RNA-circularizing nucleic acid molecules and uses thereof

Abstract

This invention discloses an RNA-circularized nucleic acid molecule and its applications, relating to the field of biotechnology, and particularly to a circularized RNA molecule capable of preparing nucleic acid molecules and / or vectors that promote circRNA circularization. Its nucleotide sequence is shown in SEQ ID NO.1. The circularized RNA molecule, the nucleic acid molecule that promotes accurate circRNA circularization, and the RNA circularization vector provided by this invention enable the accurate circularization of circRNA in target plants, providing a new tool for exploring the functional study of circRNA overexpression.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a nucleic acid molecule and its applications. Background Technology

[0002] In recent years, with the continuous development of biotechnology, non-coding RNA has gradually become a rising star in plant gene function research. Unlike linear RNAs such as mRNA, tRNA, and miRNA, circular RNA (circRNA) is a new type of non-coding RNA with regulatory functions. It has a closed circular structure and lacks 3′ and 5′ ends, thus exhibiting higher stability within cells. circRNAs are widely present in the transcriptomes of eukaryotes, and studies have shown that they have high sequence conservation, are specifically expressed in cellular tissues, and play an important role in regulating gene expression and regulating plant growth and development.

[0003] It is well known that circRNA formation depends on flanking endogenous introns and inversely complementary sequences. Therefore, some studies have utilized flanking endogenous introns of circRNAs while introducing inversely complementary sequences, adding an inversely complementary sequence upstream and downstream of the circRNA sequence. After plasmid transfection into cells, a linear RNA chain consisting of upstream + circRNA + downstream is transcribed first, and then circularization is induced by the inverse complementary pairing of upstream and downstream sequences (Gao et al. 2019; Liu et al. 2025). Although these studies can indeed induce circRNA circularization using flanking endogenous introns and artificially created inversely complementary sequences, there is still a lack of plant-derived vectors with specific sequences that can ensure accurate circularization of different circRNAs. This also affects the functional study of plant circRNAs. Summary of the Invention

[0004] This invention addresses the problem of the lack of plant-derived vectors that ensure accurate circularization of circRNAs in existing technologies, which hinders research on plant circRNA genes. It provides a circular RNA and nucleic acid molecule. The RNA circularization vector provided by this invention can offer a nucleic acid molecule that promotes accurate circularization of circular RNA, enabling the accurate circularization of circRNAs in target plants and providing a new tool for exploring the function of circRNAs after overexpression.

[0005] To achieve the technical objective of this invention, the first aspect of this invention provides a circular RNA molecule capable of preparing nucleic acid molecules and / or vectors that promote the circularization of circRNA, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] The circular RNA is formed by back splicing of exons 3, 4 and 5 of the parent gene.

[0007] In particular, the circular RNA is derived from alfalfa plants and is 525 bp in length.

[0008] To achieve the technical objective of this invention, a second aspect of this invention provides a primer set capable of identifying the primers for the circular RNA of claim 1, the nucleotide sequences of which are shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.

[0009] To achieve the technical objective of this invention, a third aspect of this invention provides a vector for overexpressing the circular RNA molecule of claim 1, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0010] The gDNA sequence of the circular RNA molecule is shown in SEQ ID NO.7.

[0011] To achieve the technical objective of this invention, a fourth aspect of this invention provides a nucleic acid molecule capable of cyclizing a biologically active target RNA molecule, the nucleotide sequence of which is shown in SEQ ID NO.8.

[0012] The aforementioned nucleic acid molecules are derived from the flanking sequence of alfalfa circRNA1780.

[0013] Specifically, the aforementioned nucleic acid molecules are derived from the forward repeat sequence of the flanking sequence of alfalfa circRNA1780.

[0014] In particular, the size of the aforementioned nucleic acid molecule is 245 bp.

[0015] To achieve the technical objective of this invention, a fifth aspect of this invention provides an RNA circularization vector having a nucleotide sequence as shown in SEQ ID NO.9, which can accurately circularize a target circRNA gene to form a circular RNA molecule.

[0016] To achieve the technical objective of this invention, a sixth aspect of this invention provides a primer set capable of constructing an RNA circularization vector, the nucleotide sequences of which are shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13.

[0017] To achieve the technical objective of this invention, a sixth aspect of this invention provides a method for preparing circular RNA, comprising:

[0018] Circular RNA precursor molecules were prepared using the above-mentioned circularized RNA;

[0019] The target circRNA gene was circularized to obtain a circular target RNA.

[0020] To achieve the technical objective of this invention, a seventh aspect of this invention provides a method for preparing circular RNA, comprising: using the above-mentioned nucleic acid molecules or vectors to circularize a target circRNA gene to obtain circular target RNA.

[0021] To achieve the technical objective of the present invention, an eighth aspect of the present invention provides a method for expressing proteins in cells, comprising transfecting the aforementioned circular RNA into the cells.

[0022] Preferably, the cells are eukaryotic cells.

[0023] To achieve the technical objective of this invention, a ninth aspect of this invention provides the application of the above-described circular RNA in circular gene research.

[0024] The gene research includes gene function studies and the discovery of new cyclic genes.

[0025] Specifically, the circular gene is a circular RNA derived from plants.

[0026] To achieve the technical objective of this invention, the tenth aspect of this invention provides a primer for identifying circular RNA;

[0027] The primer sequences, as shown in SEQ ID NO.14-17, are capable of identifying the circALDH7B4 gene; or,

[0028] The primers are those shown in SEQ ID NO.18-21, capable of identifying the circGORK gene; or,

[0029] The primers are as shown in SEQ ID NO.22-25, which are capable of identifying the circ175 gene of alfalfa;

[0030] The genomic nucleotide sequence of the alfalfa circ175 is shown in SEQ ID NO.26, and its nucleotide sequence is shown in SEQ ID NO.27.

[0031] Beneficial effects:

[0032] The circular RNA molecule provided by this invention can provide nucleic acid molecules or vectors that promote accurate circularization of circRNA, providing important support for accurate circularization and overexpression of circRNA. It achieves the technical effect of accurately circularizing circRNA of target plants using forward repeat sequences, changing the current two formation methods that require direct backsplice and lariat precursor to drive circularization, and providing a new tool for exploring the gene function of circRNA overexpression. Attached Figure Description

[0033] Figure 1 This describes the circular structure and verification of circRNA1780.

[0034] Figure 2 This is a graph showing the results of the RNase R tolerance test for circRNA1780;

[0035] Figure 3 This is the expression result of the overexpression vector of circRNA1780 in tobacco;

[0036] Figure 4 This is the expression result of the overexpression vector of circRNA1780 in alfalfa;

[0037] Figure 5 This is the ring structure of circALDH7B4 and its verification;

[0038] Figure 6 This is the expression result of the overexpression vector of circALDH7B4 in tobacco;

[0039] Figure 7 This relates to the ring structure of circGORK and its verification.

[0040] Figure 8 This is the expression result of the circGORK overexpression vector in tobacco;

[0041] Figure 9 This relates to the ring structure of circ175 and its verification.

[0042] Figure 10 This is the expression result of the overexpression vector of circ175 in tobacco. Detailed Implementation

[0043] In this application, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms related to nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology, as used herein, are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0044] As used herein, the terms “circRNA” or “circular RNA” are used interchangeably and refer to polynucleotides that form a circular structure by covalent bonds. As a type of single-stranded RNA, unlike linear RNA, it contains a covalently closed continuous loop, lacking a 5' cap and a 3' tail. circRNAs exhibit greater stability and resistance to intracellular and extracellular RNases than mRNAs, making them suitable for delivering critical payloads persistently expressed within them.

[0045] As used herein, the term "exon" refers to a nucleotide sequence present in a nucleic acid molecule that, after introns are removed during transcription, forms the mature form of an RNA molecule. Exons can be translated into proteins (e.g., in the case of messenger RNA (mRNA)).

[0046] As used herein, the term "nucleotide" refers to ribonucleotides, deoxyribonucleotides, their modified forms, or analogues thereof.

[0047] As used herein, a “nucleic acid molecule” refers to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) typically linked together by phosphodiester bonds. As used herein, the term “nucleic acid molecule” is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA. The terms “polynucleotide” and “nucleic acid molecule” are used interchangeably herein to describe polymers of any length (e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, or more bases) composed of nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and which can be produced enzymatically or synthetically, and which can hybridize with naturally occurring nucleic acids in a sequence-specific manner similar to that of two naturally occurring nucleic acids. Naturally occurring nucleic acids are composed of nucleotides, including guanine, cytosine, adenine, thymine, and uracil (G, C, A, T, and U, respectively).

[0048] As used in this article, the term "from" refers to the original source of nucleic acid molecules, but does not limit the method of preparation of nucleic acid molecules, such as chemical synthesis or recombination.

[0049] As used in this study, divergent primers (hereinafter referred to as "divergent primers") are primers whose ends face the target sequence of the PCR reaction, that is, the 5' end of the primer faces the 3' end of the target sequence. This type of primer design can be used to amplify two adjacent regions of the target sequence. Convergent primers (hereinafter referred to as "convergent primers") are primers whose ends face the same direction as the target sequence of the PCR reaction, that is, the 5' end of the primer corresponds to the 5' end or 3' end of the target sequence. This type of primer design can be used to amplify a longer region of the target sequence.

[0050] Example 1: Obtaining the circRNA1780 molecule

[0051] Reverse splicing of exons 3, 4, and 5 of the MtrunA17_Chr4g0001151 gene from alfalfa resulted in a 525 bp circRNA, the molecular structure of which is shown below. Figure 1 As shown, its sequence is shown in SEQ ID NO.1.

[0052] Based on the nucleotide sequence of the 525 bp circRNA, divergent primers AGGCTGCACCTGAAGAGGAG (as shown in SEQ ID NO.3) and AAGAGGGACTCTAATTGTTTATA (as shown in SEQ ID NO.4) were designed; convergent primers GCCACCTCCGGCAGAGATTT (as shown in SEQ ID NO.5) and GCAGCCTTCTCTTCCTCAGCA (as shown in SEQ ID NO.6) were also designed. The resulting 525 bp circRNA was subjected to PCR and sequencing to obtain the sequence shown in SEQ ID NO.6. Figure 1 The circular RNA structure shown is based on Figure 1 The band results show that the present invention can obtain gDNA and cDNA amplification bands using convergent primers, but can obtain cDNA bands using divergent primers. It is evident that the 525bp circRNA obtained in this application can be amplified in both forward and reverse directions, confirming its circular structure. Its gDNA sequence is shown in SEQ ID NO. 7.

[0053] To verify its circular RNA structure, this application tested its stability using an RNase R tolerance assay. The experimental methods and conditions followed conventional methods in the art, and this invention does not impose any limitations. The electrophoresis results of the RNase R tolerance cleavage test for circRNA are shown below. Figure 2 As shown, according to Figure 2 The results show that circRNA1780 exhibits clear bands after RNase R treatment, indicating that it is more stable than linear RNA.

[0054] Example 2: Construction of circRNA1780 overexpression vector

[0055] To construct an overexpression vector for circRNA1780, in one embodiment of this invention, pCAMBIA1302 was used as the vector. Specifically, pCAMBIA1302 was digested with Nco I and Eco065 I, followed by gel electrophoresis and recovery. The digestion, gel electrophoresis, and recovery methods are conventional in the art and are not limited by this invention. Then, the endogenous introns containing the flanking structures of circRNA1780, totaling 1524 bp, were expressed using primers C1780-OE-F (AACACGGGGGACTCTTGACCGTATATTATATAATATATATATTCTAT) and C1780-OE-R (...).

[0056] Using alfalfa genomic DNA as a template, a 1524 bp fragment was obtained by high-fidelity enzyme PCR. The reaction system for high-fidelity enzyme PCR was performed according to conventional methods in the art, or according to the instructions of commercially available high-fidelity enzymes, and this invention is not limited thereto. Finally, the fragment was ligated into the enzyme-digested vector via seamless cloning to generate the final vector circ1780-pCAMBIA1302, the sequence of which is shown in SEQ ID NO.2.

[0057] To analyze whether the constructed vector could produce the corresponding circRNA, we transformed the plasmid circ1780-pCAMBIA1302, which contained circRNA1780 overexpression, into Agrobacterium GV3101, obtained infected Agrobacterium fluid, and injected it into tobacco leaves. DNA and RNA were extracted, and the PCR and sequencing analysis results are as follows: Figure 3 As shown, according to Figure 3 The results show that both forward and reverse amplification can obtain clear bands, and the fragment size is correct, confirming that the vector constructed in this embodiment can express and produce 525bp circRNA1780.

[0058] Example 3: Expression of circRNA1780 in alfalfa plants

[0059] In one embodiment of the present invention, Agrobacterium tumefaciens liquid containing the circRNA1780 overexpression plasmid circ1780-pCAMBIA1302 was genetically transformed into alfalfa to obtain alfalfa plant material overexpressing circRNA1780. DNA and RNA were extracted from the plant material, and PCR and sequencing analyses were performed. The PCR primers (C780-con-F:GCCACCTCCGGCAGAGATTT (as shown in SEQ ID NO. 5) and

[0060] C780-con-R: GCAGCCTTCTCTTCCTCAGCA (as shown in SEQ ID NO. 6), the PCR product is 267 bp; C780-div-F: AGGCTGCACCTGAAGAGGAG (as shown in SEQ ID NO. 3) and C780-div-R1: AAGAGGGACTCTAATTGTTTATA (as shown in SEQ ID NO. 4), the PCR product is 145 bp. The PCR methods can be designed and obtained according to conventional methods in the art, and this invention is not limited thereto. Sequencing analysis results are as follows: Figure 4 As shown, according to Figure 4 The results show that the same verification results as in Example 2 were obtained, demonstrating that our constructed circRNA1780 overexpression was stably expressed in alfalfa plants and consistently produced the correct circular RNA.

[0061] RNA molecules.

[0062] Example 4: Obtaining RNA-circularized nucleic acid molecules

[0063] Based on the studies in Examples 1-3, it was found that overexpression of the circRNA1780 constructed in this application, both transient expression in tobacco and stable expression in alfalfa, could produce the correct circular RNA molecules. To obtain nucleic acid molecules that could promote RNA circularization, we analyzed the specific flanking sequences of circRNA1780 and screened for the 245bp forward repeat sequence CATATATTTTTTTGGTTTGAATTGAATGTTATATGTATATGTATATACACTAGTAAAATATGTTAGAACCGATCATGTGCTTGAGGATTGGAGTAGGACAATAGTCCATACTTTGTTTAAGAAAAATAAGGAAATGCAAATAAACAAGAGGAAATAAAATAGTTTTTATGTTCTCAAATGAAATTTGTGCTAAATATCAAATCAAACAAAATGAGTTAGCTAACAAAATCAGCATGTATATACAG (as shown in SEQ ID NO. 8). Experiments were designed to verify this, and the results showed that removing any 245bp fragment from the flanking sequence did not produce the correct circRNA1780 circular molecule. It is evident that the 245bp forward repeat sequence discovered in this invention is crucial for the circularization of circRNA molecules.

[0064] Example 5: Construction of circRNA overexpression vector

[0065] Using primers F: AACACGGGGGACTCTTGACCGTATATTATATAATATATATATTCTAT (as shown in SEQ ID NO. 10) and R: TACAGGCCTGTCAGATCTCTGTATATACATGCTGATTTTG (as shown in SEQ ID NO. 11), fragment 1 was obtained by high-fidelity enzyme PCR with the circRNA1780 overexpression vector as a template. Then, primers f: CAGAGATCTGACAGGCCTGTACTGTCCAATTCCAATATT (as shown in SEQ ID NO. 12) and R: GGGGAAATTCGAGCTGGTCACCCTGCAGTGGCCACCGACACAAG (as shown in SEQ ID NO. 11) were used. As shown in NO.13, using the circRNA1780 overexpression vector as a template, fragment 2 was obtained by high-fidelity enzyme PCR. Fragments 1 and 2 were recovered by gel extraction, then mixed in equal amounts as a template. High-fidelity enzyme PCR was performed using primers F and R to obtain a full-length fragment of 799 bp. After gel extraction, the fragment was ligated into the pCAMBIA1302 vector using a seamless cloning method. The pCAMBIA1302 vector was digested with Nco I and Eco065 I to finally generate the circRNA overexpression vector (as shown in SEQ ID NO.13). As shown in NO.9, part of its core sequence is as follows: GTATATTATATAATATATATATTCTATTATAAGAAGTTTGATCTTACATATATTTTTTTGGTTTGAATTGAATGTTATATGTATATGTATATACACTAGTAAAATATGTTAGAACCGATCATGTGCTTGAGGATTGGAGTAGGACAATAGTCCATACTTTGTTTAAGAAAAATAAGGAAATGCAAATAAACAAGAGGAAATAAAATAGTTTTTATGTTCTCAAATGAAATTTGTGCTAAATATCAAATCAAACAAAATGAGTTAGCTAACAAAATCAGCATGTATATACAG AGATCT GAC AGGCCTGTACTGTCCAATTCCAATATTTAACAAATTCAATTGATTTAACCATGGTTGGTTCAGCCGTTTTTTTTACTATACTAGGTCGTGTGTAAGAAATTGCGCAATTTATTTGATTGAACCAATGTATTTATTACGCGCGGAATATCATGTCGGGTTTTTTACTATAATATATATATTCTATTATAAGAAGTTTGAAGTTTGATCTTGCATATATTTTTTTGGTTTGAATTGAATGTTATATGTATATGTA TATACACTAGTAAAATATGTTAGAACCGATCATGTGCTTGAGGATTGGAGTAGGACAATAGTCCATACTTTGTTTAAGAAAAATAAGGAAATGCAAATAAACAAGAGGAAATAAAATAGTTTTTATGTTCTCAAATGAAATTTGTGCTAAATATCAAATCAAACAAAATGAGTTAGCTAACAAAATCAGCATGTATATACAGATTATATAATATACTCACAGCTCTTGTGTCGGTGGCCACTGCAG.

[0066] Application Example 1: Overexpression Analysis of the Arabidopsis thaliana circALDH7B4 Gene

[0067] The circALDH7B4 gene was overexpressed using the circRNA overexpression vector constructed in Example 5. The specific steps are as follows:

[0068] After digesting the circRNA overexpression vector constructed in Example 5 with Bgl II and Stu I enzymes, the gene sequence of Arabidopsis thaliana circALDH7B4 was ligated into the vector using a seamless cloning method. After ligation, there were no Bgl II and Stu I restriction sites.

[0069] The plasmid containing circALDH7B4 overexpression was transformed into Agrobacterium GV3101 and injected into tobacco leaves. DNA and RNA were extracted. PCR products using circ7B4-div-F: GCTTTGGAGGCGAGAAAGC (as shown in SEQ ID NO.14) and circ7B4-div-R: GACCTTGAGGAACCGAGTTGT (as shown in SEQ ID NO.15) yielded a 129 bp product. PCR products using circ7B4-con-F: AGATGCTGCTGTCGTCAAAGA (as shown in SEQ ID NO.16) and circ7B4-con-R: GACCTTGAGGAACCGAGTTGT (as shown in SEQ ID NO.17) yielded a 186 bp genomic product and a 107 bp cDNA product. PCR and sequencing analysis results are as follows: Figure 5 and 6 As shown in the table, the results confirm that the correct 248bp circALDH7B4 sequence can be generated, and its sequence is as follows:

[0070] TCATTTGGAGAGGCTGTTGCGATAAACAACTCGGTTCCTCAAGGTCTAAGC

[0071] AGCTCGATATTCACTCGGAACCCTGAAAACATCTTCAGGTGGATCGGACCA

[0072] CTGGGAAGTGACTGTGGCATTGTGAATGTGAACATACCGACGAATGGAGCT

[0073] GAGATTGGTGGAGCTTTTGGAGGCGAGAAAGCGACTGGTGGTGGTCGTGAAGCTGGAAGCGACTCATGGAAACAGTACATGCGTCGATCGACTTG.

[0074] Among them, the gDNA sequence of circALDH7B4 is TCATTTGGAGAGGCTGTTGCGATAAACAACTCGGTTCCTCAAGGTCTAAGCAGCTCGATATTCACTCGGAACCCTGAAAACATCTTCAGGTGGATCGGgtaagtgctataaaaacaacaacttaactcaatctttccgaccaatataatctactgaaattaa ttttaatgtgatggtttactactagACCACTGGGAAGTGACTGTGGCATTGTGAATGTGAACATACCGACGAATGGAGCTGAGATTGGTGGAGCTTTTGGAGGCGAGAAAGCGACTGGTGGTGGTCGTGAAGCTGGAAGCGACTCATGGAAACAGTACATGCGTCGATCGACTTG, the CDS sequence is.

[0075] Application Example 2: Overexpression Analysis of the Arabidopsis circGORK Gene

[0076] The circGORK gene was overexpressed using the circRNA overexpression vector constructed in Example 5. The specific steps are as follows:

[0077] After digesting the circRNA overexpression vector with Bgl II and Stu I enzymes, the Arabidopsis thaliana circGORK gene sequence was ligated into the vector using a seamless cloning method. After ligation, there were no Bgl II and Stu I restriction sites.

[0078] We transformed Agrobacterium GV3101 containing the circGORK overexpression plasmid, injected it into tobacco leaves, and extracted DNA and RNA. PCR was performed using circGORK-div-F: ACCAACACGTATTGCTTTCC (as shown in SEQ ID NO.18) and circGORK-div-R: TCCATGGGAGTGAACAATGA (as shown in SEQ ID NO.19), and using circGORK-con-F: TTTCTTCCGCGGTCTGCCTG (as shown in SEQ ID NO.20) and circGORK-con-R: TCCCAAGGGAAGCAACCGATG (as shown in SEQ ID NO.21). The PCR and sequencing analysis results are as follows: Figure 7 and Figure 8As shown in the figure, the RNA circularization vector provided in Example 5 of this invention can be used to obtain the circGORK gene, confirming that it can generate the correct 289bp circGORK gene, whose nucleotide sequence is: GTGGTACAAGGCATGGGAAATGTTTATATTGGTGTGGGCAATATACTCCTCATTGTTCACTCCCATGGAGTTTGGTTTCTTCCGCGGTCTGCCTGAGAGACTCTTTGTACTTGACATTGTTGGTCAGATCGCGTTCTTGGTCGATATTGTTCTTCAGTTCTTTGTTGCCTATCGCGATACTCAGACCTACCGGACTGTCTACAAACCAACACGTATTGCTTTCCGGTACTTGAAGTCGCATTTTCTCATGGATTTCATCGGTTGCTTCCCTTGGGATCTTATTTATAAG.

[0079] Among them, the circGORK genome sequence is GTGGTACAAGGCATGGGAAATGTTTATATTGGTGTGGGCAATATACTCCTCATTGTTCACTCCCATGGAGTTTGGTTTCTTCCGCGGTCTGCCTGAGAGACTCTTTGTACTTGACATTGTTGGTCAGATCGCGTTCTTGGTCGATATTGTTCTTCAGTTCTTTGTTGCCTATCGCGATA CTCAGACCTACCGGACTGTCTACAAACCAACACGTATTGCTTTCCGgtcagttatatatattagactcttacaatcattttaaccatttctacttatatagcctttaatggaggaagtatgtctttcatcagGTACTTGAAGTCGCATTTTCTCATGGATTTCATCGGTTGCTTCCCTTGGGATCTTATTTATAAG.

[0080] Application Example 3: Overexpression Analysis of the Alfalfa circ175 Gene

[0081] The circ175 gene was overexpressed using the circRNA overexpression vector constructed in Example 5. The specific steps are as follows:

[0082] After digesting the circRNA overexpression vector with Bgl II and Stu I enzymes, the circ175 gene sequence of alfalfa was ligated into the vector using a seamless cloning method. After ligation, there were no Bgl II and Stu I restriction sites.

[0083] We transformed Agrobacterium GV3101 containing the circ175 overexpression plasmid, injected it into tobacco leaves, and extracted DNA and RNA. We then performed PCR using C175-div-F:GGGCCCCTGCATACTCGTTC (as shown in SEQ ID NO.22) and C175-div-R:CCTCCGCACCCACCTTTGAG (as shown in SEQ ID NO.23); and C175-con-F:GAAGGGAGCTCCAACTACACCA (as shown in SEQ ID NO.24) and C175-con-R:CTGAGATTTTATCGCCGCAATG (as shown in SEQ ID NO.25). The PCR and sequencing analysis results are as follows: Figure 9 and Figure 10As shown in the results, the RNA circularization vector provided in Example 5 of this invention can be used to obtain the alfalfa circ175 gene, confirming that the correct 523bp circ175 gene can be generated, and its sequence is (as shown in SEQ ID NO.27).

[0084] Among them, the circ175 genomic sequence is GAAGGGAGCTCCAACTACACCATTGATAACTATTGCTATTACAAAGCTAATATCTGAAGTTCTTGAGAGGAACAAATTACCTGGGGCAATATTCACCTCTTTCTGTGGAGGTGCTGATATTGGGCAGGCAATATCGAAAGACAGACGCATTCCACTGGTTTCATTTACCGGAAGCTCAAAGGTAGTTTCTTTGAGTACTTTATCTCTAAACATAATTAACCAAATTGAATTGGATGTACCATTTTCTACTTTGTATTTAAAATGTAGTTGTCACCCGAGTTTCTCTGTGCGTGCAGTAATGGCATTAGGTCTTATTACAATAACTATTAATAAAGATATGTAATATTTAACACCCTATAGCATCATCTTGCTAGTGCAAATACCTCTATTTCTCAGAGATTGTGTAATCATGTATATCTGGCCTCCTATTCATACATGCTATGTTGAGATACAGTGCCGAATGTTCACTTCAGTATCATTTGTAGATTAAAATCTACGTATTGAGCTGATAATGAAATGTTCCTATTGTAGGTGGGTGCGGAGGTACAGAAAACAGTGAATGAAAGATTTGGCAAATGCTTACTGGAGTTAAGTGGTAACAATGCAATTATAGTCATGGATGATGCAGATATAAAACTGGCTGTACGCTCTATTTTCTTTGCAGCTGTGGGTACTGCTGGTCAGCGGTGCACAACCTGCCGTAGACTGGTATTAAAAACAATTTTGAAAAATTGAGTTCACAGTTGTAAAGCATCCATGTTGTTTCTGTGAGGTATATTTAATATTCAATTTTGGCTGCTTTGCATGCAGTTTCTGCATGAGAGTATTTACGCAAATGTACTAGATCAACTTGCCGGACTTTACAAACAAGTCAAAATTGGGAATCCCCTGGAGAAAGGGACTCTTGTTGGGCCCCTGCATACTCGTTCTTCCGTGGAAAACTACAAGAAGGGCATTGCGGCGATAAAATCTCAG (such as SEQ IDShown in NO.26).

[0085] As can be seen from the circularization experiments in Application Examples 1-3, the vectors constructed using the nucleic acid molecules of this application can achieve precise circularization expression of genes for multiple circRNA molecules from different plants, which will greatly promote the functional study of plant circRNAs.

[0086] The above description is merely a preferred embodiment to aid in understanding the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any alterations or modifications made by those skilled in the art based on this description without departing from the spirit of the present invention should also fall within the scope of the present invention.

Claims

1. A nucleic acid molecule that promotes the circularization of circRNA, which is capable of circularizing a target RNA molecule with biological activity, and its nucleotide sequence is shown in SEQ ID NO.

8.

2. An RNA circularization vector that has a nucleotide sequence shown in SEQ ID NO.9 and is capable of circularizing a target RNA molecule with biological activity.

3. An RNA circularization method, after digesting the RNA circularization vector described in claim 2 with enzymes, connecting the target gene into the vector by seamless cloning to achieve the RNA circularization of the target gene; Among them, The enzyme digestion uses Bgl II and Stu I to digest the RNA circularization vector described in claim 2.

4. A method for expressing circular RNA in cells, which includes transfecting the RNA circularization vector described in claim 2 into cells.

5. The method for expressing circular RNA in cells according to claim 4, wherein The cells are eukaryotic cells.

6. The application of the nucleic acid molecule described in claim 1, the RNA circularization vector described in claim 2, and the RNA circularization method described in claim 3 in the research of circular genes.

7. The application of the RNA circularization vector described in claim 2 and the RNA circularization method described in claim 3 in circularizing the Arabidopsis thaliana circALDH7B4 gene.

8. The application according to claim 7, and the circularized Arabidopsis thaliana circALDH7B4 gene is identified using the primers shown in SEQ ID NO.14-17.

9. Use of the RNA cyclization vector according to claim 2 and the RNA cyclization method according to claim 3 for cyclizing Arabidopsis circGORK genes.

10. The application according to claim 9, wherein the cyclized Arabidopsis thaliana circGORK gene is identified using the primers shown in SEQ ID NO. 18-21.

11. The application of the RNA circularization vector described in claim 2 and the RNA circularization method described in claim 3 in circularizing the Medicago sativa circ175 gene.

12. The application according to claim 11, and the circularized Medicago sativa circ175 gene is identified using the primers shown in SEQ ID NO.22-25.

Images