CN115074364A - Circular RNA molecule circRNA928 and coding gene and application thereof - Google Patents

Circular RNA molecule circRNA928 and coding gene and application thereof Download PDF

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CN115074364A
CN115074364A CN202210728370.1A CN202210728370A CN115074364A CN 115074364 A CN115074364 A CN 115074364A CN 202210728370 A CN202210728370 A CN 202210728370A CN 115074364 A CN115074364 A CN 115074364A
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circrna
molecule
circrna928
nucleic acid
plant
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梁超琼
李健强
罗来鑫
任博文
苗朔
刘�文
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China Agricultural University
Shaanxi Academy of Forestry
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Shaanxi Academy of Forestry
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    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
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    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
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Abstract

The invention discloses a circular RNA molecule circRNA928 and a coding gene and application thereof. The nucleotide sequence of the circRNA molecule is SEQ ID No.1 in a sequence table. The circRNA of the invention has a regulating effect on plant disease resistance ways. The disease-resistant function of the circRNA has great significance for the cultivation of disease-resistant cucumber varieties and is beneficial to the enrichment of vegetable breeding resources.

Description

Circular RNA molecule circRNA928 and coding gene and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to circular RNA molecule circRNA928 and a coding gene and application thereof.
Background
Non-coding RNAs (ncRNAs) including micro RNA (miRNA), small interfering RNA (siRNA), circular RNA (circular RNA) and long-chain non-coding RNA (lncRNA) can participate in the expression regulation of protein coding genes at the transcription level and the post-transcription level. The difference from most linear RNAs is that circRNAs have covalently closed circular structures. As research progresses, types of circRNA accumulate, and the three main types are exon circRNA, intron circRNA, and exon-intron circRNA, respectively. In addition, there are some other types of circular RNAs, such as fusion-gene-derived circular RNAs (fusion-circular RNAs, f-circular RNAs), read-through circular RNAs formed by transcription and read-through of polymerase II (rt-circular RNAs), circular RNAs with overlapping regions and different sites, so-called mutually inclusive circular RNAs (circular included circular RNAs), and the like.
The circRNA has stable structure and rich varieties, is specifically expressed in cells and tissues, and plays an important role in regulating gene expression and plant growth and development. For example, Tomato leaves infected with yellow leaf curl virus (TYLCV) express 32 and 83 circRNAs specifically, respectively, compared to the control group, and the expression level of the circRNAs after virus infection is lower than that of the control group; transgenic rice overexpressing circR5g05160 can improve the disease resistance to rice blast (Magnaporthe oryzae); 1934 and 44 circRNAs which can respond to salt stress and generate differential expression are respectively identified in cucumber root and leaf samples; under high temperature stress conditions, 7 circrnas in cucumber can competitively bind 114 differentially expressed mirnas, thereby interfering with the regulation of 359 downstream target mrnas (involved in phytohormone signaling, plant-pathogen interaction, and glutathione metabolism process).
Disclosure of Invention
The invention aims to provide circRNA related to plant disease resistance and application thereof.
The name of the circRNA928 is the exon type circRNA molecule provided by the invention, and the nucleotide sequence of the circRNA molecule is SEQ ID No. 1. Wherein SEQ ID No.1 consists of 523 nucleotides.
Biological materials related to circRNA928 are also within the scope of the invention.
The biological material associated with circRNA928 is an expression cassette comprising a nucleic acid molecule encoding said circRNA molecule.
Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
In one embodiment of the invention the nucleic acid molecule encoding said circRNA molecule is as SEQ ID No.2 or SEQ ID No. 3.
The biological material associated with circRNA928 is a recombinant vector comprising a nucleic acid molecule encoding said circRNA molecule, or said expression cassette.
The biological material associated with circRNA928 is a recombinant microorganism comprising a nucleic acid molecule encoding said circRNA molecule, or said expression cassette, or said recombinant vector.
The biological material associated with circRNA928 is a transgenic plant cell line comprising a nucleic acid molecule encoding said circRNA molecule, or said expression cassette, or said recombinant vector, or said recombinant microorganism.
The circRNA molecule, the expression cassette or the recombinant vector or the recombinant microorganism or the transgenic plant cell line can be used to modulate plant disease resistance.
In a specific embodiment of the present invention, the expression cassette is a DNA capable of forming a circRNA molecule after transcriptional processing in a host cell, and the DNA may include not only a promoter for initiating transcription of a nucleic acid molecule encoding the circRNA molecule, but also a terminator for terminating transcription of a nucleic acid molecule encoding the circRNA molecule. Further, the expression cassette may also include an enhancer sequence. Promoters useful 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 of cauliflower mosaic virus 35S; suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV35S terminator.
In an embodiment of the invention, the promoter in the expression cassette which initiates transcription of the nucleic acid molecule encoding the circRNA molecule is CaMV35S promoter, and the terminator which terminates transcription of the nucleic acid molecule encoding the circRNA molecule is agrobacterium nopaline synthase terminator (NOS terminator).
In embodiments of the invention, the vector may be a plasmid, a phage, or a viral vector.
The recombinant vector can be an expression vector for expressing the circRNA molecule, in particular to a recombinant expression vector containing SEQ ID No.2 or SEQ ID No.3, and the starting vector can be pCAMBIA 1304.
In a specific embodiment of the invention, the recombinant vector is pCAMBIA1304-circRNA 928; the pCAMBIA1304-circRNA928 is a recombinant vector obtained by integrating 923bp DNA fragment (DNA fragment shown in SEQ ID No. 3) between Xba I and BstE II sites of pCAMBIA1304 vector. The DNA molecule shown in SEQ ID No.3 is a sequence obtained by adding 200bp flanking introns to the upstream and downstream of the coding sequence (shown in SEQ ID No.2) of the circRNA molecule.
The invention provides a method for breeding transgenic plants with reduced resistance, which comprises the step of introducing the circRNA molecule, or the nucleic acid molecule for coding the circRNA molecule, or the expression cassette containing the nucleic acid molecule for coding the circRNA molecule, or the recombinant vector containing the nucleic acid molecule for coding the circRNA molecule into a receptor plant to obtain transgenic plants with lower disease resistance than the receptor plant.
In the method for breeding transgenic plants with reduced resistance, specifically, the method is to introduce a nucleic acid molecule encoding the circRNA molecule into a receptor plant, screen to obtain transgenic plants over-expressing the circRNA molecule, and obtain transgenic plants with lower disease resistance than the receptor plant.
In a particular embodiment of the invention, the nucleic acid molecule encoding said circRNA molecule is introduced into the recipient plant via the recombinant vector pCAMBIA1304-circRNA 928.
In the method for producing a transgenic plant having reduced resistance, the nucleic acid molecule for expressing a circRNA molecule, the expression cassette or the recombinant vector is introduced into the recipient plant by using the recombinant microorganism.
The disease resistance or resistance is preferably resistance to CGMMV (Cucumber green mosaic virus).
In an embodiment of the present invention, the recombinant microorganism may be yeast, bacteria, algae, or fungi. The bacteria may be agrobacterium; the agrobacterium may specifically be EHA105 agrobacterium.
In a specific embodiment of the invention, in the method for breeding transgenic plants with reduced resistance, the recombinant microorganism is EHA105 agrobacterium containing the recombinant vector pCAMBIA1304-circRNA 928.
In the above method, the transgenic plant exhibits a lower disease resistance than the recipient plant exhibited in all or part of the following A1) -A3):
A1) the accumulation amount of CGMMV (Cucumber green mottle mosaic virus) RNA in the transgenic plant is higher than that of the receptor plant;
A2) the accumulation amount of the CGMMV coat protein in the transgenic plant is higher than that of the receptor plant;
A3) the transgenic plant leaves have more chlorosis and yellowness spots than the recipient plant.
In embodiments of the invention, the transgenic plant cell line does not include propagation material of the plant.
In the embodiment of the invention, the regulation of plant disease resistance is the reduction of plant disease resistance, and is embodied in all or part of the following B1) -B3):
B1) when the expression level of circRNA928 in the plant protoplast is increased, the accumulation amount of CGMMV RNA in the plant is increased;
B2) when the expression level of circRNA928 in the plant protoplast is increased, the accumulation amount of CGMMV coat protein in the plant is increased;
B3) when the expression level of circRNA928 in the plant is increased, the number of chlorosis and yellowing spots on the leaves of the plant is increased.
In the above method, the plant is a dicotyledonous plant; the dicotyledonous plant can be cucumber; the cucumber can be specifically a Xintai Mici cucumber.
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 the progeny thereof. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include protoplasts, seeds, whole plants, and cells.
The invention adopts a plurality of biological means such as bioinformatics prediction, a molecular cloning technology, agrobacterium-mediated transformation, real-time fluorescence quantitative PCR, Western blot and the like, takes cucumbers over-expressing circRNA928 as research objects, and cucumber transferred into an empty vector pCAMBIA1304 as a contrast, firstly over-expresses the circRNA928, observes chlorosis and etiolation spots on leaves after diseases occur by measuring the virus RNA accumulation amount and the coat protein accumulation amount after CGMMV (cucumber green mottle mosaic virus) inoculation, and researches the influence of the circRNA928 on the damage degree of infected plants from the aspect of molecular biology. Compared with the control group plants, the disease resistance of the transgenic plants over-expressing the circRNA928 is proved to be lower than that of the control group plants, and the circRNA928 is related to the plant disease resistance and can be used for regulating the disease resistance of target plants.
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Figure 1 is a quantitative analysis of circRNA928 in cucumber protoplasts overexpressing circRNA 928.
Fig. 2 is an analysis of CGMMV RNA accumulation in cucumber protoplasts overexpressing circRNA 928.
Fig. 3 is an analysis of CGMMV Coat Protein (CP) accumulation in cucumber protoplasts overexpressing circRNA 928.
EV in FIGS. 1-3 represents an Empty vector (Empty vector).
OE in FIGS. 1-3 represents overexpression (Over expression).
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Agrobacterium tumefaciens EHA105 in the examples described below was purchased from Shanghai Diego Biotechnology, Inc.
Agrobacterium-competent cells GV3101 in the examples described below were purchased from Shanghai-Weidi Biotech Ltd.
The inbred line "Xintai Mici" of cucumber in the following examples was purchased from the Xintai Mici cucumber stock farm, Xintai City, Shandong province.
The overexpression vector pCAMBIA1304 in the following examples was purchased from Hunan Fenghui Biotech Co., Ltd.
The sequence of the circRNA928 in the invention is an RNA molecule shown as SEQ ID No.1 in a sequence table.
The coding gene sequence of the circRNA928 is a DNA molecule shown as SEQ ID No.2 in a sequence table.
The nucleic acid sequence used for constructing the vector for over-expressing circRNA928 in the invention is a DNA molecule shown as SEQ ID No.3 in a sequence table. The sequence is specifically a sequence obtained by adding 200bp flanking introns to the upstream and downstream of the sequence shown in SEQ ID No. 2.
The invention takes the cucumber transformed with the circRNA928 as a research object, takes the wild type Xintai Murray cucumber as a contrast, researches the influence of the circRNA928 on the damage degree of the infected plant from the perspective of molecular biology by detecting the accumulation amount of virus RNA and the content of virus protein in a receptor plant challenging to inoculate cucumber green mottle mosaic virus, and proves that the circRNA928 regulates and controls the disease resistance way of the plant. The invention has great significance for revealing the disease-resistant function of the circRNA928 and the cultivation of disease-resistant cucumber varieties, and is beneficial to enriching vegetable breeding resources.
Example 1 application of circRNA928 in regulation and control of cucumber disease resistance
Construction and identification of cucumber overexpressing circRNA928
An overexpression vector pCAMBIA1304-circRNA928 is constructed by adopting the Gateway technology, genetic transformation of cucumber is carried out by adopting an agrobacterium tumefaciens mediated protoplast method, and the obtained cucumber with the circRNA928 is detected by quantitative PCR and Western blot. The method comprises the following specific steps:
1. construction of cucumber overexpressing circRNA928
(1) Construction of overexpression vectors
The length of the artificially synthesized gene fragment for expressing the circRNA928 is 923bp (a DNA fragment shown in SEQ ID No. 3), and the gene fragment is formed by adding an upstream 200bp flanking intron sequence to the 5 'end of a DNA coding sequence (SEQ ID No.2) of the circRNA928 and adding a downstream 200bp flanking intron sequence to the 3' end of the DNA coding sequence.
A DNA fragment having a size of 923bp (DNA fragment shown in SEQ ID No. 3) containing a sequence expressing circRNA928 was constructed into an entry vector pENTR (purchased from Invitrogen) by the Gateway technique, and then Gateway LR clone was used TM II enzyme Mix (from Invitrogen) A DNA fragment of 923bp in size was incorporated by homologous recombination into the over-expression vector pCAMBIA1304 (between Xba I and BstE II sites) by LR reaction to give the over-expression vector pCAMBIA1304-circRNA 928.
(2) Construction of recombinant bacterium
And (2) introducing the overexpression vector pCAMBIA1304-circRNA928 constructed in the step (1) into the Agrobacterium tumefaciens EHA105 to obtain a recombinant strain EHA105/pCAMBIA1304-circRNA928 containing the overexpression vector pCAMBIA1304-circRNA 928.
(3) Obtaining of cucumber overexpressing circRNA928
Cucumber protoplasts were prepared according to established procedures and incubated with plasmid DNA containing the overexpression vector pCAMBIA1304-circRNA928, respectively (protoplast PEG-mediated genetic transformation), as follows:
accelerating germination of cucumber seeds: selecting a proper amount of Xintai honey thorn cucumber seeds, sterilizing for 20s by using 75% ethanol, removing the ethanol, washing for 4-5 times by using sterilized water, removing residual ethanol, pouring out the sterilized water after washing, adding 2% -3% sodium hypochlorite to sterilize the cucumber seeds, putting the cucumber seeds into a shaking table at 150rpm for 6min to sterilize, removing the sodium hypochlorite, washing for 4-5 times by using the sterilized water, removing the residual sodium hypochlorite, pouring out the sterilized water after washing, putting the sterilized seeds on a moist sterile filter paper sheet, and accelerating germination for 24h under dark conditions.
Secondly, carrying out sub-detoxification treatment on cucumber seeds: cucumber Green Mottle Mosaic Virus (CGMMV) infectious clone is used as a poison source, infiltration liquid is used for resuscitation and heavy suspension, and the seed for germination acceleration is detoxified by a negative pressure method. Sowing seeds with poison in a plastic flowerpot, culturing in a plant growth incubator in a laboratory, controlling the temperature at 25 ℃, the photoperiod at 16h/8h day/night and the humidity at 40-60%, and watering as required during the growth of the plants.
Material selection and enzymolysis: taking out flat and healthy cucumber cotyledons with the growth cycle of 7-12 days. Cutting the cucumber cotyledon into 0.5-1.0mm strips by using a sharp surgical blade, placing the strips into prepared cellulose enzymolysis solution (20mL), cutting about 40 pieces of cucumber cotyledon for each sample, and performing enzymolysis for 4-5h on a shaker at 40 rpm.
TABLE 1 preparation of cellulase hydrolysate
Figure BDA0003713975090000071
Obtaining protoplasts: after the enzymolysis is finished, adding the equal volume of W5 solution pre-cooled in advance into the enzymolysis liquid for enzymolysis of the cucumber cotyledon, mixing uniformly to stop the reaction, and filtering the enzymolysis product by using a nylon membrane pre-wetted by W5. Centrifuging for 2min at 4 ℃ at 200g, discarding the supernatant, adding 10mL of W5 solution for resuspension (precooling), centrifuging for 2min at 4 ℃ at 200g, discarding the supernatant, repeatedly retaining a little of the supernatant for resuspension, placing on ice, and standing for 30 min.
TABLE 2 preparation of W5 solution
Figure BDA0003713975090000081
Converting:
a. according to the experimental requirements, the protoplast after ice bath in a 50mL round-bottom centrifuge tube is subpackaged into 2mL centrifuge tubes, 150g is centrifuged for 2min, the supernatant is discarded, and an appropriate amount of MMG solution is used for resuspending the protoplast to make the concentration of the protoplast be about 2 multiplied by 10 per milliliter 6 And (4) respectively.
TABLE 3 preparation of MMG solution
Figure BDA0003713975090000082
b. Add 20. mu.g of plasmid containing the overexpression vector pCAMBIA1304-circRNA928 at a concentration of 1. mu.g/. mu.L to a new 2mL centrifuge tube and add 100. mu.L of protoplasts (ca. 2X 10) 5 One) gently mixed.
c. Adding 120 μ L of 20% PEG 4000 solution, gently mixing until no layering occurs, placing horizontally, and inducing the mixture to transform for about 15 min.
d. To the induction sample was added 600. mu.L of W5 solution at room temperature and gently mixed to terminate the conversion reaction. Centrifuge at 150g for 2min at 25 ℃ and discard the supernatant. Adding 500. mu.L WI solution to suspend the cucumber protoplast again, culturing at room temperature in the dark for about 24h, and collecting the transformed cucumber protoplast.
TABLE 4 preparation of WI solutions
Figure BDA0003713975090000083
Secondly, disease resistance analysis of cucumber overexpressing circRNA928
And detecting the expression level of circRNA928 and the accumulation amount of CGMMV RNA in the transformed cucumber protoplast by adopting a qRT-PCR method, and detecting the accumulation amount of CGMMV CP in the cucumber protoplast by adopting Western blot.
qRT-PCR detection is carried out by respectively adopting specific primers Divergent primers of circRNA928 and specific primers of CGMMV, and 2 -△△Ct The method calculates the relative expression quantity of the circRNA928 and CGMMV coat protein genes, and then analyzes the expression condition. The sequence of the upstream primer of circRNA928 is 5'-CAGTTTTCTGCAATCTGTTGTTGT-3', and the sequence of the downstream primer is 5'-CAGACCCGAGACACTGTACC-3'; the upstream primer sequence of the reference gene EF-1a is 5'-ACTGGTGGTTTTGAGGCTGGT-3', and the downstream primer sequence is 5'-CTTGGAGTATTTGGGTGTGGT-3'; the upstream primer sequence of the internal reference gene Ubiquitin is 5'-CTAATGGGGAGTGGGGAAGTA-3', and the downstream primer sequence is 5'-GTCTGGATGGACAATGTTGAT-3'; the qPCR primer sequence of the CGMMV coat protein gene is upstream 5'-ACAGCCGCTAGGGCTGAGATA-3' and downstream 5'-CCAATGAGCAAACCGTTCGAT-3'.
The results show that: in cucumber overexpressing circRNA928 (OE-circRNA928), the expression level of circRNA928 was increased 1.34 fold (fig. 1), the accumulation amount of CGMMV RNA 6.63 fold (fig. 2), and the accumulation amount of CGMMV CP protein 2.19 fold (fig. 3) compared to the empty vector control (EV). The chlorosis, etiolation spots on cucumber leaves overexpressing circRNA928 were more than the empty vector control.
In conclusion, compared with the control group plants, the disease resistance of the transgenic plants over-expressing circRNA928 is lower than that of the control group plants, which indicates that the circRNA928 is the RNA related to the plant disease resistance and can be used for regulating the disease resistance of the target plants.
Sequence listing
<110> university of agriculture in China
<120> circular RNA molecule circRNA928 and coding gene and application thereof
<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 aattgtttat ttgctgctga 120
tcagatcata attcaaaatg gttttatgtt tatggaataa tgtatacatt acatttgctt 180
tcttgttctt tatgtttcag gttgagggca ggaaatgtta tttttaatta tagatactac 240
aacaacagat tgcagaaaac tgaagtaact gaagactttt cctgtccatt ttgcttgatc 300
aaatgtgcaa gctttaaggg cttgagatgc cacttgttat cattacacga tctcttcaac 360
tttgaatttt 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 (9)

1. A circRNA molecule is characterized in that the nucleotide sequence of the circRNA molecule is SEQ ID No.1 in a sequence table.
2. Nucleic acid molecule encoding the circRNA molecule of claim 1, preferably the sequence of the encoding gene is SEQ ID No.2 of the sequence list or SEQ ID No.3 of the sequence list.
3. An expression cassette comprising a nucleic acid molecule encoding the circRNA molecule of claim 1.
4. A recombinant vector comprising a nucleic acid molecule encoding the circRNA molecule of claim 1, or the expression cassette of claim 3.
5. A recombinant microorganism comprising 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. A transgenic plant cell line comprising 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, or the recombinant microorganism of claim 5.
7. Use of the circRNA molecule of claim 1, or 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, or the recombinant microorganism of claim 5, or the transgenic plant cell line of claim 6 for modulating disease resistance in a plant.
8. A method for breeding a transgenic plant with reduced resistance comprising introducing into a recipient plant a nucleic acid molecule encoding the circRNA molecule of claim 1, or an expression cassette comprising a nucleic acid molecule encoding the circRNA molecule of claim 1, or a recombinant vector comprising a nucleic acid molecule encoding the circRNA molecule of claim 1, or a recombinant microorganism comprising a nucleic acid molecule encoding the circRNA molecule of claim 1, to obtain a transgenic plant with lower disease resistance than the recipient plant; the disease resistance is preferably resistance to cucumber green mottle mosaic virus.
Preferably, the plant is a dicot.
9. Use of the circRNA molecule of claim 1, a nucleic acid molecule encoding the circRNA molecule of claim 1, or the method of claim 8, in the creation of a plant model with reduced disease resistance; preferably, the disease resistance is preferably resistance to cucumber green mottle mosaic virus.
CN202210728370.1A 2022-06-25 2022-06-25 Circular RNA molecule circRNA928 and coding gene and application thereof Pending CN115074364A (en)

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