JP2008141960A - Protein having ribonuclease activity and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein having ribonuclease activity and useful for RNA study. <P>SOLUTION: The protein having activity for cleaving an RNA has a specific amino acid sequence or an amino acid sequence having one or several replaced, deleted, added and/or inserted amino acids in the amino acid sequence. The method for detecting the protein, the polynucleotide encoding the protein, the transformant containing the polynucleotide, and the method for treating a nucleic acid including a step for cleaving the RNA by using the protein having the ribonuclease activity are also provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a protein having RNase activity, for example, a protein having an activity of recognizing an adenine (A) base and cleaving a ribonucleotide chain and the like and use thereof.

In recent years, the function of RNA as well as DNA in organisms has been studied. In particular, RNA-degrading enzymes (RNases) that have base-specific cleavage activity are widely used for the determination of RNA molecule secondary structure and the study of protein-RNA interactions. Among currently available endo-type RNases, RNase A that cleaves single-stranded RNA molecules is the 3 ′ side of pyrimidine bases (U and C), RNase T1 is the 3 ′ side of guanine base (G), and RNaseT2 is A And 3 ′ side of U is cut. It is known that RNase V1 and RNase III specifically cleave double-stranded RNA molecules, and RNase H specifically degrades RNA / DNA double-stranded RNA strands (Non-patent Document 1).
NUCLEASES, Edited by Linn, SM and Roberts, RJ, Cold Spring Harbor Laboratory (1982)

  In order to determine the secondary structure of RNA molecules, it is necessary to cleave RNA with some selectivity, but in addition to these, RNases that can be used to determine the secondary structure of RNA molecules, etc. It has not been found yet. An RNase that recognizes the cleavage site in a base-specific manner has not yet been found. Moreover, none of these various RNases recognized adenine base (A) with high selectivity.

  Accordingly, an object of the present invention is to provide a protein having RNase activity useful for RNA research. Another object of the present invention is to provide a protein having an enzyme activity that recognizes an adenine base and cleaves an RNA chain. Furthermore, an object of the present invention is to provide a protein having an enzyme activity that recognizes adenine bases with high selectivity and cleaves RNA. Another object of the present invention is to provide use of such an RNA-degrading enzyme. A polynucleotide for expressing a protein having such enzymatic activity, and the polynucleotide can be expressed so that the protein can be expressed. It is another object of the present invention to provide a transformant to be used, and another object of the present invention is to provide a nucleic acid analysis kit and treatment method using such an enzyme.

  The inventors of the present invention have been studying proteins that are ubiquitous in plants and have RNA binding activity. As a result, the PPR motif (Pentatrico Peptide Repeat: PPR), which is considered to be involved in RNA processing in cell organs, has been studied. The present inventors have found that DYW domains that can be linked and domains similar to the DYW function as RNA-cleaving activity, that is, RNA degradation enzymes. In addition, the inventors have also found that this domain has a highly selective cleavage activity for adenine bases. The present invention has been made based on these findings, and according to the present invention, the following means are provided.

According to the present invention,
The following features:
(A) The amino acid sequence according to any of SEQ ID NO: 1 and SEQ ID NO: 4, or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in these amino acid sequences.
And a protein having the activity of cleaving RNA.

Furthermore, the protein of the present invention has the following characteristics:
(B) It has a cytochrome C heme binding motif CxxCH.
Can have. The protein of the present invention has the following characteristics:
(C) An amino acid sequence provided in the C-terminal region of a natural protein having a PPR motif, or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in these sequences.
Can also be included.

In the protein of the present invention, the amino acid sequence has an E value obtained by performing a homology search using any one of the amino acid sequences described in SEQ ID NO: 1 and SEQ ID NO: 4 as a query sequence (preferably 1. 0 × 10 ⁻¹⁰ or less) or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in these sequences. The amino acid sequence is preferably the amino acid sequence described in any one of SEQ ID NO: 1 and SEQ ID NO: 4.

The protein of the present invention can be a monocotyledonous or dicotyledonous protein. The RNA cleavage activity is preferably an endo type, and more preferably the RNA cleavage activity is base-specific. The RNA cleaving activity is preferably an activity that cleaves site-specifically on the 5 'side of a ribonucleotide having an adenine base.

  According to the present invention, there is also provided a method for screening an RNase, comprising any one of the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 6, or one or more of these sequences. There is also provided a method comprising: preparing a protein having an amino acid sequence in which amino acids are substituted, deleted, added and / or inserted; and detecting the presence or absence of RNA cleavage activity for the prepared protein. In the protein preparation step, the amino acid sequence having an E value equal to or less than a predetermined value obtained by performing a homology search using the amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 6 as a query sequence Alternatively, it can be a step of preparing a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in these sequences.

  Furthermore, according to the present invention, there is provided a method for producing an RNase, wherein the amino acid sequence according to any one of SEQ ID NO: 1 and SEQ ID NO: 4, or one or more amino acids are substituted in these amino acid sequences, There is also provided a method characterized in producing a protein comprising a deleted, added and / or inserted amino acid sequence and a cytochrome C heme binding motif CxxCH.

  According to the present invention, there is also provided a nucleic acid analysis kit comprising any one of the above-described proteins. Further, according to the present invention, a polynucleotide encoding any one of the above-described proteins is also provided. There is also provided a transformant that retains any one of the above-described proteins so that the polynucleotide can be expressed, and a method for treating a nucleic acid, comprising the step of cleaving RNA using any of the above-described proteins. This treatment method can include any one of two or more steps of separating and purifying nucleic acid, extracting RNA, and detecting RNA fragments.

The protein of the present invention has the following characteristics:
(A) The amino acid sequence according to any of SEQ ID NO: 1 and SEQ ID NO: 4, or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in these amino acid sequences.
And has an activity of cleaving RNA.

  The protein of the present invention (hereinafter simply referred to as the present protein) can function as an enzyme having RNA cleavage activity by having the amino acid sequence. Moreover, since the amino acid sequence is an amino acid sequence provided at the C-terminus of a natural protein that has a PPR motif and is considered to be involved in RNA processing, it is useful for studies such as RNA functional analysis. In particular, since RNA can be cleaved specifically for adenine bases, it is useful for accurate determination of base sequences in RNA and the like. In addition, the protein of the present invention can be said to be a protein having an amino acid sequence associated with the DYW motif, which is a C-terminal sequence of the PPR protein, and having RNA cleavage activity.

The nucleic acid analysis kit of the present invention (hereinafter simply referred to as the present analysis kit) can contain the present protein. This analysis kit is useful for extraction and separation of RNA, DNA, etc., and RNA sequence and functional analysis.

  In addition, the transformant of the present invention (hereinafter simply referred to as the present transformant) can hold the polynucleotide encoding the protein so that the protein can be expressed. This transformant is convenient for supplying a protein having RNase activity. Furthermore, according to the polynucleotide of the present invention encoding the present protein (hereinafter simply referred to as the present polynucleotide), a construct or the present protein capable of producing a useful RNase efficiently or in any host can be obtained. An expressible transformant can be provided. Furthermore, according to the nucleic acid processing method of the present invention, by using the present protein as an RNA-degrading enzyme, RNA can be decomposed to perform unprecedented nucleic acid separation and purification, RNA metabolism research, and the like.

  Hereinafter, proteins, nucleic acid analysis kits, nucleic acid processing methods, polynucleotides, transformants, and the like that are embodiments of the present invention will be described in detail.

(protein)
The protein can comprise an amino acid sequence having one or more characteristics. One amino acid sequence that can be included in the present protein can be the sequence described in SEQ ID NO: 1. The amino acid sequence shown in SEQ ID NO: 1 is encoded by Arabidopsis thaliana (ecotype columbia) unknown protein gene at2g02980 (MATDB: http://mips.gsf.de/proj/thal/db/index.html) This is a sequence consisting of a part of a protein consisting of 603 amino acids having a PPR motif (PPR protein) (SEQ ID NO: 3). Specifically, it is a sequence consisting of 109 amino acids from the 495th position to the 603rd position on the C-terminal side of the amino acid sequence shown in SEQ ID NO: 3. Although PPR protein is considered to be related to RNA processing, it has not been confirmed before this application that its C-terminal region has RNase activity.

  Another amino acid sequence that can be provided by the present protein is the sequence set forth in SEQ ID NO: 4. The amino acid sequence described in SEQ ID NO: 4 (part of Os5g30710 (OS_M23), database: TIGR Rice Genome Annotation (http://www.tigr.org/tdb/e2k1/osa1/) is derived from rice (Oryza sativa) The amino acid sequence contained in the natural protein (Os5g30710 (OS_M23). The protein containing this amino acid sequence is also a PPR protein having a PPR motif.

  The present protein is not limited to these two amino acid sequences, and can further comprise other amino acid sequences. That is, the present protein can also have an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence described in any of SEQ ID NO: 1 and SEQ ID NO: 4. . That is, these two amino acid sequences are amino acid sequences having one or more amino acid mutations selected from amino acid substitution, deletion, insertion and addition (hereinafter also referred to as modified sequences). The number of amino acid mutations is not particularly limited, but is preferably 20 or less, more preferably 10 or less. More preferably, it is 5 or less.

Introduction of site-specific mutations into the amino acid sequence can be performed by methods known to those skilled in the art. Methods well known to those skilled in the art for preparing DNA encoding proteins with altered amino acid sequences include, for example, the site-directed mutagenesis method (Kramer W & Fritz HJ: Methods Enzymol 154: 350,
1987). Also in nature, it is possible that the amino acid sequence of the encoded protein is mutated due to the mutation of the base sequence.

  Another example of such a modified sequence is encoded by a DNA that hybridizes with a base sequence encoding the original amino acid sequence or a DNA comprising a part thereof (or a complementary strand thereof). An amino acid sequence is mentioned. For example, the amino acid sequence described in SEQ ID NO: 1 is encoded by the base sequence described in SEQ ID NO: 2. The amino acid sequence described in SEQ ID NO: 4 is encoded by the base sequence described in SEQ ID NO: 5. DNA based on these base sequences can be used.

Such modified sequences can be obtained by hybridization techniques (Southern EM: J Mol Biol 98: 503, 1975) or polymerase chain reaction (PCR) techniques (Saiki
RK, et al: Science 230: 1350, 1985, Saiki
RK, et al: Science 239: 487, 1988). That is, using the DNA described in SEQ ID NO: 2 or a part thereof or a complementary strand thereof as a probe, or using an oligonucleotide that specifically hybridizes to the base sequence of the genomic DNA of such a protein as a primer, It is usually possible for a person skilled in the art to obtain DNA by isolating DNA from plants of the above by hybridization.

The DNA encoding such a protein can be preferably obtained by performing a hybridization reaction under stringent conditions. In the present invention, stringent hybridization conditions are 6M urea, 0.4%
Refers to hybridization conditions of SDS, 0.5 × SSC or equivalent stringency. Higher stringency conditions such as 6M urea, 0.4%
It is expected that DNA with higher homology can be isolated under conditions of SDS and 0.1 × SSC. Note that high homology means sequence identity of at least 50% or more, preferably 70% or more, more preferably 90% or more, and most preferably 95% or more in the entire amino acid sequence. The identity of amino acid sequences and base sequences can be determined using BLAST or the like.

The modified sequence in which an amino acid mutation is introduced into the amino acid sequence described in any one of SEQ ID NO: 1 and SEQ ID NO: 4 is an arbitrary sequence using the amino acid sequence described in any of SEQ ID NO: 1 and SEQ ID NO: 4 as a query sequence. The E value (Expected value, E-value; expected value) obtained by homology search of the database can be an amino acid sequence having a predetermined value or less. The E value is generally defined in the homology search program to be used. Regarding the E value in the present invention, if it is defined in the general definition in the homology search method or individually in the specific homology search method to be used, it follows the definition.

The smaller the E value, the higher the similarity to the sequence described in any of SEQ ID NO: 1 and SEQ ID NO: 4. The E value is preferably 1.0 × 10 ⁻¹⁰ or less. In general, if the E value is 1.0 × 10 ⁻¹⁰ or less, it can be said that it is unlikely that accidental similarity to the amino acid sequence described in SEQ ID NO: 1 or the like. Such an amino acid sequence is considered to have the same or similar activity functionally as the protein having the amino acid sequence described in SEQ ID NO: 1. Preferably, the E value is 1.0 × 10 ⁻¹⁵ or less. By providing an amino acid sequence having a smaller E value, it becomes easier to obtain the same and equivalent activity as the protein consisting of the amino acid sequence described in SEQ ID NO: 1 or the like.

The homology search can be based on local pairwise alignment by dynamic programming on the query sequence. This kind of homology search program can be appropriately selected from those disclosed or provided through the Internet or the like. For example, SSEARCH (http://www.ddbj.nig.ac.jp/search/ssearch-j.html, etc.); BLAST (Proc.
Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc
Natl Acad Sci USA 90: 5873, 1993, http://www.ncbi.nlm.nih.gov/BLAST/), PSI-BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ etc.) FASTA (http://www.ebi.ac.uk/fasta33/) and the like can be used. Of these, BLAST and PSI-BLAST can be preferably used.

  The database to be searched is not particularly limited. For example, it can be used by appropriately selecting from those prepared on a site that publishes a homology search program. It is also possible to use a database that is disclosed separately from the program or is not disclosed. Many similar sequences can be extracted from this protein using a database containing eukaryotes, particularly plant genomes or proteins, or a part (motif) thereof. This is because the amino acid sequence of this protein corresponds to or is often similar to the C-terminal region of a PPR protein having a PPR motif, and many PPR proteins are contained in plants. PPR protein is contained in both dicotyledonous plants such as Arabidopsis thaliana and monocotyledonous plants such as rice (Oryza sativa), and it is preferable to search a protein database of such plants.

  In the homology search, the amino acid sequence described in any one of SEQ ID NO: 1 and SEQ ID NO: 4 is used as a query sequence, and the base sequence encoding the amino acid sequence is used as a query sequence. It is also included to obtain an amino acid sequence having an E value equal to or less than a predetermined value by searching for. In this case, for example, various homology search methods included in BLAST or the like may be used as necessary.

For example, the amino acid sequence extracted by homology search (BLAST) using SEQ ID NO: 1 as a query sequence includes the amino acid sequence described in SEQ ID NO: 4 (Os5g30710 (OS_M23), database: TIGR Rice Genome Annotation ( http://www.tigr.org/tdb/e2k1/osa1/), ^E value 3 × 10 ⁻²⁷ ).

  In addition, this protein has one or more amino acids in an amino acid sequence having an E value equal to or less than a predetermined value when a homology search is performed using the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 4 as a query sequence. May include amino acid sequences substituted, deleted, added and / or inserted. That is, it may contain a modified sequence of the extracted amino acid sequence. The number of such amino acid mutations is not particularly limited, but is preferably 20 or less, more preferably 10 or less. More preferably, it is 5 or less.

The amino acid sequence that the protein can have may also be based on a consensus sequence obtained by multiple alignment of the PPR protein family. The method used for multiple alignment for obtaining a consensus sequence and the method for obtaining a consensus sequence are not particularly limited. For example, ClustalW: http://align.genome.jp/; HMMER (Hidden Markov Model): http://hmmer.wustl.edu/;
MultiAlin: http://prodes.toulouse.inra.fr/multalin/multalin.html; mkdom / xdom:
Various methods such as http://prodes.toulouse.inra.fr/prodom/xdom/ can be adopted. Once a consensus sequence was obtained, modified sequences that maintain highly conserved amino acids in the consensus sequence and amino acids in the predicted active and binding sites were mutated to enhance interaction at these sites. Modified sequences can be obtained. For example, as an example of such a consensus sequence, one obtained from a multiple alignment of 85 PPR protein families of Arabidopsis thaliana using the HMMER matrix (C-terminal DYW motif) is disclosed (FIG. 1 and SEQ ID NO: : 6) (The Plant Cell,
Vol. 16, 2089-2103, August 2004, Fig. 2). In FIG. 1, conserved amino acids are shown in capital letters, and particularly well conserved amino acids are shown in bold.

  Another characteristic of the amino acid sequence that can be provided by this protein is that it has a cytochrome C heme binding motif CxxCH (C: cysteine, H: histidine, x: any amino acid). This motif is known as a metal coordination motif. Retaining this motif is preferred for RNase activity and its adenine base specificity. This motif can be located, for example, in a sequence corresponding to positions 69 to 72 in SEQ ID NO: 1. The motif can be located at about 40 amino acids from the C-terminal side, for example, 35 to 45 amino acids from the C-terminal side. The presence or absence of the motif can be confirmed by various known motif databases such as PROSITE (http://au.expasy.org/prosite/). This motif is also provided in the amino acid sequences described in SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 6 already exemplified.

Yet another characteristic of the amino acid sequence that can be provided by the present protein includes an amino acid sequence located in the C-terminal region of a natural protein (PPR protein) having a PPR motif or a modified sequence thereof. The PPR (pentatricopepetide repeat) motif is a 35 amino acid conserved sequence that is repeated in plants and is known to be localized in chloroplasts and mitochondria (Biochem Soc Trans. 2004 Aug). ; 32 (Pt 4): 571-4.). The presence or absence of the PPR motif can be known from a domain database such as PFAM (http://www.sanger.ac.uk/Software/Pfam/) (Accession No. PF01535). A consensus sequence of 35 amino acids is shown in FIG. 2 and SEQ ID NO: 7. In FIG. 2, well-conserved amino acids are shown in capital letters. All of the amino acid sequences described in SEQ ID NO: 1 and SEQ ID NO: 4 are sequences located in the C-terminal region of such PPR protein. The meaning of the modified sequence in the modified sequence of the amino acid sequence located in the C-terminal region of the PPR protein also applies here.

Such a protein can have an amino acid sequence in a natural protein of monocotyledonous plants and dicotyledonous plants and an amino acid sequence that is a modified sequence thereof because PPR proteins are present in a large amount in chloroplasts and mitochondria of plants. Although plant species is not particularly limited, for example, monocotyledonous plants include rice, wheat, millet, etc., and dicotyledonous plants include Arabidopsis, soybean, peanut, sesame, rapeseed, cottonseed, sunflower, safflower, Potato, sweet potato and the like.

The protein can have RNA cleavage activity. The RNA cleavage activity of this protein may be in any form as long as it can cleave RNA. The RNA cleavage activity of this protein is preferably RNA specific. If it is RNA specific, it does not act on DNA, so it can be easily separated from DNA and the like, and the DNA sequence can be maintained. Further, an exo type or an end type may be used, but an end type is preferred. The end type is useful for RNA functional analysis and the like. In the present specification, the RNA cleavage activity may be any as long as it cleaves an oligonucleotide or polynucleotide having an RNA chain at or near the cleavage site. Therefore, DNA / RNA hybrids, DNA-RNA chimeras, nucleotide chains containing modified bases and other modifications and additions can be included as cleavage targets.

The protein preferably has base-specific RNA cleavage activity. In this specification. Having a base-specific RNA cleaving activity means having an activity of cleaving a bond at a predetermined position with respect to a specific base recognized by recognizing one or more specific bases. I mean. In the present invention, an adenine base is preferably recognized. Furthermore, it is preferable to recognize an adenine base and cleave RNA on the 5 'side thereof. Conventionally, no RNase has been found that recognizes and cleaves adenine bases with high specificity, and is useful for research and other various applications.

  When the EDTA is in a predetermined concentration range, in other words, the present protein preferably exhibits RNA cleavage activity in the presence of a predetermined concentration of divalent metal ions. By providing such a divalent metal ion concentration dependency, the activity can be controlled by the divalent metal ion concentration or the addition concentration of a chelating agent such as EDTA. Preferably, the present protein has an optimal range for the divalent metal ion concentration in exerting its RNA cleavage activity. For example, a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 does not have RNA cleavage activity in the absence of a chelating agent such as EDTA and in the presence of a high concentration, but a concentration of 20 mM or more, preferably 25 mM or more. In addition, high RNA cleaving activity can be exhibited at a concentration of 70 mM or less, preferably 62.5 mM or less. Further, for example, the concentration of divalent metal ions such as magnesium ions is preferably about 1 mM or more and 10 mM or less, more preferably, the lower limit is 2 mM and the upper limit is 5 mM.

  The RNA cleavage activity can be measured by a known method. In general, the substrate RNA and the protein may be brought into contact under an appropriate medium. The substrate RNA can be placed with an appropriate label such as a radioactive element. After reacting at an appropriate temperature for a certain period of time, RNA is extracted and the degree of RNA degradation can be detected by electrophoresis on a urea-denatured polyacrylamide gel or the like. When measuring site-specific cleavage activity, a complementary DNA is prepared for the degradation reaction product, and a DNA ladder is prepared from the DNA that is the template for the substrate RNA. A cleavage site can be identified by separating by gel electrophoresis or the like.

  In addition, as long as this protein can exhibit RNA cutting | disconnection activity, it may be provided with the other amino acid sequence. Various amino acid sequences can be added depending on the production convenience and intended use.

(Method for producing this protein)
When the present protein is a natural protein or a part thereof, it can also be obtained by extracting the protein from an organism such as a plant retained in nature or an organelle fraction where the protein is localized, followed by separation and purification, etc. it can. Moreover, even if this protein is all or a part of a natural protein, or a non-natural protein, it can also be obtained chemically or genetically. When obtained by a genetic engineering technique, a transformant is produced in a cell-free manner using a polynucleotide encoding this protein or by introducing this polynucleotide into an appropriate host, and the transformant is transformed into the host. Alternatively, it can be expressed outside the host.

(Screening method and production method of RNase)
The screening method for the RNase of the present invention comprises substitution, deletion, addition and / or insertion of one or a plurality of amino acids in any one of the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4 or in these sequences. A step of preparing a protein having the prepared amino acid sequence, and a step of detecting the presence or absence of RNA cleavage activity for the prepared protein. According to this screening method, a protein (RNA degrading enzyme) having RNA cleavage activity can be obtained efficiently. In addition, the protein preparation step includes the amino acid sequence having an E value equal to or lower than a predetermined value obtained by performing a homology search using the amino acid sequence of any one of SEQ ID NO: 1 and SEQ ID NO: 4 as a query sequence, or these It can also be set as the process of preparing protein provided with a modification arrangement | sequence. Since the amino acid sequences described in SEQ ID NO: 1 and SEQ ID NO: 4 are DYW sequences whose enzyme activity has been confirmed, amino acid sequences having equivalent activity can be efficiently extracted by using these sequences as query sequences. be able to. Furthermore, in the RNase screening method of the present invention, an amino acid sequence having an E value equal to or less than a predetermined value may be extracted by performing a homology search using the amino acid sequence set forth in SEQ ID NO: 6 as a query sequence. . Since the sequence described in SEQ ID NO: 6 is a consensus sequence of the DYW motif, a protein having an amino acid sequence having an E value equal to or less than a predetermined value can be selected as a screening target for RNA cleavage activity. Furthermore, when an amino acid sequence having a cytochrome C heme binding motif is prepared, RNase can be screened more effectively.

In the protein preparation step, for example, a protein having an amino acid sequence having an E value equal to or lower than a predetermined value in a predetermined homology search is extracted, and the protein is genetically engineered using a polynucleotide or the like encoding the protein. You can also.

In preparing a protein having an amino acid sequence having an E value equal to or less than a predetermined value, the various homology search programs already described can be used. Further, the E value is preferably 1.0 × 10 ⁻¹⁰ or less, and more preferably 1.0 × 10 ⁻¹⁵ or less. In addition, the various aspects already described for the modified sequence can be applied to the protein prepared by this method. The presence or absence of RNA cleavage activity can be measured using methods well known to those skilled in the art, such as the methods already described.

  Proteins that have been detected to have RNA-cleaving activity by such RNase screening can be obtained from natural organisms or artificially obtained in the same manner as in the production of this protein described above. You can also. The RNase is also an amino acid sequence having an E value equal to or lower than a predetermined value obtained by performing a homology search using the amino acid sequence of any one of SEQ ID NO: 1 and SEQ ID NO: 4 as a query sequence, or a modified sequence thereof And a protein comprising a cytochrome C heme-binding motif CxxCH. This is because such a protein can exhibit RNA cleavage activity with a high probability.

(Nucleic acid analysis kit)
The present analysis kit can include the present protein in a kit configuration. The protein included in the kit may be included in any form. For example, it is preferable that the protein is included as a lyophilized product in consideration of storage stability. In addition, the present analysis kit may include other RNases, DNases, and the like, enzymes and reagents necessary for nucleic acid separation and purification, analysis, and the like.

(Polynucleotide)
The polynucleotide can have a region encoding the protein. Examples of the base sequence of the polynucleotide encoding this protein include SEQ ID NO: 2 and SEQ ID NO: 5. The codon usage is not particularly limited as long as the base sequence in the polynucleotide encodes the present protein.

  The polynucleotide may be in the form of a virus such as an expression cassette, vector, plasmid, artificial chromosome, and infectious Agrobacterium, which includes a regulatory region such as a promoter so that the polynucleotide can be expressed.

(Transformant)
In the present transformant, the present polynucleotide can be used as a transformant capable of expressing the present protein. In addition to prokaryotic cells such as Escherichia coli and lactic acid bacteria, the present transformant can be hosted by yeast, plant cultured cells and plants. The plant body includes a plant individual, all types and forms of cells that can constitute the plant individual, tissues and organs that are part of a plant solid, and germ cells. Moreover, as a part of a plant individual, its propagation medium (seed, rhizome, fruit, cutting ear, etc.) is also included.

  In order for the transformant to retain the present polynucleotide capable of expressing the present protein, the present polynucleotide is preferably linked under the control of an appropriate promoter. Further, it is preferable that an appropriate terminator is also connected. In addition, an element effective for enhancing expression can be appropriately provided.

  Such transformants not only produce this protein in large quantities, but are also useful for RNA / DNA metabolism studies.

  Moreover, this transformant may be constructed so that the expression of this protein can be suppressed. For example, an amino acid sequence having an E value equal to or lower than a predetermined value by performing a homology search using the amino acid sequence described in SEQ ID NO: 1 or the amino acid sequence described in SEQ ID NO: 4 as a query sequence, which is inherent in plants or the like, or the amino acid sequence Or a protein having a cytochrome C heme-binding motif may be constructed so that expression of the protein can be suppressed. Such transformants are useful for RNA / DNA metabolism studies. As an aspect of suppressing the expression of this protein, in addition to the destruction of the gene encoding the present protein and the transcription and translation of the gene, the mutation is introduced and expressed as an incomplete protein, etc. Decrease or inactivate or express a substance that interacts with the protein and decreases the action of the protein.

  For example, as a method for suppressing the expression of a specific gene, in addition to knocking out a specific gene, expression can be suppressed for mRNA using an antisense method, a co-suppression method, an RNA interference method, a ribozyme method, or the like. Introducing and retaining a nucleic acid construct.

  In addition, a transformant can be produced by employing a method known to those skilled in the art. When the transformant is a plant body, for example, in order to create a transformant capable of expressing the protein, a construct capable of expressing the protein is constructed in the plant cell, and the construct is introduced into the plant cell. The plant body may be regenerated from the plant cell. The vector is not specifically limited. Also, use a vector having a promoter (eg, cauliflower mosaic virus 35S promoter) for constitutive gene expression in plant cells, or a vector having a promoter inducibly activated by an external stimulus. You can also. The cells into which the construct is introduced can include all types of plant cells that can be regenerated into plants. Examples thereof include cultured cells, protoplasts, shoot primordia, polyblasts, hairy roots, callus, and sections of specific organs such as leaves. Plant transformants can convert cells into plants by carrying out a predetermined regeneration step. The method of regeneration varies depending on the type of plant, but various known methods can be used. For introduction of nucleic acid constructs into plant cells, various methods known to those skilled in the art such as polyethylene glycol method, electroporation method (electroporation method), Agrobacterium-mediated method, and particle gun method can be used. . Further, in the particle gun method, for example, a product from Bio-Rad can be used. Regeneration of plant bodies from transformed plant cells can be performed by methods known to those skilled in the art depending on the type of plant cells. In addition, offspring can be obtained from the regenerated plant body by sexual reproduction or asexual reproduction. It is also possible to obtain a propagation material (for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) from the plant body, its descendants or clones, and mass-produce the plant body based on them. Is possible.

(Nucleic acid processing method)
This treatment method can include a step of cleaving RNA using a protein. This treatment method can cleave RNA and fragment RNA. According to this cutting step, for example, an RNA fragment can be generated in a test tube, and a material for searching for the function of the RNA can be obtained. In addition, since RNA is cleaved in a base-specific manner, the structure of RNA to be cleaved can be estimated by analyzing the generated RNA fragment.

  This treatment method may further comprise a step of separating and purifying nucleic acids such as DNA, a step of extracting RNA, and electrophoresis of the cleaved RNA fragment after RNA cleavage. Or a step of detecting by MALDI or the like.

  In the nucleic acid separation and purification step, the separation and purification target is preferably a sample in which RNA and DNA may coexist. Such a sample may be a sample extracted from a living body, a reaction sample when DNA is synthesized in a test, or the like. For example, when RNA and DNA coexist, the present protein does not act on DNA, and thus DNA can be easily purified from coexisting RNA as an uncut fragment. On the other hand, since this protein acts on RNA, RNA can be isolated as a cleaved fragment. In such a separation and purification step, it is convenient to immobilize this protein on an appropriate solid phase. Also, a column packed with such a solid phase may be used. It is preferable that the nucleic acid separation and purification step is provided after the RNA cleavage step.

  In the step of extracting RNA, the extraction target is preferably a biological sample. By extracting such RNA and then cleaving with this protein, RNA metabolism in vivo can be studied. The RNA extraction step is preferably prepared prior to the RNA cleavage step.

  Furthermore, the step of detecting an RNA fragment generated after RNA cleavage may be analyzed by electrophoresis, microarray, MALDI or the like after preparing the RNA fragment itself or complementary DNA.

  Hereinafter, although an example is given and the present invention is explained concretely, the present invention is not limited to the following examples.

(Example 1: Preparation of novel RNA-degrading enzyme, T-DYW and measurement of RNA-degrading activity)
(Preparation of genomic DNA from Arabidopsis thaliana)
Arabidopsis thaliana (ecotype Columbia) was cultured in Murashige sukuk medium (containing 2% sucrose and 0.5% Gellangam) for 3 weeks. The green leaves (0.5 g) of the cultured plant were extracted with phenol / chloroform, and then ethanol was added to insolubilize the DNA. Dissolve the recovered DNA in 100 μl of TE solution (10 mM Tris-HCl (pH 8.0), 1 mM EDTA), add 10 units of RNase A (DNase-free, Takara Bio Inc.), and then at 37 ° C for 30 minutes. Reacted. Thereafter, the reaction solution was again extracted with phenol / chloroform, and then DNA was collected by ethanol precipitation. 10 μg of DNA was obtained.

(Cloning of gene encoding DYW motif)
Preparation of genomic DNA from Arabidopsis thaliana was performed by the method described in Example 1 above. Arabidopsis genome information database (MATDB:
Refer to the sequence information posted at http://mips.gsf.de/proj/thal/db/index.html) and amplify the DNA sequence corresponding to the DYW motif of the protein gene at2g02980 having the DYW motif. Oligonucleotide primers (02980DYW-F, 02980_m-R; described in SEQ ID NOs: 8 and 9) were prepared. The oligonucleotide primer forward primer 02980DYW-F and reverse primer 02980_m-R have Eco
RI and SalI sequences were added. EcoRI and SalI sequences were incorporated so that the resulting clones could be used to cut out the insert sequence by restriction enzyme treatment.

The DNA fragment containing at2g02980 is Prime Star (Takara Bio Inc.) in a 50 μl reaction solution containing 100 ng of genomic DNA and the above primers in 25 cycles of 95 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 30 seconds.
Was amplified as a DNA extender by PCR. As a result, amplification of a DNA fragment of 339 base pairs was confirmed. The obtained DNA fragment was cloned using the pBAD / Thio-TOPO vector (Invitrogen). The 3 'overhanging adenosine of the DNA fragment required for cloning
It was added by reacting the unit TaKaRa Ex-Taq (Takara Bio Inc.) with 2.5 mM ATP at 70 ° C. for 10 minutes. A DNA sequence encoding the cloned DYW motif was determined, confirmed to be a sequence homologous to the DNA sequence corresponding to the DYW motif (SEQ ID NO: 2) on the database, and named plasmid pTrx_DYW.

(Preparation of T-DYW protein)
The obtained plasmid pTrx_DYW was transformed into Escherichia coli LMG194 strain (Invitrogen). This Escherichia coli was cultured at 37 ° C. in 4,000 ml of RM medium containing 0.2% glucose containing ampicillin at a concentration of 100 μg / ml (2 liter Erlenmeyer flask containing 1 liter medium, total of 4 tubes). When the turbidity of the culture solution reached an absorbance at a wavelength of 600 nm of 0.5, an inducer, L-arabinose, was added to a final concentration of 0.2%, and the culture was further continued for 4 hours. After collection by centrifugation, the cells are mixed with 200 ml of buffer A containing 1 mg / ml lysozyme (50 mM Tris / HCl pH 8.0, 500 mM KCl, 2
suspended in mM imidazole, 10 mM MgCl ₂ , 0.5% Triton X100, 10% glycerol), and the cells were disrupted by ultrasonic disruption and freeze lysis. After centrifugation at 15,000 xg for 20 minutes, the supernatant was recovered as a crude extract. This crude extract was applied to a column packed with a nickel column resin (ProBond A, Invitrogen) equilibrated with buffer A.

Column chromatography is 20
After thoroughly washing with buffer A containing mM imidazole, 200
This was performed by a two-step concentration gradient in which the target protein was eluted with buffer A containing mM imidazole. When the obtained recombinant protein was confirmed by SDS polyacrylamide gel electrophoresis, it was detected as a 30 kDa protein. This was named T-DYW protein (SEQ ID NO: 10). This protein is a fusion protein comprising the amino acid sequence described in SEQ ID NO: 1, the amino acid sequence of thioredkin for enhancing solubility on the N-terminal side, and the histidine tag sequence on the C-terminal side. 600 μg of T-DYW protein was obtained per liter of RM medium. Add 1 ml of purified fraction containing T-DYW protein to 1 liter of Buffer E (20 mM Tris-HCl pH 7.9, 60
After dialysis against mM KCl, 12.5 mM MgCl ₂ , 0.1 mM EDTA, 17% glycerol, 2 mM DTT), a purified preparation of T-DYW protein was obtained.

(Preparation of substrate RNA)
As the substrate RNA, RNA having 500 bases from the start codon of Arabidopsis thaliana chloroplast ndhB gene was used. The RNA used as the substrate RNA was named NB500 (SEQ ID NO: 11). A DNA fragment for synthesizing NB500 was obtained by using the above-mentioned Arabidopsis genomic DNA 10 using oligonucleotide primers ndhB-F and ndhB-R (described in SEQ ID NOs: 12 and 13).
Prime 50 μl reaction solution containing ng as template DNA at 25 cycles of 95 ° C for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 30 seconds
Amplification was performed by PCR using Star (Takara Bio Inc.) as a DNA elongation enzyme. The NdhB-F primer has a T7 promoter sequence to synthesize substrate RNA in vitro, and the ndhB-R primer has a nonspecific degradation by 3 '→ 5' exonuclease on the 5 'end. A base sequence that forms a stem-loop structure was added to prevent the above. The obtained DNA fragment was developed by agarose gel and then purified by cutting out from the gel. NTP mix (10 nmol GTP, CTP, ATP, 0.5 nmol UTP), 4 μl [ ³² P] α-UTP (GE Healthcare, 3000 Ci / mmol), T7 RNA polymerase (Takara Bio) The substrate RNA was synthesized by reacting 20 μl of the reaction solution containing 2) at 37 ° C. for 60 minutes. Substrate RNA was extracted with phenol / chloroform and precipitated with ethanol, and the entire amount was developed by denaturing 6% polyacrylamide gel electrophoresis containing 6 M urea and exposed to X-ray film for 60 seconds to detect ³² P-labeled RNA. ³² P-labeled RNA was excised from the gel and immersed in 200 μl of gel eluate (0.3 M sodium acetate, 2.5 mM EDTA, 0.01% SDS) at 4 ° C. for 12 hours to elute the RNA from the gel. Of the eluted RNA, 1 μl of radioactivity was measured, and the total amount of synthesized RNA was calculated. After ethanol precipitation, RNA was dissolved in ultrapure water so as to be 8,250 cpm / μl (1 fmol / μl). This preparation method usually yields about 50 μl of 8,250 cpm / μl RNA.

(RNA degradation activity of T-DYW protein)
RNA degradation activity in the presence or absence of T-DYW protein was examined. Reaction solution containing 50 mM EDTA (10 mM Tris / hydrochloric acid pH 7.9, 30
mM KCl, 6 mM MgCl ₂ , 2
mM DTT, 8% glycerol, 0.0067% of
Triton X-100) 20 μl of the above 1 fmol of substrate RNA (NB500) and 50 ng to 1.5 μg of T-DYW protein were mixed and reacted at 25 ° C. for 30 minutes. After the reaction, NB500 RNA was extracted by phenol / chloroform extraction and ethanol precipitation. RNA was developed on a denaturing 6% polyacrylamide gel containing 6 M urea, and the gel was dried after electrophoresis. The radioactivity of RNA in the gel was measured with a bioimaging analyzer BAS2000 (Fuji Film). The results are shown in FIG. 3 below.

As is clear from FIG. 3, when the T-DYW protein and RNA were mixed, the degradation activity of the substrate RNA was observed depending on the protein concentration. As a control experiment, cp28, which is an RNA binding protein, was used as a T-DYW vector, and a recombinant protein prepared in an expression system was used as T-28 (SEQ ID NO: 14). Was not observed. RNA degradation was not observed even in the absence of protein (no protein).

(Effect of EDTA on RNA degradation activity of T-DYW protein)
Many RNases require divalent metal ions for their RNase activity. In order to verify the effect of divalent metal ions contained in the purified T-DYW sample on the RNA degradation activity, EDTA was added to measure the RNA degradation activity of the T-DYW protein. 0 to 200
50 μl NB500 RNA and 50 μl in 20 μl reaction solution containing mM EDTA
ng T-DYW protein was mixed and reacted at 25 ° C. for 30 minutes. RNA extraction, electrophoresis, and detection after the reaction were performed in the same manner as described in Example 1. The results are shown in FIG.

As shown in FIG. 4, the RNA degradation activity of T-DYW protein was confirmed only in the presence of 50 mM EDTA (left side of FIG. 4). In order to verify the optimal EDTA concentration, the EDTA concentration was changed more finely. Under these reaction conditions, RNA degradation activity was detected at a concentration of 12.5 mM to 62.5 mM, and the most efficient in the presence of 25 mM EDTA. RNA degradation activity was detected (in FIG. 4). Since the RNA-degrading activity of T-DYW protein was not observed with high-concentration EDTA that sufficiently chelates divalent metal ions in the reaction solution, divalent metal ions affected the RNA-degrading activity of T-DYW protein. It was suggested to give. As a control experiment, 100 units of RNase, a known RNase
A similar experiment was performed using T1 (Ambion). As a result, RNase T1 showed the maximum RNA degradation activity when EDTA was not added, and also showed RNA degradation activity even in the presence of a high concentration of EDTA (FIG. 4 right). In addition, it is clear that T-DYW and RNase T1 have different recognition bases for RNA degradation of both proteins because the pattern of RNA degradation intermediate products is different. It has already been clarified that RNase T1 cleaves 3 ′ of cytosine residues in RNA.

(Nucleic acid specificity of enzyme activity of T-DYW protein)
The nucleic acid cleavage activity of T-DYW protein was measured using RNA, single-stranded DNA, and double-stranded DNA as substrates. ³² P-labeled double-stranded DNA uses primers ndhB-F and ndhB-R and template DNA NB500 DNA, dNTP mix (10 nmol dATP, dGTP, TTP, 0.5 nmol dCTP), 4 μl
[ ³² P] α-dCTP (GE Healthcare, 3000 Ci / mmol) was prepared by performing a PCR reaction with a solution containing Ex-Taq (Takara Bio). PCR reaction was performed in the same manner as in Example 1. The resulting DNA fragment was purified by denaturing 6% polyacrylamide gel electrophoresis and then eluting the DNA from the gel. Single-stranded DNA was prepared by treating double-stranded DNA at 90 ° C. for 3 minutes and then rapidly cooling. T-DYW protein and NB500 RNA (RNA), single-stranded NB500 DNA (ssDNA), double-stranded NB500 DNA (dsDNA) are mixed, and under the same conditions as in Example 1, the cleavage activity to each substrate nucleic acid is observed. Examined. The results are shown in FIG.

As shown in FIG. 5, the T-DYW protein showed cleavage activity only to RNA. As control experiments, 100 units of RNase T1 (Ambion), which is a known RNase, 10 units of DNase, which is a known DNase
Using I (RNase-free, Takara Bio Inc.), the nucleic acid specificity of the enzyme activity was examined. As a result, RNase T1 exhibited a cleavage activity on RNA and DNase I exhibited a cleavage activity only on DNA. Therefore, it is clear that T-DYW protein exhibits RNA-specific nucleic acid degradation activity.

(RNA cleavage site of T-DYW)
The degradation intermediate product of RNA degraded by T-DYW protein can be detected as a clear band (FIG. 5). This is a phenomenon observed with endonucleases that cleave RNAs such as RNase T1 and RNase A, which are known RNases. Therefore, in order to verify the characteristics of the RNA residue cleaved by the T-DYW protein, the 5 ′ end of the cleaved RNA was determined by the primer extension method. After reacting under the same conditions as in Example 1 using 10 fmol of unlabeled ndhB RNA and 1 μg of T-DYW protein, RNA was extracted from the reaction solution.

Next, 0.4 pmol of the ndh-R primer labeled with [ ³² P] at the 5 ′ end and the recovered RNA were mixed, and complementary DNA was synthesized using reverse transcriptase (ReverTraAce, TOYOBO). At the same time, a DNA ladder for identifying the cleaved base was prepared. The DNA ladder was prepared by Thermo Sequenase Primer Cycle Sequencing kit (GE Healthcare) using ndh-R primer labeled with [ ³² P] at the 5 ′ end and NB500 as template DNA. The obtained complementary DNA and DNA ladder were electrophoresed on a 6% polyacrylamide gel containing 6M urea. The gel was dried after electrophoresis. The radioactivity of RNA in the gel was measured using BAS2000 (Fuji
Film). The result is shown in FIG. 6 below.

As shown in FIG. 6, several 5 ′ ends of RNA cleaved by T-DYW protein were detected. The position of the obtained 5 ′ end (the 5 ′ end of NB500 is defined as 1) and the base are shown. Since all the 5 ′ ends obtained were adenosine residues, it was found that T-DYW is an RNase that selectively cleaves the 5 ′ side of adenosine residues in RNA.

(Preparation and degradation activity of rice-derived DYW protein)
First, rice (Oryza
DNA preparation from sativa, Nipponbare) was carried out in the same manner as in Example 1. Next, referring to the sequence information published in the rice genome information database (http://www.tigr.org/tdb/e2k1/osa1/overview.shtml) using the prepared rice genomic DNA as a template, Oligonucleotide primers (Os5g30710-F, Os5g30710-R for amplifying the DNA sequence of the protein gene Os5g30710 having a motif having high homology with the DYW motif sequence (SEQ ID NO: 1) in the protein (AT2980 (At2g02980); The DNA fragment containing Os5g30710 was amplified (SEQ ID NO: 17) and cloned into the pBAD / Thio-TOPO vector (Invitrogen) using the T-type described in Example 1. The same procedure as in the cloning of the DYW protein gene was performed, and the resulting plasmid was named pTrx-osDYW, and a rice-derived protein T-osDYW was prepared using this plasmid pTrx-osDYW (SEQ ID NO: 18). Preparation of Park protein was carried out analogously to the method described in Example 1.

The RNA degradation activity of the T-osDYW protein was performed in the same manner as the measurement of the RNA degradation activity of the T-DYW protein described in Example 1 using 50 ng of T-osDYW protein. The result is shown in FIG.

  As is clear from FIG. 7, the RNA-degrading activity similar to that of the tuna-derived T-DYW was also measured in the rice-derived T-osDYW protein. As a control, RNA degradation activity in the absence of protein (lane, no protein) and protein consisting only of thioredkin and histidine tag (50 ng, control, SEQ ID NO: 19) prepared from pBAD-Thio-TOPO vector without insert Was measured, but no RNA degradation was observed.

(Preparation of mutant DYW protein and RNA degradation activity)
T-DYW and T-osDYW proteins have a metal coordination motif, CxxCH, in their amino acid sequences (C, cysteine; H, histidine; x, any amino acid). In order to verify the action of this motif in RNA degradation, mutant T-DYW protein and T-DYW_M protein were prepared by the following method. Mutation was performed by replacing the amino acid sequence CxxCH in the T-DYW protein with GxxGH (G, glycine). A detailed preparation method is shown below.

Using pTrx-DYW described in Example 1 as a template, first, two DNA fragments, D1 and D2, were oligonucleotide primers 02980DYW-F (SEQ ID NO: 8), 02980M2-R (SEQ ID NO: 21), 02980M2- It was prepared by PCR under the same conditions as in Example 1 using F (SEQ ID NO: 20) and 02980_m-R (SEQ ID NO: 9). Next, by PCR using oligonucleotide primers 02980DYW-F (SEQ ID NO: 8) and 02980_m-R (SEQ ID NO: 9), using the resulting mixture of D1 and D2 DNA fragments as a template A DNA fragment encoding the mutant DYW protein was obtained (SEQ ID NO: 22). Cloning of the DNA fragment into the pBAD-Thio / TOPO vector and preparation of the recombinant protein T-DYW_M (SEQ ID NO: 23) were performed in the same manner as in Example 1 described above.

The RNA degradation activity of the obtained T-DYW_M protein was performed in the same manner as the measurement of the RNA degradation activity of the T-DYW protein described in Example 1. The obtained result is shown in FIG.

As can be seen from FIG. 8, RNA degradation activity could be observed with T-DYW protein (50 ng), but T-DYW_M protein (50 ng) with mutations introduced into two cysteine residues in T-DYW protein. ) RNA degradation activity was not observed. As a control, RNA degradation activity was measured in the absence of protein (lane, -protein) and only with the tag protein of the pBAD / Thio-TOPO vector (no 50 ng insert, SEQ ID NO: 19), but RNA degradation was observed. There wasn't. This indicates that the amino acid sequence of CxxCH, which is a metal coordination motif, plays an important role in the RNA degradation activity of T-DYW protein.

SEQ ID NO: 8, 9, 12, 13, 15, 16, 20, 21: Primer SEQ ID NO: 10, 14, 18, 19, 23: Fusion protein SEQ ID NO: 17, 22: DNA encoding the fusion protein

The figure which shows an example of the consensus sequence of the DYW motif found in PPR protein. The figure which shows an example of the 35 amino acid motif in PPR protein. The figure which shows the RNA cleavage activity of T-DYW protein prepared in Example 1. FIG. The figure which shows the effect of EDTA with respect to the RNA cleavage activity of T-DYW protein prepared in Example 1. FIG. The figure which shows the nucleic acid specificity of the RNA cleavage activity of T-TYW protein prepared in Example 1. FIG. The figure which shows that the RNA cleavage activity of T-TYW protein prepared in Example 1 is base specific. The figure which shows the RNA cleavage activity of the rice DYW protein prepared in Example 2. FIG. The figure which shows the RNA cleavage activity of the DYW protein which introduce | transduced the variation | mutation prepared in Example 3. FIG.

Claims (18)

The following features:
(A) The amino acid sequence according to any of SEQ ID NO: 1 and SEQ ID NO: 4, or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in these amino acid sequences.
Have
A protein having an activity of cleaving RNA. In addition, the following features:
(B) Contains cytochrome C heme binding motif CxxCH.
The protein according to claim 1, comprising: In addition, the following features:
(C) an amino acid sequence provided in the C-terminal region of a natural protein having a PPR motif, or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in these sequences.
The protein according to claim 1 or 2, wherein The amino acid sequence is a sequence having an E value of 1.0 × 10 ⁻¹⁰ or less obtained by performing a homology search using any one of the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 4 as a query sequence, or a sequence thereof The protein according to any one of claims 1 to 3, which is an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the sequence.   The protein according to any one of claims 1 to 4, wherein the natural protein is a protein of a monocotyledonous plant or a dicotyledonous plant.   The protein according to any one of claims 1 to 5, wherein the RNA cleavage activity is an endo type.   The protein according to any one of claims 1 to 6, wherein the RNA cleavage activity is base-specific.   The protein according to any one of claims 1 to 7, wherein the RNA cleavage activity is an activity of site-specific cleavage of the 5 'side of a ribonucleotide having an adenine base. A method for screening an RNase,
Any of the amino acid sequences described in SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 6, or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in these sequences Preparing a protein;
Detecting the presence or absence of RNA cleavage activity for the prepared protein;
A method comprising:   In the protein preparation step, the amino acid sequence having an E value equal to or less than a predetermined value obtained by performing a homology search using the amino acid sequence of any one of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 6 as a query sequence Alternatively, the method according to claim 9, which is a step of preparing a protein protein having an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in these sequences. A method for producing an RNase comprising:
Amino acid sequence according to any of SEQ ID NO: 1 and SEQ ID NO: 4, or an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in these amino acid sequences, and a cytochrome C heme binding motif Producing a protein comprising CxxCH.   A nucleic acid analysis kit comprising the protein according to claim 1.   A polynucleotide encoding the protein according to claim 1.   The transformant which hold | maintains the polynucleotide of Claim 13 so that expression of the protein in any one of Claims 1-9 is possible.   A method for treating a nucleic acid, comprising a step of cleaving RNA using the protein according to claim 1.   The method according to claim 15, comprising the step of separating and purifying the nucleic acid.   16. The method of claim 15, comprising the step of extracting RNA.   The method according to claim 15, comprising a step of detecting an RNA fragment after RNA cleavage.