Long non-coding RNA lncRNA109897 that promotes resistance to anthracnose in walnuts and its application

Abstract

The application discloses long-chain non-coding RNA lncRNA109897 for promoting walnut resistance to anthracnose and application thereof. The application provides an RNA molecule, which is a lncRNA and is named as LncRNA109897, as shown in sequence 1 of a sequence table. The application also provides a DNA molecule, which is a DNA molecule for coding LncRNA109897 and is named as LncRNA109897 gene, as shown in sequence 3 of the sequence table. The application also protects an anti-disease plant breeding method, which comprises the following steps: introducing the LncRNA109897 gene into a starting plant to obtain a transgenic plant with higher disease resistance than the starting plant. The application has great application and popularization value for plant anthracnose resistance breeding, in particular, walnut anthracnose resistance breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a long non-coding RNA lncRNA109897 that promotes resistance to anthracnose in walnuts and its applications. Background Technology

[0002] Walnut (Juglans regia L.) is an important woody oilseed tree species in northern my country and is considered a "superfood of the 21st century." Walnut anthracnose, caused by *Colletotrichum gloeosporioides* (Penz.) Penz. and Sacc., is a catastrophic disease in my country's walnut production. Its prevalence leads to the drop of unripe walnuts, reduces the production of high-quality nuts, and negatively impacts the economic benefits of the walnut industry. Therefore, in-depth research into walnut disease resistance-related genes and elucidation of the mechanisms of resistance to *Colletotrichum gloeosporioides* are crucial for molecular design breeding of anthracnose-resistant walnuts and for accelerating genetic improvement of walnut disease resistance. This is both a requirement of the national seed industry strategy and an urgent need for the upgrading and transformation of the walnut industry.

[0003] Long non-coding RNAs (lncRNAs) are a class of mRNAs ranging in length from 200 to 100,000 nt. Initially considered transcriptional byproducts or transcriptional "noise" in protein-coding genes, they lack protein-coding function. New research shows that some lncRNAs with small open reading frames (sORFs, <300 nt in length) can also encode short peptides with biological functions. lncRNAs often act as recruiters, guides, trappers, or signal molecules for target genes, exerting their functions through epigenetic, transcriptional, post-transcriptional, and translational regulation, playing a crucial role in plant disease resistance. Summary of the Invention

[0004] The purpose of this invention is to provide a long non-coding RNA lncRNA109897 that promotes resistance to anthracnose in walnuts and its applications.

[0005] This invention provides an RNA molecule, a lncRNA named LncRNA109897, as shown in Sequence 1 of the sequence listing.

[0006] The present invention also provides a DNA molecule for encoding LncRNA109897, named the LncRNA109897 gene, as shown in sequence 3 of the sequence listing.

[0007] This invention also protects the use of the LncRNA109897 or LncRNA109897 gene in promoting plant disease resistance.

[0008] Increased LncRNA109897 content leads to increased plant disease resistance.

[0009] Increased expression of the LncRNA109897 gene leads to increased plant disease resistance.

[0010] This invention also protects recombinant expression vectors, expression cassettes, transgenic cell lines, transgenic plant tissues, or recombinant microorganisms containing the LncRNA109897 gene.

[0011] This invention also protects the use of the LncRNA109897 or LncRNA109897 gene in the breeding of transgenic plants with enhanced disease resistance.

[0012] The present invention also protects the use of substances for inhibiting LncRNA109897 or substances for inhibiting the LncRNA109897 gene in the cultivation of plants with reduced disease resistance.

[0013] This invention also protects the use of the LncRNA109897 or LncRNA109897 gene as a repressive target in the cultivation of plants with reduced disease resistance.

[0014] This invention also protects a method for breeding disease-resistant plants, comprising the following steps: introducing the LncRNA109897 gene into a starting plant to obtain a transgenic plant with higher disease resistance than the starting plant.

[0015] The LncRNA109897 gene is introduced into the starting plant via a recombinant expression vector.

[0016] The recombinant expression vector was constructed using a plant expression vector as the starting vector.

[0017] The plant expression vector may specifically be the pRI-101 vector.

[0018] The recombinant expression vector may specifically be the recombinant plasmid 35S::LncRNA109897. Compared with the pRI-101 vector, the only difference of the recombinant plasmid 35S::LncRNA109897 is that the small fragment between the NdeI and EcoRI restriction sites in the pRI-101 vector is replaced by a double-stranded DNA molecule as shown in Sequence 1 of the sequence listing.

[0019] This invention also protects a method for breeding susceptible plants, comprising the following steps: introducing a substance for inhibiting LncRNA109897 or a substance for inhibiting the LncRNA109897 gene into a starting plant to obtain a transgenic plant with lower disease resistance than the starting plant.

[0020] The substance used to inhibit the LncRNA109897 gene can be an interference vector or a combination of interference vectors targeting the LncRNA109897 gene. The interference vector is the recombinant plasmid TRV::LncRNA109897. The interference vector combination is the recombinant plasmid TRV::LncRNA109897 and the pTRV1 vector. Compared to the pTRV2 vector, the only difference of the recombinant plasmid TRV::LncRNA109897 is that the small fragment between the EcoRI and KpnI restriction sites in the pTRV2 vector is replaced by a double-stranded DNA molecule as shown in Sequence 2 of the sequence listing.

[0021] The substance used to suppress the LncRNA109897 gene can be a TRV virus used to silence the LncRNA109897 gene. The TRV virus used to silence the LncRNA109897 gene contains a double-stranded DNA molecule as shown in sequence 2 of the sequence listing.

[0022] The disease resistance mentioned refers to resistance to anthrax.

[0023] The susceptibility mentioned refers to susceptibility to anthrax.

[0024] The disease resistance mentioned refers to resistance to walnut anthracnose.

[0025] The susceptibility mentioned refers to susceptibility to walnut anthracnose.

[0026] The disease resistance mentioned refers to resistance to plant diseases caused by *Colletotrichum gloeosporioides*.

[0027] The susceptibility refers to susceptibility to plant diseases caused by *Colletotrichum gloeosporioides*.

[0028] The specific type of *Colletotrichum gloeosporioides* can be *Colletotrichum gloeosporioides* strain m9.

[0029] Any of the plants mentioned above are either monocotyledons or dicotyledons.

[0030] Any of the plants mentioned above belong to the Juglandaceae family.

[0031] Any of the plants mentioned above belong to the genus Juglans.

[0032] Any of the plants mentioned above may be '4-23' walnut germplasm or 'B37' walnut germplasm.

[0033] This invention has significant application and promotion value for anthracnose-resistant plant breeding, especially for anthracnose-resistant walnut breeding. Attached Figure Description

[0034] Figure 1The results are from Example 2, which showed the relative expression level of the LncRNA109897 gene.

[0035] Figure 2 This is the result of detecting the relative expression level of the LncRNA109897 gene in step four of Example 3.

[0036] Figure 3 This is an exemplary result of the disease resistance test in step four of Example 3.

[0037] Figure 4 This is the result of detecting the relative expression level of the LncRNA109897 gene in step five of Example 3.

[0038] Figure 5 This is an exemplary result of the disease resistance test in step five of Example 3. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0040] Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Unless otherwise specified, the quantitative experiments in the following examples are all performed in triplicate, and the results are averaged. The '4-23' walnut germplasm is an anthracnose-resistant variety among existing walnut germplasms. The 'B37' walnut germplasm is an anthracnose-susceptible variety among existing walnut germplasms.

[0041] Walnut germplasm of '4-23' and 'B37', pRI-101 vector (i.e., "pRI-101vector" in the literature), pTRV1 vector, pTRV2 vector, and *C. gloeosporioides* strain m9 (i.e., "strain 'm9' of *C. gloeosporioides*" in the literature) are all described in the following literature: Zhou R., Dong Y., Liu X., Feng S., Wang C., Ma X., Liu J., Liang Q., Bao Y., Xu S., Lang X., Gai S., Yang KQ, Fang H. *JrWRKY21 interacts with JrPTI5L to activate the expression of JrPR5L for resistance to *C. gloeosporioides* in walnut. *Plant Journal*, 2022, (111): 1152-1166.

[0042] Example 1: Discovery of the LncRNA109897 gene

[0043] The inventors discovered a new long non-coding RNA in the mature leaves of '4-23' walnut plants infected with Colletotrichum gloeosporioides, named LncRNA109897, as shown in Sequence 1 of the sequence listing.

[0044] Example 2: Correlation between LncRNA109897 gene expression and disease resistance substances

[0045] I. Obtaining the test sample

[0046] The detached leaves were from the '4-23' walnut plant and the 'B37' walnut plant.

[0047] (1) Preparation of pathogenic bacteria cake

[0048] The *Colletotrichum gloeosporioides* m9 strain was inoculated onto solid PDA medium and incubated statically at 28°C for 7–12 days. Then, holes were punched using a 5 mm diameter punch to obtain pathogenic bacterial pellets.

[0049] (2) Take the detached leaves and spray them with 75% ethanol aqueous solution for disinfection. After the ethanol evaporates, make a cross-shaped cut on the back of each leaf with a blade. Then place the leaves on a solid agar plate (with the front of the leaves in contact with the agar plate) and place a pathogenic fungus cake at the cross-shaped cut position on the back of the leaves.

[0050] Solid agar plate: A plate formed by solidification of 15 g / L agar aqueous solution.

[0051] (3) After completing step (2), place the plate with the leaves on it at 24°C and incubate under alternating light and dark conditions (16h light / 8h darkness). The incubation time is set to 24 hours, 48 ​​hours, 72 hours or 120 hours respectively.

[0052] Set up 5 repeat processes.

[0053] The test samples obtained are as follows:

[0054] Leaves without pathogenic fungal cakes were taken as the 0hpi control.

[0055] After 24 hours of cultivation, diseased parts of the leaves were taken and used as 24hpi treatment.

[0056] After 48 hours of cultivation, diseased parts of the leaves were taken and used as 48hpi treatment.

[0057] After 72 hours of cultivation, diseased parts of the leaves were taken and used as 72hpi treatment.

[0058] After 120 hours of cultivation, unaffected parts (distal parts) of the leaves were taken and treated as 120 hpi.

[0059] II. Detect the relative expression level of the LncRNA109897 gene in the test samples.

[0060] Total RNA was extracted and reverse transcribed to obtain cDNA. Using cDNA as a template and the Jr18S gene as an internal reference gene, the relative expression level of the LncRNA109897 gene in the test samples was detected.

[0061] The primers used to detect the LncRNA109897 gene are as follows:

[0062] F1: 5'-CCCAAGCATATCTCAAACAA-3';

[0063] R1: 5'-CAAGGAGTGAAGTGATGATAC-3'.

[0064] The primers used to detect the internal reference gene are as follows:

[0065] F2: 5'-GGTCAATCTTCTCGTTCCCTT-3';

[0066] R2: 5'-TCGCATTTCGCTACGTTCTT-3'.

[0067] See results Figure 1 .

[0068] Example 3: Transient transformation of the LncRNA109897 gene

[0069] I. Construction of overexpression vectors

[0070] 1. Synthesize the double-stranded DNA molecule shown in Sequence 1 of the sequence listing.

[0071] 2. Using the double-stranded DNA molecule obtained in step 1 as a template, perform PCR amplification using primers composed of F3 and R3, and recover the PCR amplification product.

[0072] F3: 5'-CCATATGATGAGTTCAGTTTATGTCACTTCTAGTATATTT-3';

[0073] R3: 5'-G GAATTC GTAAATTTGAAGCCTATACACAAATTGG-3'.

[0074] 3. Take the PCR amplification product obtained in step 2, and perform double digestion with restriction endonucleases NdeI and EcoRI, and recover the digested products.

[0075] 4. Take the pRI-101 vector and perform double digestion with restriction endonucleases NdeI and EcoRI to recover approximately 10.4 kb of the vector backbone.

[0076] 5. Ligate the enzyme digestion product recovered in step 3 and the vector backbone recovered in step 4 to obtain recombinant plasmid 35S::LncRNA109897. Recombinant plasmid 35S::LncRNA109897 has been sequenced and verified. Compared with the pRI-101 vector, the only difference of recombinant plasmid 35S::LncRNA109897 is that the small fragment between the NdeI and EcoRI restriction sites in the pRI-101 vector is replaced by a double-stranded DNA molecule as shown in Sequence 1 of the sequence listing.

[0077] II. Construction of Interference Carriers

[0078] 1. Synthesize the double-stranded DNA molecule shown in Sequence 2 of the sequence listing.

[0079] 2. Using the double-stranded DNA molecule obtained in step 1 as a template, perform PCR amplification using primers composed of F4 and R4, and recover the PCR amplification product.

[0080] F4: 5'-G GAATTC ATGGCTACTGGTAGTGTATCATCACTT-3';

[0081] R4: 5'-GG GGTACC GTGACCTACTGTATTGATTTCCATAGG-3'.

[0082] 3. Take the PCR amplification product obtained in step 2, and perform double digestion with restriction endonucleases EcoRI and KpnI, and recover the digested products.

[0083] 4. Take the pTRV2 vector and perform double digestion with restriction endonucleases EcoRI and KpnI to recover the vector backbone of approximately 9.6 kb.

[0084] 5. The enzyme digestion products recovered in step 3 and the vector backbone recovered in step 4 were ligated to obtain the recombinant plasmid TRV::LncRNA109897. The recombinant plasmid TRV::LncRNA109897 has been sequenced and verified. Compared with the pTRV2 vector, the only difference of the recombinant plasmid TRV::LncRNA109897 is that the small fragment between the EcoRI and KpnI restriction sites in the pTRV2 vector was replaced by a double-stranded DNA molecule as shown in Sequence 2 of the sequence listing.

[0085] III. Preparation of Infection Solution

[0086] Infection buffer: containing 10 mM MgCl2, 10 mM MES and 266.7 μM acetylsylcholine, with the remainder being water.

[0087] 1. Prepare the infection solution for the overexpression experimental group and the corresponding infection solution for the control group.

[0088] (1) The recombinant plasmid 35S::LncRNA109897 was introduced into Agrobacterium GV3101 to obtain recombinant Agrobacterium.

[0089] (2) Take the recombinant Agrobacterium cells obtained in step (1), suspend them in infection buffer, and obtain OD. 600nm The bacterial culture with a concentration of 1.2 is the overexpression test group infection solution, named 35S::LncRNA109897 infection solution.

[0090] (3) The pRI-101 vector was introduced into Agrobacterium GV3101 to obtain recombinant Agrobacterium.

[0091] (4) Take the recombinant Agrobacterium cells obtained in step (3), suspend them in infection buffer, and obtain OD. 600nm The bacterial solution with a concentration of 1.2 is the control group infection solution, named 35S::00 infection solution.

[0092] 2. Prepare the infection solution for the interference test group and the corresponding infection solution for the control group.

[0093] The recombinant plasmid TRV::LncRNA109897 was introduced into Agrobacterium GV3101 to obtain recombinant Agrobacterium, named recombinant Agrobacterium A. The bacterial cells of recombinant Agrobacterium A were resuspended in infection buffer to obtain OD. 600nm The bacterial solution with a concentration of 1.2 is named bacterial solution A.

[0094] The pTRV2 vector was introduced into Agrobacterium GV3101 to obtain recombinant Agrobacterium, named recombinant Agrobacterium B. The bacterial cells of recombinant Agrobacterium B were suspended in infection buffer to obtain OD. 600nm The bacterial solution with a concentration of 1.2 is named bacterial solution B.

[0095] The pTRV1 vector was introduced into Agrobacterium GV3101 to obtain recombinant Agrobacterium, named recombinant Agrobacterium C. The bacterial cells of recombinant Agrobacterium C were suspended in infection buffer to obtain OD. 600nm The bacterial solution with a concentration of 1.2 is named bacterial solution C.

[0096] Equal volumes of bacterial suspension A and bacterial suspension C were mixed to obtain the interference test group infection solution, which was named TRV::LncRNA109897 infection solution.

[0097] Equal volumes of bacterial suspension B and bacterial suspension C were mixed to obtain the control group infection solution, which was named TRV::00 infection solution.

[0098] Prepare the inoculum fresh and let it stand in the dark for 3 hours before use.

[0099] IV. Transient transfection (overexpression of lncRNA109897)

[0100] Test infection solutions: 35S::LncRNA109897 infection solution or 35S::00 infection solution.

[0101] Walnut plant: 'B37' walnut plant.

[0102] 1. Sow seeds in flowerpots filled with growing medium and cultivate normally until the walnut plants sprout 14-21 days after sprouting (at this time, the plant height is 20-30cm and has 12-17 leaves).

[0103] 2. After completing step 1, pull the plant out by the roots, invert the plant and immerse all the leaves in the test infection solution. Place the plant and the container holding the infection solution in the same airtight container and vacuum treat it (0.8MPa, 15min) to allow the recombinant Agrobacterium to penetrate into the plant leaves through the vacuum. Then, replant the plant in the original flowerpot and culture it at 24℃ with alternating light and dark conditions (16h light / 8h darkness) for 5 days.

[0104] Two plants were infected with 35S::LncRNA109897 infection solution. The two plants were named 35S::LncRNA109897-1 and 35S::LncRNA109897-2, respectively.

[0105] One plant was infected with 35S::00 infection solution. The plant was named the 35S::00 plant.

[0106] 3. After completing step 2, remove all leaves (6-9 leaves) from one side of each plant and detect the relative expression level of the LncRNA109897 gene.

[0107] The detection method is the same as step two of Example 2.

[0108] See results Figure 2 Transient overexpression of the LncRNA109897 gene resulted in an approximately 5-6 fold increase in LncRNA109897 gene expression levels.

[0109] 4. After completing step 2, remove all leaves (6-9 leaves) from the other side of each plant for disease resistance testing.

[0110] Methods for detecting disease resistance:

[0111] (1) Preparation of pathogenic bacteria cake

[0112] The *Colletotrichum gloeosporioides* m9 strain was inoculated onto solid PDA medium and incubated statically at 28°C for 7–12 days. Then, holes were punched using a 5 mm diameter punch to obtain pathogenic bacterial pellets.

[0113] (2) Take leaves and spray them with 75% ethanol aqueous solution for disinfection. After the ethanol evaporates, make a cross-shaped cut on the back of each leaf with a blade. Then place the leaves on a solid agar plate (with the front of the leaves in contact with the agar plate) and place a pathogenic fungus cake at the cross-shaped cut on the back of the leaves.

[0114] Solid agar plate: A plate formed by solidification of 15 g / L agar aqueous solution.

[0115] (3) After completing step (2), place the plate with the leaves on it and culture it at 24°C with alternating light and dark conditions (16h light / 8h darkness) for 5 days.

[0116] (4) After completing step (3), take a leaf, take a picture of the front of the leaf and count the diameter of the pathogenic bacteria spots.

[0117] See results Figure 3 . Figure 3 The top image is a photograph, and the bottom image shows the statistical results of the pathogenic bacterial plaque diameter. Transient overexpression of the LncRNA109897 gene significantly improved the plant's resistance to the pathogenic bacteria.

[0118] The results were consistent after three or more repeated experiments.

[0119] V. Transient transfection (inhibiting LncRNA109897)

[0120] Test infection solutions: TRV::LncRNA109897 infection solution or TRV::00 infection solution.

[0121] Walnut plant: '4-23' walnut plant.

[0122] The experimental method is the same as step four.

[0123] Two plants were infected with TRV::LncRNA109897 infection solution. The two plants were named TRV::LncRNA109897-1 and TRV::LncRNA109897-2, respectively.

[0124] One plant was infected with TRV::00 infection solution. The plant was named the TRV::00 plant.

[0125] The relative expression level of the LncRNA109897 gene is shown in the figure. Figure 4 Transient silencing of the LncRNA109897 gene resulted in an 11-fold downregulation of its expression level.

[0126] The results of the disease resistance test are shown in Figure 5 . Figure 5 The top image is a photograph, and the bottom image shows the statistical results of the pathogenic bacterial plaque diameter. Transient silencing of the LncRNA109897 gene significantly reduced the plant's resistance to the pathogen.

[0127] The results were consistent after three or more repeated experiments.

[0128] The results of this embodiment indicate that the LncRNA109897 gene positively regulates the resistance of walnut leaves to Colletotrichum gloeosporioides.

[0129] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A DNA molecule, as shown in SEQ ID NO:

3.

2. The application of the DNA molecule of claim 1 in promoting plant disease resistance; wherein the disease resistance is resistance to walnut anthracnose; and wherein the plant is walnut.

3. A recombinant expression vector, expression cassette, transgenic cell line, transgenic plant tissue, or recombinant microorganism containing the DNA molecule described in claim 1.

4. The application of the DNA molecule of claim 1 in the cultivation of transgenic plants with enhanced disease resistance; wherein the disease resistance is resistance to walnut anthracnose; and wherein the plant is walnut.

5. A method for breeding disease-resistant plants, comprising the following steps: introducing the DNA molecule of claim 1 into a starting plant to obtain a transgenic plant with higher disease resistance than the starting plant; wherein the disease resistance is resistance to walnut anthracnose; and wherein the plant is a walnut.

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