WO2022174563A1 - 水稻根分泌多肽pep1及其编码基因和应用 - Google Patents

水稻根分泌多肽pep1及其编码基因和应用 Download PDF

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WO2022174563A1
WO2022174563A1 PCT/CN2021/112192 CN2021112192W WO2022174563A1 WO 2022174563 A1 WO2022174563 A1 WO 2022174563A1 CN 2021112192 W CN2021112192 W CN 2021112192W WO 2022174563 A1 WO2022174563 A1 WO 2022174563A1
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gene
plant
seq
development
polypeptide
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PCT/CN2021/112192
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French (fr)
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毛传澡
蒙福宁
向丹
王奥迪
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浙江大学
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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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A01H6/4636—Oryza sp. [rice]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/40—Liliopsida [monocotyledons]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P21/00—Plant growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates to the field of biotechnology, in particular to the application of a plant root secreted polypeptide and its encoding gene in regulating the growth and development of plant roots.
  • Roots are important underground organs of plants, which can not only fix plants, but also absorb nutrients, water and other substances from the soil for the growth and development of plants.
  • the root system of plants can also monitor various signal molecules in the soil environment, such as soil moisture content, nutrient levels, and some plant hormones and signals, so as to improve the adaptability of plants to their growing environment (Meng et al. .2019).
  • Plant roots are mainly divided into two categories, tap root system and fibrous root system.
  • Rice is a monocotyledonous model plant, and its root system is a fibrous root system, which is mainly composed of seed roots and adventitious roots, as well as lateral roots and root hairs on seed roots and adventitious roots (Rebouillat et al.
  • the size of plant root meristem is also regulated by the CLE (CLAVATA3(CLV3)/EMBRYO SURFROUNDING REGION(ESR)-related) family of polypeptides.
  • CLE CLAVATA3(CLV3)/EMBRYO SURFROUNDING REGION(ESR)-related
  • Type A CLE polypeptides play a role in root meristem development, while type B CLE polypeptides are involved in root vascular bundle development (Cock and McCormick 2001; Whitford et al. 2008).
  • the type A CLE polypeptide CLE40 is required for columnar cell differentiation, and the loss-of-function mutant cle40 exhibits an irregularly shaped root tip due to the delayed differentiation of columnar cells, thus showing a short root phenotype (Stahl et al. 2009 ).
  • Other CLE polypeptides (CLE26, CLE40, CLE45) control primary root growth by regulating primary epithelium formation during vascular bundle development (Depuydt et al. 2013; Rodriguez-Villalon et al. 2014; Rodriguez-Villalon et al. 2015; Hazak et al. 2017).
  • the tyrosine sulfated peptides PSK and PSY1 can promote main root growth by regulating cell expansion in the elongation/meristem (Amano et al. 2007; Kwezi et al. 2011; Hartmannet et al. 2014; Ladwiget et al.2015).
  • Kjeldahl Integrity Factor 1 (CIF1) polypeptide is essential for Kjeldahl band formation, as CIF1 loss-of-function mutants exhibit severe growth retardation at high iron concentrations, but the CIF1 polypeptide is implicated by the use of exogenously applied CIF1 After processing, these defects can be fully recovered (Nakayama et al. 2017).
  • Benfey PN (2012) Toward a Systems Analysis of the Root. Cold Spring Harbor Symposia on Quantitative Biology 77, 91 (Benfey PN (2012) A Guide to the Analysis of Plant Root Systems. Cold Spring Harbor Symposium on Quantitative Biology 77, 91).
  • the technical problem to be solved by the present invention is to provide a polypeptide related to regulating plant root development and its encoding gene and application.
  • the present invention provides a use of a polypeptide for regulating plant root development: for treating plants, the root length of the plants after the treatment becomes shorter or longer;
  • polypeptide is any of the following:
  • amino acid sequence is shown in SEQ ID NO: 1 (Sequence Listing NO: 1);
  • the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is a polypeptide derived from the polypeptide that has undergone substitution and/or deletion and/or addition of one or several amino acid residues and is related to the development of plant roots.
  • amino acid sequence of the precursor protein of the polypeptide is shown in SEQ ID NO: 2;
  • the protein shown in SEQ ID NO: 2 is a protein derived from the protein shown in SEQ ID NO: 2 with substitution and/or deletion and/or addition of one or several amino acid residues and is related to plant development;
  • the present invention provides another application of regulating plant root development gene: for constructing transgenic plants, the root length of the transgenic plants is shortened or lengthened;
  • the gene is any of the following:
  • the nucleotide sequence of the gene coding region is shown in SEQ ID NO: 3;
  • the gene shown in SEQ ID NO: 3 has undergone one or several nucleotide substitutions and/or deletions and/or additions and is a gene derived therefrom that is relevant to plant development.
  • the plant is rice.
  • the recombinant expression vector I-OsPEP1 overexpression vector obtained by inserting the DNA molecule shown in SEQ ID NO: 3 into the multiple cloning site of plasmid pCAMBIA1300.
  • the gene DNA fragment shown in SEQ ID NO: 3 is connected to the transition vector pBSSK-in in the forward and reverse directions, and then inserted into the plasmid pCAMBIA1300 to obtain the recombinant expression vector II---OsPEP1 inhibition expression vector.
  • the polypeptide (PEP1) provided by the present invention, its precursor protein, and the encoding gene are from the japonica rice Xiushui 134 (Oryza sativa L.ssp. Japonica cv. Xiushui134), which is the following (a) or (b) polypeptide, and its precursor protein , or encoding gene, is the following (a), (b), (c), (d), (e) or (f) polypeptide, protein, or encoding gene:
  • the plant development is reflected in the plant root length trait.
  • tags including but not limited to those shown in Table 1 can be attached to the amino-terminus or carboxyl-terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1.
  • Poly-His 2-10 (usually 6) HHHHHH (SEQ ID NO: 5) FLAG 8 DYKDDDDK (SEQ ID NO: 6) Strep-tagII 8 WSHPQFEK (SEQ ID NO: 7) c-myc 10 EQKLISEEDL (SEQ ID NO: 8)
  • the PEP1 in the above (a) can be obtained by artificial synthesis, or by first synthesizing its encoding gene and then biologically expressing it.
  • the encoding gene of PEP1 in the above (a) can be obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 3, and/or carrying out one or several base pairs of missense. mutated, and/or ligated to the coding sequence of the tags shown in Table 1 at its 5' and/or 3' ends.
  • the gene encoding the protein also belongs to the protection scope of the present invention.
  • the gene encoding the protein (OsPEP1) can be a DNA molecule of the following 1) or 2) or 3):
  • the recombinant expression vector, expression cassette, transgenic cell line or recombinant bacteria containing the gene or its antisense gene all belong to the protection scope of the present invention.
  • the recombinant expression vector containing the OsPEP1 gene can be constructed by using the existing plant expression vector.
  • the plant expression vectors include, but are not limited to, such as binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, and the like.
  • the plant expression vector may also contain the 3' untranslated region of the exogenous gene, ie, containing the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression.
  • the poly(A) signal can guide the addition of poly(A) to the 3' end of the mRNA precursor, such as, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmid genes (such as nopaline synthase Nos gene), plant genes (eg soybean storage protein gene) 3'-terminal transcribed untranslated regions have similar functions.
  • any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, including but not limited to cauliflower mosaic virus (CAMV) 35S promoter, Maize ubiquitin promoters, which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct plant expression vectors, enhancers, including translation enhancers or transcription enhancers, can also be used , These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence.
  • the sources of the translational control signals and initiation codons are extensive, either natural or synthetic. Translation initiation regions can be from transcription initiation regions or structural genes.
  • the plant expression vector used can be processed, such as adding, including but not limited to, genes that can be expressed in plants and encode enzymes or light-emitting compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical reagent-resistant marker genes (such as herbicide-resistant genes) and the like.
  • GUS gene luciferase gene
  • antibiotic markers with resistance gentamicin marker, kanamycin marker, etc.
  • chemical reagent-resistant marker genes such as herbicide-resistant genes
  • the recombinant expression vector can specifically be the following (I) or (II):
  • the plasmids pCAMBIA1300 and pBSSK-in in (I) or (II) are both engineered (published).
  • the present invention also protects a method for cultivating transgenic plants, which is to introduce the gene or gene fragment into the target plant by forward and reverse connection to the transition vector pBSSK-in, and the transgenic plant is the root length of the target plant compared with that of the target plant. Altered transgenic plants.
  • the OsPEP1 gene or gene fragment provided by the present invention is connected to the transition vector pBSSK-in in forward and reverse directions and then introduced into plant cells, so that the root length can be changed.
  • the expression vector carrying the OsPEP1 gene or its partial gene forward and reverse ligation can be used to transform plant cells by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electrical conductivity, and Agrobacterium-mediated Or tissue, and the transformed plant tissue is grown into a plant.
  • the transformed plant host can be a grass plant, such as rice (eg, Xiushui 134).
  • the method When cultivating a transgenic plant whose root length is longer than that of the target plant; the method is to introduce (1) the recombinant expression vector into the target plant to obtain a transgenic plant with shortened roots.
  • the method When cultivating a transgenic plant whose root length is shorter than that of the target plant; the method is to introduce (II) the recombinant expression vector into the target plant to obtain a transgenic plant with shortened roots.
  • the present invention discovers a new rice root secreted polypeptide PEP1 and its encoding gene OsPEP1, and obtains a recombinant expression vector containing the encoding gene or a partial fragment of the gene that is connected to the transition vector pBSSK-in in forward and reverse directions.
  • the recombinant vector transforms the target plant, and the transgenic plant with altered root length can be obtained. Therefore, OsPEP1 can be used as a potential molecular breeding tool to improve plant yield by improving root length of plants.
  • the present invention isolates and identifies a polypeptide PEP1 that controls rice root growth, and determines its encoding gene OsPEP1 (LOC_Os11g09560) and precursor protein OsPEP1. That is, the present invention isolates and identifies a polypeptide PEP1 that regulates plant root development and its encoding gene OsPEP1. Exogenous application of PEP1 to treat plants can inhibit the root development of plants. And a recombinant expression vector containing the coding gene or part of the DNA of the gene is obtained, and a plant transformed with the recombinant expression vector can obtain a transgenic plant with altered root growth and development. Therefore, the polypeptide can be used as a plant growth regulator, and its encoding gene and precursor protein can be used as a potential molecular breeding target for crop improvement, and can improve crop yield by regulating the growth and development of crop roots.
  • OsPEP1 LOC_Os11g09560
  • precursor protein OsPEP1 precursor protein
  • Fig. 1 is that gene OsPEP1 (LOC_Os11g09560) encodes a protein containing five amino acids (SDFDR, SEQ ID NO: 1) in Example 1;
  • A is the amino acid sequence of the protein encoded by the gene OsPEP1 (MGEKERRLRVEGWMGRTEMIDRRRQRLHSGERERRLCVRKRMGS S DFDR GARFGGVDDGRLGEGTKRCEEMVGAIWDVGFERDNPDRSTRNEDVNISW, SEQ ID NO: 2), and the polypeptide PEP1 with 5 amino acid residues is underlined;
  • B is the LC-MS analysis of polypeptide substances secreted by the roots of wild-type rice plants
  • C is Ser-Asp-Phe-Asp-Arg (PEP1) (SEQ ID NO: 1), a wild-type rice plant root secreted polypeptide substance identified by LC-MS/MS with a retention time of 12.25 minutes;
  • D is LC-MS analysis of LOC_Os11g09560 overexpressing transgenic rice plant root secreted polypeptide substances
  • E is Ser-Asp-Phe-Asp-Arg (PEP1) (SEQ ID NO: 1), a transgenic rice plant overexpressing LOC_Os11g09560 with a retention time of 12.26 minutes, identified by LC-MS/MS, which is a polypeptide substance secreted by the root system;
  • Fig. 2 is the functional analysis of root secreted polypeptide PEP1 in Example 1;
  • A is the phenotype of wild-type plants treated with different concentrations of PEP1 for 7 days, and the scale bar is 5 cm;
  • B is the main root length statistics of wild-type plants treated with different concentrations of PEP1 for 7 days; the error is represented by ⁇ SD (the number of measured samples ⁇ 15); different letters represent significant differences at the 0.01 level (one-way ANOVA, LSD method);
  • C is the root apical elongation zone (upper left) and meristem (lower left) of wild-type plants not treated with PEP1 (-PEP1) or treated with PEP1 at a concentration of 10 -6 M (+PEP1) for 2 days after 2 days Longitudinal section phenotype of zone (top right) and meristem (bottom right), scale bar is 100 ⁇ m;
  • D is the statistical result of the length of the meristem in figure C; the error is expressed as ⁇ SD (the number of measurement samples ⁇ 15); ***P ⁇ 0.001 (T test);
  • E is the statistical result of the length of cells in the elongation zone in the figure C; the error is expressed as ⁇ SD (the number of measurement samples ⁇ 15); ***P ⁇ 0.001 (T test);
  • Fig. 3 is the expression profile of gene OsPEP1 in embodiment 2;
  • A is the expression level of the gene OsPEP1 detected by qRT-PCR in rice roots, stems, leaves, and young ears, and the root, stem, leaf, and leaf sheath samples were taken from 7-day-old rice seedlings;
  • B is the GUS staining results of proOsPEP1::GUS transgenic plants at 7 days of age, and the scale bar is 1 cm;
  • C is the GUS staining of the taproot of the proOsPEP1::GUS transgenic plant of 2-day-old seedlings, and the scale bar is 0.1 cm;
  • E is the longitudinal section GUS staining of the main root meristem in C, the scale bar is 100 ⁇ m;
  • Fig. 4 is the phenotypic analysis of OsPEP1 overexpression transgenic plants in Example 2;
  • A is the phenotype of 7-day-old seedlings of WT and OsPEP1 overexpressing pig gene lines OE1, OE2 and OE3 (bar is 5 cm);
  • B is the root length statistics of the corresponding lines in Figure A, and the error is represented by SD (the number of individuals in the statistics is greater than 30), ***P ⁇ 0.001 (T test);
  • C is the expression level of the gene OsPEP1 detected by qRT-PCR in the corresponding plant in Figure A, and the error is represented by ⁇ SD (three biological replicates);
  • D is the longitudinal phenotype of the root tip elongation zone (top) and meristem (bottom) of the wild-type WT and OsPEP1-overexpressing transgenic plants OE2 treated with 10 -6 M PEP1 for 2 days, the bar is 100 ⁇ m ;
  • E is the statistical result of the length of the apical meristem in the D figure, the error is represented by ⁇ SD (measured sample size ⁇ 15); different letters represent significant differences at the 0.01 level (one-way ANOVA, LSD method);
  • F is the statistical result of the cell length in the elongation zone in the D figure, the error is represented by ⁇ SD (the number of measurement samples ⁇ 15); different letters represent significant differences at the 0.01 level (one-way ANOVA, LSD method);
  • Fig. 5 is the phenotypic analysis of OsPEP1 suppressing expression transgenic plants in Example 2;
  • A is the phenotype of Ri1, Ri2 and Ri3 plants of WT and OsPEP1 overexpressing pig gene lines at 7 days of seedling age (bar is 5 cm);
  • B is the root length statistics of the corresponding lines in Figure A, and the error is represented by SD (the number of individuals in the statistics is greater than 30), ***P ⁇ 0.001 (T test);
  • C is the expression level of the gene OsPEP1 detected by qRT-PCR in the corresponding plant in Figure A, and the error is represented by ⁇ SD (three biological replicates);
  • D is the longitudinal phenotype of the root tip elongation zone (top) and meristem (bottom) of the wild-type WT and OsPEP1 overexpressing transgenic plants Ri2 treated with 10 -6 M PEP1 for 2 days, the bar is 100 ⁇ m ;
  • E is the statistical result of the length of the apical meristem in the D figure, the error is represented by ⁇ SD (measured sample size ⁇ 15), and different letters represent significant differences at the 0.01 level (one-way ANOVA, LSD method);
  • F is the statistical result of the cell length in the elongation zone in the D panel; the error is expressed as ⁇ SD (measured sample size ⁇ 15), and different letters indicate significant differences at the 0.01 level (one-way ANOVA, LSD method).
  • composition of the hydroponic medium is shown in Table 2.
  • the gene expression level detection results in the following examples, unless otherwise specified, are all based on the target gene expression level of the wild-type plant Xiushui 134 as 1, and the target gene expression levels of other plants are compared with those of the wild-type plant. .
  • the medium was collected for subsequent concentration, extraction and precipitation of root exudates.
  • the specific method is as follows: first, the culture solution (500 ml) obtained in the previous step is concentrated 20 times by using a rotary evaporator. Then, 20 ml of chlorophenol containing 1% NEM (N-ethylmorpholine, N-ethylmorpholine) was added and shaken for 1 minute at room temperature, centrifuged at 10,000 g for 10 minutes, the organic phase was collected, and 20 times the volume of acetone was added to precipitate overnight at room temperature. Then centrifuge at 10000g for 10 minutes, collect the precipitate, wash with acetone 3-4 times, and then use vacuum drying to powder. Finally, the powder was sent to the company (Zhongke Xinsheng) to identify 234 rice root secreted polypeptides using liquid chromatography tandem mass spectrometry (LP-MS/MS).
  • LP-MS/MS liquid chromatography tandem mass spectrometry
  • the secreted polypeptide has a signal peptide sequence at its N-terminus.
  • the present invention uses the SignalP 4.1 server in the HMM webpage to screen 704 protein-coding genes with a signal peptide at the N-terminus (P ⁇ 0.75).
  • the present invention uses EXCEL2003 to remove protein coding genes containing 6 or more cysteines to obtain 416 potential rice polypeptide coding candidate genes.
  • the present invention By comparing the 234 root secreted polypeptides obtained by the above method 1 and the 416 hypothetical rice polypeptide-encoding genes obtained by the method 2, the present invention found that a new polypeptide (Ser-Asp-Phe-Asp-Arg, SEQ ID NO. :1) and its candidate coding gene (LOC_Os11g09560) was identified (A in Figure 1). In order to further confirm that LOC_Os11g09560 is the gene encoding the polypeptide, the present invention uses LP-MS/MS to detect the root exudates of transgenic plants overexpressing the gene.
  • the present invention uses different concentrations of artificially synthesized PEP1 to treat rice seedlings (the concentration is as described in A in Figure 2, the treatment method is to start treatment after sowing, and the treatment time is 7 days), and it is found that exogenous application of PEP1 can significantly inhibit the growth of rice taproot Elongation (A and B in Figure 2).
  • the present invention observed the slice structure of these rice root tips, and found that after PEP1 treatment, the length of the meristematic zone and the length of cells in the elongation zone of rice root tips were significantly shortened, but There was no significant change in its quiescent central region (shown in the meristem section of the root apex, C in Fig. 2-E in Fig. 2).
  • cDNA template 0.1 ⁇ l
  • the PCR procedure is as follows:
  • Cooling 40°C for 30 seconds
  • the DNA of Xiushui 134 rice was extracted, and the DNA was used as a template for PCR amplification to prepare the first 2kb nucleotide sequence of the gene OsPEP1 promoter.
  • the primers for PCR amplification are as follows:
  • the obtained PCR product was inserted between the SalI and BamHI restriction sites of the vector pBI101.3-GUSplus modified in our laboratory by the method of recombinant cloning (Lv et al., 2014) to obtain the proOsPEP1::GUS vector.
  • the vector was verified to be correct by sequencing.
  • the constructed overexpression vector was transferred into Agrobacterium EHA105 for transforming wild-type plants, with reference to conventional steps, as follows:
  • the air-dried callus was transferred to the selection medium containing 300 mg/L carbenicillin sodium and the corresponding selection pressure for the first round of selection, and cultured at 28° C. for 14 days in the light;
  • the mRNA of Xiushui 134 rice was extracted and reverse transcribed into cDNA, and the cDNA was used as a template for PCR amplification to prepare the OsPEP1 sequence (DNA shown in SEQ ID NO: 3).
  • the primers for PCR amplification are as follows:
  • the obtained PCR product was inserted between the SacI and PstI restriction sites of the vector pCAMBIA1300 (Lv et al., 2014) modified in our laboratory by recombinant cloning to obtain an OsPEP1 overexpression vector.
  • the OsPEP1 overexpression vector was verified to be correct by sequencing.
  • the OsPEP1 overexpression vector constructed in the above step 1 was transferred into Agrobacterium EHA105 for transformation of rice Xiushui 134, and 32 positive transgenic plants were obtained.
  • the specific steps were identical to step 2 of Example 2.
  • the mRNA of rice Xiushui 134 was extracted and reverse transcribed into cDNA, and the long cDNA was used as a template for PCR amplification to prepare the partial DNA sequence of OsPEP1:
  • the primers for PCR amplification are as follows:
  • the cloned PCR product was connected with T carrier (purchased from TAKARA company), and the connected plasmid was digested with PstI, BamH I and Pst I, Sal I respectively to obtain two fragments; the two fragments were connected together into the pBSSK-in carrier (Wang et al. 2019), in two steps.
  • pBSSK-in was first digested with PstI and BamHI, and then ligated with a fragment, then digested with Nsi I and Sal I, followed by another fragment.
  • the OsPEP1 inhibitory expression vector constructed in the above step 3 was transferred into Agrobacterium EHA105 for transformation of rice Xiushui 134, and 53 positive transgenic plants were obtained.
  • the specific steps are identical to the second step in Embodiment 2.
  • the present invention compares 234 rice root secreted polypeptides and 416 rice secreted polypeptide candidate coding genes identified by LC-MS/MS, and combines genetic experiments to find a method that regulates rice taproot development.
  • the root secreted polypeptide PEP1 (Ser-Asp-Phe-Asp-Arg, SEQ ID NO: 1) and its encoding gene OsPEP1 (LOC_Os11g09560) was determined.
  • the physiological experiment results of the present invention show that exogenous application of PEP1 inhibits rice root elongation, and the genetic experiments of the present invention show that overexpression or inhibition of expression can inhibit rice root elongation.

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Abstract

提供了一个调控植物根发育的多肽PEP1及其编码基因OsPEP1。外源施加PEP1处理植物，可抑制植物的根发育。含有该编码基因或该基因部分DNA的重组表达载体，用重组表达载体转化的植物，可以得到根生长发育改变的转基因植物。该多肽可作植物生长调节剂，其编码基因以及前体蛋白可以作为一个潜在的分子育种靶运用于作物的改良，通过调控作物根生长发育来提高作物产量。

Description

水稻根分泌多肽PEP1及其编码基因和应用
本申请要求于2021年2月20日提交中国专利局、申请号为202110194477.8、发明名称为“水稻根分泌多肽PEP1及其编码基因和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物技术领域，特别涉及一种植物根分泌多肽及其编码基因在调控植物根生长与发育中的应用。
背景技术
根是植物重要的地下器官，其不仅可以固定植株，而且还可以从土壤中吸收营养、水分等物质供植物的生长发育。此外，植物的根系还可以监测土壤环境中的各种信号分子，比如土壤中的水分含量，营养物质的水平以及一些植物激素及信号等，以提高植物对其生长环境的适应性(Meng et al.2019)。植物根系主要分为两大类，分别为直根系和须根系。水稻是单子叶模式植物，其根系为须根系，主要由种子根和不定根及种子根和不定根上的侧根和根毛组成(Rebouillat et al.2009；Coudert et al.2010)。研究表明根冠比高，发达的根系有助于提高水稻的抗旱能力及产量(Fukai et al.1995；Gowda et al.2011)。之前研究表明，水稻根系的生长发育受到外源信号与内源遗传程序的严格调控(Benfey et al.2012；Orman-Ligeza et al.2013)。
之前的研究结果表明，多肽可以作为外源的信号分子调控拟南芥的根系发育(Oh et al.,2018)。外源施加人工合成的多肽根茎生长因子1(RGF1)可以回复tpst-1突变体中根尖分生区缺陷的表型，表明RGF1是维持根尖干细胞所必需的(Matsuzaki et al.2010)。进一步的突变分析表明，RGF/GLV通过转录后调控机制控制根分生组织中生长素外排载体PINFORMED 2的丰度和运输动力学,进而调控植物根尖分生区的大小(Whitford et al.2012)。植物根分生组织的大小也受多肽CLE(CLAVATA3(CLV3)/EMBRYO SURFROUNDING REGION(ESR)-related)家族的调控。拟南芥中有32个CLE多肽编码基因，其编码的多肽可分为两组：A型CLE和B型CLE。A型CLE多肽在根分生组织发育中起作用，而B型CLE多肽与根维管束发育有关(Cock and McCormick 2001；Whitford et al.2008)。例如A型CLE多肽CLE40是柱状细胞分化所必需的，功能丧失的突变体cle40由于柱状细胞的延迟分化而表现出具有不规则形状的根尖，从而表现为短根表型(Stahl et al.2009)。其他另一些的CLE多肽(CLE26，CLE40，CLE45)通过调节维管束发育过程中原上皮形成来控制主根的生长(Depuydt et al.2013；Rodriguez-Villalon et al.2014；Rodriguez-Villalon et al.2015；Hazak et al.2017)。此外，酪氨酸硫酸化肽PSK与PSY1可通过调节伸长区/分生区中的细胞扩增来促进主根生长(Amano et al.2007；Kwezi et al.2011；Hartmannet et al.2014；Ladwiget et al.2015)。凯氏带完整性因子1(CIF1)多肽对于凯氏带的形成是必不 可少的，因为CIF1功能丧失的突变体在高铁浓度下表现出严重的生长发育迟缓，但是通过使用外源施加CIF1多肽处理后，可以完全回复这些缺陷(Nakayama et al.2017)。
与拟南芥相比，多肽调控水稻根系生长发育的机制尚不清楚。所以分离鉴定水稻根系生长发育相关的多肽，确定这些多肽的编码基因对进一步全面了解水稻及其他单子叶植物根系发生发育的机理，获得优良基因以培育新的水稻品种是十分重要和必要的。
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发明内容
本发明要解决的技术问题是提供一种调控植物根发育相关多肽及其编码基因和应用。
为了解决上述技术问题，本发明提供一种调控植物根发育多肽的用途：用于处理植物，所述处理后植物的根长度变短或者变长；
所述多肽为以下任一：
氨基酸序列如SEQ ID NO:1(序列表NO:1)所示；
SEQ ID NO:1所示氨基酸序列组成的多肽经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物根发育相关的由其衍生的多肽。
作为本发明的调控植物根发育多肽的用途的改进：
多肽的前体蛋白的氨基酸序列如SEQ ID NO:2所示；
或者，将SEQ ID NO:2所示的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物发育相关的由其衍生的蛋白质；
本发明提供另一种调控植物根发育基因的用途：用于构建转基因植物，所述转基因植物的根长度变短或者变长；
所述基因为以下任一：
基因编码区的核苷酸序列如SEQ ID NO:3所示；
或者，将SEQ ID NO:3所示的基因经过一个或几个核苷酸的取代和/或缺失和/或添加且与植物发育相关的由其衍生的基因。
作为本发明的调控植物根发育基因的用途的改进：
所述植物为水稻。
作为本发明基因用途的改进：
使用不同浓度的多肽处理植物，得到长度变短或者变长的植物；
作为本发明基因用途的进一步改进：
将重组表达载体Ⅰ导入目的植物中，得到长度变短的转基因植物；
将SEQ ID NO:3所示DNA分子插入质粒pCAMBIA1300的多克隆位点得到的重组表达载体Ⅰ—OsPEP1过表达载体。
作为本发明基因用途的进一步改进：
将重组表达载体Ⅱ导入目的植物中，得到长度变短的转基因植物；
将SEQ ID NO:3所示基因DNA片段通过正向与反向连接到过渡载体pBSSK-in后插入质粒pCAMBIA1300得到的重组表达载体Ⅱ---OsPEP1抑制表达载体。
本发明技术方案具体如下：
本发明提供的多肽(PEP1)，其前体蛋白，以及编码基因来自粳稻秀水134(Oryza sativa L.ssp.Japonica cv.Xiushui134)，是如下(a)或(b)的多肽，其前体蛋白，或者编码基因，是如下(a)，(b)，(c)，(d)，(e)或(f)的多肽，蛋白，或者编码基因：
(a)由SEQ ID NO:1所示氨基酸序列组成的多肽；
(b)将SEQ ID NO:1所示氨基酸序列组成的多肽经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物根发育相关的由其衍生的多肽；
(c)由SEQ ID NO:2所示的氨基酸序列组成的蛋白质；
(d)将SEQ ID NO:2的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物发育相关的由其衍生的蛋白质；
(e)由SEQ ID NO:3所示的核苷酸序列组成的基因；
(f)将SEQ ID NO:3的基因经过一个或几个核苷酸的取代和/或缺失和/或添加且与植物发育相关的由其衍生的基因；
所述植物发育体现在植株根长性状上。
为了使(a)中的PEP1便于纯化，可在由SEQ ID NO:1所示的氨基酸基酸序列组成的蛋白质的氨基末端或羧基末端连接上包括但不限于如表1所示的标签。
表1标签的序列
标签 残基 序列
Po1y-Arg 5-6(通常为5个) RRRRR(SEQ ID NO:4)
Poly-His 2-10(通常为6个) HHHHHH(SEQ ID NO:5)
FLAG 8 DYKDDDDK(SEQ ID NO:6)
Strep-tagII 8 WSHPQFEK(SEQ ID NO:7)
c-myc 10 EQKLISEEDL(SEQ ID NO:8)
上述(a)中的PEP1可人工合成，也可先合成其编码基因，再进行生物表达得到。上述(a)中的PEP1的编码基因可通过将SEQ ID NO:3所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5'端和/或3'端连上表1所示的标签的编码序列得到。
所述蛋白的编码基因也属于本发明的保护范围。
所述蛋白的编码基因(OsPEP1)可为如下1)或2)或3)的DNA分子：
1)、其编码序列是SEQ ID NO:3所示的DNA分子，即基因的编码区的核苷酸序列如SEQ ID NO:3所示；
2)、在严格条件下与1)限定的DNA序列杂交且编码相同功能蛋白质的DNA分子；
3)、与1)或2)限定的DNA序列具有90％以上同源性，且编码相同功能蛋白质的DNA分子。
含有所述基因或其反义基因的重组表达载体、表达盒、转基因细胞系或重组菌均属于本发明的保护范围。
可用现有的植物表达载体构建含有OsPEP1基因的重组表达载体。
所述植物表达载体包括但不限于如双元农杆菌载体和可用于植物微弹轰击的载体等。所述植物表达载体还可包含外源基因的3'端非翻译区域，即包含聚腺苷酸信号和任何其它参与mRNA加工或基因表达的DNA片段。所述聚腺苷酸信号可引导聚腺苷酸加入到mRNA前体的3'端，如包括但不限于农杆菌冠瘿瘤诱导(Ti)质粒基因(如胭脂合成酶Nos基因)、植物基因(如大豆贮藏蛋白基因)3'端转录的非翻译区均具有类似功能。
使用OsPEP1构建重组植物表达载体时，在其转录起始核苷酸前可加上任何一种增强型启动子或组成型启动子，包括但不限于如花椰菜花叶病毒(CAMV)35S启动子、玉米的泛素启动子(ubiquitin)，它们可单独使用或与其它植物启动子结合使用；此外，使用本发明的基因构建植物表达载体时，还可使用增强子，包括翻译增强子或转录增强子，这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的，可以是天然的，也可以是合成的。翻译起始区域可以来白转录起始区域或结构基因。
为了便于对转基因植物细胞或植物进行鉴定及筛选，可对所用植物表达载体进行加工，如加入包括但不限于可在植物中表达的编码可产生颜色 变化的酶或发光化合物的基因(GUS基因、荧光素酶基因等)、具有抗性的抗生素标记物(庆大霉素标记物、卡那霉素标记物等)或是抗化学试剂标记基因(如抗除草剂基因)等。从转基因植物的安全性考虑，可不加任何选择性标记基因，直接以逆境筛选转化植株。
所述重组表达载体具体可为如下(I)或(II)：
(I)将SEQ ID NO:3所示DNA分子插入质粒pCAMBIA1300-的多克隆位点得到的含有上述基因的重组表达载体；
(II)将SEQ ID NO:3所示基因DNA片段通过正向与反向连接到过渡载体pBSSK-in后插入质粒pCAMBIA1300得到的重组表达载体；
(I)或(II)中的所述质粒pCAMBIA1300与pBSSK-in都是改造得到的(已公开)。
本发明还保护一种培育转基因植物的方法，是将所述基因或基因的片段通过正反向连接到过渡载体pBSSK-in后导入目的植物中，所述转基因植物为与目的植物相比根长度改变的转基因植物。
利用任何一种可以引导外源基因在植物中表达的载体，将本发明所提供的OsPEP1基因或基因的片段通过正反向连接到过渡载体pBSSK-in后导入植物细胞，可获得根长改变的转基因细胞系及转基因植株。携带有OsPEP1基因或其部分基因正反向连接的表达载体可通过使用Ti质粒、Ri质粒、植物病毒载体、直接DNA转化、显微注射、电导、农杆菌介导等常规生物学方法转化植物细胞或组织，并将转化的植物组织培育成植株。被转化的植物宿主可为禾本科植物，如水稻(如秀水134)。
当培育根长大于目的植物的转基因植物时；所述方法是将(I)所述重组表达载体导入目的植物中，得到根变短的转基因植物。当培育根长小于目的植物的转基因植物时；所述方法是将(II)所述重组表达载体导入目的植物中，得到根变短的转基因植物。
本发明发现了一个新的水稻根分泌多肽PEP1及其编码基因OsPEP1，并获得了含有该编码基因或该基因的部分片段通过正向与反向连接到过渡载体pBSSK-in的重组表达载体，用重组载体转化目的植物，可以得到根长改变的转基因植物。因此OsPEP1可以作为一种潜在的分子育种工具，通过改良植物的根长来提高植物产量。
综上所述，本发明分离、鉴定了一个控制水稻根生长的多肽PEP1，并确定了其编码基因OsPEP1(LOC_Os11g09560)以及前体蛋白OsPEP1。即，本发明分离鉴定了一个调控植物根发育的多肽PEP1及其编码基因OsPEP1。外源施加PEP1处理植物，可抑制植物的根发育。并获得了含有该编码基因或该基因部分DNA的重组表达载体，用重组表达载体转化的植物，可以得到根生长发育改变的转基因植物。因此该多肽可作植物生长调节剂，其编码基因以及前体蛋白可以作为一个潜在的分子育种靶运用于作物的改良，通过调控作物根生长发育来提高作物产量。
说明书附图
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1为实施例1中基因OsPEP1(LOC_Os11g09560)编码一个含有五个氨基酸(SDFDR，SEQ ID NO:1)的蛋白；
A为基因OsPEP1编码蛋白的氨基酸序列(MGEKERRLRVEGWMGRTEMIDRRRQRLHSGERERRLCVRKRMGS S DFDRGARFGGVDDGRLGEGTKRCEEMVGAIWDVGFERDNPDRSTRNEDVNISW，SEQ ID NO:2)，5个氨基酸残基的多肽PEP1用下划线标出；
B为LC-MS分析野生型水稻植株根系分泌多肽物质；
C为LC-MS/MS鉴定得到的滞留时间为12.25分钟的野生型水稻植株根系分泌多肽物质Ser-Asp-Phe-Asp-Arg(PEP1)(SEQ ID NO:1)；
D为LC-MS分析LOC_Os11g09560过量表达转基因水稻植株根系分泌多肽物质；
E为LC-MS/MS鉴定得到的滞留时间为12.26分钟的LOC_Os11g09560过量表达转基因水稻植株根系分泌多肽物质Ser-Asp-Phe-Asp-Arg(PEP1)(SEQ ID NO:1)；
图2为实施例1中根分泌多肽PEP1的功能分析；
A为不同浓度PEP1处理7天后野生型植株的表型，标尺为5厘米；
B为不同浓度PEP1处理7天后野生型植株主根长度统计；误差用±SD表示(测量样本数量≥15)；不同的字母表示0.01水平的显著性差异(单因素方差分析，LSD方法)；
C为野生型植株未使用PEP1(-PEP1)处理的根尖伸长区(左上)与分生区(左下)或者使用浓度为10 -6M PEP1处理(+PEP1)2天后的根尖伸长区(右上)与分生区(右下)的纵切表型，标尺为100微米；
D为C图中的分生区长度的统计结果；误差用±SD表示(测量样本数量≥15)；***P<0.001(T检验)；
E为C图中伸长区细胞长度的统计结果；误差用±SD表示(测量样本数量≥15)；***P<0.001(T检验)；
图3为实施例2中基因OsPEP1的表达谱；
A为qRT-PCR检测基因OsPEP1在水稻根、茎、叶、以及幼穗中的表达量，根、茎、叶、叶鞘样品取自7天苗龄水稻苗；
B为7天苗龄的proOsPEP1::GUS转基因植株的GUS染色结果，标尺为1厘米；
C为2天苗龄的proOsPEP1::GUS转基因植株主根的GUS染色，标尺为0.1厘米；
D为C图中主根伸长区的横切GUS染色图，标尺为100μm；
E为C图中主根分生区区的纵切GUS染色图，标尺为100μm；
图4为实施例2中OsPEP1过表达转基因植株的表型分析；
A为7天苗龄的WT与OsPEP1过表达猪基因株系OE1、OE2和OE3植株表型(标尺为5厘米)；
B为A图中相应株系的根长统计，误差用SD用表示(统计的个体数目大于30)，***P<0.001(T检验)；
C为qRT-PCR检测A图中相应植株中基因OsPEP1的表达量，误差用±SD表示(三次生物学重复)；
D为野生型WT与OsPEP1过量表达转基因植株OE2在浓度为10 -6M PEP1处理2天的根尖伸长区(上)与分生区(下)与的纵切表型，标尺 为100微米；
E为D图中的根尖分生区长度的统计结果，误差用±SD表示(测量样本数量≥15)；不同的字母表示0.01水平的显著性差异(单因素方差分析，LSD方法)；
F为D图中伸长区细胞长度的统计结果，误差用±SD表示(测量样本数量≥15)；不同的字母表示0.01水平的显著性差异(单因素方差分析，LSD方法)；
图5为实施例2中OsPEP1抑制表达转基因植株的表型分析；
A为7天苗龄的WT与OsPEP1过表达猪基因株系Ri1、Ri2和Ri3植株表型(标尺为5厘米)；
B为A图中相应株系的根长统计，误差用SD用表示(统计的个体数目大于30)，***P<0.001(T检验)；
C为qRT-PCR检测A图中相应植株中基因OsPEP1的表达量，误差用±SD表示(三次生物学重复)；
D为野生型WT与OsPEP1过量表达转基因植株Ri2在浓度为10 -6M PEP1处理2天的根尖伸长区(上)与分生区(下)与的纵切表型，标尺为100微米；
E为D图中的根尖分生区长度的统计结果，误差用±SD表示(测量样本数量≥15)，不同的字母表示0.01水平的显著性差异(单因素方差分析，LSD方法)；
F为D图中伸长区细胞长度的统计结果；误差用±SD表示(测量样本数量≥15)，不同的字母表示0.01水平的显著性差异(单因素方差分析，LSD方法)。
具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
水培培养基的成分如表2所示。
表2水培培养基的成分
Figure PCTCN2021112192-appb-000001
Figure PCTCN2021112192-appb-000002
以下实施例中的基因表达量检测结果，如无特殊说明，均是以野生型植株秀水134的目的基因表达量为1，其它植株的目的基因表达量与野生型植株的目的基因表达量进行比较。
实施例1、水稻多肽PEP1的获得及其功能研究
一、水稻多肽PEP1及其编码基因的获得
1、水稻根部分泌多肽的获得：
选取30粒左右饱满的野生型水稻(秀水134)种子，用0.5％稀硝酸进行破休眠处理16-22小时后，用自来水清洗2-3次，加适量自来水浸泡种子于37℃的培养箱中催芽约两天至露白。在这期间每天早晚各换水一次。最后将露白的种子播于浮在营养液(水培培养基)上的尼龙网纱上，并置于人工气候室中培养。人工气候室的培养条件：光照为14小时，平均昼夜温度为30℃/22℃，光照强度保持在200μmol/m 2s 1，湿度为60％。培养10天后，收集培养基用于后续根分泌物质的浓缩、提取以及沉淀。具体方法为：首先使用旋转蒸发仪将上一步获得的培养液(500ml)浓缩20倍。然后加入含有1％NEM(N-ethylmorpholine，N-乙基吗啉)的20ml的氯酚室温振荡1分钟后，10000g离心10分钟，收集有机相，加入20倍体积丙酮室温过夜沉淀。然后10000g离心10分钟，收集沉淀，使用丙酮清洗3-4次，再使用真空干燥至粉末。最后将粉末送往公司(中科新生命)使用液相色谱串联质谱(LP-MS/MS)方法鉴定得到234个水稻根分泌多肽。
2、水稻分泌型多肽编码候选基因的获得。
之前的研究结果表明，拟南芥中一些小信号多肽(PSK，PSY1，CLV3/CLE)编码基因的产物大都为长度在70-110氨基酸，且不富含半胱氨酸的蛋白质。所以本发明推测如果水稻中的某些基因编码的蛋白具这些类特点，那么该基因有可能是类似多肽的候选编码基因。基于这种猜测，本发明从水稻基因组注释数据库( http://rice.plantbiology.msu.edu/index.shtml,2017年3月)中一共下载得到了66343个蛋白编码基因，然后利用MICROSOFT WORD 2003与EXCEL 2003得到了12678个长度在50-150氨基酸的蛋白编码基因。分泌型的多肽在其N端有一段信号肽序列，本发明使用HMM网页中的SignalP 4.1服务器筛选得到704个N末端具有信号肽蛋白编码基因(P≥0.75)。最后本发明使用EXCEL2003去除了含有6或者更多半胱氨酸的蛋白编码基因，得到416个潜在的水稻多肽编码候选基因。
3、水稻根分泌多肽PEP1及其编码基因的确定。
通过比对上述方法1获得的234个根分泌多肽和方法2获得的416个假想的水稻多肽编码基因，本发明发现得到了一个新的多肽(Ser-Asp-Phe-Asp-Arg，SEQ ID NO:1)以及确定了其候选编码基因(LOC_Os11g09560)(图1中的A)。为了进一步地确认LOC_Os11g09560是该多肽的编码基因，本发明使用LP-MS/MS检测了该基因过表达转基因植株根系的分泌物，结果发现，与野生型的根系分泌物质中的该多肽的峰值相比，该基因过量表达转基因植株的根系分泌物质中该多肽峰值明显增强(图1中的B-图1中的E)。这些结果表明，基因LOC_Os11g09560确实是该多肽的编码基因，此后将该多肽(Ser-Asp-Phe-Asp-Arg，SEQ ID NO:1)命名为PEP1，其编码基因(LOC_Os11g09560)命名为OsPEP1。
二、水稻多肽PEP1的功能研究
本发明使用不同浓度人工合成的PEP1处理水稻幼苗(浓度如图2中的A所述，处理方式为播种后开始处理，处理时间为7天)，结果发现外源施加PEP1可以显著抑制水稻主根的伸长(图2中的A和B)。为了明确PEP1抑制水稻根伸长的细胞学基础，本发明观察了这些水稻根尖的切片结构，结果发现PEP1处理后，水稻根尖的分生区长度与伸长区细胞的长度显著缩短，但是其静止中心区域没有显著变化(根尖的分生区切片所示，如图2中的C-图2中的E)。
实施例2、水稻多肽PEP1编码基因OsPEP1的表达谱研究
一、qRT-PCR检测基因OsPEP1在水稻不同组织的表达
以野生型秀水134水稻为材料，在正常营养液(水培培养基)中培养7天，取其根、茎、叶及叶鞘，然后待植株将要抽穗时候，取其幼穗。所有样品于液氮中速冻并研磨，分别抽提总RNA并进行逆转录获得相应的cDNA。用qRT-PCR方法检测OsPEP1在各组织中的表达。结果表明，OsPEP1基因在本发明所检测的不同组织中都有表达，在根中表达量较高(图3中的A)。检测引物序列如下：
5'-GGCGTGGATGACGGGAGACT-3'(SEQ ID NO:9)；
5'-TACATCCTCATTCCTCGTTG-3'(SEQ ID NO:10)，
其反应体系与程序如下
2×Master:2.5μl
cDNA模板:0.1μl
Primer-F(10μM):0.1μl
Primer-R(10μM)：0.1μl
H 2O:2.2μl
Total:5μl；
PCR程序如下：
起始:95℃1分钟
扩增:95℃10秒
58℃10秒
72℃20秒45循环
溶解曲线:95℃5秒
65℃1分钟
97℃降温处理，直到65℃
冷却:40℃30秒；
二、GUS染色检测OsPEP1在水稻不同组织的表达
提取秀水134水稻的DNA，以此DNA为模板进行PCR扩增制备基因OsPEP1启动子前2kb核苷酸序列。PCR扩增的引物如下：
5'-GCATGCCTGCAGGTCGACGTTTCTCAGCTACGCCCCTG-3'(SEQ ID NO:11)；
5'-CCATGGTACCGTGGATCCCCGGAGCGCAGCCGTCGTCT-3'(SEQ ID NO:12)，
将获得PCR产物利用重组克隆的方法插入到本实验室改造的载体pBI101.3-GUSplus的SalI和BamHI酶切位点之间(Lv etal.,2014)，得到proOsPEP1::GUS载体。载体经过测序检验正确。将构建的过表达载体转入农杆菌EHA105，用于转化野生型植株，参照常规步骤，具体如下：
(1)取培养好的菌液500μl于1.5ml离心管中，室温，4000rmp，离心2分钟，去上清。用含200μmol/L乙酰丁香酮的30ml AAM感菌液制成悬浮液，菌液终浓度OD 600为0.01；将长到一定大小(约为1立方厘米)的80～120个水稻愈伤组织挑出，放入农杆菌悬浮液，在水平摇床上摇晃5分钟；
(2)将愈伤组织取出，置于无菌的滤纸上沥干30到40分钟；
(3)将愈伤组织置于有一张无菌滤纸的共培养基上，25℃暗培养3天；
(4)上述的愈伤组织取出，再用无菌水清洗5到6次，其间需不停的振荡。用含300mg/L羧苄青霉素钠(Carb)的无菌水清洗2遍，每次在水平摇床上摇晃30分钟。最后置于无菌滤纸上沥干2小时；
(5)将晾干的愈伤转入含300mg/L羧苄青霉素钠和相应筛选压力的选择培养基上进行第一轮选择，28℃，光照培养14天；
(6)将长有抗性愈伤的初始愈伤转到含300mg/L羧苄青霉素钠和相应筛选压力的培养基上进行第二轮选择，28℃，光照培养，直到颗粒性的抗性愈伤组织长出(约14天左右)；
(7)挑取从不同愈伤来的颜色鲜黄的抗性愈伤3到5颗，移入装有分化培养基的塑料广口瓶中，用封口膜封好，放入恒温(25℃)培养室中(光周期：16小时光照)，等待分化成苗(约40天)；
(8)待苗长至3厘米左右，用剪刀从苗基部剪去老根和愈伤组织，放入生根培养基中壮苗(约1周)。将根部和茎叶分化得较完好的苗从试管挑出(苗长至试管顶部，就要及时开盖)，打开封口膜，加入适量蒸馏水或无菌水(防止培养基长菌)，炼苗2到3天，然后洗去琼脂，移栽到温室的水培条件中生长。利用抗赤霉素基因的引物检测转基因植物，引物序列如下:
5'-ATGAAAAAGCCTGAACTCACC-3'(SEQ ID NO:13)；
5'-CTATTCCTTTGCCCTCGGACG-3'(SEQ ID NO:14)，
在得到的T2代转移基因水稻中，选择代表性株系(GUS染色阳性的转基因植株)进行GUS染色研究OsPEP1在水稻不同组织中的表达情况。结果表明基因OsPEP1主要在水稻根部表达，尤其是根尖的根冠区域、以及分生区和成熟区的外表皮(图3中的B-图3中的E)。
实施例3、水稻多肽PEP1编码基因OsPEP1的功能研究
一、OsPEP1过表达重组载体的构建
提取秀水134水稻的mRNA并逆转录为cDNA，以此cDNA为模板进行PCR扩增制备OsPEP1序列(SEQ ID NO:3所示DNA)。PCR扩增的引物如下:
5'-ACGGGGGACGAGCTCATGGGAGAGAAGGAGCGGAG-3'(SEQ ID NO:15)；
5'-GACTCTAGAGGATCCCAACTGATGTTTACATCCTCA-3'(SEQ ID NO:16)，
将获得PCR产物利用重组克隆的方法插入到本实验室改造的载体pCAMBIA1300(Lv et al.,2014)的SacI与PstI酶切位点之间，得到OsPEP1过表达载体。OsPEP1过表达载体经过测序检验正确。
二、OsPEP1过表达水稻的获得
将上述步骤一构建的OsPEP1过表达载体转入农杆菌EHA105，用于转化水稻秀水134，得到32株阳性转基因植株，具体步骤等同于实施例2的步骤二。
在得到的T2代转移基因水稻中，挑选三个代表性转基因株系(OsPEP1过量表达的独立转基因植系)(OE1，OE2，OE3)进行表型分析。结果表明，与野生型相比，转基因株系的株高其根长度显著缩短(见图4中的A和图4中的B)。用实时定量PCR的方法对水稻秀水134与OsPEP1过表达转基因植株中(OE1，OE2，OE3)中内源OsPEP1相对表达量进行分析(引物为:GGCGTGGATGACGGGAGACT(SEQ ID NO:9)；TACATCCTCATTCCTCGTTG(SEQ ID NO:10))，结果表明，OsPEP1的过表达转基因株系的主根长度与OsPEP1表达量正相关(见图4中的C)。此外本发明观察了这些OsPEP1过表达转基因水稻根尖的切片结构，与对照植株秀水134(WT)相比，OsPEP1过表达转基因水稻根尖的分生区长度与伸长区细胞的长度显著缩短，外源的PEP处理不能恢复OsPEP1过表达转基因水稻根尖缺陷表型至野生型水平(见图4中的D-图4中的F)。
三、OsPEP1抑制表达重组载体的构建
提取水稻秀水134的mRNA并逆转录为cDNA，以此长cDNA为模板进行PCR扩增制备OsPEP1的部分DNA序列：
ACTCGGGAGAGAGGGAGCGCAGATTGTGCGTGAGGAAACGGATGGGAAGCAGCGATTTCGATCGAGGGGCGCGATTTGGGGGCGTGGATGACGGGAGACTGGGAGAGGGGACGAAGCGGTGTGAGGAGATGGTGGGAGCGATTTGGG(SEQ ID NO:17)。
PCR扩增的引物如下：
5'-ACTCGGGAGAGAGGGAGCGC-3'(SEQ ID NO:18)；
5'-CCCAAATCGCTCCCACCATC-3'(SEQ ID NO:19)，
将克隆到的PCR产物与T载体(购自TAKARA公司)连接，连接好的质粒分别用PstI、BamH I以及Pst I、Sal I酶切得到两片段；两片段一同连入pBSSK-in载体(Wang et al.2019)，分两步进行。pBSSK-in先用PstI、BamH I酶切，连上一个片段后，再Nsi I、Sal I酶切，连另一片段。最后用Sac I、Sal I将两片段以及intron切下，连入相同酶切的植物双元载体pCAMBIA1300中(Lv etal.,2014)，得到OsPEP1抑制表达载体。OsPEP1抑制表达载体经过测序检验正确。
四、OsPEP1抑制表达水稻的获得
将上述步骤三构建的OsPEP1抑制表达载体转入农杆菌EHA105，用于转化水稻秀水134，得到53株阳性转基因植株。具体步骤等同于实施例2的步骤二。
在得到的T2代转移基因水稻中，挑选三个代表性株系(OsPEP1抑制表达的独立转基因植株)(Ri1，Ri2，Ri3)进行表型分析。结果表明，与野生型相比其根长度显著缩短(见图5中的A和图5中的B)。用实时定量PCR的方法对秀水134与OsPEP1抑制表达转基因植株(Ri1，Ri2，Ri3)中内源OsPEP1相对表达量进行分析(引物为:GGCGTGGATGACGGGAGACT(SEQ ID NO:9)；TACATCCTCATTCCTCGTTG(SEQ ID NO:10))，结果表明，基因OsPEP1的抑制表达转基因株系的主根长度与OsPEP1表达量正相关(见图5中的C)；此外本发明观察了这些OsPEP1抑制表达转基因水稻根尖的切片结构，与对照秀水134(WT)相比，OsPEP1抑制表达转基因水稻根尖的分生区长度与伸长区细胞的长度显著缩短，外源的PEP处理可以恢复OsPEP1抑制表达转基因水稻根尖缺陷表型至野生型水平(见图5中的D-图5中的F)。
综合以上的结果，本发明将通过LC-MS/MS鉴定得到的234个水稻根系分泌多肽和416个水稻分泌型多肽候选编码基因的对比研究，并结合遗传学实验，找到了一个调控水稻主根发育的根分泌多肽PEP1(Ser-Asp-Phe-Asp-Arg，SEQ ID NO:1)并确定了其编码基因OsPEP1(LOC_Os11g09560)。本发明的生理实验结果表明外源施加PEP1抑制水稻根伸长，本发明的遗传学实验表明，过量表达或抑制表达均会抑制水稻根伸长。这些结果表明，水稻根分泌多肽可能作为一个信号分子在水稻根发育中起着重要作用，可以通过调控OsPEP1的表达量控制水稻根的生长发育。
最后，还需要注意的是，以上列举的仅是本发明的若干个具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。本领域的技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。

Claims (12)

  1. 调控植物根发育多肽的用途，其特征是：用于处理植物，所述处理后植物的根长度变短或者变长；
    所述的调控植物根发育多肽为以下任一：
    氨基酸序列如SEQ ID NO:1所示；
    SEQ ID NO:1所示氨基酸序列组成的多肽经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物根发育相关的由其衍生的多肽。
  2. 根据权利要求1所述的调控植物根发育多肽的用途，其特征是：
    该多肽的前体蛋白的氨基酸序列如SEQ ID NO:2所示；
    或者，将SEQ ID NO:2所示的蛋白质经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物发育相关的由其衍生的蛋白质；
  3. 调控植物根发育基因的用途，其特征是：用于构建转基因植物，所述转基因植物的根长度变短或者变长；
    所述的调控植物根发育基因为以下任一：
    基因编码区的核苷酸序列如SEQ ID NO:3所示；
    或者，将SEQ ID NO:3所示的基因经过一个或几个核苷酸的取代和/或缺失和/或添加且与植物发育相关的由其衍生的基因。
  4. 根据权利要求3所述的调控植物根发育基因的用途，其特征是：
    所述植物为水稻。
  5. 根据权利要求4所述的调控植物根发育基因的用途，其特征是：
    使用不同浓度的多肽处理植物，得到长度变短或者变长的植物。
  6. 根据权利要求3～5任一所述的基因的用途，其特征是：
    将重组表达载体Ⅰ导入目的植物中，得到长度变短的转基因植物；
    将SEQ ID NO:3所示核苷酸序列的DNA分子插入质粒pCAMBIA1300的多克隆位点得到的重组表达载体Ⅰ—OsPEP1过表达载体。
  7. 根据权利要求3～5任一所述的基因的用途，其特征是：
    将重组表达载体Ⅱ导入目的植物中，得到长度变短的转基因植物；
    将SEQ ID NO:3所示核苷酸序列的基因DNA片段通过正向与反向连接到过渡载体pBSSK-in后插入质粒pCAMBIA1300得到的重组表达载体Ⅱ---OsPEP1抑制表达载体。
  8. 含调控植物根发育基因或其反义基因的重组表达载体；所述调控植物根发育基因为以下任一：
    基因编码区的核苷酸序列如SEQ ID NO:3所示；
    或者，将SEQ ID NO:3所示的基因经过一个或几个核苷酸的取代和/或缺失和/或添加且与植物发育相关的由其衍生的基因。
  9. 根据权利要求8所述的重组表达载体，其特征在于，所述重组表达载体具体可为如下(I)或(II)：
    (I)将SEQ ID NO:3所示DNA分子插入质粒pCAMBIA1300的多克隆位点得到的含有上述基因的重组表达载体；
    (II)将SEQ ID NO:3所示基因DNA片段通过正向与反向连接到过渡载体pBSSK-in后插入质粒pCAMBIA1300得到的重组表达载体。
  10. 含调控植物根发育基因或其反义基因的表达盒，所述调控植物根发育基因为以下任一：
    基因编码区的核苷酸序列如SEQ ID NO:3所示；
    或者，将SEQ ID NO:3所示的基因经过一个或几个核苷酸的取代和/或缺失和/或添加且与植物发育相关的由其衍生的基因。
  11. 含调控植物根发育基因或其反义基因的转基因细胞系，所述调控植物根发育基因为以下任一：
    基因编码区的核苷酸序列如SEQ ID NO:3所示；
    或者，将SEQ ID NO:3所示的基因经过一个或几个核苷酸的取代和/或缺失和/或添加且与植物发育相关的由其衍生的基因。
  12. 含调控植物根发育基因或其反义基因的重组菌，所述调控植物根发育基因为以下任一：
    基因编码区的核苷酸序列如SEQ ID NO:3所示；
    或者，将SEQ ID NO:3所示的基因经过一个或几个核苷酸的取代和/或缺失和/或添加且与植物发育相关的由其衍生的基因。
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