Key gene for resisting paeonia black spot by increasing lignin content and application thereof

Abstract

The application discloses a key gene for resisting Podosphaera leucotricha by increasing lignin content and application thereof, the key gene is PlWAT1, the sequence of the PlWAT1 is shown as SEQ ID NO. 1, and the sequence of a silencing gene of the PlWAT1 is shown as SEQ ID NO. 2. After overexpression of the PlWAT1 gene, the tobacco plant height and stem thickness increase, and the stem xylem width becomes wide; the silencing gene can be used for regulating the growth of Paeonia lactiflora, after silencing, the Paeonia lactiflora plant height and stem thickness decrease, the stem xylem width becomes narrow, the lesion area is larger after back grafting of Alternaria alternata, and the enzyme activity is significantly reduced. The application can regulate the Paeonia lactiflora plant height, stem thickness and lignin content by overexpression or silencing of the gene, the resistance to Podosphaera leucotricha, and different Paeonia lactiflora varieties with different stem height, thickness and hardness can be bred according to the demand, so the application has important application value in the Paeonia lactiflora molecular breeding process.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a key gene that resists peony black spot disease by increasing lignin content and its application. Background Technology

[0002] Peony (Paeonia lactiflora Pall.) is a perennial herbaceous plant belonging to the genus Paeonia (Paeonia L.) of the family Paeoniaceae. It is one of China's traditional famous flowers, with a long history of cultivation and profound cultural significance. In ancient Chinese culture, peonies are often endowed with auspicious meanings, symbolizing beauty, love, and longing. As early as the *Classic of Poetry*, there is a record of "men and women, they jest with each other, giving each other peonies," indicating that peonies were used to express emotions in ancient times. Furthermore, peonies and peonies are considered the "two wonders of flowers," with the peony being the "king of flowers" and the peony the "prime minister of flowers," both holding important positions in horticultural culture. In the international flower market, the sales of cut peonies have been increasing year by year, especially in the Amsterdam flower auction market in the Netherlands, where their popularity has surpassed some traditional cut flower varieties. While the flowering period of peonies is relatively short, through variety selection and improvements in cultivation techniques, their viewing period can be extended to some extent. Peony stem bending and peony diseases affect the commercial value of peonies, and consequently, the healthy development of the peony cut flower industry.

[0003] As a vital organ of plants, the stem's strength is a key factor influencing its bending, breaking, and lodging. Stem strength is primarily determined by the strength of the secondary cell walls within the stem cells. Chemically, these walls are mainly composed of cellulose and lignin. Lignin, a class of phenolic compounds primarily deposited in lignified cell walls, exists in a highly cross-linked form within the secondary cell walls, thus enhancing their strength and providing mechanical support for the entire plant. Within the plant's cell walls, lignin enhances the plant's resistance to pathogens by providing physical barriers and chemical defenses. Studies have shown that increased lignin content can improve a plant's defense against certain pathogens. Lignin participates in the plant's immune response and forms a physical barrier against invading pathogens. The biosynthesis of lignin monomers is genetically regulated; increased transcriptional abundance of these genes can enhance a plant's defense against fungi. Lignin accumulation can also affect pathogen mobility, thereby limiting their spread within the plant. Summary of the Invention

[0004] Purpose of the invention: In view of the shortcomings of the existing technology, the purpose of this invention is to provide a key gene for resisting peony black spot disease by increasing lignin content. By regulating the expression of the PlWAT1 gene, the plant height, stem diameter and lignin content of plants such as tobacco and peony can be controlled, thereby regulating the resistance of tobacco and peony to black spot disease.

[0005] The present invention also provides vectors for the aforementioned key genes and their applications.

[0006] Technical solution: In order to achieve the above objectives, the present invention provides a key gene for promoting plant growth and increasing lignin content, wherein the key gene is PlWAT1, and its nucleotide sequence is shown in SEQ ID NO.1.

[0007] The plants mentioned include tobacco and peony.

[0008] The promotion of plant growth and increase of lignin content refers to increasing the height of tobacco plants, stem diameter, stem xylem width, and secondary wall thickness of sclerenchyma cells; or increasing the height of peony plants, stem diameter, xylem thickness, and lignin content.

[0009] The silencing gene of the PlWAT1 gene described in this invention is TRV-PlWAT1, whose nucleotide sequence is shown in SEQ ID NO.2.

[0010] The present invention contains either the PRI101 overexpression vector of the key gene or the TRV silencing vector of the silencing gene.

[0011] The present invention contains host cells containing the PRI101 overexpression vector or TRV silencing vector described above.

[0012] The overexpression vector is the PRI101 vector.

[0013] The gene silencing vector is a TRV silencing vector.

[0014] The host cell is based on Agrobacterium as the starting strain.

[0015] The application of the key genes or silent genes described in this invention in regulating plant growth and lignin content.

[0016] The application of the key genes or silenced genes described in this invention in regulating plant resistance to black spot disease.

[0017] Among them, when the PlWAT1 gene was transferred into tobacco plants, transgenic tobacco plants that overexpressed the PlWAT1 gene showed increased plant height and stem diameter, wider stem xylem, thicker secondary cell walls, smaller area of ​​Alternaria solani lesions, and significantly increased enzyme activity.

[0018] Among them, transferring the TRV-PlWAT1 gene into peony plants and silencing the peony PlWAT1 gene resulted in a decrease in peony plant height and stem diameter, a narrowing of stem xylem width, a reduction in the thickness of secondary cell walls of sclerenchyma cells, an increase in the area of ​​Alternaria solani lesions after reinoculation, and a significant decrease in enzyme activity.

[0019] The PlWAT1 gene provided by this invention can be used to regulate the growth of tobacco and peony. Overexpression of the PlWAT1 gene increases the lignin content in tobacco, reduces the area of ​​Alternaria re-inoculation lesions, and significantly increases enzyme activity. This indicates that the PlWAT1 gene is a key gene regulating peony plant height, stem diameter, and xylem formation. Furthermore, silencing the PlWAT1 gene reduces plant height and stem diameter, narrows the stem xylem width, decreases lignin content, reduces the thickness of secondary cell walls in sclerenchyma cells, increases the area of ​​Alternaria re-inoculation lesions, and significantly reduces enzyme activity. By overexpressing or silencing the PlWAT1 gene to regulate peony plant height, stem diameter, and lignin content, and thus regulates peony resistance to black spot disease, it is possible to breed peony varieties with different stem heights, thicknesses, and hardnesses that are disease-resistant, thus having important application value in peony molecular breeding.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0021] This invention marks the first cloning of the novel PlWAT1 gene from peony. By transferring PlWAT1 into tobacco plants, transgenic tobacco plants overexpressing the PlWAT1 gene exhibited increased plant height and stem diameter, wider xylem width, increased lignin content, thicker secondary cell walls, smaller Alternaria alternata lesions, and significantly increased enzyme activity. Furthermore, a silencing gene for PlWAT1 was obtained, which can silence the PlWAT1 gene in peony. This silenced the peony plant height and stem diameter, narrower xylem width, reduced lignin content, thinner secondary cell walls, larger Alternaria alternata lesions, and significantly reduced enzyme activity. Therefore, by regulating the expression level of the PlWAT1 gene in peony, it is possible to cultivate peony varieties with different stem heights, thicknesses, and hardness, as well as disease resistance, according to specific needs. This has significant application value in peony molecular breeding. Attached Figure Description

[0022] Figure 1 The following are examples of PlWAT1 gene cloning and vector construction: a: PlWAT1 gene cloning; b: PlWAT1 gene overexpression vector construction; c: PlWAT1 gene silencing vector construction.

[0023] Figure 2 Phenotypic observations of PlWAT1-overexpressing tobacco plants: a: Phenotype of PlWAT1-overexpressing plants; bc: Morphological indicators of PlWAT1-overexpressing plants; d: Lignin content of PlWAT1-overexpressing plants; e: Paraffin sections of PlWAT1-overexpressing plants (Xv: xylem vessels; Xy: xylem; Xf: xylem fibers); f: Microstructure of PlWAT1-overexpressing stems.

[0024] Figure 3The effects of VIGS silencing of the PlWAT1 gene on plant phenotype are as follows: a: Effect of PlWAT1 gene silencing on plant phenotype; b: Expression level of PlWAT1 in PlWAT1-silenced plants; cd: Morphological parameters of PlWAT1-silenced plants; e: Lignin content of PlWAT1-silenced plants; f: Paraffin sections of PlWAT1-silenced plants (Xv: xylem vessels; Xy: xylem; Xf: xylem fibers).

[0025] Figure 4 The effects of PlWAT1 overexpression in tobacco re-inoculation with Alternaria alternifolia: a: Phenotype of PlWAT1 overexpression plants after re-inoculation with Alternaria alternifolia; b: Superoxide dismutase activity in PlWAT1 overexpression plants after re-inoculation with Alternaria alternifolia; c: Peroxidase activity in PlWAT1 overexpression plants after re-inoculation with Alternaria alternifolia; d: Malondialdehyde content in PlWAT1 overexpression plants after re-inoculation with Alternaria alternifolia; e: Catalase activity in PlWAT1 overexpression plants after re-inoculation with Alternaria alternifolia.

[0026] Figure 5 The effects of plWAT1 gene silencing followed by re-inoculation with Alternaria alternata on peonies are as follows: a: Phenotype of peonies after plWAT1 gene silencing followed by re-inoculation with Alternaria alternata; b: Image of maximum photochemical efficiency (Fv / Fm) of chlorophyll fluorescence after plWAT1 gene silencing followed by re-inoculation with Alternaria alternata; c: Superoxide dismutase activity after plWAT1 gene silencing followed by re-inoculation with Alternaria alternata; d: Peroxidase activity after plWAT1 gene silencing followed by re-inoculation with Alternaria alternata; e: Malondialdehyde content after plWAT1 gene silencing followed by re-inoculation with Alternaria alternata; f: Catalase activity after plWAT1 gene silencing followed by re-inoculation with Alternaria alternata. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0029] In the examples, all raw materials are known conventional materials, among which TRV1 and TRV2 are known carriers (An R2R3-MYB network modulates stem strength by regulating lignin biosynthesis and secondary cell wall thickening in herbaceous peony[J].The Plant Journal,2023,113(6):1237-1258).

[0030] Example 1

[0031] Cloning PlWAT1

[0032] (1) Based on peony miRNA-seq data, genes were screened, and primers for the PlWAT1 gene were designed artificially. The forward primer (F primer) was 5'-ATGGCTATTGGATTCTTTTACTATGGT-3', and the reverse primer (R primer) was 5'-CTTGGCATAACTGTGGCATTCAG-3'. Peony leaf cDNA template was used for amplification.

[0033] (2) PCR amplification was performed using Phanta Max Super-Fidelity DNA Polymerase (Novizan, China). The PCR system is as follows:

[0034]

[0035] Gently mix the above mixture, briefly centrifuge at low speed, and then place it in a standard PCR instrument. Set the following program:

[0036]

[0037] Electrophoresis: The gene amplification product from the PCR instrument is retrieved, and an appropriate amount of the product is spotted onto a 1% agarose gel using an electrophoresis apparatus. After approximately 20 minutes, the product is removed and observed using an imaging system to obtain the target fragment. Figure 1 a).

[0038] (3) Ligation reaction of purified fragments and cloning vector

[0039] Following the instructions of the 5min TA / Blunt-Zero Cloning Kit (Novizan, China), the gel-recovered product was ligated to the cloning vector. The specific system is as follows:

[0040]

[0041] Mix the solutions in the system in a microtube and react at room temperature for 5 minutes. After the reaction is complete, place on ice until ready to use.

[0042] (4) Escherichia coli transformation

[0043] Referring to the Trans1-T1 Phage Resistant Chemically Competent Cell product instructions (TransGold, China), the ligated product was mixed with competent cells, and after ice bath, heat shock, and recovery, an appropriate amount was spread on LB plates, the plates were inverted, and cultured overnight at 37°C.

[0044] (5) Positive clone screening and sequencing analysis

[0045] Single colonies were selected from the screening culture plate and inoculated into LB liquid medium. The culture was incubated overnight at 37°C and 250 rpm. The recombinant transformants were directly detected by PCR using the overnight culture as a template.

[0046] Reaction system:

[0047]

[0048]

[0049] Reaction procedure:

[0050]

[0051] Clones that tested positive by bacterial culture PCR were sent to Shanghai Sangon Biotech Co., Ltd. (Shanghai) for sequencing and identification. Plasmids were extracted, and the CDS sequence of PlWAT1 was determined to be 663 bp, as shown in SEQ ID NO.1. A 300 bp silencing gene for PlWAT1 was selected from the CDS sequence as TRV-PlWAT1, and its nucleotide sequence is shown in SEQ ID NO.2. This sequence can be effectively ligated with the silencing vector TRV2.

[0052] Example 2

[0053] Construction of plant expression vectors for PlWAT1

[0054] (1) In this experiment, the TaKaRa QuickCut restriction enzyme (TaKaRa, Japan) pRI101 vector, TRV2 vector, CDS sequence of PlWAT1, and the silenced gene of PlWAT1 were used for enzyme digestion experiments. The specific reaction system is as follows:

[0055]

[0056] After mixing all solutions in the system, the mixture was briefly centrifuged and incubated in a 37°C water bath for 1 hour to end the enzyme digestion reaction. The enzyme digestion bands were observed by agarose gel electrophoresis. Subsequently, the target gene and vector fragments were digested and recovered separately for subsequent vector ligation reactions.

[0057] (2) Following the instructions for TaKaRa T4 DNALigase (TaKaRa, Japan), the expression vector recovered after the double enzyme digestion reaction was ligated with the target DNA fragment product. The system is as follows:

[0058]

[0059]

[0060] The solutions in the system were mixed in a microtube and reacted in a metal bath at 16°C for 5-6 hours.

[0061] PCR testing confirmed the successful construction of the PlWAT1 overexpression vector and silencing vector, named pRI101-PlWAT1-OE and TRV2:PlWAT1( Figure 1 (b and c).

[0062] (3) Transformation of Agrobacterium tumefaciens

[0063] Following the instructions for the GV3101 / EHA105 Chemically Competent Cell product (TransGold, China), the expression vector plasmid constructed in step (2) was mixed with GV3101 Agrobacterium competent cells. The cells were then incubated sequentially on ice for 30 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and on ice for 5 min before adding culture medium and shaking. An appropriate amount was spread onto LB agar plates and incubated upside down at 28°C. Single colonies were picked from the plates, and an appropriate amount of LB liquid medium was added. After incubation for 48 h, the bacterial culture was sequenced to obtain Agrobacterium containing the pRI101-PlWAT1-OE and TRV2:PlWAT1 vectors.

[0064] Example 3

[0065] Genetic transformation of pRI101-PlWAT1-OE and TRV2:PlWAT1 gene silencing

[0066] 1. Preparation and transformation of bacterial culture

[0067] The obtained positive Agrobacterium tumefaciens bacterial suspension was prepared into an infection solution. An appropriate amount of the preserved Agrobacterium tumefaciens bacterial suspension was activated on a YEB plate (containing 50 mg / L Kana and 50 mg / L Rif) to pick single colonies. The suspension was incubated at 28°C with shaking. The turbid bacterial suspension was then added to 20 mL of liquid YEB (containing 50 mg / L Kana and 50 mg / L Rif) and incubated at 28°C with shaking until the OD value reached 1.2. The bacterial cells were collected by centrifugation at 25°C for 10 min. The supernatant was discarded, and 20 mL of antibiotic-free liquid YEB was added for elution. The bacterial cells were collected by centrifugation at 25°C for 10 min, and 20 mL of antibiotic-free liquid YEB was added for elution. The bacterial cells were collected by centrifugation at 25°C for 10 min, and then resuspended in liquid (containing 10 mM MES, MgCl, and 200 μM AS) until the OD600 of the bacterial cells was 1.2. After the resuspended suspension was allowed to stand for 1 h for activation, it could be used for infection.

[0068] Select appropriately sized tissue-cultured large-leaf tobacco leaves, cut them into 1cm x 1cm squares, immerse them in bacterial solution for 5–10 minutes, then wash and place them into MS medium (containing 2 mg / L). -1 6-BA, 100 mg·L -1 Cb and 25 mg·L -1 Hyg), cultured in the dark for 2–3 days, then under normal management conditions; when larger resistant buds differentiate, transfer to 1 / 2 MS medium (containing 0.3 mg·L⁻¹). -1 IBA, 50 mg·L -1 Cb and 8 mg·L -1 Hyg); Continue cultivation until the root system is well developed, then transplant into a pot, and continue cultivation until flowering before conducting subsequent experiments.

[0069] Because the TRV2 vector requires the assistance of the TRV1 vector to function, and the TRV1 vector is an empty vector that does not require gene ligation, equal volumes of bacterial cultures containing empty TRV1 and empty TRV2 vectors were mixed as the control TRV. Equal volumes of bacterial cultures containing empty TRV1 vector and the target gene (TRV2:PlWAT1) were mixed as the experimental group. Peonies with five buds were selected. The mixed bacterial culture was added to a vacuum pump. The peony roots were washed, trimmed, and placed in a vacuum filter, ensuring the trimmed wounds were immersed in the bacterial culture. The container was sealed, and Vaseline was evenly applied. Vacuum filtration was performed at 0.06 MPa for 20 minutes. After removal, the roots were washed and planted in pots, incubated in the dark for 3 days, and then cultured under normal conditions. Subsequent experiments were conducted after 5 weeks of culture.

[0070] 2. Detection and phenotypic observation of transgenic materials

[0071] Phenotypic observations of the transgenic tobacco plants after flowering revealed that the overexpressing plants had significantly higher plant height and stem diameter than the wild type. Figure 2ac). Results from acid washing analysis of lignin levels showed that the lignin content in transgenic plants was significantly higher than that in wild-type plants. Figure 2 d) Further observation using histochemical staining of paraffin sections revealed that overexpression of the PlWAT1 gene increased the width of the xylem in the tobacco plant stem. Figure 2 e). Scanning electron microscopy results showed that PlWAT1 gene treatment significantly increased the xylem diameter and the number of sclerenchyma cell layers in the vascular tissue of tobacco stems compared to the control. Figure 2 f).

[0072] Using PrimeScript TM Real-time quantitative PCR was performed using the Reverse Transcriptase Reagent Kit (TaKaRa, Japan) to determine the expression level of PlWAT1 in silent plants. Phenotypic observation of silent plants revealed that they were significantly shorter than wild-type plants. Figure 3 a). Quantitative results showed that the expression level of PlWAT1 was significantly reduced in silent plants ( Figure 3 b). Silencing the PlWAT1 gene significantly reduced the plant height and stem diameter of peony plants. Figure 3 cd). Lignin content also decreased significantly ( Figure 3 e). Paraffin section results showed that VIGS silencing of the PlWAT1 gene significantly reduced the xylem width of peony plants. Figure 3 f). These results indicate that the PlWAT1 gene plays an important role in regulating peony plant height, stem diameter, xylem thickness, and lignin content.

[0073] Example 4

[0074] Live inoculation experiments were conducted on transgenic plants and silent plants.

[0075] 1. Preparation of bacterial culture and reinoculation experiment

[0076] First, the Alternaria strain (conventional wild type) was activated and cultured on PDA solid medium for 14 days. Then, a spore suspension was prepared using sterile water and its concentration was adjusted to 1×10⁻⁶. 7 10 μl of spore suspension was prepared for use. The leaves of the transgenic plants were punctured with a sterile needle to form wounds. 10 μl of spore suspension was dripped into each wound. After all plants were re-inoculated, the leaves and transparent sealed bags were sprayed with enough sterile water according to the conditions for spore germination. The plants were then placed at 28°C and cultured for 48 h. The disease situation was observed for subsequent experiments.

[0077] 2. Live animal reconnection experiment

[0078] Second-generation transgenic tobacco was planted, and healthy, disease-free leaves were selected for in vivo re-inoculation experiments. The results showed that the transgenic plants had significantly smaller diseased areas compared to wild-type plants. Figure 4 a). Regarding the activities of the three enzymes SOD, POD, and CAT, the transgenic plants showed significantly higher activities than the wild-type plants. Figure 4 bc,e), while the MDA content was also significantly higher than that of the wild type ( Figure 4 d) These results indicate that overexpression of the PlWAT1 gene can significantly enhance plant resistance to black spot disease.

[0079] Following the same method described above, a live inoculation experiment was conducted. After silencing the PlWAT1 gene and inoculating with Alternaria for 48 hours, the results showed that the leaf lesion area of ​​the experimental group of peony was significantly larger than that of the control group. Figure 5 a) is consistent with the results in imaging of chlorophyll fluorescence maximum photochemical efficiency (Fv / Fm). Figure 5 (b) In terms of the activities of the three enzymes SOD, POD, and CAT, the activities of the experimental group plants were significantly lower than those of the control group. Figure 5 cd,f), while the MDA content was also significantly lower than that of the control group ( Figure 5 e) These results indicate that silencing the PlWAT1 gene weakens peony resistance to black spot disease. The results of this invention found that heterologous expression of the PlWAT1 gene significantly increases tobacco plant height and stem diameter, widens stem xylem, thickens secondary cell walls, reduces the area of ​​Alternaria re-inoculation lesions, and enhances resistance to black spot disease. This suggests that the PlWAT1 gene is a regulatory gene for peony growth and resistance to black spot disease, and its overexpression improves peony growth and development (such as plant height and stem diameter), lignin content, and resistance to black spot disease.

Claims

1. A key gene that promotes tobacco growth and increases lignin content, characterized in that, The key gene is PlWAT1 The gene, whose nucleotide sequence is shown in SEQ ID NO.

1.

2. A PRI101 overexpression vector containing the key gene described in claim 1.

3. A host cell containing the PRI101 overexpression vector of claim 2.

4. The application of the key gene described in claim 1 in regulating plant growth and lignin content; PlWAT1 Genes are transferred into plants and overexpressed. PlWAT1 The genetically modified tobacco plants exhibit increased plant height and stem diameter, wider stem xylem, and thicker secondary cell walls; the plant in question is tobacco.

5. The application of the key gene described in claim 1 in improving the resistance of plants to black spot disease; PlWAT1 Genes are transferred into plants and overexpressed. PlWAT1 The area of ​​Alternaria solani lesions in plants inoculated with the gene was smaller, and the activities of SOD, POD and CAT enzymes were significantly increased. The plant in question was tobacco.

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