Plant-derived Argonaute protein and application thereof

Abstract

The invention discloses a plant-derived Argonaute protein and application thereof, and belongs to the technical field of gene editing and detection. The plant-derived Argonaute protein is specifically a protein with an amino acid sequence as shown in SEQ ID NO.1, or a protein which has at least 80% of homology with the amino acid sequence as shown in SEQ ID NO.1 and has corresponding functions; the Argonaute protein specifically cleaves target nucleic acid under mediation of guide nucleic acid, the guide nucleic acid is guide DNA or guide RNA, and the target nucleic acid is a DNA target or an RNA target. Compared with the reported eukaryotic Argonaute protein derived from unicellular organisms, the Argonaute protein used in the invention is derived from rice, has greater application potential in the field of animal and plant gene editing, not only has RNase activity and activity of cutting a DNA target by utilizing guide RNA, but also can cut the DNA target by utilizing guide DNA, so that the scheme of the invention has good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene editing and detection, and specifically relates to an Argonaute protein derived from the monocotyledonous plant rice and an application thereof. Background Art

[0002] Argonaute (Ago) protein family members are widely present in organisms and can be divided into eukaryotic Argonaute (eAgo) and prokaryotic Argonaute (pAgo) according to their sources. eAgos plays an important role in the RNA interference pathway in organisms and is the functional core of the RNA-induced silencing complex RISC. It plays the role of a biological scaffold in mediating the complementary pairing of the guide strand and the target. It can cut complementary targets through the activity of eAgos' own nuclease or recruit other protein molecules to act on the target. eAgos regulates gene expression by targeted RNA cleavage, inhibiting mRNA translation, DNA methylation, etc., participates in DNA repair, and reduces the mobility of transposons to maintain the integrity of the genome. It plays an important role in growth, development, reproduction, resistance to pathogens, and coping with stress.

[0003] At present, the physiological function of pAgos in vivo is not as clear as that of eAgos, and there is a lack of reports on the activity of pAgos in eukaryotes, probably because it is difficult to form an effective pAgo-guide nucleic acid complex in vivo. Most eAgos have specific RNA cleavage activity in vitro, but there are few reports on specific DNA cleavage activity.

[0004] eAgos have great diversity in genetic evolution classification and enzymology. Currently, eAgos that study the properties of nuclease cleavage mainly come from single-cell eukaryotic microorganisms. For eAgos from multicellular plants and animals, the main research is on physiological functions in vivo. There is no detailed research on in vitro biochemical properties such as interactions with DNA. Currently, there is no application of animal and plant eAgos for gene editing, modification, and molecular detection. Therefore, characterizing eAgos from multicellular organisms is expected to provide more nucleic acid manipulation tools for use in plants and animals. Summary of the invention

[0005] In view of this, the present invention aims to explore an eAgo from multicellular eukaryotic organisms that can be used in the fields of gene editing, modification and molecular detection, and to clarify its specific application scheme, so as to provide a new optional molecular tool for molecular detection under complex conditions and gene editing and modification of plants and animals.

[0006] The first aspect of the present invention discloses a plant-derived Argonaute protein and its complex with a guide nucleic acid for use in nucleic acid editing and detection, wherein the Argonaute protein is derived from the monocotyledonous model organism rice, specifically: The protein whose amino acid sequence is shown in SEQ ID NO.1; or A protein having at least 80% sequence homology, preferably at least 90% sequence homology, more preferably at least 95% sequence homology to the amino acid sequence shown in SEQ ID NO.1 and having corresponding functions.

[0007] In the above application, Argonaute protein specifically cuts the target nucleic acid under the mediation of the guide nucleic acid, wherein the guide nucleic acid is a guide DNA or / and a guide RNA, the target nucleic acid is a DNA target or / and an RNA target, and the target nucleic acid contains a sequence complementary to the guide nucleic acid. Among them, the RNA target has no higher-order structure, or has a higher-order structure, or is double-stranded RNA, or is in vitro transcribed RNA, or is viral genomic RNA, or is mRNA, or other RNA in the cell; the DNA target is a synthetic single-stranded DNA, or is double-stranded DNA, or is a plasmid DNA; it can be cell genomic DNA, or it can be other DNA in the cell.

[0008] It is understood that the Argonaute protein used in the present invention can be obtained by natural extraction or heterologous expression. Moreover, the sequence of the nucleic acid molecule encoding the Argonaute protein of the present invention is shown in SEQ ID NO.2 or has at least 80% homology with the sequence shown in SEQ ID NO.2.

[0009] Preferably, in the above application, the length of the guide DNA is 10-40 nt; wherein, the optimal length of the Argonaute protein of the present invention when cleaving a DNA target under the mediation of the guide DNA is 13 nt, and the optimal length when cleaving an RNA target under the mediation of the guide DNA is 15 nt.

[0010] Preferably, in the above application, the length of the guide RNA is 12-40 nt; wherein, the optimal length of the Argonaute protein of the present invention when cleaving a DNA target under the mediation of the guide RNA is 19 nt, and the optimal length when cleaving an RNA target under the mediation of the guide RNA is 18 nt.

[0011] Preferably, in the above application, the 5' end of the guide nucleic acid has a phosphorylation modification.

[0012] Preferably, in the above application, the first nucleotide at the 5' end of the guide DNA is thymine (T), and the first nucleotide at the 5' end of the guide RNA is uracil (U).

[0013] In the above application, the complex formed by the Argonaute protein and the guide nucleic acid has the activity of cleaving the target nucleic acid at 25-60° C. Specifically, when the target nucleic acid is a DNA target, the cleavage site is between the 10th and 11th bases from the 5' end to the 3' end of the guide nucleic acid complementary to the DNA target; when the target nucleic acid is an RNA target, the cleavage site is between the 9th and 10th bases from the 5' end to the 3' end of the guide nucleic acid complementary to the RNA target.

[0014] Preferably, in the above application, the complex formed by the Argonaute protein and the guide nucleic acid cuts the target nucleic acid at 41-60°C. Experimental data show that under the mediation of guide DNA, the Argonaute protein can effectively cut the DNA target at 25-45°C, with 41°C being the best; under the mediation of guide RNA, the Argonaute protein can effectively cut the DNA target at 25-55°C, with 50°C being the best; when cutting RNA targets, the guide DNA or guide RNA can effectively cut the RNA target at 25-60°C, with 45°C being the best.

[0015] Preferably, in the above application, the target nucleic acid contains a sequence that is complementary to the guide nucleic acid, specifically: The guide nucleic acid has a nucleotide sequence that is completely complementary to the target nucleic acid; or, The guide nucleic acid has a nucleotide sequence that has a single-base or double-base mismatch with the target nucleic acid, wherein the single-base mismatch does not occur at the first nucleotide at the 5' end of the guide nucleic acid.

[0016] In addition, it is understood that the specific cleavage of the target nucleic acid by the Argonaute protein under the mediation of the guide nucleic acid in the present invention includes the following situations: Argonaute protein cuts the target nucleic acid in vitro, and its application process can be: incubating Argonaute protein with guide nucleic acid to form an eAgo complex, the eAgo complex contacts the target nucleic acid, and the target nucleic acid has a nucleotide sequence that is complementary to the guide nucleic acid sequence, so the Argonaute protein cuts the target at a specific site.

[0017] When Argonaute protein cuts the target nucleic acid in the cell, its application process can be: incubating the Argonaute protein with the guide nucleic acid to form an eAgo complex, or introducing a vector containing the guide nucleic acid and the Argonaute protein gene into plant cells or animal cells by transformation, transfection or transduction, and then regulating cell expression through targeted cutting.

[0018] The second aspect of the present invention provides a nucleic acid cleavage system, which at least comprises: Argonaute protein, specifically a protein having an amino acid sequence as shown in SEQ ID NO.1, or a protein having at least 80% homology, preferably at least 90% homology, more preferably at least 95% homology to the amino acid sequence shown in SEQ ID NO.1, and having corresponding functions; Guide nucleic acid, with phosphorylation modification at the 5' end; The target nucleic acid can be targeted and cleaved by Argonaute protein under the guidance of guide nucleic acid; Divalent metal cations.

[0019] In the nucleic acid cleavage system of the present invention, Argonaute protein binds to the guide nucleic acid to form an eAgo complex. After the complex contacts the target nucleic acid, if the target nucleic acid has a nucleotide sequence that is complementary to the guide nucleic acid sequence, the Argonaute protein cleaves the target nucleic acid (i.e., RNA target and / or DNA target) at a specific site under the guidance of the guide nucleic acid recognition. Based on this cleavage system, Argonaute protein can be used to achieve nucleic acid detection and editing.

[0020] Preferably, in the above nucleic acid cleavage system, the divalent metal ion is selected from Mg 2+ , Mn 2+ 、Co 2+ , Ca 2+ More preferably, the divalent metal cation is Mn 2+ Mg 2+ , and the concentration of divalent metal cations is 0.5-60 mM. Specifically, experimental data show that when Argonaute protein cuts DNA targets, it can use Mn 2+ 、Co 2+ Efficient cleavage of DNA targets, where 40 mM Mn 2+ When the guide RNA is mediated, Mn 2+ Mg 2+ Efficient cleavage of DNA targets, where 0.5 mM Mn 2+ When Argonaute protein cuts RNA target, it can utilize Mn under the guidance of guide DNA. 2+ Mg 2+ Efficient cleavage of RNA targets, where 60 mM Mg 2+ When the guide DNA is mediated, Mg 2+ , Mn 2+ , Ca 2+ Efficient cleavage of DNA targets, where 60 mM Mg 2+ Best time.

[0021] Preferably, in the above nucleic acid cleavage system, the pH of the nucleic acid cleavage system is 3-10.5.

[0022] Based on the Argonaute protein used in the present invention, the third aspect of the present invention provides a silent mutant by mutating the nuclease active site of the sequence shown in SEQ ID NO.1, and the mutant completely loses the cleavage activity. The active sites are positions 833, 874, 906 and 1045 of the sequence shown in SEQ ID NO.1; relative to the wild type, the mutant mutates the aspartic acid (D) at the first and third positions to alanine (A).

[0023] The mutants provided by the present invention can be fused with other effector proteins or combined with labeled guide nucleic acids, thereby expanding the application direction of the Argonaute protein of the present invention. For example, fluorescently labeled guide DNA is loaded into the mutant, and the mutant guides the DNA to bind to its complementary target. Since the DNA has a fluorescent label, the entire process can be visualized; in addition, the ability of the fluorescently labeled guide DNA to bind to the RNA target can also be used to achieve specific visualization of miRNA.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides an Argonaute protein that can cut a target nucleotide sequence (i.e., a target nucleic acid) under the guidance of a nucleic acid chain, and a complex formed by the Argonaute protein and a guide nucleic acid. The Argonaute protein is derived from the plant rice and is different from Argonaute from other eukaryotic sources. The Argonaute protein has the activity of cutting not only RNA targets but also DNA targets, indicating that it has application potential in nucleic acid editing and detection.

[0025] The Argonaute protein provided by the present invention has binding activity to phosphorylated guide nucleic acids (i.e., single-stranded DNA (ssDNA) or RNA), and has nuclease activity to RNA targets or DNA targets. Therefore, when the Argonaute protein binds to the guide nucleic acid and eAgo to form an eAgo complex, it undergoes a site-specific cleavage reaction with the target nucleic acid, and the site specificity can be adjusted by selecting a guide nucleic acid with a specific nucleotide sequence.

[0026] The eAgo complex of the present invention can effectively use 13-15 nt guide DNA and / or 18-19 nt guide RNA to specifically cleave DNA targets or RNA targets, especially when ssDNA is used as a guide to cleave RNA targets, it has high activity. Compared with guide RNA, guide DNA is cheaper and has a shorter synthesis time, which can save costs.

[0027] The Argonaute protein used in the present invention does not have the activity of non-specific incidental cleavage that CRISPR-related proteins need to rely on special nucleotide sequences near the target site to recognize and bind to the target, and the guide nucleic acid design is more convenient without considering site restrictions. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The phylogenetic tree diagram of OsAgo18 in the present invention and some reported model organism Ago proteins; Figure 2 A schematic diagram of the sequence alignment of OsAgo18 in the present invention and the characterized Agos; Figure 3 The TBE-PAGE gel electrophoresis diagrams of OsAgo18 cleaving target nucleic acids mediated by different types of guide nucleic acids in Example 1 of the present invention, wherein A is the electrophoresis diagram when cleaving DNA targets, and B is the electrophoresis diagram when cleaving RNA targets; Figure 4 Schematic diagram of the cleavage of the guide nucleic acid and the target nucleic acid used in Example 1 of the present invention and a diagram of the product detection results, wherein A is a schematic diagram of the cleavage site when cleaving a DNA target, B is a schematic diagram of the cleavage site when cleaving an RNA target, C is a TBE-PAGE gel electrophoresis diagram of the product obtained by OsAgo18 cleaving a DNA target under the mediation of a phosphorylated guide nucleic acid, and D is a TBE-PAGE gel electrophoresis diagram of the product obtained by OsAgo18 cleaving an RNA target under the mediation of a phosphorylated guide nucleic acid; Figure 5 This is a TBE-PAGE gel electrophoresis diagram of OsAgo18 cleavage of target nucleic acid mediated by guide nucleic acids of different lengths in Example 2 of the present invention; Figure 6 This is a TBE-PAGE gel detection result diagram of the product obtained by OsAgo18 cleaving the target using a guide with a phosphate group at the 5' end under different divalent metal ion conditions in Example 3 of the present invention; Figure 7 This is a TBE-PAGE gel detection result diagram of the products obtained by OsAgo18 cleaving the target using a guide with a phosphate group at the 5' end under different magnesium ion or manganese ion concentration conditions in Example 3 of the present invention; Figure 8 This is a TBE-PAGE gel detection result diagram of the products obtained by OsAgo18 cleaving the target using a guide with a phosphate group at the 5' end under different temperature conditions in Example 4 of the present invention; Fig. 9 This is a TBE-PAGE gel detection result diagram of the products obtained by OsAgo18 cleaving the DNA target using the guide with a phosphate group at the 5' end under different 5' end nucleotide conditions in Example 5 of the present invention; Fig.10Figure 2 is a TBE-PAGE gel detection result of the product obtained by OsAgo18 cleaving the RNA target using the guide with a phosphate group at the 5' end under different 5' end nucleotide conditions in Example 5 of the present invention Fig.11 Schematic diagram of guide DNA and guide RNA for single-base or double-base mutation in Example 6 of the present invention; Fig.12 This is a TBE-PAGE gel detection result diagram of the products obtained by OsAgo18 cleavage of the target mediated by the guide DNA / RNA of single-base or double-base mutation in Example 6 of the present invention. DETAILED DESCRIPTION

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The term "comprises" and any variation thereof in the specification and claims of the present invention are intended to cover non-exclusive inclusions.

[0030] In view of the lack of eAgos from multicellular organisms in the prior art, and the lack of research on eAgos from multicellular plants and animals for gene editing, modification and molecular detection, the present invention aims to explore eAgos from plants and animals, thereby providing more potential tool enzymes for gene editing, modification and detection of multicellular eukaryotic organisms. Through a large number of screening and verification, the present invention obtained an Argonaute protein from the monocotyledonous model plant rice, namely OsAgo18, which has the potential for application in nucleic acid editing and molecular detection, and its silent mutant OsAgo18-DM can be used for gene modification.

[0031] Existing studies have shown that rice has 19 Argonaute proteins. According to the classification method of Arabidopsis Argonaute, rice Argonaute can be divided into six major groups, and Ago18 is a group unique to monocots. These Ago proteins participate in RNA silencing and play an important role in rice growth, development, reproduction, disease resistance, stress resistance and other aspects. The amino acid sequence of OsAgo18 screened by the present invention is shown in SEQ ID NO.1, and the results of constructing a phylogenetic tree with other eAgos from model organisms such as rice, Arabidopsis, mouse, corn, and fruit fly are shown in Figure 1 As shown, several Ago proteins with the highest homology are shown in Table 1 (none of the eAgos shown in the characterization was confirmed to have specific cleavage activity).

[0032] Table 1

[0033] The experimental data of the present invention show that OsAgo18 from the plant rice has four types of cutting activities, specifically binding to DNA guide to cut DNA targets, binding to RNA guide to cut DNA targets, binding to DNA guide to cut RNA targets, and binding to RNA guide to cut RNA targets, indicating its application potential in nucleic acid editing, and multiple binding and cutting activities ensure its wide range of applications.

[0034] It is understandable that OsAgo18 in the present invention can be a naturally extracted protein or a recombinant protein obtained by heterologous expression.

[0035] The sequence of OsAgo18 was compared with that of the characterized Ago. Figure 2 As shown, the region where the catalytic quartet is located is shown, indicating that the catalytic active sites of OsAgo18 are positions 833, 874, 906 and 1045 of the sequence shown in SEQ ID NO. 1. The first and third amino acids of the catalytic quartet DEDH are mutated from D to A to construct a silent mutant OsAgo18-DM.

[0036] The technical solution of the present invention will be described clearly and completely below in conjunction with specific embodiments. It should be understood that the embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0037] The OsAgo18 used in the following examples was obtained by heterologous expression, and its preparation method was as follows: the target gene encoding the OsAgo18 protein (sequence as shown in SEQ ID NO.2) was cloned into an expression plasmid, which was transformed into the competent E. coli ArcticExpress (DE3) pRARE2 and induced for expression, and OsAgo18 was obtained by a series of purification methods such as nickel ion affinity chromatography, heparin, molecular sieve, etc. The preparation method of the silent mutant OsAgo18-DM refers to OsAgo18.

[0038] If no specific techniques or conditions are specified in the following examples, all of them are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions; if the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0039] Example 1 This example demonstrates the cleavage activity and restriction site of OsAgo18 and the types of guide nucleic acids that are suitable for it.

[0040] In this example, a single-stranded DNA with a length of 45 nucleotides is used as a DNA target, and its 5' end is labeled with FAM fluorescence, and its sequence is shown in SEQ ID NO.3; a single-stranded RNA with a length of 45 nucleotides is used as an RNA target, and its 5' end is labeled with FAM fluorescence, and its sequence is shown in SEQ ID NO.4; a DNA with a length of 18 nucleotides is used as a guide DNA, and its 5' end has a phosphate group or a hydroxyl group, and its sequence is shown in SEQ ID NO.5; an RNA with a length of 18 nucleotides is used as a guide RNA, and its 5' end has a phosphate group or a hydroxyl group, and its sequence is shown in SEQ ID NO.6.

[0041] The specific operations are as follows: Dilute the protein to 10 pmol / μL for later use. Dilute the guide DNA, guide RNA, DNA target, and RNA target to 1 pmol / μL for later use. Prepare 5 mM and 400 mM Mn 2+ and 600 mM Mg 2+ Prepare enzyme digestion reaction buffer containing 100 mM NaCl, 10 mM PIPES-NaOH (pH 7.5), and 5% (v / v) glycerol for later use.

[0042] Prepare a 10 μL reaction system: add 1 μL protein, 1 μL guide nucleic acid, 2 μL enzyme digestion buffer, 1 μL divalent metal ion solution, 1 μL target nucleic acid, 0.2 μL RNase inhibitor, and DEPC water to make up. Specifically, when the guide nucleic acid is guide DNA and the target nucleic acid is DNA target, the divalent metal ion solution is 400 mM Mn 2+ And Mn 2+ The final concentration in the system is 40 mM; when the guide nucleic acid is guide RNA and the target nucleic acid is DNA target, the divalent metal ion solution is 5 mM Mn 2+ And Mn 2+ The final concentration in the system is 0.5 mM; when the guide nucleic acid is a guide DNA or guide RNA and the target nucleic acid is an RNA target, the divalent metal ion solution is 600 mM Mg 2+ And Mg 2+ The final concentration in the system is 60 mM.

[0043] Without adding target nucleic acid, the reaction system was first incubated at 37°C for 10 minutes to load the guide nucleic acid on eAgo, and then the corresponding target nucleic acid was added and reacted for 1 hour; after the reaction was completed, 2× RNA loading buffer (containing 95% formamide, 18 mM EDTA, 0.025% SDS and 0.025% bromophenol blue) of the same volume as the reaction system was added, mixed evenly and heated at 95°C for 5 minutes to terminate the reaction. The reaction product was electrophoresed with 20% denatured TBE-PAGE gel, stained with SYBR Gold (Invitrogen), and analyzed with Gel Doc TM The gel was photographed using XR+ (Bio-Rad).

[0044] The results are as follows Figure 3-4 As shown: ① No product band was found in the control group results of cleaving the target nucleic acid after incubation without OsAgo18, indicating that the product band was formed due to the nuclease activity of OsAgo18 ( Figure 3 ); ②OsAgo18 can use guide DNA or guide RNA with a phosphate group at the 5' end to cut DNA targets and RNA targets ( Figure 3 ); ③OsAgo18 specifically produces a 34 nt product when using an 18 nt guide DNA or guide RNA to cleave a DNA target, so the cleavage site is between the 10th and 11th bases from the 5' end to the 3' end of the guide nucleic acid that is complementary to the DNA target ( Figure 4 ); ④OsAgo18 specifically produces a 35 nt product when using an 18 nt guide DNA or guide RNA to cleave an RNA target, so the cleavage site is between the 9th and 10th bases from the 5' end to the 3' end of the guide nucleic acid that is complementary to the RNA target ( Figure 4 ); ⑤OsAgo18-DM does not have the ability to bind to the guide nucleic acid and cut the target nucleic acid.

[0045] Example 2 This example tests the effect of guide nucleic acid length on OsAgo18 cleavage activity. The test process includes the following: Prepare 1 pmol / μL of guide DNA or guide RNA of different lengths with a phosphate group at the 5' end; the target nucleic acid in this example is the same as in Example 1, and the design method of guide nucleic acids of different lengths is the same as in Example 1. Referring to the reaction system and method of Example 1, 1 μL of the prepared guide nucleic acids of different lengths are added to the reaction system to verify their activity in cutting DNA targets and RNA targets.

[0046] Test results such as Figure 5 As shown: Figure 5 A indicates that OsAgo18 can cleave DNA targets under the guidance of guide DNA of 9-30 nt in length, wherein the length of the guide DNA is preferably 13 nt; Figure 5 B shows that OsAgo18 can cleave DNA targets under the guidance of guide RNAs of 12-40 nt in length, where the length of the guide RNA is preferably 19 nt; Figure 5 C shows that OsAgo18 can cleave RNA targets under the guidance of guide DNAs of 9-40 nt in length, where the length of the guide DNA is preferably 15 nt; Figure 5 D shows that OsAgo18 can cleave RNA targets under the guidance of 14-25 nt long guide RNA, where the length of the guide RNA is preferably 18 nt. The above results show that the length of the guide nucleic acid has an effect on the activity of OsAgo18 in recognizing and cleaving targets.

[0047] Example 3 This example tests the effect of divalent metal ions and their concentrations in the reaction system on the cleavage activity of OsAgo18. The test process includes the following: (1) Influence of divalent metal ions.

[0048] Prepare 50 mM Fe 2+ 、Co 2+ 、Ni 2+ , Cu 2+ 、Zn 2+ , Ca 2+ , Mn 2+ Mg 2+ According to the results of Example 2, 1 pmol / μL of 13 nt 5'-end phosphorylated guide DNA, 15 nt 5'-end phosphorylated guide DNA, 18 nt 5'-end phosphorylated guide RNA and 19 nt 5'-end phosphorylated guide RNA were prepared respectively.

[0049] Different configured divalent metal ions were used, and 13 nt guide DNA was used to target and cleave DNA targets, 19 nt guide RNA was used to target and cleave DNA targets, 15 nt guide DNA was used to target and cleave RNA targets, and 18 nt guide RNA was used to target and cleave RNA targets. Referring to the reaction system and method in Example 1, 1 μL of different divalent metal ions was added to the reaction system (i.e., the corresponding final concentration was 5 mM), and the activity of OsAgo18 in recognizing and cleaving targets using phosphorylated guide nucleic acids under different divalent metal ion conditions was measured.

[0050] Test results such as Figure 6 As shown: Figure 6 A indicates that OsAgo18 can be 2+ , Mn 2+ Under the conditions, the guide DNA is used to effectively cut the DNA target. Figure 6 B shows that OsAgo18 can be 2+ Mg2+ Guide RNA effectively cuts DNA targets under these conditions. Figure 6 C showed that OsAgo18 can be 2+ Mg 2+ Under the same conditions, the guide DNA is used to effectively cleave the RNA target. Figure 6 D indicates that OsAgo18 can be expressed in Mg 2+ , Ca 2+ , Mn 2+ Guide RNA is used to efficiently cleave RNA targets under certain conditions.

[0051] (2) Mn 2+ Mg 2+ Effect of divalent metal ion concentration.

[0052] Configure Mn in the concentration range of 0~1 M 2+ Mg 2+ Ionic solution, different concentrations of divalent metal ions are used in the reaction system, and the guide length of the enzyme cleavage reaction is the same as step (1). Referring to the reaction system and method of Example 1, 1 μL of ionic solution is added to the reaction system, and its final ion concentration range is 0~100 mM, and the activity of OsAgo18 in recognizing and cleaving the target using phosphorylated guide nucleic acid under different concentrations of divalent metal ions is measured.

[0053] Test results such as Figure 7 As shown: The concentration of divalent metal ions has an effect on the cleavage activity of OsAgo18, and the 2+ Mg 2+ The target can be cleaved in the range of 0.5-100 mM ion concentration; specifically, the optimal Mn for OsAgo18 to cleave the DNA target under the guidance of the guide DNA is 2+ The optimal Mn concentration for OsAgo18 to cleave DNA targets under the guidance of guide RNA is 40 mM. 2+ The optimal Mg concentration for OsAgo18 to cleave DNA targets under the guidance of guide RNA was 0.5 mM. 2+ The optimal Mg concentration for OsAgo18 to cleave RNA targets under the guidance of guide DNA or guide RNA was 30 mM. 2+ The concentration is 60 mM.

[0054] Example 4 This example tests the effect of reaction temperature on the cleavage activity of OsAgo18. The test process includes the following: 13 nt guide DNA was used to target and cleave DNA targets, 19 nt guide RNA was used to target and cleave DNA targets, 15 nt guide DNA was used to target and 18 nt guide RNA was used to target and cleave RNA targets, and the reaction system and method of Example 1 were referred to. After OsAgo18 and the guide nucleic acid were incubated to form a complex, the corresponding target was added and reacted at 25-75°C, respectively, to determine the activity of OsAgo18 in recognizing and cleaving the target using the phosphorylated guide under temperature conditions.

[0055] Test results such as Figure 8 As shown: OsAgo18 can bind to guide DNA and cleave DNA targets at 25~45℃, among which the cutting activity at 41℃ is relatively high; it can bind to guide RNA and cleave DNA targets at 25~55℃, among which the cutting activity at 50℃ is relatively high; it can bind to guide DNA or guide RNA and cleave RNA targets at 25~60℃, among which the cutting activity at 45℃ is relatively high.

[0056] Example 5 This example tests the effect of the 5' end nucleotide type on the cleavage activity of OsAgo18. The test process includes the following: Synthesize 13 nt phosphorylated guide DNA and 15 nt phosphorylated guide DNA with the first nucleotide base of A, T, C and G at the 5' end, and 18 nt phosphorylated guide RNA and 19 nt phosphorylated guide RNA with the first nucleotide base of A, U, C and G at the 5' end, corresponding to the complementary 45 nt DNA target and RNA target. Except for the first nucleic acid base at the 5' end, the rest of the guide chain is the same. Dilute the synthesized nucleic acid to 1 pmol / μL.

[0057] In the reaction system, the guides with different 5' terminal nucleotide types were used to cut complementary targets, and 13 nt guide DNA was used to cut DNA targets, 19 nt guide RNA was used to cut DNA targets, 15 nt guide DNA was used to cut RNA targets, and 18 nt guide RNA was used to cut RNA targets. Referring to the reaction system and method of Example 1, 1 μL of 1 pmol / μL guide nucleic acid was added to the reaction system for incubation with OsAgo18, and then 1 μL of 1 pmol / μL complementary target was added to the system for reaction, and the corresponding time gradient (DNA target: 0 min, 30 min, 60 min, 90 min, 120 min; RNA target: 0 min, 5 min, 15 min, 30 min, 60 min) of the reaction at 37°C was used to measure the activity of OsAgo18 in recognizing and cutting targets under the time gradient using phosphorylated guide nucleic acids with different 5' terminal nucleotides.

[0058] Test results such as Figure 9-10 As shown: the 5' terminal nucleotide of the guide nucleic acid has an effect on the cutting activity. When the 5' terminal nucleotide of the guide DNA is T or the 5' terminal nucleotide of the guide RNA is U, the cutting effect of the target nucleic acid is best. After 90 minutes of the targeted cutting DNA reaction, the cutting rate slows down, while after 30 minutes of the targeted cutting RNA reaction, the reaction rate enters a plateau phase.

[0059] Example 6 This example tests the effect of guide base mutation on OsAgo18 cleavage activity. The test process includes the following: Guide DNA and guide RNA for synthesizing single-base or double-base mutations (m1, m2, m3, m4, m5, m6, m7, m8, m9, m10, m11, m12, m13, m14, m15, m16, m17, m18, m7m8, m8m9, m8m9, m9m10, m10m11, m11m12, m12m13, m13m14, for mutations, refer to Fig.11 as shown).

[0060] Referring to the reaction system and method of Example 1, the guide nucleic acid before and after mutation was used for detection, and the results were as follows: Fig.12 As shown: The results of the influence of base mutation in the guide DNA on OsAgo18 cleavage of DNA targets are shown in Fig.12 As shown in A, the results show that compared with no mismatch, the OsAgo18 cleavage activity was significantly improved when single base mutations occurred at positions 2-10, 14, and 15, and double base mutations occurred at positions 13-14. Among them, the OsAgo18 cleavage efficiency was most significantly improved when there was a mismatch at position 8, and the OsAgo18 cleavage activity was the weakest when there was a base mutation at position 1.

[0061] The results of the influence of base mutation guide RNA on OsAgo18 cleavage of DNA targets are shown in Fig.12 As shown in B, the results show that compared with no mismatch, the OsAgo18 cleavage activity was significantly improved when the 2nd and 4-8th bases were mutated, while the OsAgo18 cleavage activity was weaker when the 1st, 9th and 13th bases were mutated.

[0062] The results of the influence of base mutation in the guide DNA on OsAgo18 cleavage of RNA targets are shown in Fig.12 As shown in C, the results showed that compared with no mismatch, the OsAgo18 cleavage activity was significantly reduced when single base mutations at positions 1 and 12 and double base mutations at positions 7-14 occurred.

[0063] The results of the influence of base mutation guide RNA on OsAgo18 cleavage of target RNA are as follows Fig.12As shown in D, the results showed that compared with no mismatch, the OsAgo18 cleavage activity was significantly reduced when single base mutations occurred at positions 1 and 4, and double base mutations occurred at positions 8-9, 11-12, and 12-13.

[0064] Example 7 In this case, the cleavage activity of OsAgo18 in cells was detected. The detection process included the following: Cells expressing firefly luciferase and Renilla luciferase were constructed, with the firefly luciferase gene as the reporter gene and the Renilla luciferase gene as the reference gene. At the same time, multiple phosphorylated guide DNAs and guide RNAs that can complement the mRNA transcribed from the firefly luciferase gene were synthesized.

[0065] The cells were cultured to a cell density of 70%-80%, and the OsAgo18 protein was incubated with the guide nucleic acid and then introduced into the cells using a protein transfection reagent. After culturing for a period of time, the cells were lysed and the activities of firefly luciferase and Renilla luciferase were detected by fluorescence.

[0066] Calculate the ratio of the firefly luciferase detection value to the Renilla luciferase detection value, and compare whether there is a significant difference between cells transfected with the protein complex and cells that have not been transfected. When the ratio of transfected cells is much smaller than that of untransfected cells, it indicates that OsAgo18 has cleavage activity in cells and can regulate cell expression. The test results in this example show that OsAgo18 has cleavage activity in cells.

[0067] In summary, the plant-derived OsAgo18 protein provided by the present invention can not only target and cleave RNA targets, but also DNA targets under medium temperature conditions using a guide RNA or guide DNA with a phosphate group at the 5' end. In addition, the OsAgo18-DM protein provided by the present invention can specifically modify genetic material. Therefore, the OsAgo18 protein can be effectively applied to many fields of biotechnology such as gene editing, nucleic acid detection and gene modification.

[0068] It should be noted that the above embodiments are only part of the embodiments of the present invention rather than all the embodiments, and are only used to illustrate the technical solution of the present invention rather than to limit it. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. Application of a plant-derived Argonaute protein and a complex formed by the protein and the guide nucleic acid in nucleic acid editing and detection; It is characterized in that The Argonaute protein is a protein whose amino acid sequence is shown in SEQ ID NO.1, or a protein having at least 80% homology with the amino acid sequence shown in SEQ ID NO.1 and having corresponding functions.

2. The use according to claim 1, It is characterized in that The Argonaute protein specifically cuts the target nucleic acid under the mediation of the guide nucleic acid, wherein the guide nucleic acid is a guide DNA or / and a guide RNA, the target nucleic acid is a DNA target or / and an RNA target, and the target nucleic acid contains a sequence that is complementary to the guide nucleic acid.

3. The use according to claim 2, It is characterized in that The length of the guide DNA is 10-40 nt, and the length of the guide RNA is 12-40 nt.

4. The use according to claim 3, It is characterized in that The 5' end of the guide nucleic acid has a phosphorylation modification.

5. The use according to claim 4, It is characterized in that The first nucleotide at the 5' end of the guide DNA is thymine, and the first nucleotide at the 5' end of the guide RNA is uracil.

6. The use according to claim 2, It is characterized in that The Argonaute protein has the activity of cleaving target nucleic acid at 25-60°C.

7. The use according to claim 2, It is characterized in that The guide nucleic acid has a nucleotide sequence that is completely complementary to the target nucleic acid, or has a nucleotide sequence that has a single or double base mismatch with the target nucleic acid.

8. A nucleic acid cleavage system, It is characterized in that At least: Argonaute protein, specifically a protein having an amino acid sequence as shown in SEQ ID NO.1, or a protein having a sequence with at least 80% homology to the amino acid sequence as shown in SEQ ID NO.1 and having corresponding functions; Guide nucleic acid, with phosphorylation modification at the 5' end; The target nucleic acid can be targeted and cleaved by the Argonaute protein under the mediation of the guide nucleic acid; Divalent metal cations.

9. The nucleic acid cleavage system according to claim 8, It is characterized in that The divalent metal ion is selected from Mg 2+ , Mn 2+ 、Co 2+ , Ca 2+ One or more of .

10. The nucleic acid cleavage system according to claim 8, It is characterized in that The pH of the nucleic acid cleavage system is 3-10.5.

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