence LhNCED3 The endogenous ABA content in the TRV2-LhNCED3 group was 5.31 ng·g -1 , decreased by 46.6%. LhNCED3 The gene may inhibit lily's resistance to gray mold by enhancing ABA synthesis.

Claims

1. Use of a compound in improving lily's resistance to gray mold, characterized in that: The compound is any one of the following: (1) Used to separate lily LhNCED3 gene silencing sequences; (2) Used to separate lily LhNCED3 Gene knockout sequence.

2. The use according to claim 1, characterized in that Lily LhNCED3 The CDS sequence of the gene is shown in SEQ ID NO.

1.

3. The use according to claim 2, characterized in that Lily LhNCED3 The promoter sequence of the gene is shown in SEQ ID NO.

2.

4. The use according to any one of claims 1 to 3, characterized in that The compound is used for LhNCED3 The sequence of gene silencing, the sequence of the specific fragment used for gene silencing is shown in SEQ ID NO.

3.

5. The use according to claim 4, characterized in that The specific fragment of gene silencing was cloned into a plant expression vector and transformed into lily using Agrobacterium, so that LhNCED3 Gene silencing.

6. A method for improving lily's resistance to gray mold, characterized in that: Lily in LhNCED3 Gene silencing or knockout.

7. The method according to claim 6, characterized in that Lily LhNCED3 The CDS sequence of the gene is shown in SEQ ID NO.

1.

8. The method according to claim 7, characterized in that The sequence of the specific fragment used for gene silencing is shown in SEQ ID NO.

3.

9. The method according to claim 8, characterized in that The specific fragment of gene silencing was cloned into a plant expression vector and transformed into lily using Agrobacterium, so that LhNCED3 Gene silencing.

10. A method for screening lilies with high resistance to gray mold, characterized in that: By detecting the LhNCED3 The gene expression levels are high and low, and the lily plants with lower expression levels have higher resistance to gray mold.

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