CN108676075B - Method for guiding protein molecules into plant cell nucleus - Google Patents
Method for guiding protein molecules into plant cell nucleus Download PDFInfo
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- CN108676075B CN108676075B CN201810554472.XA CN201810554472A CN108676075B CN 108676075 B CN108676075 B CN 108676075B CN 201810554472 A CN201810554472 A CN 201810554472A CN 108676075 B CN108676075 B CN 108676075B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8221—Transit peptides
Abstract
A method for introducing a protein molecule into the nucleus of a plant cell, in order to find a specific amino acid sequence which can be recognized and bound by a transporter complex, thereby transporting the protein molecule carrying the sequence into the nucleus; the invention adopts a section of specific amino acid sequence to guide protein molecules to enter plant cell nucleuses, thereby enabling the protein molecules to exert the corresponding functions of the protein molecules in the cell nucleuses; the one letter code of the amino acid sequence is: qanrkvslqryrekrdr; the three-letter code is: GlnALAAsnAAArgLysValSerLeuGlnArgTyrArgGluLysArgLysAspArg; due to the degeneracy of triplet codons, the above amino acid sequences can correspond to an infinite number of nucleotide sequences, one of which is shown below, for example: CAGGCGAACAGAAAGGTATCTTTG CAAAGATATCGTGAAAAGCGGAAAGACAGA, respectively; all nucleic acid sequences corresponding to this amino acid sequence have the same function.
Description
Technical Field
The invention relates to the technical field of plant molecular biology and plant genetic engineering, in particular to a method for guiding protein molecules to enter plant cell nucleuses.
Background
A cell contains a variety of organelles and a variety of protein molecules, and the specific molecule is required to reach a specific location in the cell, particularly into a specific organelle, to achieve its function. For example, transcription factors need to enter the nucleus to bind to transcriptional regulatory regions of a particular gene in the genome to initiate transcriptional expression of the gene. In biological research, a specific protein molecule is sometimes required to be sent into a cell nucleus to research the function of the protein molecule, or in the application of genetic engineering technology, the specific protein molecule must enter the cell nucleus to realize the function of the protein molecule. To achieve this, one approach is to have the protein molecule carry a signal or sequence that leads into the nucleus, as long as this sequence is present, the nuclear transport mechanism within the cell is recognized and the protein molecule is transported from the cytoplasm to the nucleus by the transporter complex. This sequence may be at the N-or C-terminus or in the middle of the protein molecule. A plurality of protein sequences for introducing into the nucleus exist in the cell, and the invention determines a novel sequence which can lead protein molecules into the plant nucleus.
Disclosure of Invention
The present invention aims to overcome the above-mentioned drawbacks of the prior art and to provide a method for introducing protein molecules into the nucleus of plant cells, which method seeks a specific amino acid sequence which is recognized and bound by a transporter complex, thereby transporting the protein molecules carrying this sequence into the nucleus.
The invention adopts a section of specific amino acid sequence to guide protein molecules to enter plant cell nucleuses, thereby enabling the protein molecules to exert the corresponding functions of the protein molecules in the cell nucleuses; such a signal or sequence is determined and can be placed in a fusion fashion at the N-or C-terminus or in the middle of the protein molecule. The one letter code of the amino acid sequence is: qanrkvslqryrekrdrr, whose three-letter code is: Gln-Ala-Asn-Arg-Lys-Val-Ser-Leu-Gln-Arg-Tyr-Arg-Glu-Lys-Arg-Lys-Asp-Arg. Consisting of 18 amino acids. The corresponding nucleotide consists of 54 and is easy to connect with target gene sequence to prepare fusion protein with nuclear entry signal. The target gene may be a binding protein encoding DNA or an endonuclease encoding DNA, etc. Due to the degeneracy of triplet codons, the above amino acid sequences can correspond to an infinite number of nucleotide sequences, one of which is shown below, for example: CAGGCGAACAGAAAGGTATCTTTGCAAAGATATCGTGAAAAGCGGAAAGACAGA, respectively; all nucleic acid sequences corresponding to this amino acid sequence have the same function.
The sequence has the function of introducing the whole molecule into the nucleus of a plant cell at the N-terminal or C-terminal of the protein molecule sequence or in the middle of the sequence. The protein molecule carrying the specific sequence enters the nucleus of the plant cell, so that the protein molecule can exert the corresponding function of the plant cell nucleus, and mainly comprises the steps of combining with the genome DNA to activate or inhibit the expression of genes, or specifically shearing the genome DNA to complete genome editing and the like. Combining the sequence with a nucleic acid sequence coding green fluorescent protein, cloning the sequence into the middle of a CaMV35S promoter and an NOS terminator to form a green fluorescent protein expression unit carrying a nuclear signal, and after the gene is expressed in a plant cell, the green fluorescent protein accumulates in the cell nucleus due to the existence of the nuclear signal, as shown in figures 1, 2 and 3, while the green fluorescent protein molecules which are not carried into the nuclear signal do not accumulate in the plant cell nucleus, as shown in figures 4, 5 and 6.
The present invention is mainly suitable for plant gene engineering, and features that a new sequence for leading protein molecule to enter plant cell nucleus is invented. The sequence is capable of being recognized and bound by a transporter complex, thereby transporting the protein molecule carrying the sequence into the nucleus.
Drawings
FIG. 1 is a diagram showing the position of the nucleus after staining with the nucleic acid dye DAPI;
FIG. 2 is a graph showing the accumulation of green fluorescent protein molecules in the nucleus of cells transformed in the same field;
FIG. 3 is a diagram showing epidermal cells of onion in white light in the same field of view;
FIG. 4 is a diagram showing the position of the nucleus after staining with the nucleic acid dye DAPI;
FIG. 5 is a graph showing the distribution of green fluorescent protein molecules in cells transformed in the same field;
FIG. 6 is a diagram showing epidermal cells of onion in white light in the same field of view.
Detailed Description
FIGS. 1, 2 and 3 show that the fusion of the nuclear import guide sequence and the nucleic acid sequence encoding green fluorescent protein is expressed under the drive of CaMV35S promoter, and the fusion protein is aggregated in the cell nucleus. The plasmid containing the expression unit was transferred into epidermal cells of onion using a gene gun, and 5 days later, it was observed under a microscope at 200X and recorded photographically.
As a control, FIGS. 4, 5 and 6 show that, in the present invention, no green fluorescent protein carried into the nuclear sequence is expressed, and the expressed green fluorescent protein molecule is not aggregated in the nucleus under the drive of CaMV35S promoter. The plasmid containing the expression unit was transferred into epidermal cells of onion using a gene gun, and 5 days later, it was observed under a microscope at 200X and recorded photographically.
The fusion mode of the nucleic acid sequence of the nuclear signal in the invention and the nucleic acid sequence of any protein molecule needing to be introduced into the nucleus can be completed by methods such as gene synthesis, connection of different DNA fragments by using DNA ligase or overlap-PCR amplification, and the fusion part can be at the N end or C end of the protein molecule or any part in the middle. When the sequence is placed between protein molecules, care is taken to maintain the functional integrity of the protein molecules so that the function is not affected or destroyed by changes in the protein structure. When expressed as a protein in plant cells, the fused molecule will be transported into the nucleus, due to the nuclear-entry signal or sequence carried. For example, in genome editing techniques, a specific endonuclease called molecular scissors must enter the nucleus of a cell to cut genomic DNA, a sequence encoding the specific endonuclease is fused to a sequence of the present invention, and the fused nucleic acid sequence is constructed in an expression unit, i.e., between a suitable promoter and terminator. When the expression unit sequence is introduced into the nucleus of a plant recipient or into the genome, the fused gene is transcribed and then translated into a protein in the ribosome. Since the protein molecule carries a nuclear entry leader sequence or a nuclear entry signal sequence, the molecule enters the nucleus to perform its intended function.
Claims (2)
1. A method for leading protein molecules to enter plant cell nucleuses is characterized in that a specific amino acid sequence is adopted to lead the protein molecules to enter the plant cell nucleuses, so that the protein molecules can exert the corresponding functions of the protein molecules in the cell nucleuses; the plant cell nucleus is an onion cell nucleus; the protein molecule is green fluorescent protein;
the one letter code of the amino acid sequence is: qanrkvslqryrekrdrr, whose three-letter code is: Gln-Ala-Asn-Arg-Lys-Val-Ser-Leu-Gln-Arg-Tyr-Arg-Glu-Lys-Arg-Lys-Asp-Arg.
2. The method of claim 1, wherein the sequence has the function of introducing the whole molecule into the nucleus of a plant cell at both the N-terminus and the C-terminus of the protein molecule sequence.
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|---|---|---|---|---|
| US20030084482A1 (en) * | 2001-06-08 | 2003-05-01 | Gerald Hall | Production of proteins in plants |
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