Novel nuclease

Abstract

The invention discloses a novel nuclease with 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 a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biotechnology, and specifically relates to a novel nuclease having an amino acid sequence as shown in SEQ ID NO: 1 and its application as a tool enzyme in molecular biology. Background Art

[0002] Corn (Zea mays L.) is an important food and economic crop in my country and is also one of the classic model plants for studying the synthesis of endosperm storage substances. Corn endosperm is the main storage site for nutrients, containing 70% starch and 10% protein [1]. Amyloplasts, as the main site of starch synthesis in corn endosperm, are differentiated from the proplastids. At the initial stage of grain filling, a large number of proplastids proliferate and differentiate to prepare for starch biosynthesis. Therefore, corn endosperm can be used as an excellent model system for studying plastid development. Plastids (including proplastids, chloroplasts, amyloplasts and chromoplasts, etc.) are semi-autonomous organelles that originated from the endosymbiosis of cyanobacteria in non-photosynthetic eukaryotes and are widely involved in photosynthesis, as well as the synthesis of fats, pigments, hormones and carbohydrates [2]. During the evolution of plastids, a large number of genes were horizontally transferred to the host cell nuclear genome, resulting in the continuous reduction of the plastid genome, and ultimately only the key genes involved in photosynthesis, chloroplast biogenesis and gene expression regulation were retained [3]. Plastids usually have multiple copies of the genome, and these DNAs together with proteins form the nucleoid [4]. Plastids cannot be synthesized de novo in the cytoplasm and can only be inherited and replicated through the maternal cytoplasm. Therefore, plastids have a continuity of development and accumulation of genetic variation in organisms [2]. In order to maintain the normal function of plastids, the plastid genome must be replicated and separated with high fidelity during the proliferation process, and nucleases are of great significance to the stability of the plastid genome. Summary of the Invention

[0003] In our research on high-quality maize gene discovery, we screened the maize inbred line B73 using EMS chemical mutagenesis, resulting in a unique floury endosperm mutant. Compared to the wild-type, this mutant exhibited fewer slightly floury endosperm apex mutations in heterozygous inbred ears. Further selfing of this mutant revealed a complete absence of endosperm. Whole-genome pooled sequencing identified the candidate gene, Zm00001d047988 (GenBank: AQL08261.1). The mutation occurs at the splice site between exon 12 and an intron, resulting in a GA transition at position 7399 in SEQ ID NO:4, impairing proper intron splicing. This gene encodes a plastid-localized 5'-3' exonuclease (PEN1), the amino acid sequence of which is shown in SEQ ID NO:5. In order to study the properties of this enzyme, we tried to directly express the full-length protein PEN1 in E. coli, but found that it was expressed in inclusion bodies; we removed the chloroplast localization signal at its N-terminus (i.e., amino acids 1-92) to form its mutant PEN1mut, and tried it, and found that PEN1mut was still expressed in inclusion bodies. Then, we established a stable and efficient prokaryotic protein purification system for PEN1mut through codon optimization technology, and finally achieved the stable expression of PEN1mut in E. coli fermentation supernatant, and established a stable protein purification system in the laboratory. Then we designed a series of nucleic acid substrates for enzyme activity analysis. The study found that PEN1mut can be expressed in Mn 2+ The ability to efficiently cleave substrates with diverse DNA and RNA structures in the presence of α-terminal ...

[0004] The first aspect of the present invention provides a nuclease, which is a plant-specific 5'-3' exonuclease, selected from the following polypeptides:

[0005] (a) A polypeptide with an amino acid sequence as shown in SEQ ID NO: 1, which is a mutant of the 5'-3' exonuclease protein PEN1 (plastid-localized 5'-3' exonuclease) encoded by gene Zm00001d047988 (GenBank: AQL08261.1), designated as PEN1mut;

[0006] (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);

[0007] (c) a conservative variant polypeptide derived from (a) that has 95% or more homology, preferably 96% or more homology, preferably 97% or more homology, preferably 98% or more homology, and more preferably 99% or more homology with the polypeptide sequence defined in (a), and has the function of the polypeptide of (a); or

[0008] (d) A derivative polypeptide having a sequence containing the polypeptide sequence described in (a) or (b) or (c).

[0009] The above function refers to the 2+ Nuclease functions that efficiently cleave DNA and / or RNA in the presence of a nucleic acid include, but are not limited to, endonucleases and exonucleases, such as 5'-3' exonucleases.

[0010] SRIMLVDGTSMMYRSYYKILAQLQHGQLEHADGNGDWVLTIFKALSLLLDMLEFIPSHAAVVFDHDGVPYGHYTAMPSKECHMAKGMTFRHMLYPAYKSNRTPTPDTVVQGMQYLKASIKAMSIKVIEVPGVEADDVIGTLAINSVSAGYKVRIVSPDKDFFQILSP SLRLLRIAPRGSGMVSFGVEDFVKRYGPLKPSQFVDVVALSGDKADNIPGVEGIGDINAVKLISKFGSLDNLLKSVDEVEDERIKQALISHSEQAILCKNLATLRSDLPHYMVPFKTADLVFKKPQDDGEKFIKLLRALEAYAEGSSVNPIIRRAAYLWNKLKS(SEQ ID NO:1).

[0011] The second aspect of the present invention provides a polynucleotide, wherein the polynucleotide is selected from the group consisting of:

[0012] (A) a polynucleotide encoding the polypeptide described above;

[0013] (B) a polynucleotide encoding a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1;

[0014] (C) a polynucleotide whose nucleotide sequence is shown in SEQ ID NO: 2, which is a gene encoding the nuclease PEN1mut suitable for prokaryotic and eukaryotic expression;

[0015] (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;

[0016] (E) A nucleotide sequence complementary to the nucleotide sequence described in any one of (A) to (D).

[0017] AGCCGTATCATGCTGGTTGATGGTACCTCCATGATGTACCGTAGCTACTACAAGATTCTGGCACAGCTGCAACACGGTCAGCTGGAACACGCTGATGGTAACGGCGACTGGGTACTGACCATCTTCAAAGCACTGTCCCTGCTGCTGGATATGCTGGAATTCATCCCGTCCCACGCGGCTGTTGTTTTCGATCACGATGGCGTTCCATACGGTCACTATACTGCGATGCCGAGCAAAGAATGCCATATGGCAAAAGGTATGACCTTCCGCCATATGCTGTACCCGGCTTACAAATCCAATCGTACTCCGACCCCTGACACTGTCGTTCAGGGCATGCAGTACCTGAAAGCGTCTATTAAGGCGATGAGCATTAAAGTTATCGAAGTCCCGGGTGTCGAGGCTGATGATGTTATCGGTACCCTGGCTATCAATAGCGTGTCCGCGGGCTATAAAGTGCGCATCGTTTCCCCGGATAAAGACTTCTTCCAGATTCTGTCCCCGTCTCTGCGTCTGCTGCGTATTGCTCCTCGTGGTTCCGGTATGGTTAGCTTCGGTGTAGAAGATTTCGTTAAACGTTATGGCCCGCTGAAGCCGTCTCAATTCGTCGATGTTGTGGCTCTGAGCGGCGACAAGGCGGATAACATCCCAGGCGTTGAAGGCATCGGCGACATTAACGCCGTGAAACTGATCTCTAAATTCGGTTCCCTGGATAATCTGCTGAAATCCGTCGACGAAGTAGAGGACGAGCGCATTAAACAGGCTCTGATCAGCCACTCTGAACAAGCAATTCTGTGCAAAAATCTGGCCACCCTGCGTTCCGATCTGCCGCATTACATGGTTCCGTTCAAAACTGCAGACCTGGTGTTCAAAAAACCGCAGGATGACGGCGAAAAGTTTATTAAGCTGCTGCGTGCGCTGGAAGCCTATGCCGAAGGCAGCTCCGTTAACCCGATCATTCGTCGTGCCGCCTACCTGTGGAATAAACTGAAATCCTGA(SEQID NO:2).

[0018] The third aspect of the present invention provides a DNA molecule comprising the polynucleotide as described above, for example, an expression cassette / expression frame of the polypeptide PEN1mut.

[0019] A fourth aspect of the present invention provides a recombinant plasmid comprising the DNA molecule described above, wherein the recombinant plasmid is an overexpression vector formed by cloning the DNA molecule described above into a plasmid vector suitable for expression in an industrial microorganism, wherein the industrial microorganism is selected from bacteria or yeasts commonly used for heterologous expression of target proteins, such as Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Vibrio natriuresis, Pichia pastoris, and baker's yeast;

[0020] In consideration of industrial production of PEN1mut, for example, production of PEN1mut by fermentation of engineered Escherichia coli, the plasmid vector used to construct the above-mentioned recombinant plasmid is preferably a pET vector series, such as a pET-28a-Sumo vector.

[0021] In one embodiment, the nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO: 3.

[0022] A fifth aspect of the present invention provides an engineered microorganism, characterized in that it is a transformant comprising the recombinant plasmid described above, and can be used to produce a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1 by fermentation.

[0023] A sixth aspect of the present invention provides a method for preparing the polypeptide PEN1mut described above, which comprises producing the polypeptide PEN1mut by fermentation of the engineered microorganism described above.

[0024] The seventh aspect of the present invention provides the use of the above-mentioned polypeptide such as PEN1mut, the above-mentioned polynucleotide, the above-mentioned DNA molecule or the above-mentioned recombinant plasmid as a tool enzyme.

[0025] In one embodiment, the above-mentioned use refers to the use of the tool enzyme as a molecular biology tool enzyme in gene editing technology, as an endonuclease and exonuclease in the preparation of 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.

[0026] As known to those skilled in the art, the application fields of the gene editing technology include genetic engineering modification of microorganisms, plants, animals and other living organisms.

[0027] This study reports the cytological phenotype of the 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), the prokaryotic protein expression and purification of the polypeptide encoded by the mutant, PEN1mut, and the mechanism of PEN1mut's enzymatic activity. As a highly efficient nuclease, PEN1mut has the potential to be a tool in molecular biology and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The phenotypes of wild-type B73 and pen1 mutant are shown. a: wild-type B73, pen1 / + self-pollinated ears, and pen1 F2 Phenotype of self-pollinated ears; b: wild type B73, pen1 / + self-pollinated ears segregated pen1 F2 and pen1 F2 Images of semi-thin section analysis of endosperm from inbred kernels.

[0029] Figure 2 Figure 1 shows the genetic location analysis of Pen1. (a) BSA sequencing of wild-type and mutant lines isolated from F2 lines of pen1 / + self-crosses revealed a distinct peak on chromosome 9. (b) The splice site at the junction of the 12th exon and intron of the candidate gene Pen1 was altered to GA, resulting in intron splicing failure.

[0030] Figure 3 Figure 1 shows the subcellular localization analysis of PEN1. Figures ab and b show the subcellular localization of PEN1-GFP. Red indicates chloroplast autofluorescence, and DAPI is used to label the nucleus and chloroplast DNA. The C-terminus of PEN1 was fused to GFP and expressed in tobacco leaf epidermal cells. Figure c shows immunofluorescence analysis of PEN1.

[0031] Figure 4 The figure shows the expression and purification of the PEN1mut prokaryotic protein. Molecular exclusion chromatography and SDS-PAGE analysis of PEN1mut are shown. M represents a protein marker.

[0032] Figure 5 Images of PEN1mut enzyme activity assays are shown. (a) PEN1mut activity against different DNA substrates; (b) PEN1mut activity against different RNA substrates. Red lines represent RNA bases, black lines represent DNA bases, and solid black circles represent streptavidin.

[0033] Figure 6The structural map of the recombinant plasmid SUMO-PEN1 overexpressing PEN1mut is shown. DETAILED DESCRIPTION

[0034] EMS (Ethylmethanesulfonate) is a commonly used chemical mutagen and a DNA ethylating agent. It can induce a high-density series of allelic point mutations. It has the advantages of high efficiency, low negative effects, and easy operation. It has been widely used in plant-related genetic research and mutation breeding.

[0035] In the study of genes related to maize endosperm development and biosynthesis, we used EMS chemical mutagenesis on the maize inbred line B73 and obtained a unique maize kernel mutant through extensive genetics. Its heterozygous self-pollinated ears separated and produced a small number of slightly powdery kernels at the endosperm top (pen1 F2 ), and pen1 F2 Self-pollinated offspring (pen1 F3 ) completely lacks endosperm. Compared with the wild type, pen1 F2 The number of starch bodies in pen1 was significantly reduced, while F3 Amyloplasts are virtually absent in the endosperm of B73. Genomic comparison revealed a mutation in the gene Zm00001d047988 (GenBank: AQL08261.1), encoding a 5'-3' exonuclease. We mapped this locus to a 1Mb interval on chromosome 9. Within this interval, only PEN1 harbors a single SNP at the intron splicing site, resulting in abnormal intron splicing. PEN1 is localized to chloroplasts, where it aggregates in tiny punctate patterns. To investigate PEN1, we attempted to prokaryotically express its CDS in Escherichia coli, but found that PEN1 was expressed only in inclusion bodies. We then deleted the N-terminal chloroplast localization signal (i.e., amino acids 1-92) to create a mutant, PEN1mut. We found that PEN1mut expression was still localized in inclusion bodies. We codon-modified the PEN1mut protein and inserted it into the pET-28a-Sumo vector. Prokaryotic expression and purification yielded a highly purified target protein, PEN1mut. We conducted a detailed study on the enzyme activity of PEN1mut and found that PEN1mut can 2+In the presence of 5'-3' exonucleases, PEN1mut efficiently cleaves substrates of diverse DNA and RNA structures. Therefore, PEN1mut is a highly efficient nuclease that can be expressed and purified in large quantities, and can also serve as a nucleic acid tool enzyme for molecular biology research. Furthermore, given the wide range of nuclease applications, PEN1mut has the potential to be applied in a wider range of fields, not just as a 5'-3' exonuclease. This includes applications as an endonuclease and exonuclease in the preparation of biochemical agents, as a DNA enzyme and RNA enzyme in the pharmaceutical and food industries, and in the preparation of small molecule nucleic acid drugs and nutritional additives.

[0036] Since the mutant PEN1mut retains the function of wild-type PEN1, for the sake of convenience, wild-type PEN1 and its mutant PEN1mut are sometimes collectively referred to as PEN1 in this article. Those skilled in the art can easily understand the meaning of each "PEN1" and their differences in different descriptions. For example, the accompanying drawings uniformly use the labeling method of PEN1 and gene pen1.

[0037] Nucleases are categorized as endonucleases and exonucleases. Exonucleases remove nucleotides from the free 5' or 3' ends of DNA. Exonucleases are found in eukaryotic cells, prokaryotes, and the venom of certain organisms. They are divided into three classes and act in the 3'-5' direction of DNA / RNA chains, removing single nucleotides to form sticky ends. 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 sole active component of the N-terminal fragment of DNA polymerase I. Its primary function is to remove the RNA primer from the 5' end of newly synthesized DNA, allowing polymerase activity to fill the resulting gap. DNA polymerase I is the only Escherichia coli DNA polymerase to possess both 3'-to-5' and 5'-to-3' exonuclease activities. Pol II and Pol III can both polymerize DNA and excise fragments in the 3'-to-5' direction, but they lack 5'-to-3' exonuclease activity. 3' to 5' activity can only remove a single nucleotide at a time, while 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 key roles in DNA and RNA repair, replication, and transcription.

[0038] In recent years, exonucleases, as important biological tools, have demonstrated tremendous potential in disease treatment and gene editing. Exonucleases can be used in gene editing to repair errors and damage in DNA and RNA chains. Based on the repair mechanism of exonucleases, researchers can design specific exonuclease analogs to repair genetic diseases caused by gene mutations. This gene editing technology has been widely used in the research and treatment of various genetic diseases, offering new hope for the treatment of related diseases. Furthermore, exonucleases can also be used as drug targets for diseases associated with DNA and RNA damage. Inhibitors targeting exonucleases have been developed for the treatment of various cancers and genetic diseases. These inhibitors can inhibit the growth and spread of tumor cells by interfering with the normal function of exonucleases, providing new insights into cancer treatment.

[0039] Those skilled in the art can expect that it is reasonable that some conservative variant polypeptides of the polypeptide PEN1 have the same nuclease function.

[0040] As used herein, the term "conservative variant polypeptide" refers to a polypeptide that substantially retains the same biological function or activity as the polypeptide. The "conservative variant polypeptide" may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group 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 prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or protein sequence used to purify the polypeptide. For example, (1) a polypeptide having the amino acid sequence shown in the polypeptide PEN1 (1) A polypeptide having the function of polypeptide PEN1 formed by substitution, deletion or addition of one or more (such as 1-20, preferably 1-10; more preferably 1-5; more preferably 1-3) amino acid residues; (2) A polypeptide having more than 50% (preferably more than 60%; more preferably more than 70%; more preferably more than 80%; more preferably more than 85%; more preferably more than 90%; more preferably more than 95%; more preferably more than 98%; more preferably more than 99%) identity with the sequence shown in polypeptide PEN1; or (3) A polypeptide formed by adding a tag sequence to the N or C terminus of the polypeptide PEN1, or adding a signal peptide sequence to its N terminus. According to the teachings of this article These fragments, derivatives and analogs are well known to those skilled in the art. The term "variation" or "mutation" includes but is not limited to replacement, deletion, insertion, chemical modification of amino acid residues, preferably forward mutations, i.e. mutations that improve 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 with similar side chains, and therefore generally involve replacing an amino acid in a polypeptide with an amino acid in the same or similar amino acid defined class. However, as used herein, if a conservative mutation may instead be aliphatic to aliphatic, non-polar to non-polar, polar to polar, acidic to acidic, basic to basic, Conservative mutations do not include substitutions from hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue. Common examples of conservative substitutions are known in the art and include: substitutions between aromatic amino acids F, W, and Y; substitutions between hydrophobic amino acids L, I, and V; substitutions between polar amino acids Q and N; substitutions between basic amino acids K, R, and H; substitutions between acidic amino acids D and E; and substitutions between hydroxyl amino acids S and T. Furthermore, A, V, L, or I can be conservatively mutated to another aliphatic residue or another non-polar residue. Exemplary conservative substitutions can be made, for example, according to the following table.

[0041]

[0042]

[0043] It is well known that the expression results of the same nucleotide sequence in different microbial hosts often vary greatly. To optimally express PEN1 in Escherichia coli, the most commonly used bacterium in genetic engineering, we codon-optimized the PEN1 expression gene.

[0044] Codon optimization is a kind of technology that can be used for making protein expression in organism maximized by increasing the translation efficiency of gene of interest.Different organisms usually illustrate the special preference of one of some codons for encoding identical amino acids due to mutation tendency and natural selection.For example, in fast-growing microorganisms such as Escherichia coli, the composition of its respective genome tRNA library is reflected by optimizing codon.Therefore, in fast-growing microorganisms, the low frequency codon of amino acid can be used for identical amino acid but the codon replacement of high frequency.Therefore, the expression of optimized dna sequence dna is improved in fast-growing microorganisms.

[0045] In order to express the nuclease SEQ ID NO: 1 in Escherichia coli, the present invention performs codon optimization on the encoding gene thereof. After optimization, the encoding gene of the nuclease SEQ ID NO: 1 is SEQ ID NO: 2.

[0046] Furthermore, in order to achieve large-scale production of the polypeptide PEN1, PEN1 can be produced by fermentation of industrial microorganisms such as Escherichia coli, and its encoding gene, such as the polynucleotide SEQ ID NO: 2, is used as an exogenous gene to construct a gene expression cassette or expression construct as a DNA molecule. The expression cassette / expression construct is operably linked to a plasmid vector by subcloning to obtain a recombinant plasmid, and the recombinant plasmid is then transformed into a host cell to obtain a transformant, i.e., a genetically engineered bacterium or a recombinant bacterium.

[0047] The terms "recombinant bacteria (strain)" and "(genetically) engineered bacteria (strain)" herein have the same meaning, and both refer to strains containing a PEN1 gene overexpression vector.

[0048] In the description of the technical solutions of the present invention, the term "and / or" as used in phrases 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). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0049] For simplicity, the term "PEN1" may sometimes be used interchangeably with the name of its encoding gene (DNA), such as the polypeptide PEN1. Those skilled in the art will understand that these refer to different types of substances in different contexts. Their meanings are readily understood by those skilled in the art based on the context. For example, when describing the protein function or class of 5'-3' exonuclease, "PEN1" refers to the protein; when describing a gene, "PEN1" refers to the gene encoding the protein.

[0050] As used herein, the "expression cassette" or "gene expression cassette" refers to a gene expression system that contains all the necessary elements required to express the target protein PEN1, which generally includes the following elements: a promoter, a gene sequence encoding a polypeptide, and a terminator; in addition, it may optionally include a signal peptide coding sequence, etc.; these elements are operably connected.

[0051] As used herein, the term "expression construct" or "expression construct" refers to a recombinant DNA molecule comprising a 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).

[0052] As used herein, the terms "exogenous" or "heterologous" refer to the relationship between two or more nucleic acid or protein sequences from different sources, or the relationship between a protein (or nucleic acid) and a host cell from different sources. For example, a nucleic acid is exogenous to a host cell if the combination of nucleic acid and host cell does not normally occur in nature. A particular sequence is "exogenous" to the cell or organism into which it is inserted.

[0053] As used herein, the terms "operably linked" or "operably connected" refer to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, a promoter region is placed at a specific position relative to a target gene, such as the nucleic acid sequence of SEQ ID NO: 2, such that transcription of the nucleic acid sequence is directed by the promoter region. Thus, the promoter region is "operably linked" to the nucleic acid sequence.

[0054] The nucleic acid constructs of the present invention can be manipulated in a variety of ways to ensure expression of the PEN1 polypeptide. The nucleic acid constructs can be manipulated prior to insertion into a vector, depending on the specific expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are well known in the art.

[0055] In certain embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a knock-in vector. The nucleic acid sequence SEQ ID NO:2 of the present invention can be cloned into many types of vectors, for example, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Cloning vectors can be used to provide the coding sequence of the protein or polypeptide of the present invention. The expression vector can 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 SEQ ID NO:2 of the present invention to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration into eukaryotic cells. Typical expression vectors contain expression control sequences that can be used to regulate the expression of the desired nucleic acid sequence.

[0056] Methods well known to those skilled in the art can be used to construct nucleic acid constructs. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operatively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of such promoters include the lac or trp promoters of Escherichia coli; the lambda phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoter, the LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, or, for E. coli and Agrobacterium, tetracycline resistance, ampicillin resistance, or chloramphenicol resistance, etc.

[0057] The present invention will be further described in detail below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0058] Example

[0059] The examples involve the addition amounts, contents and concentrations of various substances, wherein the percentages mentioned are by mass unless otherwise specified.

[0060] In the examples herein, if no specific description is given for the reaction temperature or the operating temperature, the temperature generally refers to room temperature (15-30° C.).

[0061] The molecular biology experiments in the embodiment include plasmid construction, enzyme digestion, competent cell preparation, transformation, etc., mainly with reference to " Molecular Cloning Laboratory Manual " (3rd edition), J. Sambrook, DW Russell (U.S.) wrote, Huang Peitang et al. translated, Science Press, Beijing, 2002) carried out. For example, competent cell transformation method and competent preparation method are all carried out with reference to " Molecular Cloning Laboratory Manual " (3rd edition) Chapter 1, page 96. Specific experimental conditions can be determined by simple experiments if necessary.

[0062] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. These conditions can be adjusted through simple experiments if necessary.

[0063] EMS mutagenesis, self-pollination, and hybridization of corn were carried out according to conventional breeding methods.

[0064] BSA sequencing analysis was performed by Shanghai Ouyi Biotechnology Co., Ltd.

[0065] The primer synthesis and gene sequencing in the examples were all completed by Shanghai Boshang Biotechnology Co., Ltd.

[0066] Example 1: Discovery of genes related to maize endosperm formation and development by EMS mutagenesis and phenotypic analysis

[0067] We used EMS mutagenesis to identify genes that affect maize endosperm development and biosynthesis.

[0068] The maize inbred line B73 was subjected to EMS mutagenesis and an endosperm filling mutant was isolated. The EMS-mutated contemporary M0 seeds were self-pollinated and harvested from single ears for phenotypic observation. Figure 1 As shown, this mutant (i.e., pen1) is a unique floury endosperm mutant. Compared with wild-type B73, fewer mutants with slightly floury endosperm tips were isolated from Pen1 heterozygous self-pollinated ears (pen1 F2 ), the offspring of the mutant continued to self-pollinate (i.e., pen1 F3 ), characterized by complete absence of endosperm ( Figure 1 We also performed cytological analysis using semi-thin sections. Compared with the wild type, the number of starch granules in the mutant was significantly reduced, and almost no starch granules were observed in the endosperm ( Figure 1 (b)

[0069] Shanghai Ouyi Biotechnology Co., Ltd. was commissioned to conduct whole-genome sequencing analysis of the mutant and screen for mutant genes.

[0070] Example 2: Genetic location analysis of mutant genes

[0071] 100 wild-type and mutant strains were isolated from the mutant (i.e., pen1) and the F2 population of B73, and mixed into wild-type and mutant pools, respectively, for mixed pool sequencing. Sequencing data were analyzed using the MutMap method, see Figure 2 , the results showed that only a significant peak appeared on chromosome 9 ( Figure 2 (a) Genomic comparison revealed that the candidate gene is Zm00001d047988 (GenBank: AQL08261.1, nucleotide sequence shown in SEQ ID NO: 4), encoding a plant-specific 5'-3' exonuclease containing 422 amino acid residues. The mutant exhibits a gene mutation at the splice site at the junction of exon 12 and intron (position 7399 in SEQ ID NO: 4) to GA, resulting in intron failure.

[0072] Example 3: Subcellular localization analysis of mutant genes

[0073] 1300-35S-AtRNH1C-TDT and 1300-35S-Pen1-GFP were constructed and colocalized in tobacco epidermal cells. Colocalization signals were analyzed using Fiji. PEN1-Flag transgenic plants were constructed, and their chloroplasts were isolated and analyzed by immunofluorescence. Different chloroplast components of PEN1-Flag transgenic plants were isolated and analyzed by immunoblotting.

[0074] See also Figure 3 Subcellular localization analysis confirmed that the mutant gene was localized in the plastid, so the 5'-3' exonuclease encoded by the gene was named PEN1 (plastid-localized 5'-3'exonuclease, PEN1), and the mutant gene was correspondingly called Pen1.

[0075] Figure 3 Figures ab show subcellular localization images of PEN1-GFP. Red indicates chloroplast autofluorescence, and DAPI is used to label nuclei and chloroplast DNA. The C-terminus of PEN1 was fused to GFP and expressed in tobacco leaf epidermal cells. Figure 3 C shows the immunofluorescence analysis image of PEN1.

[0076] Example 4: Construction of PEN1 Escherichia coli engineered bacteria

[0077] To investigate the properties of PEN1, we attempted to express its CDS gene in E. coli. However, we found that the expressed PEN1 was only localized in inclusion bodies, making it difficult to isolate and purify. We then removed the chloroplast localization signal at its N-terminus (i.e., amino acids 1-92), creating a mutant, PEN1mut, with the amino acid sequence shown in SEQ ID NO:1. We found that PEN1mut expression was still localized in inclusion bodies.

[0078] To achieve in vitro secretion of PEN1mut, we optimized the amino acid sequence of PEN1mut, SEQ ID NO: 1, based on the codon preference of E. coli, and obtained its coding gene sequence, SEQ ID NO: 2. We commissioned Shanghai Boshang Biotechnology Co., Ltd. to perform full gene synthesis and cloned it into the plasmid pET-28a-Sumo (this plasmid was designed by our laboratory using Pet-28a as the backbone) to obtain the recombinant plasmid SUMO-PEN1. The plasmid structure map is shown in the figure. Figure 6 The nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO: 3.

[0079] The correctly sequenced recombinant plasmid SUMO-PEN1 was electroporated into the competent host Escherichia coli Rosetta (DE3), and positive clones were screened to construct recombinant Escherichia coli expressing PEN1mut.

[0080] Example 5: Expression and purification of PEN1mut prokaryotic protein

[0081] A single colony of the recombinant E. coli PEN1mut constructed in Example 4 was selected and inoculated into 5 mL of liquid LB medium containing 50 μg / mL kanamycin sulfate and 25 μg / mL chloramphenicol. The cells were cultured overnight at 37°C and 250 rpm. The next day, the inoculum was transferred to a shake flask containing 200 mL of liquid TB medium at a volume concentration of 1% v / v. The cells were cultured at 37°C and 250 rpm for 2-3 hours. When the OD600 reached 0.6-0.8, 0.1 mM IPTG was added for induction for 16 hours. The cells were collected by centrifugation at 6,000 rpm for 5 minutes and evenly suspended in buffer A (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5 mM β-mercaptoethanol, 20 mM imidazole, 1 mM PMSF, 5% v / v glycerol) and lysed using an Avestin emulsion flex-c5 at 4°C. The lysate was centrifuged at 75,000 g for 60 min at 4°C, and the supernatant was affinity purified using Ni-NTA agarose. Contaminants were eluted with 20 CV (column volumes) of buffer A and eluted with SUMOstar protease in 5 CV of buffer A at 4°C. Eluted PEN1mut was dialyzed overnight against buffer B (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM β-mercaptoethanol, 1 mM PMSF, 1% v / v glycerol). PEN1 was further purified using a Q HP column (HiTrap Q HP 5-mL, Cytiva) using Q HP buffer A (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% v / v glycerol, 1 mM DTT) and Q HP buffer B (20 mM Tris-HCl pH 7.5, 1000 mM NaCl, 1% v / v glycerol, 1 mM DTT). Fractions containing PEN1mut were collected and further purified using HiLoad 16 / 600 Superdex 75 pg (Cytiva). Fractions containing PEN1mut were concentrated to 5.0 mg / mL and stored at -80°C until use.

[0082] Figure 4 Shown are images of molecular exclusion chromatography and SDS-PAGE analysis of the PEN1mut protein.

[0083] Example 6: PEN1mut enzyme activity test analysis

[0084] PEN1mut nuclease activity was tested using substrate modifications including / 6FAMdT / , '5-P, and Biotin-TEG, and purified using 2×HPLC. The substrates were diluted to 100 μM in RNase-free annealing buffer (50 mM Tris-HCl (pH 7.4), 200 mM NaCl). DNA and RNA substrates were annealed using a PCR instrument (annealing protocol: 95°C for 5 minutes, followed by a 0.1°C decrease every 8 seconds to 25°C (700 cycles), and then stored at 12°C). Double-stranded substrates required blocking the free 5' end with streptavidin to prevent PEN1 from cleaving at non-target sites.

[0085] The PEN1mut digestion reaction system consisted of 20 mM Tris-HCl (pH 7.4), 10 mM MnCl2, and 1 mM DTT. The substrate concentration was 25 nM, and the PEN1 concentration gradient was 0, 6.25, 12.5, 25, and 50 pM. The prepared reaction system was incubated at 37°C for 15 minutes. Two volumes of reaction termination buffer (90% formamide, 5 mM EDTA) were added, and the reaction was incubated at 98°C for 10 minutes, followed by rapid cooling on ice. Electrophoresis was performed on a high-resolution 15% / 8 M urea denaturing PAGE sequencing gel (50W) and detected using a Cytiva Typhoon 5.

[0086] See also Figure 5 The results of the nuclease test showed that PEN1mut can efficiently cut DNA and RNA, indicating that it has the enzymatic activity of DNA enzyme (DNase) and RNA enzyme (RNase), and is not limited to 5'-3' nuclease activity, and can be widely used as a tool enzyme.

[0087] The above embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the protection scope of the present invention.

[0088] References

[0089] [1] Yang, T., Wu, XG, Wang, WQ, and Wu, YR (2023). Regulation of seedstorage protein synthesis in monocot and dicot plants: Acomparative review. MolPlant 16, 145-167.

[0090] [2]Choi,H.,Yi,T.,and Ha,S.H.(2021).Diversity of Plastid Types andTheir Interconversions.Front Plant Sci 12,692024.

[0091] [3]Zimorski,V.,Ku,C.,Martin,W.F.,and Gould,S.B.(2014).Endosymbiotictheory for organelle origins.Curr Opin Microbiol 22,38-48.

[0092] [4]Pfalz,J.,and Pfannschmidt,T.(2013).Essential nucleoid proteins inearly chloroplast development.Trends Plant Sci 18,186-194。

Claims

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 95% or more homology with the polypeptide sequence defined in (a) and has the functions of the polypeptide of (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 Comprising the polynucleotide according to claim 2.

4. A recombinant plasmid, characterized in that The recombinant plasmid 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. A microbial engineering bacterium, characterized in that: The transformant comprises the recombinant plasmid as claimed in claim 4 and is used for producing a polypeptide having 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 as claimed in 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 a nuclease and exonuclease in the preparation of 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.

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