CN120424906A - A new type of nuclease - Google Patents

Abstract

The present invention discloses a novel nuclease having an amino acid sequence as shown in SEQ ID NO: 1, which can efficiently cut DNA and RNA in the presence of Mn ²⁺ , can be used as a tool enzyme in molecular biology, and has broad application prospects.

Description

Novel nuclease

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a novel nuclease with an amino acid sequence shown as SEQ ID NO. 1 and application of the nuclease as a molecular biological tool enzyme.

Background

Corn (Zea mays l.) is an important food and commercial crop in our country and is one of the classical-mode plants that study endosperm storage material synthesis. Corn endosperm is the main storage site for nutrients, comprising 70% starch and 10% protein [1]. Amyloplasts are produced by the differentiation of the proplastids as the primary site of starch synthesis in the corn endosperm. In the initial stage of grouting, a large number of proplastids proliferate and differentiate in preparation for starch biosynthesis, so that corn endosperm can be used as an excellent model system for researching plastid development. Plastids (including proplastids, chloroplasts, amylosomes, and chromosomes, etc.) are a semi-autonomous organelle originating from the endosymgenesis of cyanobacteria in non-photosynthetic eukaryotes, widely involved in photosynthesis, and synthesis of fat, pigments, hormones, and carbohydrates [2]. Plastids are accompanied by a large number of gene levels transferred to the host cell nuclear genome during evolution, resulting in a progressive decrease in plastid genome, ultimately retaining only the key genes involved in photosynthesis, chloroplast biogenesis and regulation of gene expression [3]. Plastids typically have multiple copies of the genome, and these DNA's together with proteins make up the pseudocore [4]. Plastids cannot be synthesized de novo in the cytoplasm, but can only inherit to offspring and replicate through the cytoplasm of the parent. Thus, plastids have a continuum of development and accumulation of genetic variation in organisms [2]. In order to maintain normal function of plastids, the plastid genome must replicate and isolate with high fidelity during proliferation, whereas nucleases have important implications for the stability of the plastid genome.

Disclosure of Invention

In the research of the high-quality corn gene mining subject, a unique pink endosperm mutant is obtained by carrying out EMS chemical mutagenesis screening on a corn inbred line B73. Compared with the wild type, the mutant has less slightly powdery top endosperm separated from heterozygous selfing clusters, and the offspring of the mutant which continue selfing show complete loss of endosperm. Ext> byext> wholeext> genomeext> poolext> sequencingext> analysisext>,ext> aext> candidateext> geneext>,ext> Zmext> 00001ext> dext> 047988ext> (ext> GenBankext>:ext> AQLext> 08261.1ext>)ext>,ext> wasext> lockedext>,ext> andext> mutationext> ofext> theext> geneext> occurredext> atext> theext> cleavageext> siteext> atext> theext> junctionext> ofext> theext> 12ext> thext> exonext> andext> intronext>,ext> i.e.ext>,ext> theext> transitionext> ofext> Gext> -ext> Aext> atext> positionext> 7399ext> inext> nucleotideext> sequenceext> SEQext> IDext> NOext>:ext> 4ext>,ext> resultingext> inext> thatext> theext> intronext> thereofext> couldext> notext> beext> properlyext> cleavedext>.ext> The gene codes plastid-localized5'-3' exonuclease (plastid-localized 5'-3' exonuclease, PEN 1), and the amino acid sequence is shown as SEQ ID NO. 5. In order to study the properties of the enzyme, we tried to directly prokaryotic express the full-length protein PEN1 by using escherichia coli, but found that the full-length protein PEN1 is expressed in inclusion bodies, and tried to remove the localization signal (namely amino acids 1-92) of chloroplast at the N end to form mutant PEN1mut, and found that PEN1mut is expressed in inclusion bodies. Next, we established a stable and efficient prokaryotic protein purification system for PEN1mut by means of codon optimization technology, and finally realized that PEN1mut can be stably expressed in E.coli fermentation supernatant, and established a stable protein purification system in a laboratory. A series of nucleic acid substrates were then designed for enzyme activity analysis. The PEN1mut was found to be capable of cleaving substrates of different DNA and RNA structures with high efficiency in the presence of Mn ²⁺, which suggests that PEN1mut has a broad range of nuclease activity. In conclusion, this patent reports a plant-specific 5'-3' exonuclease PEN1 mutant PEN1mut, which has a broad nuclease activity and is expected to be a molecular biological tool enzyme. Based on the research results, the invention comprises the following technical scheme.

In a first aspect the invention provides a nuclease which is a plant-specific 5'-3' exonuclease selected from the group consisting of:

(a) The polypeptide with the amino acid sequence shown as SEQ ID NO. 1 is a mutant of 5'-3' exonuclease protein PEN1 (plastid-localized 5'-3' exonase) encoded by a gene Zm00001d047988 (GenBank: AQL 08261.1), and is named PEN1mut;

(b) A conservative variant polypeptide which is formed by substitution, deletion or addition of one or more amino acid residues of the amino acid sequence SEQ ID NO. 1 and has the function of the polypeptide (a) and is derived from the polypeptide (a);

(c) A conservative variant polypeptide derived from (a) having a homology of 95% or more, preferably 96% or more, preferably 97% or more, preferably 98% or more, more preferably 99% or more, with the polypeptide sequence defined in (a) and having the function of the polypeptide of (a), or

(D) A polypeptide derived from the polypeptide sequence of (a) or (b) or (c) contained in the sequence.

The above functions refer to nuclease functions that cleave DNA and/or RNA efficiently in the presence of Mn ²⁺, including, but not limited to, endonuclease and exonuclease such as 5'-3' exonuclease functions.

SRIMLVDGTSMMYRSYYKILAQLQHGQLEHADGNGDWVLTIFKALSLLLDMLEFIPSHAAVVFDHDGVPYGHYTAMPSKECHMAKGMTFRHMLYPAYKSNRTPTPDTVVQGMQYLKASIKAMSIKVIEVPGVEADDVIGTLAINSVSAGYKVRIVSPDKDFFQILSPSLRLLRIAPRGSGMVSFGVEDFVKRYGPLKPSQFVDVVALSGDKADNIPGVEGIGDINAVKLISKFGSLDNLLKSVDEVEDERIKQALISHSEQAILCKNLATLRSDLPHYMVPFKTADLVFKKPQDDGEKFIKLLRALEAYAEGSSVNPIIRRAAYLWNKLKS(SEQ ID NO:1).

In a second aspect, the invention provides a polynucleotide selected from the group consisting of:

(A) A polynucleotide encoding a polypeptide as described above;

(B) A polynucleotide for coding a polypeptide with an amino acid sequence shown as SEQ ID NO. 1;

(C) The polynucleotide with the nucleotide sequence shown as SEQ ID NO. 2 is a coding gene PEN1mut of nuclease PEN1mut suitable for prokaryotic expression and eukaryotic expression;

(D) A polynucleotide having a nucleotide sequence having a homology of 95% or more, preferably 96% or more, preferably 97% or more, preferably 98% or more, more preferably 99% or more with the nucleotide sequence shown in SEQ ID NO. 2;

(E) A nucleotide sequence complementary to the nucleotide sequence of any one of (a) - (D).

AGCCGTATCATGCTGGTTGATGGTACCTCCATGATGTACCGTAGCTACTACAAGATTCTGGCACAGCTGCAACACGGTCAGCTGGAACACGCTGATGGTAACGGCGACTGGGTACTGACCATCTTCAAAGCACTGTCCCTGCTGCTGGATATGCTGGAATTCATCCCGTCCCACGCGGCTGTTGTTTTCGATCACGATGGCGTTCCATACGGTCACTATACTGCGATGCCGAGCAAAGAATGCCATATGGCAAAAGGTATGACCTTCCGCCATATGCTGTACCCGGCTTACAAATCCAATCGTACTCCGACCCCTGACACTGTCGTTCAGGGCATGCAGTACCTGAAAGCGTCTATTAAGGCGATGAGCATTAAAGTTATCGAAGTCCCGGGTGTCGAGGCTGATGATGTTATCGGTACCCTGGCTATCAATAGCGTGTCCGCGGGCTATAAAGTGCGCATCGTTTCCCCGGATAAAGACTTCTTCCAGATTCTGTCCCCGTCTCTGCGTCTGCTGCGTATTGCTCCTCGTGGTTCCGGTATGGTTAGCTTCGGTGTAGAAGATTTCGTTAAACGTTATGGCCCGCTGAAGCCGTCTCAATTCGTCGATGTTGTGGCTCTGAGCGGCGACAAGGCGGATAACATCCCAGGCGTTGAAGGCATCGGCGACATTAACGCCGTGAAACTGATCTCTAAATTCGGTTCCCTGGATAATCTGCTGAAATCCGTCGACGAAGTAGAGGACGAGCGCATTAAACAGGCTCTGATCAGCCACTCTGAACAAGCAATTCTGTGCAAAAATCTGGCCACCCTGCGTTCCGATCTGCCGCATTACATGGTTCCGTTCAAAACTGCAGACCTGGTGTTCAAAAAACCGCAGGATGACGGCGAAAAGTTTATTAAGCTGCTGCGTGCGCTGGAAGCCTATGCCGAAGGCAGCTCCGTTAACCCGATCATTCGTCGTGCCGCCTACCTGTGGAATAAACTGAAATCCTGA(SEQ ID NO:2).

In a third aspect the invention provides a DNA molecule comprising a polynucleotide as described above, for example an expression cassette/cassette for the polypeptide PEN1 mut.

In a fourth aspect, the present invention provides a recombinant plasmid comprising a DNA molecule as described above, which is an over-expression vector formed by cloning the DNA molecule as described above onto a plasmid vector suitable for expression in an industrial microorganism selected from the group consisting of bacteria or yeasts commonly used for the heterologous expression of a protein of interest, such as escherichia coli, bacillus subtilis, corynebacterium glutamicum, vibrio natrii, pichia pastoris, baker's yeast, and the like;

For the industrial production of PEN1mut, for example, PEN1mut by fermentation with E.coli engineering bacteria, the plasmid vector used for constructing the above recombinant plasmid is preferably a pET vector series such as pET-28a-Sumo vector.

In one embodiment, the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 3.

In a fifth aspect, the invention provides a microorganism engineering bacterium, which is a transformant containing the recombinant plasmid as described above and can be used for producing a polypeptide with an amino acid sequence shown as SEQ ID NO. 1 by fermentation.

In a sixth aspect the present invention provides a method of preparing a polypeptide PEN1mut as described above by fermentation of a microorganism engineering bacterium as described above to produce the polypeptide PEN1mut.

In a seventh aspect the invention provides the use of a polypeptide as described above, such as PEN1mut, a polynucleotide as described above, a DNA molecule as described above or a recombinant plasmid as described above, as a tool enzyme.

In one embodiment, the above uses refer to the use of the tool enzyme as a molecular biological tool enzyme for gene editing technology, as an endonuclease and an exonuclease for the preparation of biochemical agents, or as a DNase (DNase) and/or an RNase (RNase) for the cleavage of nucleic acids for the preparation of small molecule nucleic acid pharmaceuticals and/or nutritional additives.

As known to those skilled in the art, the application fields of the gene editing technology include genetic engineering of living organisms such as microorganisms, plants, animals, and the like.

This study reports the cytological phenotype of maize B73-derived 5'-3' exonuclease gene Zm00001d047988 (GenBank: AQL08261.1, nucleotide sequence shown in SEQ ID NO:4, amino acid sequence shown in SEQ ID NO: 5) after functional deletion, prokaryotic protein expression and purification mode of mutant-encoded polypeptide PEN1mut, and enzyme activity mechanism of PEN1 mut. Meanwhile, PEN1mut can be used as a high-efficiency nuclease and can be used as a potential tool enzyme for molecular biology, and has wide application prospect.

Drawings

FIG. 1 shows the phenotypes of wild-type B73 and pen1 mutants. Wherein a is wild type B73, pen1/+ selfing clusters and clusters phenotype of pen1 ^F2 selfing, B is a semi-thin slice analysis image of wild type B73, pen1/+ selfing clusters separation pen1 ^F2 and pen1 ^F2 selfing kernel endosperm.

FIG. 2 shows analysis of Pen1 genetic mapping. Ext> theext> wildext> typeext> andext> mutantext> separatedext> fromext> Fext> 2ext> ofext> Penext> 1ext> /ext> +ext> selfingext> areext> usedext> forext> BSAext> mixedext> poolext> sequencingext> typingext>,ext> aext> distinctext> peakext> appearsext> onext> chromosomeext> 9ext>,ext> andext> bext>,ext> theext> cuttingext> siteext> Gext> -ext> Aext> atext> theext> junctionext> ofext> theext> 12ext> thext> exonext> andext> theext> intronext> ofext> theext> candidateext> geneext> Penext> 1ext> isext> convertedext>,ext> soext> thatext> theext> intronext> cannotext> beext> cutext> correctlyext>.ext>

FIG. 3 shows subcellular localization analysis of PEN 1. Wherein, a-b, subcellular localization of PEN1-GFP, red color indicates chloroplast autofluorescence, DAPI is used for labeling nucleus and chloroplast DAN. The C end of PEN1 is fused with GFP and expressed in tobacco leaf epidermal cells, and C is PEN1 immunofluorescence analysis image.

FIG. 4 shows the purification of PEN1mut prokaryotic protein expression. Molecular exclusion chromatography and SDS-PAGE analysis of PEN1mut. Wherein M represents a protein Marker.

FIG. 5 shows PEN1mut enzyme activity assay images. Wherein a is the activity of PEN1mut on different DNA structure substrates, and b is the activity of PEN1mut on different RNA structure substrates. Red lines represent RNA bases, black lines represent DNA bases, and black filled circles represent streptavidin.

FIG. 6 shows a structural map of recombinant plasmid SUMO-PEN1 overexpressing PEN1 mut.

Detailed Description

EMS (Ethylmethanesulfonate ) is a commonly used chemical mutagen, belongs to DNA mutagen, can induce and generate high-density series allelic point mutation, has the advantages of high efficiency, small side effect, easy operation and the like, and is widely applied to related genetic research and mutation breeding work of plants at present.

In the research of maize endosperm development and biosynthesis related genes, we performed EMS chemical mutagenesis on maize inbred line B73, and obtained a unique maize kernel mutant through massive inheritance, which heterozygous inbred ear was isolated to produce a small amount of kernel with slightly pink top of endosperm (pen 1 ^F2), whereas the endosperm of the pen1 ^F2 inbred progeny (pen 1 ^F3) was completely deleted. The amount of amyloids of pen1 ^F2 was significantly reduced compared to the wild type, whereas almost no amyloids were observed in the endosperm of pen1 ^F3. By genomic comparison, the gene Zm00001d047988 encoding 5'-3' exonuclease in the B73 genome (GenBank: AQL 08261.1) was found to have been mutated. We mapped this site to the 1Mb region of chromosome 9, where only PEN1 contained a unique SNP at the intron cleavage site, resulting in an intron cleavage abnormality. PEN1 localizes to chloroplasts and exhibits tiny punctate aggregates. To study PEN1, we tried to prokaryotic express its gene CDS with E.coli, but found that PEN1 was expressed only in inclusion bodies. Thus, the localization signal of chloroplasts at the N-terminus (i.e., amino acids 1-92) was removed to form its mutant PEN1mut, and PEN1mut expression was found to remain in inclusion bodies. The PEN1mut codon is modified and synthesized, inserted into pET-28a-Sumo vector, prokaryotic expressed and purified to prepare the high purity target protein PEN1mut. We have studied the enzymatic activity of PEN1mut in detail and found that PEN1mut is capable of cleaving substrates of different DNA and RNA structures with high efficiency in the presence of Mn ²⁺. PEN1mut thus acts as a highly potent nuclease and simultaneously is capable of large amounts of expression and purification, as it can also be used as a nuclease for molecular biology research. Moreover, PEN1mut has potential to be applied to more fields based on the wide range of uses of nucleases, not limited to 5'-3' exonuclease uses only, but also includes use as endonucleases and exonucleases for preparing biochemical agents, use as dnases and rnases for preparing small-molecule nucleic acid pharmaceuticals and nutritional additives, etc. in pharmaceutical and food fields.

Since the mutant PEN1mut retains the function of the wild-type PEN1, the wild-type PEN1 and its mutant PEN1mut are sometimes referred to herein as PEN1 for convenience of description, and the meaning of each "PEN1" and their differences in the different descriptions, such as the manner in which PEN1 and gene PEN1 are labeled in the figures, will be readily understood by those skilled in the art.

Nucleases are classified into endonucleases and exonucleases. Exonucleases remove nucleotides from the free 5 'or 3' ends of DNA. Exonucleases are found in the venom of eukaryotic cells, prokaryotic cells and certain organisms. They fall into 3 classes, acting in the 3'-5' direction of the DNA/RNA strand, removing the mononucleotide, forming a cohesive end. They play a role in genetic quality control, DNA proofreading during replication, homologous binding and DNA repair, ensuring genomic stability. The 5' -3' exonuclease activity is the only active component of the N-terminal fragment of DNA polymerase I, and the main function is to remove the RNA primer at the 5' -end of newly synthesized DNA, so that the polymerase activity can fill the blank. DNA polymerase I is the only E.coli DNA polymerase having both 3 'to 5' and 5 'to 3' exonuclease activity. Both pol.II and pol.III can polymerize DNA and cleave fragments in the 3'-5' direction, but they do not have a 5'-3' exonuclease function. 3 'to 5' activity can remove only one single nucleotide at a time, whereas 5 'to 3' activity can remove a single nucleotide or up to 10 nucleotides at a time. In the fields of biochemistry and molecular biology, 3'-5' exonucleases and 5'-3' exonucleases are two important enzymes that play a key role in DNA and RNA repair, replication and transcription.

In recent years, exonucleases have shown great potential as an important biological tool in the fields of disease treatment and gene editing. Exonucleases can be used in gene editing to repair errors and lesions on DNA and RNA strands. Based on the repair mechanism of exonuclease, researchers can design specific exonuclease analogues to repair genetic diseases caused by gene mutation. The gene editing technology has been widely applied to research and treatment of various genetic diseases, and provides new hope for treatment of related diseases. In addition, exonucleases can also be used as drug targets for the treatment of diseases associated with DNA and RNA damage. Inhibitors against exonucleases have been developed for the treatment of a variety of cancers and genetic diseases. These inhibitors may be useful by interfering with the normal function of the exonuclease, to inhibit the growth and diffusion of tumor cells, and provide a new idea for the treatment of cancers.

It would be reasonable to those skilled in the art that some conservatively variant polypeptides of polypeptide PEN1 have the same nuclease function.

As used herein, the term "conservatively modified polypeptide" refers to a polypeptide that retains essentially the same biological function or activity as the polypeptide. The "conservatively altered polypeptide" may be (i) a polypeptide having one or more, preferably conservative amino acid residues, substituted or non-conservative amino acid residues, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that increases the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide. for example, (1) a polypeptide having the function of the polypeptide PEN1, which is formed by substitution, deletion or addition of one or more (e.g., 1 to 20, preferably 1 to 10, more preferably 1 to 5, still more preferably 1 to 3) amino acid residues of the polypeptide PEN1, or (2) a polypeptide having 50% or more (preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably 98% or more, still more preferably 99% or more) the identity to the polypeptide represented by the polypeptide PEN1, or (3) a polypeptide having a tag sequence added to the N-or C-terminus of the polypeptide PEN1, or having a signal peptide sequence added to the N-terminus thereof. such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein. The term "variation" or "mutation" includes, but is not limited to, substitution, deletion, insertion, chemical modification of amino acid residues, preferably a positive mutation, i.e., a mutation with increased function. The substitutions may be non-conservative substitutions, conservative substitutions or a combination of non-conservative and conservative substitutions. "conservative" amino acid substitutions or mutations refer to the interchangeability of residues having similar side chains, and thus generally include the substitution of amino acids in polypeptides with amino acids in the same or similar amino acid definition categories. However, as used herein, a conservative mutation does not include a substitution of a hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue if the conservative mutation may instead be an aliphatic to aliphatic, nonpolar to nonpolar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or residue-limiting to residue-limiting substitution. Common conditions for conservative substitutions, known in the art, include the mutual substitution between aromatic amino acids F, W, Y, the mutual substitution between hydrophobic amino acids L, I, V, the mutual substitution between polar amino acids Q, N, the mutual substitution between basic amino acids K, R, H, the mutual substitution between acidic amino acids D, E, and the mutual substitution between hydroxy amino acids S, T. Furthermore, A, V, L or I may be conservatively mutated to another aliphatic residue or another nonpolar residue. Exemplary conservative substitutions may be made, for example, according to the following table.

It is well known that the same nucleotide sequence often varies greatly in the results of expression in different microbial hosts. In order to optimally express PEN1 in E.coli, which is most commonly used in genetic engineering, we have codon optimized the PEN1 expressed gene.

Codon optimization is a technique that can be used to maximize protein expression in an organism by increasing the translational efficiency of a gene of interest. Different organisms often show a special preference for one of some codons encoding the same amino acid due to mutation propensity and natural selection. For example, in a fast-growing microorganism such as E.coli, the optimized codons reflect the composition of their respective genomic tRNA pool. Thus, in fast-growing microorganisms, the low frequency codons of an amino acid can be replaced with codons for the same amino acid but at a high frequency. Thus, the expression of the optimized DNA sequence is improved in fast growing microorganisms.

In order to express nuclease SEQ ID NO. 1 in escherichia coli, the encoding gene of the nuclease is subjected to codon optimization, and the encoding gene of the nuclease SEQ ID NO. 1 after optimization is SEQ ID NO. 2.

Further, in order to realize the large-scale production of the polypeptide PEN1, PEN1 can be produced by an industrial microorganism such as an escherichia coli engineering bacterium fermentation method, a gene expression cassette or expression construct serving as a DNA molecule is constructed by taking a coding gene such as a polynucleotide SEQ ID NO. 2 as an exogenous gene, the expression cassette/expression construct is operably connected to a plasmid vector through subcloning to obtain a recombinant plasmid, and the recombinant plasmid is transformed into a host cell to obtain a transformant, namely the genetically engineered bacterium or recombinant bacterium.

The terms "recombinant strain" and "genetically engineered strain" as used herein refer to the same meaning and refer to strains comprising a PEN1 gene overexpression vector.

In the description of the technical scheme of the present invention, the term "and/or" used in terms such as "A and/or B", "A and/or B" is intended to include both A and B, A or B, A (alone), and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments: A, B and C, A, B or C, A or B, B or C, A and B, B and C, A (alone), B (alone), C (alone).

In this context, for the sake of simplicity of description, a protein such as the polypeptide PEN1 is sometimes used in combination with the name PEN1 of its coding gene (DNA), it being understood by the person skilled in the art that they represent different substance types in the context of the description. Those skilled in the art will readily understand their meaning depending on the context and context. For example, for PEN1, the protein is referred to when describing the function or class of the 5'-3' exonuclease protein, and the gene encoding the protein when described as a gene.

As used herein, the term "expression cassette" or "gene expression cassette" refers to a gene expression system comprising all the necessary elements necessary for expression of the intended protein PEN1, typically including a promoter, a gene sequence encoding a polypeptide, a terminator, and optionally a signal peptide coding sequence, etc., which are operably linked.

As used herein, the term "expression construct" or "expression construct" refers to a recombinant DNA molecule comprising the desired nucleic acid coding sequence (e.g., SEQ ID NO: 2), which may comprise one or more gene expression cassettes. The "construct" is typically contained in an expression vector (plasmid vector).

As used herein, the term "exogenous" or "heterologous" refers to a relationship between two or more nucleic acid or protein sequences from different sources, or a relationship between a protein (or nucleic acid) from different sources and a host cell. For example, if the combination of nucleic acid and host cell is not normally naturally occurring, the nucleic acid is exogenous to the host cell. The particular sequence is "exogenous" to the cell or organism into which it is inserted.

As used herein, the terms "operably linked" or "operably linked" refer to a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, the promoter region is placed in a specific position relative to a gene of interest, e.g., the nucleic acid sequence SEQ ID NO. 2, such that transcription of the nucleic acid sequence is directed by the promoter region, whereby the promoter region is "operably linked" to the nucleic acid sequence.

The nucleic acid constructs of the present invention may be manipulated in a variety of ways to ensure expression of the polypeptide PEN 1. The nucleic acid construct may be manipulated according to the expression vector or requirements prior to insertion into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

In certain embodiments, the nucleic acid construct is a vector. The vector may be a cloning vector, an expression vector, or a gene knock-in vector. The nucleic acid sequence SEQ ID NO.2 of the present invention may be cloned into many types of vectors, for example, plasmids, phagemids, phage derivatives, animal viruses and cosmids. Cloning vectors may be used to provide the coding sequence for a protein or polypeptide of the invention. The expression vector may be provided to the cell in the form of a bacterial vector or a viral vector. Expression of the PEN1 gene is typically achieved by operably linking the nucleic acid sequence of the invention SEQ ID NO.2 to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration of eukaryotic cells. Typical expression vectors contain expression control sequences that can be used to regulate the expression of a desired nucleic acid sequence.

Methods well known to those skilled in the art can be used to construct the nucleic acid construct. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are the lac or trp promoter of E.coli, the lambda phage PL promoter, eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, LTRs from retroviruses and some other known promoters which control gene expression in prokaryotic or eukaryotic cells or viruses thereof. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In addition, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, or for tetracycline, ampicillin resistance, chloramphenicol, etc. of E.coli, agrobacterium.

The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

Examples

The examples relate to the amounts, amounts and concentrations of various substances, wherein the percentages refer to percentages by mass unless otherwise specified.

In the examples herein, if no specific description is made regarding the reaction temperature or the operation temperature, the temperature is generally referred to as room temperature (15 to 30 ℃).

The molecular biology experiments in the examples include plasmid construction, enzyme digestion, competent cell preparation, transformation, etc., and are mainly performed by referring to "molecular cloning experiment guidelines (third edition), J.Sam Broker, D.W. Lassel, huang Peitang et al, science Press, beijing, 2002). For example, competent cell transformation methods and competent preparation methods were carried out according to chapter 1, page 96 of the guidelines for molecular cloning experiments (third edition). The specific experimental conditions can be determined by simple experiments, if necessary.

The PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template suppliers. Can be adjusted if necessary by simple tests.

EMS mutagenesis, selfing, hybridization, etc. of maize are performed in accordance with conventional breeding methods.

BSA sequencing analysis was performed by Shanghai European Biolabs.

Primer synthesis and gene sequencing in the examples were all accomplished by Shanghai platinum Biotechnology Co.

Example 1 discovery of genes and phenotypic analysis associated with corn endosperm formation and development by EMS mutagenesis

We have discovered genes related to the development and biosynthesis of corn endosperm through EMS mutagenesis.

EMS mutagenesis was performed on maize inbred line B73 to isolate an endosperm-grouting mutant. EMS mutagenesis current generation M0 seeds are selfed and harvested in a single spike, and phenotype investigation is carried out. As shown in FIG. 1, the mutant (i.e., pen 1) is a unique pink endosperm mutant. Less of the slightly pinkish endosperm top mutant (Pen 1 ^F2) was isolated from Pen1 heterozygous inbred ears compared to wild-type B73, and the progeny of the mutant continued to selfe (i.e., pen1 ^F3) exhibited a complete loss of endosperm (fig. 1 a). At the same time we performed cytologic analysis using semi-thin sections, the mutant had significantly reduced amyloidogenic number compared to the wild type, while almost no amyloidogenic was observed in the endosperm (fig. 1 b).

The mutant was subjected to whole gene sequencing analysis by Shanghai European biotechnology Co.Ltd, and the mutant gene was selected.

EXAMPLE 2 genetic mapping analysis of mutant genes

100 Strains of the mutant (i.e. pen 1) and the mutant separated from the F2 group of B73 are selected and respectively mixed into a wild type pool and a mutant pool, and mixed pool sequencing is carried out. Sequencing data were analyzed by MutMap method, see FIG. 2, and the results show that only the significant main peak appears on chromosome 9 (a in FIG. 2). Ext> genomicext> comparisonext> showsext> thatext> theext> candidateext> geneext> isext> Zmext> 00001ext> dext> 047988ext> (ext> GenBankext>:ext> AQLext> 08261.1ext>,ext> nucleotideext> sequenceext> shownext> asext> SEQext> IDext> NOext>:ext> 4ext>)ext>,ext> encodesext> aext> plantext> -ext> specificext> 5ext> 'ext> -ext> 3ext>'ext> exonucleaseext> comprisingext> 422ext> aminoext> acidext> residuesext>,ext> whereasext> theext> geneticext> variationext> inext> theext> mutantext> occursext> atext> theext> cleavageext> siteext> atext> theext> junctionext> ofext> theext> 12ext> thext> exonext> andext> intronext> (ext> positionext> 7399ext> inext> SEQext> IDext> NOext>:ext> 4ext>)ext> inext> theext> transitionext> ofext> Gext> -ext> Aext>,ext> resultingext> inext> theext> inabilityext> ofext> theext> intronext> toext> beext> correctlyext> cleavedext>.ext>

EXAMPLE 3 subcellular localization analysis of mutant genes

Construction of 1300-35S-AtRNH C-TDT and 1300-35S-Pen1-GFP, co-localization in tobacco epidermal cell system, and co-localization signal analysis using Fiji. PEN1-Flag transgenic plants were constructed and chloroplasts thereof were isolated for immunofluorescence analysis. Different components of chloroplasts of PEN1-Flag transgenic plants were isolated and subjected to immunoblot analysis.

Referring to FIG. 3, the localization of the mutant gene to plastids was confirmed by subcellular localization analysis (plastid), so that the 5'-3' exonuclease encoded by this gene was designated PEN1 (plastid-localized 5'-3' exonase, PEN 1), and the mutant gene was designated Pen1 accordingly.

In FIG. 3 a-b shows subcellular localization images of PEN1-GFP, red for chloroplast autofluorescence, DAPI for labeling the nucleus and chloroplast DNA. PEN 1C-terminal fused with GFP and expressed in tobacco leaf epidermal cells, and PEN1 immunofluorescence analysis image is shown in FIG. 3C.

EXAMPLE 4 construction of PEN1 E.coli engineering bacteria

To investigate the properties of PEN1, we tried to express its gene CDS using E.coli, but it was found that the expressed PEN1 was only present in inclusion bodies and was difficult to isolate and purify. Thus, it was determined that the localization signal of chloroplast at the N-terminus thereof (i.e., amino acids 1 to 92) was removed to form a mutant PEN1mut having the amino acid sequence shown in SEQ ID NO:1, and as a result, it was found that PEN1mut was expressed still in inclusion bodies.

In order to realize in vitro secretion of PEN1mut, the coding gene sequence SEQ ID NO. 2 is obtained by codon optimization according to the amino acid sequence SEQ ID NO. 1 of PEN1mut and the codon preference of escherichia coli. The Shanghai platinum is entrusted to complete gene synthesis, cloned to plasmid pET-28a-Sumo (the plasmid is self-designed by taking pET-28a as a framework in the laboratory) to obtain recombinant plasmid SUMO-PEN1, the plasmid structure map is shown in figure 6, and the nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO: 3.

The recombinant plasmid SUMO-PEN1 with correct sequence is electrically transformed into host escherichia coli Rosetta (DE 3) to be competent, positive clones are screened, and recombinant escherichia coli expressing PEN1mut is constructed.

EXAMPLE 5 purification of the prokaryotic protein expression of PEN1mut

A single colony of the recombinant E.coli of PEN1mut constructed in example 4 was picked up, inoculated into 5mL of liquid LB medium containing 50. Mu.g/mL kanamycin sulfate and 25. Mu.g/mL chloramphenicol, and cultured overnight at 37℃and 250 rpm. The next day, the inoculated amount with 1% v/v of volume concentration is transferred to a shaking flask containing 200mL of liquid TB medium, and the liquid TB medium is subjected to bed culture for 2-3 hours at 37 ℃ and 250rpm, and when the OD600 is 0.6-0.8, 0.1mM IPTG is added for induction for 16 hours. The cells were collected by centrifugation at 6,000rpm for 5 minutes and uniformly suspended in buffer A (50 mM Tris-HCl pH7.5,500mM NaCl,5mM β -mercaptoethanol, 20mM imidazole, 1mM PMSF,5% v/v glycerol) and cell lysis was performed at 4℃using Avestin emulsion flex-C5. Lysates were centrifuged at 75,000g for 60min at 4℃and supernatants were affinity purified using Ni-NTA agarose, eluting with 20CV (column volumes) buffer A for hetero-proteins and SUMOstar protease in 5CV buffer A at 4 ℃. The eluted PEN1mut was dialyzed overnight in buffer B (20 mM Tris-HCl pH7.5,150mM NaCl,5mM. Beta. -mercaptoethanol, 1mM PMSF,1% v/v glycerol). PEN1 was further purified using a Q HP column (HITRAP Q HP-mL, cytiva) with Q HP buffer A (20 mM Tris-HCl pH7.5,150mM NaCl,1% v/v glycerol, 1mM DTT) and Q HP buffer B (20 mM Tris-HCl pH7.5,1000mM NaCl,1% v/v glycerol, 1mM DTT). The PEN1 mut-containing fraction was collected and further purified using Hilload/600 Superdex 75pg (Cytiva) and PEN1 mut-containing fraction was collected. PEN1mut was concentrated to 5.0mg/mL and stored at-80℃until use.

FIG. 4 shows molecular exclusion chromatography and SDS-PAGE analysis images of PEN1mut protein.

Example 6 PEN1 put enzyme activity test assay

The substrate modification modes used in the detection of nuclease activity of PEN1mut include/6 FAMdT/,' 5-P, biotin-TEG, and the purification method uses 2X HPLC. The solution was diluted to 100. Mu.M in RNase-free annealing buffer (50 mM Tris-HCl (pH 7.4), 200mM NaCl) for use. The DNA and RNA structural substrates were annealed using a PCR apparatus (annealing procedure: 95 ℃ C. For 5min, 0.1 ℃ C. Down to 25 ℃ C., 700 cycles, 12 ℃ C. For preservation every 8 s). Substrates of double-stranded structure require the use of streptavidin to block the free 5' end, preventing cleavage of PEN1 at non-target sites.

PEN1mut cleavage reaction system contained (20 mM Tris-HCl (pH 7.4), 10mM MnCl ₂, 1mM DTT), substrate concentration of 25nM, PEN1 concentration gradient of 0, 6.25, 12.5, 25, 50pM, the prepared reaction system was incubated at 37℃for 15min, 2 volumes of reaction stop buffer (90% formamide, 5mM EDTA) were added, and immediately after incubation at 98℃for 10min, cooling rapidly on ice. Electrophoresis detection (50W) was performed using high resolution 15%/8M urea denaturing PAGE sequencing gels, using Cytiva Typhoon detection.

Referring to FIG. 5, nuclease test results show that PEN1mut is capable of cleaving DNA and RNA with high efficiency, indicating that it has the enzyme activities of DNase (DNase) and RNase (RNase), not limited to 5'-3' exonuclease activity, and can be widely used as a tool enzyme.

The above embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention.

Reference to the literature

[1]Yang,T.,Wu,X.G.,Wang,W.Q.,and Wu,Y.R.(2023).Regulation of seed storage protein synthesis in monocot and dicot plants:Acomparative review.Mol Plant 16,145-167.

[2]Choi,H.,Yi,T.,and Ha,S.H.(2021).Diversity of Plastid Types and Their Interconversions.Front Plant Sci 12,692024.

[3]Zimorski,V.,Ku,C.,Martin,W.F.,and Gould,S.B.(2014).Endosymbiotic theory for organelle origins.Curr Opin Microbiol 22,38-48.

[4]Pfalz,J.,and Pfannschmidt,T.(2013).Essential nucleoid proteins in early chloroplast development.Trends Plant Sci 18,186-194.

Claims (10)

1. A nuclease, which is a polypeptide selected from the group consisting of: (a) a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1, designated as PEN1mut; (b) a conservative variant polypeptide derived from (a) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence SEQ ID NO: 1 and having the function of the polypeptide of (a); (c) a conservative variant polypeptide derived from (a) that has a sequence homology of more than 95% with the polypeptide defined in (a) and has the functions of the polypeptide defined in (a); or (d) A derivative polypeptide having a sequence containing the polypeptide sequence described in (a) or (b) or (c). 2. A polynucleotide selected from the group consisting of: (A) a polynucleotide encoding the polypeptide of claim 1; (B) a polynucleotide encoding a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1; (C) a polynucleotide whose nucleotide sequence is shown in SEQ ID NO: 2, which is the nucleotide sequence of a gene encoding nuclease PEN1mut; (D) a polynucleotide having a nucleotide sequence identity of ≥95%, preferably ≥96%, preferably ≥97%, preferably ≥98%, and more preferably ≥99% to the nucleotide sequence of SEQ ID NO: 2; (E) A nucleotide sequence complementary to the nucleotide sequence described in any one of (A) to (D). 3. A DNA molecule, characterized in that it comprises the polynucleotide according to claim 2. 4. A recombinant plasmid, characterized in that it comprises the DNA molecule according to claim 3, wherein the recombinant plasmid is an overexpression vector formed by cloning the DNA molecule according to claim 3 into a plasmid vector suitable for expression in industrial microorganisms. 5. The recombinant plasmid according to claim 4, wherein the plasmid vector is a pET vector series. 6. The recombinant plasmid according to claim 5, wherein the nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO: 3. 7. An engineered microorganism, characterized in that it is a transformant comprising the recombinant plasmid according to claim 4, and is used for producing a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1 by fermentation. 8. A method for preparing the polypeptide PEN1mut according to claim 1, characterized in that the polypeptide PEN1mut is produced by fermentation of the microbial engineering bacteria according to claim 7. 9. Use of the polypeptide according to claim 1, the polynucleotide according to claim 2, the DNA molecule according to claim 3 or the recombinant plasmid according to claim 4 as a tool enzyme. 10. The use according to claim 6, characterized in that the tool enzyme is used as a molecular biology tool enzyme in gene editing technology, as an endonuclease and exonuclease for preparing biochemical preparations, or as a DNA enzyme (DNase) and/or RNA enzyme (RNase) for cutting nucleic acids to prepare small molecule nucleic acid drugs and/or nutritional additives.