Key gene for resisting paeonia lactiflora black spot by increasing lignin content and application of key gene

Abstract

The invention discloses a key gene for resisting paeonia lactiflora black spot by increasing lignin content and application of the key gene. The key gene is PlWAT1, the sequence of the key gene is as shown in SEQ ID NO.1, and a silent gene of the PlWAT1 gene is TRV-PlWAT1, and the sequence of the silent gene is as shown in SEQ ID NO.2. After the PlWAT1 gene is over-expressed, the plant height and the stem diameter of the tobacco are increased, and the xylem of the stem is widened; the silent gene can be used for regulating and controlling the growth of paeonia lactiflora, the plant height and stem diameter of the silenced paeonia lactiflora are reduced, the stem xylem width is narrowed, the scab area is larger after alternaria alternata is inoculated, and the enzyme activity is remarkably reduced. The plant height, the stem diameter, the lignin content and the black spot resistance of the Chinese herbaceous peony are regulated and controlled through overexpression or silencing genes, black spot resistant Chinese herbaceous peony varieties with stems different in height, thickness and hardness can be cultivated according to requirements, and therefore the method has important application value in the Chinese herbaceous peony molecular breeding process.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and particularly relates to a key gene for resisting peony black spot disease by increasing lignin content and an application thereof. Background Art

[0002] Peony (Paeonia lactiflora Pall.) is a perennial herbaceous plant of the genus Paeonia (Paeonia L.) in the family Paeoniaceae. Peony is one of China's traditional famous flowers, with a long history of cultivation and rich cultural heritage. In ancient Chinese culture, peonies are often endowed with beautiful meanings, symbolizing beauty, love, and longing. As early as the Book of Songs, there is a record of "a man and a woman, they teased each other, and gave each other peonies," indicating that peonies were used to express affection in ancient times. Furthermore, peonies are considered one of the "two most beautiful flowers," along with the peony, with the peony being the "king of flowers" and the peony the "minister of flowers." Both hold a prominent position in horticultural culture. Sales of cut peonies have been rising annually in the international flower market, particularly at the Amsterdam Flower Auction in the Netherlands, where their popularity has surpassed that of some traditional cut flower varieties. Peonies have a relatively short flowering period, but through improved cultivar selection and cultivation techniques, their viewing period can be extended to a certain extent. Peony stem bending and peony diseases affect the commercial value of peony, and thus affect the healthy development of the peony cut flower industry.

[0003] Stem strength is a key factor affecting plant stem bending, breakage, and lodging. Plant stem strength is primarily determined by the strength of the secondary cell wall within the stem cells. Lignin is primarily composed of cellulose and lignin. Lignin is a phenolic compound primarily deposited in lignified cell walls and exists in a highly cross-linked form within the secondary wall, enhancing the secondary cell wall's strength and providing mechanical support for the entire plant. Within plant cell walls, lignin enhances plant resistance to pathogens by providing both physical and chemical defenses. Studies have shown that increased lignin content can improve plant defenses against certain pathogens. Lignin participates in plant immune responses and forms a physical barrier against invading pathogens. The biosynthesis of lignin monomers is regulated by genes, and increased transcript abundance of these genes can enhance plant defenses against fungi. Lignin accumulation can affect pathogen mobility, thereby limiting the spread of pathogens within the plant. Summary of the Invention

[0004] Purpose of the invention: In view of the deficiencies in the prior art, the purpose of the present invention is to provide a key gene for resisting peony black spot 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, peony, etc. to black spot disease.

[0005] The invention also provides a vector of the key gene and an application thereof.

[0006] Technical solution: In order to achieve the above-mentioned purpose, 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] Wherein, the plants include tobacco and peony.

[0008] The promoting of plant growth and increasing of lignin content are increasing tobacco plant height, stem diameter, stem xylem width, and secondary wall thickness of sclerenchyma cells; or increasing peony plant height, stem diameter, xylem thickness and lignin content.

[0009] The silencing gene of the P1WAT1 gene of the present invention is TRV-P1WAT1, and its nucleotide sequence is shown in SEQ ID NO.2.

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

[0011] The present invention provides a host cell containing the PRI101 overexpression vector or the TRV silencing vector.

[0012] Wherein, the overexpression vector is a PRI101 vector.

[0013] Wherein, the gene silencing vector is a TRV silencing vector.

[0014] Wherein, the host cell is Agrobacterium as the starting strain.

[0015] The key gene or silenced gene of the present invention is used to regulate plant growth and lignin content.

[0016] The key gene or silencer gene of the present invention is used to regulate the resistance of plants to black spot disease.

[0017] Among them, the PlWAT1 gene was transferred into tobacco plants. The transgenic tobacco overexpressing the PlWAT1 gene had increased plant height and stem thickness, wider stem xylem width, thicker secondary wall of sclerenchyma cells, smaller area of ​​Alternaria lesions, and significantly increased enzyme activity.

[0018] Among them, the TRV-PlWAT1 gene was transferred into peony plants and the peony PlWAT1 gene was silenced. The plant height and stem thickness of the peony were reduced, the width of the stem xylem became narrower, the thickness of the secondary wall of the thick-walled cells became smaller, the area of ​​the Alternaria lesions became larger, and the enzyme activity was significantly reduced.

[0019] The P1WAT1 gene provided by the present invention can be used for regulating tobacco and peony growth. After overexpressing the P1WAT1 gene, tobacco lignin content is increased, the area of ​​the back-joining alternaria lesion spot decreases, and enzyme activity significantly increases. This shows that the P1WAT1 gene is a key gene for regulating peony plant height, stem thickness and xylem formation. Further, after silencing the P1WAT1 gene, the drug plant height and stem thickness are reduced, the stem xylem width narrows, and the lignin content is reduced. The thickness of the secondary wall of the thick-walled cells decreases, the area of ​​the back-joining alternaria lesion spot increases, and enzyme activity significantly decreases. By regulating the overexpression or silencing of the P1WAT1 gene, peony plant height, stem thickness and lignin content and then regulating the resistance of peony to black spot disease, it is possible to cultivate peony varieties with different heights, roughness and hardness and disease resistance of the stalk according to demand, and therefore there is important application value in the peony molecular breeding process.

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

[0021] The present invention clones a new P1WAT1 gene from peony for the first time. By transferring P1WAT1 into tobacco plants, transgenic tobacco plants that overexpress the P1WAT1 gene increase plant height and stem thickness, widen the stem xylem width, increase lignin content, thicken the secondary wall thickness of sclerenchyma cells, reduce the area of ​​​​rejoining Alternaria lesions, and significantly increase enzyme activity. A silencing gene of the P1WAT1 gene was further obtained, which can silence the P1WAT1 gene of peony. The plant height and stem thickness of peony are reduced, the stem xylem width is narrowed, and the lignin content is reduced. The secondary wall thickness of sclerenchyma cells is reduced, the area of ​​​​rejoining Alternaria lesions is enlarged, and the enzyme activity is significantly reduced. Therefore, by regulating the expression amount of the P1WAT1 gene of peony, peony varieties with different heights, roughness and hardness and disease resistance can be cultivated according to demand, and therefore have important application value in the molecular breeding process of peony. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 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 effect of VIGS silencing of the PlWAT1 gene on plants a: The effect of silencing the PlWAT1 gene on plant phenotype; b: The expression level of PlWAT1 in PlWAT1-silenced plants; cd: Morphological index determination 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 Effects of PlWAT1 overexpression on tobacco grafted with Alternaria a: Phenotype of plants overexpressing PlWAT1 grafted with Alternaria; b: Superoxide dismutase activity of plants overexpressing PlWAT1 grafted with Alternaria; c: Peroxidase activity of plants overexpressing PlWAT1 grafted with Alternaria; d: Malondialdehyde content of plants overexpressing PlWAT1 grafted with Alternaria; e: Catalase activity of plants overexpressing PlWAT1 grafted with Alternaria.

[0026] Figure 5 Effects of PlWAT1 gene silencing and inoculation with Alternaria on peony a: Phenotype of peony after PlWAT1 gene silencing and inoculation with Alternaria; b: Chlorophyll fluorescence maximum photochemical efficiency (Fv / Fm) imaging after PlWAT1 gene silencing in peony and inoculation with Alternaria; c: Superoxide dismutase activity after PlWAT1 gene silencing in peony and inoculation with Alternaria; d: Peroxidase activity after PlWAT1 gene silencing in peony and inoculation with Alternaria; e: Malondialdehyde content after PlWAT1 gene silencing in peony and inoculation with Alternaria; f: Catalase activity after PlWAT1 gene silencing in peony and inoculation with Alternaria. DETAILED DESCRIPTION

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

[0028] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available. Experimental methods without specific conditions specified in the examples are generally performed under conventional conditions or the conditions recommended by the manufacturer.

[0029] The raw materials in the examples are all well-known conventional materials, among which TRV1 vector and TRV2 vector are well-known vectors (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 of PlWAT1

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

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

[0034]

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

[0036]

[0037] Gel running: Take out the gene amplification product from the PCR instrument, and use an electrophoresis instrument to spot an appropriate amount of the product on a 1% agarose gel for detection. After about 20 minutes, take it out and observe it with an imaging system to obtain the target fragment ( Figure 1 a).

[0038] (3) Ligation reaction between purified fragment and cloning vector

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

[0040]

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

[0042] (4) E. coli transformation

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

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

[0045] A single colony was selected from the screening culture plate and inoculated into LB liquid culture medium, and the culture was shaken at 37°C and 250 rpm overnight; the recombinant transformants were directly detected by PCR using the overnight culture liquid as a template.

[0046] Reaction system:

[0047]

[0048]

[0049] Reaction procedure:

[0050]

[0051] Clones that tested positive for PCR in the bacterial culture were sent to Shanghai Sangon Biotechnology 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 fragment was selected from the CDS sequence to be the silencing gene for the PlWAT1 gene, TRV-PlWAT1, with a nucleotide sequence shown in SEQ ID NO. 2. This sequence can be effectively linked to the silencing vector TRV2.

[0052] Example 2

[0053] Construction of plant expression vector of PlWAT1

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

[0055]

[0056] After mixing the various solutions in the system, they were centrifuged instantaneously and kept in a 37°C water bath for 1 hour to terminate the enzyme digestion reaction. The enzyme digestion bands were observed by agarose gel electrophoresis, and the target gene and vector fragments were then cut and recovered from the gel for subsequent vector ligation reactions.

[0057] (2) Referring to the operating instructions of TaKaRa T4 DNA Ligase (TaKaRa, Japan), the expression vector recovered after the double enzyme digestion reaction and the target DNA fragment product were ligated with each other. 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] The PlWAT1 overexpression vector and silencing vector were successfully constructed by PCR detection and named pRI101-PlWAT1-OE and TRV2:PlWAT1( Figure 1 b and c).

[0062] (3) Transformation of Agrobacterium

[0063] Referring to the operating instructions of the GV3101 / EHA105 Chemically Competent Cell product (Quanshijin, China), the expression vector plasmid constructed in step (2) was mixed with GV3101 Agrobacterium competent cells, and the cells were placed on ice for 30 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes. The cells were then added to the culture medium and shaken. An appropriate amount was spread on an LB plate and incubated in an inverted position at 28°C. A single colony on the plate was picked, and an appropriate amount of LB liquid medium was added. The cells were cultured for 48 hours, and the bacterial solution 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. Bacterial solution preparation and transformation

[0067] The positive Agrobacterium liquid obtained above was prepared into an infection liquid. An appropriate amount of the preserved Agrobacterium liquid was taken and activated on a YEB (containing 50 mg / L Kana and 50 mg / L Rif) plate to pick a single clone and incubate it with shaking at 28°C. The turbid bacterial liquid was placed in 20 mL of liquid YEB (containing 50 mg / L Kana and 50 mg / L Rif) and incubated with shaking at 28°C to OD: 1.2. The cells were collected by centrifugation at 25°C for 10 min, the supernatant was discarded, 20 mL of antibiotic-free liquid YEB was added for elution, and the cells were collected by centrifugation at 25°C for 10 min. 20 mL of antibiotic-free liquid YEB was added for elution, and the cells were collected by centrifugation at 25°C for 10 min. Liquid (containing 10 mM MES and MgCl and 200 uM AS) was added to resuspend the cells to OD600 of 1.2. After the resuspension was allowed to stand for activation for 1 hour, it could be used for infection.

[0068] Select a large-leaf tobacco plant of appropriate size for tissue culture, cut the leaves into 1cm*1cm squares, immerse them in the bacterial solution for 5-10 minutes, then wash and stick them into MS culture medium (containing 2mg·L -1 6-BA, 100mg·L -1 Cb and 25 mg·L -1 Hyg), dark culture for 2-3 days, then normal management conditions; when cultured to differentiate larger resistant buds, transfer to 1 / 2MS medium (containing 0.3mg·L -1 IBA, 50 mg L -1 Cb and 8mg·L -1 Hyg); continue to culture until the root system is well developed, then transplant it into a pot, and continue to culture until it blooms before subsequent experiments.

[0069] Because TRV2 vector needs the assistance of TRV1 vector to play a role, TRV1 vector is an empty vector that does not need to connect genes. The bacterial solution of TRV1 empty vector and TRV2 empty vector is mixed in equal volumes as control TRV, and the bacterial solution of TRV1 empty vector and target gene (TRV2:PlWAT1) is mixed in equal volumes as test group. Select the peony containing 5 scale buds, add the mixed bacterial solution in a vacuum pump, put the root of peony into vacuum filtration instrument after cleaning and pruning, immerse the wound pruned by peony root in bacterial solution, cover, evenly apply vaseline, pressure is 0.06MPa, vacuum filtration 20min, clean and plant in basin after taking out, dark culture 3d, then normal management condition culture. Carry out subsequent experiments after 5 weeks of cultivation.

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

[0071] Phenotypic observation of the transgenic tobacco after flowering revealed that the height and stem thickness of the overexpressing plants were significantly higher than those of the wild type ( Figure 2ac). The results of acid washing to determine the lignin content showed that the lignin content of the transgenic plants was significantly higher than that of the wild type ( Figure 2 d), further observations were made through paraffin sections and histochemical staining, which revealed that overexpression of the PlWAT1 gene increased the xylem width of tobacco plant stems ( Figure 2 e). Scanning electron microscopy results showed that the PlWAT1 gene treatment significantly increased the diameter width and number of thick-walled cell layers of tobacco stem vascular tissue compared with the control ( Figure 2 f).

[0072] Leveraging PrimeScript TM The expression level of PlWAT1 in silenced plants was determined by real-time quantitative PCR using the Reverse Transcriptase Reagent Kit (TaKaRa, Japan). Phenotypic observation of silenced plants revealed that silenced plants were significantly shorter than wild type plants ( Figure 3 a). Quantitative results showed that the expression level of PlWAT1 was significantly reduced in silenced plants ( Figure 3 b). After silencing the PlWAT1 gene, the plant height and stem diameter of peony plants were significantly reduced ( Figure 3 cd). The lignin content was also significantly reduced ( Figure 3 e). The results of paraffin sections showed that after VIGS silencing the PlWAT1 gene, the xylem width of peony plants was significantly reduced ( Figure 3 f). These results indicate that the PlWAT1 gene plays an important role in regulating plant height, stem diameter, xylem thickness, and lignin content in peony.

[0073] Example 4

[0074] In vivo grafting experiments on transgenic and silenced plants

[0075] 1. Bacterial liquid preparation and re-inoculation experiment

[0076] First, the Alternaria strain (conventional wild type) was activated on PDA solid medium and cultured for 14 days. Then, a spore suspension was prepared with sterile water and the concentration was adjusted to 1×10 7 / mL, set aside, use a sterile needle to pierce the leaves of the transgenic plants to form wounds, drop 10μl of spore suspension on each wound, after all are back-grafted, spray the leaves and transparent sealing bags with enough sterile water according to the conditions for spore germination, place them at 28℃ and culture for 48h, observe the disease situation for subsequent experiments.

[0077] 2. In vivo grafting experiment

[0078] The second generation of transgenic tobacco was planted and healthy leaves without lesions were selected for in vivo grafting experiments. The experimental results showed that compared with wild-type plants, the lesion area of ​​transgenic plants was significantly smaller ( Figure 4 a). From the activities of the three enzymes SOD, POD and CAT, the activities of transgenic plants were significantly higher than those of wild-type plants ( Figure 4 bc,e), and the MDA content was significantly higher than that of the wild type ( Figure 4 d), these results indicate that overexpression of the PlWAT1 gene can significantly enhance the plant's resistance to black spot disease.

[0079] The same method as above was used to conduct the inoculation experiment. After PlWAT1 gene was silenced and then inoculated with Alternaria alternata 48 hours later, the results showed that the lesion area of ​​peony leaves in the experimental group was significantly larger than that in the control group ( Figure 5 a), which is consistent with the results of chlorophyll fluorescence maximum photochemical efficiency (Fv / Fm) imaging ( Figure 5 b), from 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), and the MDA content was significantly lower than that of the control group ( Figure 5 e) These results indicate that silencing the P1WAT1 gene weakens peony resistance to black spot. The present invention found that heterologous expression of the P1WAT1 gene significantly increased tobacco plant height and stem diameter, widened stem xylem width, thickened secondary wall thickness of sclerenchyma cells, reduced the area of ​​reconnected Alternaria lesions, and increased black spot resistance. This indicates that the P1WAT1 gene is a regulatory gene for peony growth and black spot resistance, and its overexpression improves peony growth and development (such as plant height and stem diameter), lignin content, and black spot resistance.

Claims

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

1.

2. The key gene for promoting plant growth and increasing lignin content according to claim 1, characterized in that: The plants include tobacco and peony.

3. The key gene for promoting plant growth and increasing lignin content according to claim 1, characterized in that: The promoting plant growth and increasing lignin content are increasing tobacco plant height, stem diameter, stem xylem width, and sclerenchyma cell secondary wall thickness; or increasing peony plant height, stem diameter, xylem thickness, and lignin content.

4. A silencing gene of the P1WAT1 gene according to claim 1, characterized in that The silencing gene of the P1WAT1 gene is TRV-P1WAT1, and its nucleotide sequence is shown in SEQ ID NO.

2.

5. A PRI101 overexpression vector containing the key gene according to claim 1 or a TRV silencing vector containing the silencing gene according to claim 2. A host cell containing the PRI101 overexpression vector or TRV silencing vector according to claim 5 .

7. Use of the key gene according to claim 1 or the silenced gene according to claim 4 in regulating plant growth and lignin content.

8. Use of the key gene according to claim 1 or the silencer gene according to claim 4 in regulating plant resistance to black spot disease.

9. The use according to claim 8, characterized in that The PlWAT1 gene was transferred into tobacco plants. The transgenic tobacco plants overexpressing the PlWAT1 gene had increased plant height and stem thickness, wider stem xylem width, thicker secondary wall thickness of sclerenchyma cells, smaller area of ​​Alternaria lesions, and significantly increased enzyme activity.

10. The use according to claim 8, characterized in that The TRV-PlWAT1 gene was transferred into peony plants and the PlWAT1 gene was silenced. The plant height and stem diameter of the peony were reduced, the width of the stem xylem was narrowed, the thickness of the secondary wall of the sclerenchyma cells was reduced, the area of ​​the Alternaria lesions was enlarged, and the enzyme activity was significantly reduced.

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