Circular rna molecule circrna928, its encoding gene and application

Abstract

The application discloses a circular RNA molecule circRNA928, a coding gene thereof and an application. The nucleotide sequence of the circRNA molecule is SEQ ID No. 1 in the sequence listing. The circRNA has a regulation effect on a plant disease resistance pathway. The disease resistance function of the circRNA is of great significance to cultivation of a disease-resistant cucumber variety and is helpful to enrichment of vegetable breeding resources.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the circular RNA molecule circRNA928, its encoding gene, and its applications. Background Technology

[0002] Non-coding RNAs (ncRNAs), including microRNAs (miRNAs), small interfering RNAs (siRNAs), circular RNAs (circRNAs), and long non-coding RNAs (lncRNAs), participate in the expression regulation of protein-coding genes at both the transcriptional and post-transcriptional levels. Unlike most linear RNAs, circRNAs have a covalently closed circular structure. With further research, the types of circRNAs have accumulated, with the three main types being exon circRNAs, intron circRNAs, and exon-intron circRNAs. In addition, there are other types of circRNAs, such as fusion-circRNAs (f-circRNAs), read-through circRNAs (rt-circRNAs) formed by polymerase II transcription, and circRNAs with overlapping regions and different sites, called mutually inclusive circular RNAs.

[0003] circRNAs are structurally stable, diverse, and specifically expressed in cells and tissues, playing a crucial role in regulating gene expression and plant growth and development. For example, tomato leaves infected with Tomato Yellow Leaf Curl Virus (TYLCV) showed 32 and 83 circRNAs specifically expressed, respectively, compared to the control group, with the expression levels of circRNAs after infection being lower than the control. Transgenic rice overexpressing circR5g05160 enhanced resistance to Magnaphalthe oryzae. 1934 and 44 circRNAs, respectively, were identified in cucumber root and leaf samples that responded to salt stress and exhibited differential expression. Under high-temperature stress, seven circRNAs in cucumber competitively bound to 114 differentially expressed miRNAs, thereby interfering with their regulation of 359 downstream target mRNAs (involved in plant hormone signal transduction, plant-pathogen interactions, and glutathione metabolism). Summary of the Invention

[0004] The purpose of this invention is to provide a circRNA related to plant disease resistance and its application.

[0005] The exon-type circRNA molecule provided by this invention is named circRNA928, and its nucleotide sequence is SEQ ID No. 1. SEQ ID No. 1 consists of 523 nucleotides.

[0006] Biological materials related to circRNA928 are also within the scope of protection of this invention.

[0007] The biological material associated with circRNA928 is an expression cassette containing a nucleic acid molecule encoding the circRNA molecule.

[0008] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0009] In one embodiment of the present invention, the nucleic acid molecule encoding the circRNA molecule is such as SEQ ID No. 2 or SEQ ID No. 3.

[0010] The biological material associated with circRNA928 is a recombinant vector, which contains a nucleic acid molecule encoding the circRNA molecule or the expression cassette.

[0011] The biological material associated with circRNA928 is a recombinant microorganism, which contains a nucleic acid molecule encoding the circRNA molecule, or the expression cassette, or the recombinant vector.

[0012] The biological material associated with circRNA928 is a transgenic plant cell line, which contains a nucleic acid molecule encoding the circRNA molecule, or the expression cassette, or the recombinant vector, or the recombinant microorganism.

[0013] The circRNA molecule, the expression cassette, the recombinant vector, the recombinant microorganism, or the transgenic plant cell line can be used to regulate plant disease resistance.

[0014] In a specific embodiment of the present invention, the aforementioned expression cassette refers to DNA capable of forming circRNA molecules after transcriptional processing in a host cell. This DNA may include not only promoters that initiate transcription of nucleic acid molecules encoding circRNA molecules, but also terminators that terminate transcription of nucleic acid molecules encoding circRNA molecules. Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in the present invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter 35S of cauliflower mosaic virus; suitable transcription terminators include, but are not limited to: the Agrobacterium-mediated cauliflower mosaic virus (NOS terminator) and the CaMV 35S terminator of cauliflower mosaic virus.

[0015] In an embodiment of the present invention, the promoter that initiates transcription of the nucleic acid molecule encoding the circRNA molecule in the expression cassette is the CaMV35S promoter, and the terminator that terminates transcription of the nucleic acid molecule encoding the circRNA molecule is the Agrobacterium carmine synthase terminator (NOS terminator).

[0016] In embodiments of the present invention, the vector may be a plasmid, a bacteriophage, or a viral vector.

[0017] The recombinant vector may be an expression vector that expresses the above-mentioned circRNA molecule, specifically a recombinant expression vector containing SEQ ID No. 2 or SEQ ID No. 3, whose starting vector may be pCAMBIA1304.

[0018] In one specific embodiment of the present invention, the recombinant vector is pCAMBIA1304-circRNA928; pCAMBIA1304-circRNA928 is a recombinant vector obtained by integrating a 923bp DNA fragment (the DNA fragment shown in SEQ ID No. 3) into the XbaⅠ and BstEⅡ sites of the pCAMBIA1304 vector. The DNA molecule shown in SEQ ID No. 3 is a sequence obtained by adding 200bp flanking introns upstream and downstream of the coding sequence of the above-mentioned circRNA molecule (the sequence shown in SEQ ID No. 2).

[0019] The present invention provides a method for cultivating transgenic plants with reduced resistance, comprising the steps of introducing the circRNA molecule, or a nucleic acid molecule encoding the circRNA molecule, or an expression cassette containing a nucleic acid molecule encoding the circRNA molecule, or a recombinant vector containing a nucleic acid molecule encoding the circRNA molecule into a recipient plant, thereby obtaining a transgenic plant with lower disease resistance than the recipient plant.

[0020] Specifically, the method for cultivating transgenic plants with reduced resistance involves introducing a nucleic acid molecule encoding the circRNA molecule into a recipient plant, screening for transgenic plants that overexpress the circRNA molecule, and obtaining transgenic plants with lower disease resistance than the recipient plant.

[0021] In a specific embodiment of the present invention, the nucleic acid molecule encoding the circRNA molecule is introduced into the recipient plant via the recombinant vector pCAMBIA1304-circRNA928.

[0022] In the method for cultivating transgenic plants with reduced resistance, the recombinant microorganism is used to introduce the nucleic acid molecule expressing circRNA, the expression cassette, or the recombinant vector into the recipient plant.

[0023] The aforementioned disease resistance or resistance is preferably resistance to CGMMV (Cucumber greenmottle mosaic virus).

[0024] In embodiments of the present invention, the recombinant microorganism may be yeast, bacteria, algae, or fungi. The bacteria may be Agrobacterium; specifically, Agrobacterium EHA105.

[0025] In a specific embodiment of the present invention, in the method for cultivating transgenic plants with reduced resistance, the recombinant microorganism is Agrobacterium tumefaciens EHA105 containing the recombinant vector pCAMBIA1304-circRNA928.

[0026] In the above method, the disease resistance of the transgenic plant is lower than that of the recipient plant, as manifested in all or part of the following A1)-A3):

[0027] A1) The accumulation of CGMMV (Cucumber green mottle mosaic virus) RNA in the transgenic plant was higher than that in the recipient plant;

[0028] A2) The accumulation of CGMMV coat protein in the transgenic plant is higher than that in the recipient plant;

[0029] A3) The transgenic plant leaves have more chlorotic and yellow spots than the recipient plant.

[0030] In embodiments of the present invention, the transgenic plant cell lines do not include plant propagation material.

[0031] In embodiments of the present invention, the regulation of plant disease resistance is to reduce plant disease resistance, specifically manifested in all or part of the following B1)-B3):

[0032] B1) When the expression level of circRNA928 in plant protoplasts increases, the accumulation of CGMMV RNA in the plant increases;

[0033] B2) When the expression level of circRNA928 in plant protoplasts increases, the accumulation of CGMMV coat protein in the plant increases;

[0034] B3) When the expression level of circRNA928 in plants increases, the number of chlorotic and yellow spots on the leaves of the plants increases.

[0035] In the above method, the plant is a dicotyledonous plant; the dicotyledonous plant can be a cucumber; specifically, the cucumber can be a Xintai dense-thorned cucumber.

[0036] In the above method, the transgenic plant is understood to include not only the first-generation transgenic plant obtained by transforming the target plant with the gene, but also its progeny. For transgenic plants, the gene can be propagated within the species, or it can be transferred into other varieties of the same species using conventional breeding techniques, particularly commercial varieties. The transgenic plant includes protoplasts, seeds, complete plants, and cells.

[0037] This invention employs multiple biological techniques, including bioinformatics prediction, molecular cloning, Agrobacterium-mediated transformation, real-time quantitative PCR, and Western blot. Using cucumbers overexpressing circRNA928 as the research object and cucumbers transformed with the empty vector pCAMBIA1304 as a control, this study investigated the effect of circRNA928 on the degree of damage in infected plants from a molecular biological perspective. The results showed that transgenic plants overexpressing circRNA928 exhibited lower disease resistance compared to the control group, indicating that circRNA928 is an RNA associated with plant disease resistance and can be used to regulate the disease resistance of target plants. Attached Figure Description

[0038] Figure 1 Quantitative analysis of circRNA928 in cucumber protoplasts overexpressing circRNA928.

[0039] Figure 2 To analyze the accumulation of CGMMV RNA in cucumber protoplasts overexpressing circRNA928.

[0040] Figure 3 To analyze the accumulation of CGMMV coat protein (CP) in cucumber protoplasts overexpressing circRNA928.

[0041] Figures 1-3 In the middle, EV represents an empty vector.

[0042] Figures 1-3 OE in the text stands for Overexpression. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0044] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0045] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0046] The Agrobacterium tumefaciens EHA105 used in the following examples can be purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0047] The Agrobacterium competent cells GV3101 used in the following examples can be purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0048] The cucumber inbred line “Xintai Mici” used in the following examples was purchased from Xintai Mici Cucumber Seed Farm in Xintai City, Shandong Province.

[0049] The overexpression vector pCAMBIA1304 used in the following examples can be purchased from Hunan Fenghui Biotechnology Co., Ltd.

[0050] The circRNA928 sequence in this invention is the RNA molecule shown in SEQ ID No. 1 of the sequence listing.

[0051] The circRNA928 encoding gene sequence in this invention is the DNA molecule shown in SEQ ID No. 2 in the sequence listing.

[0052] The nucleic acid sequence used in this invention to construct the vector overexpressing circRNA928 is the DNA molecule shown in SEQ ID No. 3 of the sequence listing. Specifically, this sequence is obtained by adding 200 bp flanking introns upstream and downstream of the sequence shown in SEQ ID No. 2.

[0053] This invention uses cucumber transfected with circRNA928 as the research object and wild-type Xintai dense-spined cucumber as the control. By detecting the accumulation of viral RNA and the content of viral protein in recipient plants challenged with cucumber green mottle mosaic virus, the influence of circRNA928 on the degree of damage to infected plants was studied from a molecular biology perspective, demonstrating the regulation of plant disease resistance pathways by circRNA928. This invention is of great significance for revealing the disease resistance function of circRNA928 and the breeding of disease-resistant cucumber varieties, and contributes to enriching vegetable breeding resources.

[0054] Example 1: Application of circRNA928 in regulating cucumber disease resistance

[0055] I. Construction and Identification of Cucumber Overexpressing circRNA928

[0056] The overexpression vector pCAMBIA1304-circRNA928 was constructed using Gateway technology, and cucumber was genetically transformed using Agrobacterium tumefaciens-mediated protoplast method. The results of the circRNA928-transformed cucumbers were detected by quantitative PCR and Western blot. The specific steps are as follows:

[0057] 1. Construction of cucumber overexpression of circRNA928

[0058] (1) Construction of overexpression vector

[0059] The artificially synthesized gene fragment expressing circRNA928 is 923 bp in length (DNA fragment shown in SEQ ID No. 3). It is formed by adding a downstream 200 bp flanking intron sequence to the 5' end of the DNA coding sequence of circRNA928 (SEQ ID No. 2) and adding a downstream 200 bp flanking intron sequence to its 3' end.

[0060] A 923 bp DNA fragment containing the sequence expressing circRNA928 (the DNA fragment shown in SEQ ID No. 3) was constructed into the entry vector pENTR (purchased from Invitrogen) using Gateway technology, and then GatewayLR Clonase was used. TM II. Enzymen Mix (purchased from Invitrogen) was used to homologously integrate a 923 bp DNA fragment into the overexpression vector pCAMBIA1304 (between the XbaⅠ and BstEⅡ sites) via LR reaction, resulting in the overexpression vector pCAMBIA1304-circRNA928.

[0061] (2) Construction of recombinant bacteria

[0062] The overexpression vector pCAMBIA1304-circRNA928 constructed in step (1) was introduced into Agrobacterium rhizogenes EHA105 to obtain recombinant bacteria EHA105 / pCAMBIA1304-circRNA928 containing the overexpression vector pCAMBIA1304-circRNA928.

[0063] (3) Obtaining cucumbers by overexpressing circRNA928

[0064] Cucumber protoplasts were prepared according to the established method and co-incubated with plasmid DNA containing the overexpression vector pCAMBIA1304-circRNA928 (protoplast PEG-mediated genetic transformation). The specific steps are as follows:

[0065] ① Cucumber seed germination: Select an appropriate amount of Xintai dense-thorned cucumber seeds, disinfect them with 75% ethanol for 20 seconds, discard the ethanol, wash them 4-5 times with sterile water to remove residual ethanol, pour off the sterile water after washing, add 2%-3% sodium hypochlorite to disinfect the cucumber seeds, place them in a shaker at 150 rpm for 6 minutes to disinfect, discard the sodium hypochlorite, wash them 4-5 times with sterile water to remove residual sodium hypochlorite, pour off the sterile water after washing, place the disinfected seeds on moist sterile filter paper, and germinate them in the dark for 24 hours.

[0066] ② Cucumber seed infection treatment: Using an infectious clone of Cucumber green mottle mosaic virus (CGMMV) as the source, the seeds were resuspended in an infiltration solution and then infected using a negative pressure method. The infected seeds were sown in plastic pots and cultured in a laboratory plant growth chamber at 25℃, with a photoperiod of 16h / 8h day / night, and humidity of 40-60%. Watering was done as needed during the plant's growth period.

[0067] ③ Material selection and enzymatic hydrolysis: Take flat, healthy cucumber cotyledons with a growth cycle of 7-12 days. Cut the cucumber cotyledons into strips of 0.5-1.0 mm with a sharp scalpel blade and place them in the prepared cellulose enzymatic hydrolysis solution (20 mL). Take about 40 cucumber cotyledons for each sample and enzymatically hydrolyze them on a shaker at 40 rpm for 4-5 hours.

[0068] Table 1. Preparation of Cellulose Enzymatic Hydrolysate

[0069]

[0070] ④ Obtaining protoplasts: After enzymatic hydrolysis, add an equal volume of pre-cooled W5 solution to the enzymatic hydrolysate used for hydrolyzing cucumber cotyledons, mix well to terminate the reaction, and filter the hydrolysate using a nylon membrane pre-wetted with W5. Centrifuge at 200g, 4℃ for 2min, discard the supernatant, add 10mL of W5 solution to resuspend (pre-cooled), centrifuge at 200g, 4℃ for 2min, discard the supernatant, repeat once, retain a small amount of supernatant for resuspending, place on ice and let stand for 30min.

[0071] Table 2. Preparation of W5 solution

[0072]

[0073] ⑤ Transformation:

[0074] a. As required by the experiment, aliquot the protoplasts from the 50mL round-bottom centrifuge tubes (after ice bath) into 2mL centrifuge tubes, centrifuge at 150g for 2min, discard the supernatant, and resuspend the protoplasts with an appropriate amount of MMG solution to a concentration of approximately 2×10⁻⁶ / mL. 6 indivual.

[0075] Table 3. Preparation of MMG solution

[0076]

[0077] b. Add 20 μg of plasmid containing the overexpression vector pCAMBIA1304-circRNA928 at a concentration of 1 μg / μL to a new 2 mL centrifuge tube, and add 100 μL of protoplasts (approximately 2 × 10⁻⁶). 5 (One) Gently blend.

[0078] c. Add 120 μL of 20% PEG 4000 solution, mix gently until no layering occurs, place horizontally, and induce conversion of the mixture for about 15 min.

[0079] d. At room temperature, add 600 μL of W5 solution to the induction sample and mix gently to terminate the transformation reaction. Centrifuge at 150 g for 2 min at 25 °C and discard the supernatant. Resuspend the cucumber protoplasts in 500 μL of WI solution and incubate at room temperature in the dark for about 24 h. Collect the transformed cucumber protoplasts.

[0080] Table 4. Preparation of WI solution

[0081]

[0082] II. Disease resistance analysis of cucumbers overexpressing circRNA928

[0083] The expression level of circRNA928 and the accumulation of CGMMVRNA in transformed cucumber protoplasts were detected by qRT-PCR, and the accumulation of CGMMV CP in cucumber protoplasts was detected by Western blot.

[0084] qRT-PCR was performed using Divergent primers specific to circRNA928 and specific primers specific to CGMMV, respectively, and 2 -△△Ct The relative expression levels of circRNA928 and CGMMV capsid protein genes were calculated using a qPCR method, followed by expression analysis. The upstream primer sequence for circRNA928 was 5'-CAGTTTTCTGCAATCTGTTGTTGT-3', and the downstream primer sequence was 5'-CAGACCCGAGACACTGTACC-3'; the upstream primer sequence for the internal reference gene EF-1a was 5'-ACTGGTGGTTTTGAGGCTGGT-3', and the downstream primer sequence was 5'-CTTGGAGTATTTGGGTGTGGT-3'; the upstream primer sequence for the internal reference gene Ubiquitin was 5'-CTAATGGGGAGTGGGGAAGTA-3', and the downstream primer sequence was 5'-GTCTGGATGGACAATGTTGAT-3'; the qPCR primer sequences for the CGMMV capsid protein gene were 5'-ACAGCCGCTAGGGCTGAGATA-3' upstream and 5'-CCAATGAGCAAACCGTTCGAT-3' downstream.

[0085] The results showed that in cucumbers overexpressing circRNA928 (OE-circRNA928), the expression level of circRNA928 increased by 1.34 times compared to the empty vector control (EV). Figure 1 The accumulation of CGMMV RNA increased by 6.63 times. Figure 2 The accumulation of CGMMV CP protein increased by 2.19 times. Figure 3 Cucumber leaves overexpressing circRNA928 showed more chlorotic and yellow spots than the control group transduced with empty vector.

[0086] In conclusion, compared with the control group, the transgenic plants overexpressing circRNA928 showed lower disease resistance, indicating that circRNA928 is an RNA associated with plant disease resistance and can be used to regulate the disease resistance of target plants. sequence list <110> China Agricultural University <120> Circular RNA molecule circRNA928 and its encoding gene and applications <130> WHOI1220038 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 523 <212> RNA <213> Cucumber (Cucumis sativus L.) <400> 1 guugagggca ggaaauguua uuuuuaauua uagauacuac aacaacagau ugcagaaaac 60 ugaaguaacu gaagacuuuu ccuguccauu uugcuugauc aaaugugcaa gcuuuaaggg 120 cuugagaugc cacuuguuau cauuacacga ucucuucaac uuugaauuuu ggguaaccga 180 agaguaccaa gcuguaaacg uuuccaugaa gacugauguu uggaggucug agaucaucgc 240 agauggcauu gauccaaagc agcaaacauu cuucuucugc ucaaggccuc ucagacguaa 300 aaaggcuaag agcgcaguuc aaagugcaaa gcacauacau ccuuuuguug uagagucuga 360 auauucggac aaggcggaug augcucaauc uagcaaaggg cacaaugucg caccaacuau 420 gcuucaauuu gccaagacga gaaaauuauc gauugaacgu gcagacccga gacacuguac 480 ccuccuaagg aagagacagu ucuuucauuc ucacagagcu cag 523 <210> 2 <211> 523 <212> DNA <213> Cucumber (Cucumis sativus L.) <400> 2 gttgagggca ggaaatgtta tttttaatta tagatactac aacaacagat tgcagaaaac 60 tgaagtaact gaagactttt cctgtccatt ttgcttgatc aaatgtgcaa gctttaaggg 120 cttgagatgc cacttgttat cattacacga tctcttcaac tttgaatttt gggtaaccga 180 agagtaccaa gctgtaaacg tttccatgaa gactgatgtt tggaggtctg agatcatcgc 240 agatggcatt gatccaaagc agcaaacatt cttcttctgc tcaaggcctc tcagacgtaa 300 aaaggctaag agcgcagttc aaagtgcaaa gcacatacat ccttttgttg tagagtctga 360 atattcggac aaggcggatg atgctcaatc tagcaaaggg cacaatgtcg caccaactat 420 gcttcaattt gccaagacga gaaaattatc gattgaacgt gcagacccga gacactgtac 480 cctcctaagg aagagacagt tctttcattc tcacagagct cag 523 <210> 3 <211> 923 <212> DNA <213> Cucumber (Cucumis sativus L.) <400> 3 aatctggtct ggacattgaa atgtaatatc tgtatctggt tctcaggaaa ttagtttagt 60 ttgtatgtct caaaatttgt tgtggcaatt tttgtccaaa aattgttat ttgctgctga 120 tcagatcata attcaaaatg gttttatgtt tatggaataa tgtatacatt acatttgctt 180 tcttgttctt tatgtttcag gttgagggca ggaaatgtta ttttaatta tagatactac 240 aacaacagat tgcagaaaac tgaagtaact gaagactttt cctgtccatt ttgcttgatc 300 aaatgtgcaa gctttaaggg cttgagatgc cacttgttat cattacacga tctcttcaac 360 tttgaattt gggtaaccga agagtaccaa gctgtaaacg tttccatgaa gactgatgtt 420 tggaggtctg agatcatcgc agatggcatt gatccaaagc agcaaacatt cttcttctgc 480 tcaaggcctc tcagacgtaa aaaggctaag agcgcagttc aaagtgcaaa gcacatacat 540 ccttttgttg tagagtctga atattcggac aaggcggatg atgctcaatc tagcaaaggg 600 cacaatgtcg caccaactat gcttcaattt gccaagacga gaaaattatc gattgaacgt 660 gcagacccga gacactgtac cctcctaagg aagagacagt tctttcattc tcacagagct 720 caggtgaatt ccctttttat cttattattg atgtgtaaaa ttttcatggt gcacaacaga 780 tgggaagtat ttgtgcatct gtatcccatc tgtattttgt gttacttttt ccatccactt 840 tttgcattcc tttgttagtt tcgtacctgg attctgatga tatatatgct taaaaaaatt 900 ctagaaatta tcctaatttc tag 923

Claims

1. A circRNA molecule, characterized in that, The nucleotide sequence of the circRNA molecule is SEQ ID No. 1 in the sequence listing.

2. A nucleic acid molecule encoding the circRNA molecule of claim 1, wherein the sequence of the encoding gene is SEQ ID No. 2 or SEQ ID No. 3 in the sequence listing.

3. An expression box, characterized in that, The expression cassette contains a nucleic acid molecule encoding the circRNA molecule of claim 1.

4. A recombinant vector, characterized in that, The recombinant vector comprises a nucleic acid molecule encoding the circRNA molecule of claim 1, or the expression cassette of claim 3.

5. A recombinant microorganism, characterized in that, The recombinant microorganism comprises a nucleic acid molecule encoding the circRNA molecule of claim 1, or the expression cassette of claim 3, or the recombinant vector of claim 4.

6. The application of the circRNA molecule of claim 1, or the nucleic acid molecule encoding the circRNA molecule of claim 1, or the expression cassette of claim 3, or the recombinant vector of claim 4, or the recombinant microorganism of claim 5 in reducing plant disease resistance; wherein the disease resistance is resistance to cucumber green mottle mosaic virus.

7. A method for cultivating transgenic plants with reduced disease resistance, characterized in that, The method includes introducing a nucleic acid molecule encoding the circRNA molecule of claim 1, or an expression cassette containing the circRNA molecule of claim 1, or a recombinant vector containing the nucleic acid molecule encoding the circRNA molecule of claim 1, or a recombinant microorganism containing the nucleic acid molecule encoding the circRNA molecule of claim 1 into a recipient plant, to obtain a transgenic plant with lower disease resistance than the recipient plant; wherein the disease resistance is resistance to cucumber green mottle mosaic virus.

8. The application of the circRNA molecule of claim 1, the nucleic acid molecule encoding the circRNA molecule of claim 1, or the method of claim 7 in establishing a plant model with reduced disease resistance; wherein the disease resistance is resistance to cucumber green mottle mosaic virus.

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