WO2022188287A1 - Protéine pour raccourcir le stade d'épiaison du riz, et gène codant pour cette protéine et son application - Google Patents

Protéine pour raccourcir le stade d'épiaison du riz, et gène codant pour cette protéine et son application Download PDF

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WO2022188287A1
WO2022188287A1 PCT/CN2021/100543 CN2021100543W WO2022188287A1 WO 2022188287 A1 WO2022188287 A1 WO 2022188287A1 CN 2021100543 W CN2021100543 W CN 2021100543W WO 2022188287 A1 WO2022188287 A1 WO 2022188287A1
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rice
plant
hap
yield
irgsp
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PCT/CN2021/100543
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English (en)
Chinese (zh)
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周文彬
李霞
魏少博
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中国农业科学院作物科学研究所
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Priority claimed from CN202110259057.3A external-priority patent/CN113073110A/zh
Priority claimed from CN202110359893.9A external-priority patent/CN113073142B/zh
Application filed by 中国农业科学院作物科学研究所 filed Critical 中国农业科学院作物科学研究所
Publication of WO2022188287A1 publication Critical patent/WO2022188287A1/fr

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    • 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
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • 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
    • 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
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • 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 a protein used for shortening the heading stage of rice in the field of biotechnology and its encoding gene and application.
  • Heading date is one of the important agronomic traits of crops, which determines the season, regional adaptability and yield of rice. A suitable heading date is the guarantee of stable and high yield of crops. Breeding new early-maturing and high-yielding varieties has always been one of the main directions of crop genetics and breeding research. "High-yielding but not early-maturing, early-maturing but not high-yielding” is the so-called “excellent but not early, early but not excellent” phenomenon, which is a major problem encountered in the cultivation of crop varieties.
  • the growth period of crops plays a decisive role in crop yield, among which the heading date of rice is one of the most important agronomic traits that determine the yield and quality of rice.
  • Appropriate heading date plays a key role in the adaptation of rice varieties to different ecological regions and different planting seasons, and is the guarantee of stable and high crop yield.
  • the main influencing factors of heading date include cultivar differences, photoperiod and tillage methods, etc.
  • the differences of heading dates of different rice cultivars directly affect the cultivating area and seasonal adaptability and yield traits of rice cultivars.
  • Heading date and yield of rice are typical complex quantitative traits controlled by multiple genes. There are usually multiple SNP loci in a specific candidate gene, and the phenotypic traits are not caused by a single SNP, but by a specific combination of multiple SNPs. is a haplotype. Haplotype analysis of SNP information in a large number of rice germplasm resources can provide more abundant information for candidate gene research on heading date and yield of rice, as well as directional selection in the process of natural selection and artificial domestication. A more reliable reference.
  • a technical problem to be solved by the present invention is how to shorten the flowering time of plants.
  • the present invention firstly provides any of the following applications of a protein or a substance that regulates the activity or content of the protein:
  • Said protein (whose name is OsDREB1C) is the following A1), A2), A3) or A4):
  • amino acid sequence is the protein of sequence 1;
  • amino acid sequence shown in SEQ ID NO: 1 in the sequence listing has undergone the substitution and/or deletion and/or addition of one or several amino acid residues and has the same function;
  • A3 A protein derived from rice, millet, maize, sorghum, goat grass, wheat or Brachypodium bismuth and having 64% or more identity with sequence 1 and the same function as the protein described in A1);
  • A4 A fusion protein obtained by linking a tag to the N-terminus or/and C-terminus of A1) or A2) or A3).
  • amino-terminal or carboxyl-terminal of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing can be attached with the tags shown in the following table.
  • the protein in the above A2) is a protein having 64% or more identity to the amino acid sequence of the protein shown in SEQ ID NO: 1 and having the same function. Said having 64% or more identity is having 64%, having 75%, having 80%, having 85%, having 90%, having 95%, having 96%, having 97%, having 98% or having 99% % identity.
  • the protein in the above A2) can be artificially synthesized, or can be obtained by first synthesizing its encoding gene and then carrying out biological expression.
  • the coding gene of the protein in the above-mentioned A2) can be obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 2, and/or carrying out missense mutation of one or several base pairs, and/ Or the coding sequence of the tag shown in the above table is connected to its 5' end and/or 3' end.
  • the DNA molecule shown in SEQ ID NO: 2 encodes the protein shown in SEQ ID NO: 1.
  • the present invention also provides any of the following applications of the biomaterial related to OsDREB1C:
  • the biological material is any one of the following B1) to B9):
  • B2 an expression cassette containing the nucleic acid molecule of B1);
  • B3 a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2);
  • B4 a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);
  • B5 a transgenic plant cell line containing the nucleic acid molecule of B1), or a transgenic plant cell line containing the expression cassette of B2);
  • B6 a transgenic plant tissue containing the nucleic acid molecule of B1), or a transgenic plant tissue containing the expression cassette of B2);
  • B7 a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);
  • B9 An expression cassette, recombinant vector, recombinant microorganism, transgenic plant cell line, transgenic plant tissue or transgenic plant organ comprising the nucleic acid molecule of B8).
  • nucleic acid molecule of B1) may be as follows b11) or b12) or b13) or b14) or b15):
  • the coding sequence is the cDNA molecule or DNA molecule of sequence 2 in the sequence listing;
  • b14 has 73% or more identity to the nucleotide sequence defined by b11) or b12) or b13), and encodes a cDNA molecule or DNA molecule of OsDREB1C;
  • b15 hybridizes under stringent conditions to a nucleotide sequence defined in b11) or b12) or b13) or b14) and encodes a cDNA molecule or DNA molecule of OsDREB1C;
  • the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.
  • nucleotide sequence encoding the OsDREB1C protein of the present invention can easily mutate the nucleotide sequence encoding the OsDREB1C protein of the present invention using known methods, such as directed evolution and point mutation.
  • Those artificially modified nucleotides with 73% or higher identity to the nucleotide sequence of the OsDREB1C protein isolated by the present invention, as long as they encode the OsDREB1C protein and have the function of the OsDREB1C protein, are all derived from the nucleus of the present invention. nucleotide sequences and are equivalent to the sequences of the present invention.
  • identity refers to sequence similarity to a native nucleic acid sequence. “Identity” includes 73% or more, or 85% or more, or 90% or more, or 95% or more of the nucleotide sequence of the protein consisting of the amino acid sequence shown in the coding sequence 1 of the present invention. Nucleotide sequences of higher identity. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.
  • the stringent conditions may be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO 4 and 1 mM EDTA, 50°C, 2 ⁇ SSC, 0.1 Rinse in %SDS; also: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO 4 and 1mM EDTA, rinse at 50°C, 1 ⁇ SSC, 0.1% SDS; also: 50°C , hybridized in a mixed solution of 7% SDS, 0.5M NaPO 4 and 1 mM EDTA, washed at 50°C, 0.5 ⁇ SSC, 0.1% SDS; also: 50°C, in 7% SDS, 0.5M NaPO 4 and Hybridize in a mixed solution of 1mM EDTA, rinse in 0.1 ⁇ SSC, 0.1% SDS at 50°C; alternatively: hybridize in a mixed solution of 7% SDS, 0.5M NaPO
  • the above-mentioned 73% or more identity may be 80%, 85%, 90% or more than 95% identity.
  • B2 described expression cassette (OsDREB1C gene expression cassette) containing the nucleic acid molecule of coding OsDREB1C protein, refers to the DNA that can express OsDREB1C protein in host cell, this DNA can not only include the start that starts OsDREB1C gene transcription It can also include a terminator that terminates transcription of the OsDREB1C gene. Further, the expression cassette may also include enhancer sequences. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development specific promoters, and inducible promoters.
  • promoters include, but are not limited to: the constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiol acid S-methyl ester)); tomato Protease inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoter (US Pat. No. 5,187,267); tetracycline-inducible promoter (US Pat. No.
  • seed-specific promoters such as foxtail millet seed-specific promoter pF128 (CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (for example, promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety.
  • Suitable transcription terminators include, but are not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine Synthase terminators (see, eg: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al.
  • NOS terminator Agrobacterium nopaline synthase terminator
  • cauliflower mosaic virus CaMV 35S terminator tml terminator
  • pea rbcS E9 terminator nopaline and octopine Synthase terminators
  • the recombinant vector containing the OsDREB1C gene expression cassette can be constructed by using the existing expression vector.
  • the plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301 or pCAMBIA1391-Xb (CAMBIA company) and so on.
  • the plant expression vector may also contain the 3' untranslated region of the foreign 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 Agrobacterium crown gall-inducing (Ti) plasmid genes (such as nopaline synthase gene Nos), plant genes (such as soybean The untranslated regions transcribed from the 3' end of the storage protein gene) have similar functions.
  • enhancers can also be used, including translation enhancers or transcription enhancers.
  • the translation control signals and initiation codons can be derived from a wide variety of sources, either natural or synthetic.
  • the translation initiation region can be derived from a transcription initiation region or a structural gene.
  • the plant expression vector used can be processed, such as adding a gene (GUS gene, luciferase gene, luciferase gene) that can be expressed in plants encoding an enzyme that can produce color change or a luminescent compound.
  • marker genes for antibiotics such as the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, the hph gene that confers resistance to the antibiotic hygromycin , and the dhfr gene conferring resistance to methotrexate, the EPSPS gene conferring resistance to glyphosate
  • marker genes for chemical resistance such as herbicide resistance genes
  • mannose-6- which provides the ability to metabolize mannose Phosphoisomerase gene.
  • the vector may be a plasmid, cosmid, phage or viral vector.
  • the plasmid may specifically be a pBWA(V)HS vector or a psgR-Cas9-Os-containing plasmid.
  • the recombinant vector can specifically be pBWA(V)HS-OsDREB1C.
  • the pBWA(V)HS-OsDREB1C is a recombinant vector obtained by inserting the OsDREB1C encoding gene shown in SEQ ID NO: 2 in the sequence listing at the BsaI (Eco31I) restriction site of the pBWA(V)HS vector.
  • the pBWA(V)HS-OsDREB1C can overexpress the protein encoded by the OsDREB1C gene (ie, the OsDREB1C protein shown in sequence 1) under the drive of the CaMV 35S promoter.
  • the recombinant vector can be a recombinant vector prepared by using the crisper/cas9 system, which can reduce the content of OsDREB1C.
  • the recombinant vector can express sgRNA targeting the nucleic acid molecule of B1).
  • the target sequence of the sgRNA can be positions 356-374 of sequence 2 in the sequence listing.
  • the microorganisms may be yeast, bacteria, algae or fungi.
  • the bacteria can be Agrobacterium, such as Agrobacterium rhizogenes EHA105.
  • transgenic plant cell lines, transgenic plant tissues and transgenic plant organs may all include propagating material or may not include propagating material.
  • the present invention also provides any of the following methods:
  • X1 a method for cultivating a plant with an advanced flowering time, comprising expressing OsDREB1C in the recipient plant, or increasing the content or activity of OsDREB1C in the recipient plant, to obtain a target plant with an advanced flowering time;
  • X2 a method for cultivating plants with delayed flowering time, comprising reducing the content or activity of OsDREB1C in the recipient plant, or inhibiting the expression of the gene encoding OsDREB1C in the recipient plant, to obtain a target plant with delayed flowering time compared with the recipient plant;
  • X3 a method for cultivating plants with advanced flowering time and increased yield, comprising expressing OsDREB1C in the recipient plant, or increasing the content or activity of OsDREB1C in the recipient plant, to obtain a target plant with advanced flowering time and increased yield;
  • a method for cultivating plants with enhanced photosynthesis, earlier flowering time and increased yield comprising expressing OsDREB1C in recipient plants, or increasing the content or activity of OsDREB1C in recipient plants, to obtain enhanced photosynthesis, earlier flowering time and increased yield target plant.
  • the methods described in X1)-X4) can be realized by introducing the gene encoding OsDREB1C into the recipient plant and expressing the gene encoding.
  • the encoding gene can be the nucleic acid molecule of B1).
  • the encoding gene of OsDREB1C can be modified as follows first, and then introduced into the recipient plant to achieve better expression effect:
  • the promoters may include constitutive, inducible, time-sequential regulation, developmental regulation, chemical regulation, tissue-preferred and tissue-specific promoters ; the choice of promoter will vary with the temporal and spatial requirements of expression and will also depend on the target species; e.g. tissue- or organ-specific expression promoters, depending on what stage of development the receptor is desired; although the provenance of the source Many promoters for dicotyledonous plants are functional in monocotyledonous plants and vice versa, but ideally, a dicotyledonous promoter is chosen for expression in dicotyledonous plants and a monocotyledonous promoter for expression in monocots;
  • Linking with a suitable transcription terminator can also improve the expression efficiency of the gene of the present invention; for example, tml derived from CaMV, E9 derived from rbcS; any available terminator known to function in plants can be combined with The gene of the present invention is connected;
  • enhancer sequences such as intron sequences (eg from Adhl and bronzel) and viral leader sequences (eg from TMV, MCMV and AMV).
  • the OsDREB1C-encoding gene can be introduced into recipient plants using a recombinant expression vector containing the OsDREB1C-encoding gene.
  • the recombinant expression vector can specifically be the pBWA(V)HS-OsDREB1C.
  • the recombinant expression vector can be introduced into plant cells by using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation and other conventional biotechnology methods (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York). , pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition).).
  • the plant of interest is understood to include not only the first-generation plants in which the OsDREB1C protein or its encoding gene has been altered, but also its progeny.
  • the gene can be propagated in that species, and conventional breeding techniques can be used to transfer the gene into other varieties of the same species, including in particular commercial varieties.
  • the plants of interest include seeds, callus, whole plants and cells.
  • the present invention also provides a product having any of the following functions D1)-D3), the product containing OsDREB1C or the biological material:
  • the plant may be M1) or M2) or M3):
  • M2 grasses, crucifers or legumes
  • the photosynthesis can be embodied in photosynthetic rate, net photosynthetic rate, heat dissipation NPQ, maximum carboxylation efficiency and/or maximum electron transfer rate;
  • the flowering time can be reflected in the heading stage
  • the yield can be expressed in grain yield, shoot yield and/or harvest index.
  • the harvest index refers to the ratio of economic yield (grain, fruit, etc.) to biological yield when a plant is harvested.
  • the modulation can be enhancing or inhibiting, or promoting or inhibiting, or increasing or decreasing.
  • Another technical problem to be solved by the present invention is how to detect the heading stage characters of rice.
  • the present invention first provides a method for detecting the traits of heading stage of rice, the method comprising: detecting the rice haplotypes to be tested, the rice haplotypes are Hap.1, Hap.2 and Hap .3, the Hap.1 in the rice reference genome version number Os-Nipponbare-Reference-IRGSP-1.0 (IRGSP-1.0) (updated 2013.2.6, URL https://rapdb.dna.affrc.go.jp/ ) of the physical positions 1433007, 1433155, 1433229, 1433365, 1433528, 1433919, 1434216, 1434258, 1434486, 1434806
  • the nucleotides are A, A, G, C, A, G, G, C, T and G in sequence;
  • the nucleotides at positions 1433007, 1433155, 1433229, 1433365, 1433528, 1433919, 1434216, 1434258, 1434486, and 1434806 are A, G, G, C, T, G, G, C, T and G in sequence;
  • the nucleotides at positions 1433007, 1433155, 1433229, 1433365, 1433528, 1433919, 1434216, 1434258, 1434486, and 1434806 are G, G, A, G, T, A, A, T, G and T in sequence;
  • the heading date traits of the tested rice were judged according to the following steps: the homozygous test rice with haplotypes Hap2 and Hap3 had an early heading date, and the homozygous test rice with haplotype Hap1 had a late heading date.
  • the present invention also provides another method for detecting rice heading stage traits, the method comprising: detecting the rice haplotypes to be tested, the rice haplotypes are Hap.1, Hap.2 and Hap.3, and the Hap. .1 at physical location 1433007 of the Rice Reference Genome Version No.
  • the nucleotides of 1433155, 1433229, 1433365, 1433528, 1433919, 1434216, 1434258, 1434486 and 1434806 are A, A, G, C, A, G, G, C, T and G in sequence;
  • the nucleotides at positions 1433007, 1433155, 1433229, 1433365, 1433528, 1433919, 1434216, 1434258, 1434486, and 1434806 are A, G, G, C, T, G, G, C, T and G in sequence;
  • the nucleotides at positions 1433007, 1433155, 1433229, 1433365, 1433528, 1433919, 1434216, 1434258, 1434486, and 1434806 are G, G, A, G, T, A, A, T, G and T in sequence;
  • the heading date traits of the rice to be tested are judged according to the following steps: the heading date of the tested rice homozygous for the Hap1 haplotype is longer than that of the Hap.2 haplotype or the Hap.3 haplotype homozygous for the tested rice, and the Hap.2 haplotype pure The heading date of the tested rice was no different from that of the Hap.3 haplotype homozygous tested rice.
  • the present invention also provides a rice breeding method, the method comprising: detecting the rice reference genome version number Os-Nipponbare-Reference-IRGSP-1.0 (IRGSP-1.0) in the rice genomic DNA (updated date 2013.2.6, website https:/ /rapdb.dna.affrc.go.jp/) nucleotides at physical locations 1433007, 1433155, 1433229, 1433365, 1433528, 1433919, 1434216, 1434258, 1434486, 1434806, select 14333007, 1433155, 1433328, 143343529, 4
  • the nucleotides of 1433919, 1434216, 1434258, 1434486, 1434806 are A, G, G, C, T, G, G, C, T, G rice or G, G, A, G, T, A, A , T, G, T rice were bred as parents.
  • the present invention also provides the application of a substance for detecting rice haplotypes in detecting rice heading stage traits, wherein the rice haplotypes are Hap.1, Hap.2 and Hap.3, and the Hap.1 is in the rice reference genome Version number Os-Nipponbare-Reference-IRGSP-1.0 (IRGSP-1.0) (updated 2013.2.6, URL https://rapdb.dna.affrc.go.jp/) Physical location 1433007, 1433155, 1433229, 1433365, The nucleotides of 1433528, 1433919, 1434216, 1434258, 1434486, and 1434806 are A, A, G, C, A, G, G, C, T and G in sequence;
  • the nucleotides at positions 1433007, 1433155, 1433229, 1433365, 1433528, 1433919, 1434216, 1434258, 1434486, and 1434806 are A, G, G, C, T, G, G, C, T and G in sequence;
  • the nucleotides at positions 1433007, 1433155, 1433229, 1433365, 1433528, 1433919, 1434216, 1434258, 1434486, 1434806 are G, G, A, G, T, A, A, T, G, and T, in that order.
  • the present invention also provides an application of a substance for detecting rice haplotypes in preparing a product for detecting rice heading stage traits, wherein the rice haplotypes are Hap.1, Hap.2 and Hap.3, and the Hap.1 in rice Reference genome version number Os-Nipponbare-Reference-IRGSP-1.0 (IRGSP-1.0) (updated on 2013.2.6, URL https://rapdb.dna.affrc.go.jp/) Physical locations 1433007, 1433155, 1433229, The nucleotides of 1433365, 1433528, 1433919, 1434216, 1434258, 1434486, and 1434806 are A, A, G, C, A, G, G, C, T and G in sequence;
  • the nucleotides at positions 1433007, 1433155, 1433229, 1433365, 1433528, 1433919, 1434216, 1434258, 1434486, and 1434806 are A, G, G, C, T, G, G, C, T and G in sequence;
  • the nucleotides at positions 1433007, 1433155, 1433229, 1433365, 1433528, 1433919, 1434216, 1434258, 1434486, 1434806 are G, G, A, G, T, A, A, T, G, and T, in that order.
  • the detection of heading stage traits of rice can be carried out by using the substance for detection of rice haplotypes.
  • the substances for detecting rice haplotypes can be primers and/or probes capable of detecting the Hap.1, the Hap.2 and the Hap.3, and can also be used for sequencing (such as first-generation sequencing or high-pass sequencing). kits and/or instruments required for quantitative sequencing).
  • Figure 1 shows the sequence alignment results.
  • Figure 2 shows the relative expression level detection of OsDREB1C gene in transgenic rice and the sequence detection results of the target region of gene knockout rice material.
  • A Relative expression level of OsDREB1C gene
  • B gene editing site of OsDREB1C gene knockout rice.
  • Figure 3 shows photosynthesis parameters of wild-type and transgenic rice plants.
  • a - diurnal variation of photosynthesis B - diurnal variation of NPQ;
  • C - light response curve D - CO 2 response curve;
  • E - maximum CO 2 carboxylation efficiency E - maximum electron transfer rate.
  • Figure 4 shows the detection results of nitrogen uptake and utilization of wild-type and transgenic rice plants.
  • A- 15 N content in shoots B- 15 N content in roots; C- 15 N uptake efficiency; D- 15 N transport efficiency from roots to shoots; E- nitrogen content in different tissues of rice; F- different tissues of rice distribution ratio of nitrogen.
  • E and F from top to bottom, the grains, stems and leaves are in order.
  • FIG. 5 shows the detection result of Bar gene in transgenic wheat (A) and the detection result of relative expression level of OsDREB1C gene in transgenic Arabidopsis (B).
  • Figure 6 shows the phenotypes of wild type and transgenic wheat.
  • A Wild-type wheat (Fielder) and transgenic wheat phenotypes.
  • B-D Heading time (B), photosynthetic rate (C), number of grains per ear (D), 1000-grain weight (E), and grain yield per plant (F) for wild-type and transgenic lines.
  • Figure 7 is a phenotypic profile of wild-type and transgenic Arabidopsis.
  • A Arabidopsis wild-type Col-0 and transgenic line phenotypes.
  • B-D Bolting time (B), number of rosette leaves (C), and fresh shoot weight (D) of Arabidopsis wild-type and transgenic lines.
  • Figure 8 is a sequence analysis of three haplotypes.
  • Figure 9 is the heading date analysis of the three haplotypes.
  • the ordinate is heading date, Hap1, Hap2, and Hap3 represent Hap.1, Hap.2, and Hap.3, respectively, and different letters represent significant differences between haplotypes (P ⁇ 0.05, Duncan multiple range test).
  • the pBWA(V)HS vector in the following examples (Zhao et al., DEP1is involved in regulating the carbon-nitrogen metabolic balance to affect grain yield and quality in rice (Oriza sativa L.), PLOS ONE, March 11, 2019 , https://doi.org/10.1371/journal.pone.0213504), the public can obtain the biological material from the applicant, and the biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.
  • Plasmids containing psgR-Cas9-Os in the following examples Hu Xuejiao, Yang Jia, Cheng Can, Zhou Jihua, Niu Fuan, Wang Xinqi, Zhang Meiliang, Cao Liming, Chu Huangwei.
  • CRISPR/Cas9 system to edit the SD1 gene of rice.
  • Application of the CRISPR-Cas system for efficient genome engineering in plants. Mol Plant, 2013, 6( 6): 2008-2011. the public can obtain the biological material from the applicant, and the biological material is only used for repeating the relevant experiments of the present invention, and cannot be used for other purposes.
  • Example 1 OsDREB1C has the effect of improving rice photosynthetic efficiency, promoting nitrogen absorption, promoting early heading and increasing yield
  • This example provides a protein derived from Nipponbare rice, which has the functions of improving rice photosynthesis efficiency, promoting nitrogen absorption, promoting early heading and improving yield.
  • the name of the protein is OsDREB1C, and its sequence is sequence 1 in the sequence table.
  • the coding gene sequence of OsDREB1C is sequence 2, and the genome sequence is 3001-4131 of sequence 3.
  • positions 1-3000 are the promoter of OsDREB1C gene in Nipponbare genomic DNA.
  • PCR product containing the full-length CDS of OsDREB1C gene was amplified from rice Nipponbare cDNA by PCR, and the obtained PCR product was digested with BsaI (Eco31I).
  • the pBWA(V)HS vector was linked to the backbone of the vector obtained by single digestion with BsaI(Eco31I), and the resulting recombinant vector with the correct sequence was denoted as pBWA(V)HS-OsDREB1C.
  • pBWA(V)HS-OsDREB1C is a recombinant vector obtained by inserting the OsDREB1C encoding gene shown in SEQ ID NO: 2 in the sequence table at the BsaI(Eco31I) restriction site of the pBWA(V)HS vector.
  • pBWA(V)HS-OsDREB1C can The protein encoded by the OsDREB1C gene (ie the OsDREB1C protein shown in sequence 1) was expressed under the drive of the CaMV 35S promoter.
  • the primer sequences used are as follows:
  • OsDREB1C-F 5'-CAGTGGTCTCACAACATGGAGTACTACGAGCAGGAGGAGT-3' (sequence 4 in the sequence listing);
  • OsDREB1C-R 5'-CAGTGGTCTCATACATCAGTAGCTCCAGAGTGTGACGTCG-3' (sequence 5 in the sequence listing).
  • sgRNA and primers were designed according to the online design website ( http://skl.scau.edu.cn/ ), and the target sequence was determined to be 5′-AGTCATGCCCGCACGACGC-3′ (No. 356-374 of sequence 2). bits).
  • the OsDREB1C-sgRNA-F and OsDREB1C-sgRNA-R were annealed, the resulting product was digested with BsaI, and the resulting digested product was connected to the vector skeleton obtained by digesting the psgR-Cas9-Os plasmid with BsaI, and the obtained
  • the recombinant vector with the correct sequence is the OsDREB1C knockout vector, denoted as OsU3-sgRNA-OsUBI-Cas9-OsDREB1C.
  • the OsU3 promoter drives sgRNA and the OsUBI promoter drives Cas9.
  • primer sequences used are as follows:
  • OsDREB1C-sgRNA-F 5'-TGTGTGGCGTCGTGCGGGCATGACT-3' (SEQ ID NO: 6 in the sequence listing);
  • OsDREB1C-sgRNA-R 5'-AAACAGTCATGCCCGCACGACGCCA-3' (SEQ ID NO: 7 in the sequence listing).
  • the mature seeds of the japonica rice variety Nipponbare were sterilized and induced to obtain embryogenic callus.
  • the pBWA(V)HS-OsDREB1C and OsU3-sgRNA-OsUBI-Cas9-OsDREB1C obtained in step 1 were introduced into Agrobacterium EHA105, respectively, and the Bacillus-mediated rice genetic transformation method infects and co-cultivates callus, and uses resistance screening to obtain transgenic plants.
  • the screened transgenic rice obtained from pBWA(V)HS-OsDREB1C is OsDREB1C transgenic rice.
  • the transgenic rice obtained by sgRNA-OsUBI-Cas9-OsDREB1C is the OsDREB1C knockout rice material.
  • the primers used were: 5′-CATGATGATGCAGTACCAGGA-3′ (Sequence 8 in the sequence listing), 5'-GATCATCAGTAGCTCCAGAGTG-3' (sequence 9 in the sequence listing);
  • the internal reference gene is rice Ubiqutin, and the primers for the internal reference gene are: 5'-AAGAAGCTGAAGCATCCAGC-3' (sequence 10 in the sequence listing), 5 '-CCAGGACAAGATGATCTGCC-3' (sequence 11 in the sequence listing).
  • the three lines (KO1, KO2 and KO3) of OsDREB1C knockout rice material were amplified and sequenced by PCR using primer pairs capable of amplifying the target sequence and its upstream and downstream. The results showed that the target sites of these three lines were The sequence changes are shown in B in Figure 2.
  • One nucleotide deletion occurred in KO1 and KO2 one nucleotide insertion occurred in KO3, and frameshift mutations occurred in the target genes of the three lines.
  • the rice to be tested wild-type Nipponbare rice (WT), OsDREB1C overexpressing rice (OE1/OE2/OE5), OsDREB1C knockout rice (KO1/KO2/KO3).
  • the flag leaves of the rice to be tested at the heading stage were measured with a LICOR-6400XT portable photosynthesis instrument (LI-COR, USA) to measure the photosynthetic diurnal variation, light response curve and CO 2 response curve, which reflect the photosynthetic capacity of plants.
  • the determination of the diurnal variation of photosynthesis was carried out in clear and cloudless weather, and the measurement was carried out every 2-4 hours from 8:00 to 16:00.
  • the rate of photosynthesis was measured with a LI-COR 6400XT portable photosynthesis instrument, and NPQ (non-photochemical quenching) was measured with a FluorPen FP100 (PSI, Czech Republic).
  • the CO 2 concentration was set to 400 ⁇ mol mol ⁇ 1 , and the light intensity (PPFD) was 0 to 2000 ⁇ mol m ⁇ 2 s ⁇ 1 .
  • the PPFD was set at 1200 ⁇ mol m ⁇ 2 s ⁇ 1 , and the CO 2 concentration was decreased from 400 to 50 ⁇ mol mol ⁇ 1 and then increased from 400 to 1200 ⁇ mol mol ⁇ 1 again.
  • the light response curve and CO 2 response curve were both fitted by the Farquhar-von Caemmerer-Berry (FvCB) model, and the maximum carboxylation efficiency (V cmax ) and the maximum electron transfer rate (J max ) were calculated from the CO 2 response curve .
  • FvCB Farquhar-von Caemmerer-Berry
  • the rice to be tested wild-type Nipponbare rice (WT), OsDREB1C overexpressing rice (OE1/OE2/OE5), OsDREB1C knockout rice (KO1/KO2/KO3).
  • the rice seedlings to be tested that were grown in nutrient solution hydroponics for 3 weeks in the greenhouse were placed in Kimura B solution (Kimura B solution) without nitrogen (without (NH 4 ) 2 SO 4 and KNO 3 ) in advance for nitrogen starvation treatment 3 sky.
  • Kimura B solution Kimura B solution
  • the roots of seedlings were completely immersed in 0.1 mM CaSO 4 solution (solvent is deionized water) for 1 minute, and then the residual water was absorbed, and the roots were placed in a nutrient solution containing 0.5 mM K 15 NO 3 for cultivation. After 3 hours, the roots of the seedlings were soaked in 0.1 mM CaSO 4 solution for 1 minute, and the residual water was absorbed.
  • the nutrient solution used is as follows:
  • the heading date of rice was detected, and the rice to be tested: wild-type Nipponbare rice (WT), OsDREB1C overexpressing rice (OE1/OE2/OE5), and OsDREB1C knockout rice (KO1/KO2/KO3).
  • the heading date of each tested rice was counted under the Beijing field planting conditions.
  • the statistical method is as follows: 3 plots of each type of rice to be tested are planted in duplicate and randomly arranged, and 50% of the plants in the plot are recorded as heading when the ears of 50% of the plants are exposed from the flag leaf sheaths, and the heading period is the experience from sowing to heading. days.
  • the aboveground dry weight and grain yield of rice were detected.
  • the rice to be tested wild-type Nipponbare rice (WT), OsDREB1C overexpressing rice (OE1/OE2/OE5), and OsDREB1C gene knockout rice (KO1/KO2/KO3).
  • each type of rice to be tested was planted in 3 replicate plots and randomly arranged. After the rice grains were mature, the grain yield per plant, the plot grain yield, and the dry weight of the aboveground straw were measured and calculated.
  • Harvest index is the ratio of rice grain yield per plant to above-ground biomass (the sum of above-ground straw dry weight and grain yield per plant).
  • the method for measuring the dry weight of aboveground straw is as follows: after the rice is mature, after removing the grains from the straw per plant, put it into a nylon mesh bag and dry it to constant weight at 80°C, and then weigh the sample. 20-30 replicates of each rice material were taken for statistical analysis.
  • the measurement method of grain yield is as follows: threshing and removing the shriveled grains per plant of rice is the grain yield per plant, and 20-30 single plants of each rice material are repeated for statistical analysis.
  • the side row was removed, and the 30 rice plants in the middle were taken to measure the grain weight as the yield of one plot, and 3 plots were repeated for statistical analysis.
  • the measurement method of rice quality is as follows: the rice grains harvested in the Beijing field experiment in 2019 were used for rice quality analysis after natural storage for 3 months. The results showed that the grain yield of rice overexpressing OsDREB1C was significantly higher than that of the wild type, and the difference was significant.
  • the brown rice rate, milled rice rate, and whole milled rice rate were all significantly higher than those of the wild type, and the chalkiness and chalky grain rate were reduced, which improved the appearance quality. , while the amylose and protein content had no significant effect.
  • the yield per plant and plot of OsDREB1C knockout rice were significantly lower than those of the wild type, and the above-ground biomass was increased, which directly led to a 22.4-37.7% reduction in the harvest index compared with the wild type.
  • the results indicated that OsDREB1C gene transfer into rice can greatly improve the yield, rice quality and harvest index of transgenic rice.
  • OsDREB1C and its encoded gene can regulate the grain yield, rice quality and harvest index of rice.
  • PCR was used to amplify the CDS full-length of OsDREB1C gene from rice Nipponbare cDNA, and the obtained PCR recovery product was combined with pWMB110 vector (Huiyun Liu, Ke Wang, Zimiao Jia, Qiang Gong,Zhishan Lin,Lipu Du,Xinwu Pei,Xingguo Ye,Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system,Journal of Experimental Botany,Volume 71,Issue 4,7 February 2020, Pages 1337–1349, https://doi.org/10.1093/jxb/erz529)
  • the backbone of the vector obtained by double digestion with BamHI and SacI was linked, and the resulting recombinant vector with the correct sequence was denoted as pWMB110-OsDREB1C.
  • pWMB110-OsDREB1C is a recombinant vector obtained by replacing the DNA fragment between the BamHI and SacI recognition sites of the pWMB110 vector with the OsDREB1C coding gene shown in SEQ ID NO: 2 in the sequence listing.
  • pWMB110-OsDREB1C can express the OsDREB1C gene under the drive of the UBI promoter.
  • the encoded protein ie, the OsDREB1C protein shown in SEQ ID NO: 1).
  • the primer sequences used are as follows:
  • Fielder-OsDREB1C-OE-F 5′-CAGGTCGACTCTAGA GGATCC ATGGAGTACTACGAGCAGGAG-3′ (sequence 12 in the sequence listing);
  • Fielder-OsDREB1C-OE-R 5'-CGATCGGGGAAATTC GAGCTC TCAGTAGCTCCAGAGTGTGAC-3' (sequence 13 in the Sequence Listing).
  • pGWBs gateway binary vectors
  • the CDS sequence of OsDREB1C was transferred into the final vector pGWB5, and the resulting recombinant vector with correct sequence was denoted as pGWB5-OsDREB1C.
  • pGWB5-OsDREB1C can express the protein encoded by the OsDREB1C gene (ie, the OsDREB1C protein shown in sequence 1) under the drive of the CaMV 35S promoter.
  • the primer sequences used are as follows:
  • OsDREB1C-CDS-F 5'-CACCATGGAGTACTACGAGCAGGAG-3' (sequence 14 in the sequence listing);
  • OsDREB1C-CDS-R 5'-GTAGCTCCAGGTGTGACGTC-3' (sequence 15 in the sequence listing).
  • Embryogenic callus was obtained by inducing young embryos of wheat cultivar Fielder after disinfection.
  • the pWMB110-OsDREB1C obtained in step 1 was introduced into Agrobacterium C58C1, and the callus was infected and co-cultured by Agrobacterium-mediated wheat genetic transformation. , using resistance screening to obtain transgenic plants, the screened OsDREB1C transgenic wheat material. Due to the high similarity between OsDREB1C and its own homologous genes in wheat, the expression level of OsDREB1C gene in rice could not be detected by qRT-PCR method. Therefore, using the wheat wild-type Fielder as a control, the PCR method was used to detect whether the Bar gene of the vector existed in the cDNA of transgenic wheat.
  • the primers used were: 5'-CAGGAACCGCAGGAGTGGA-3' (sequence 16 in the sequence table), 5'-CCAGAAACCCACGTCATGCC-3' (Sequence 17 in the Sequence Listing).
  • the results of electrophoresis showed that the Bar gene was present in all three lines (TaOE-5, TaOE-8 and TaOE-9) of OsDREB1C transgenic wheat, but not in the wild type, indicating that these three lines were all transgenic Transgenic material of the pWMB110-OsDREB1C vector (A in Figure 5).
  • the pGWB5-OsDREB1C obtained in step 1 was introduced into Agrobacterium GV3101, and transformed into Arabidopsis wild-type Col-0 by the Agrobacterium-mediated flower soaking method.
  • OsDREB1C transgenic Arabidopsis material For OsDREB1C transgenic Arabidopsis material.
  • Arabidopsis wild-type Col-0 as a control, the relative expression level of OsDREB1C gene at RNA level in OsDREB1C transgenic Arabidopsis was detected by qRT-PCR method.
  • the primers used were: 5′-CATGATGATGCAGTACCAGGA-3′ (Sequence Listing Middle sequence 18), 5'-GATCATCAGTAGCTCCAGAGTG-3' (sequence 19 in the sequence table); the internal reference gene is Arabidopsis Actin, and the internal reference gene primer is: 5'-GCACCACCTGAAAGGAAGTACA-3' (sequence 20 in the sequence table), 5' - CGATTCCTGGACCTGCCTCATC-3' (sequence 21 in the Sequence Listing).
  • the tested materials wild-type wheat (Fielder), overexpressing OsDREB1C wheat (TaOE-5, TaOE-8 and TaOE-9).
  • the greenhouse temperature was controlled at 22-24°C, and the sunshine length was 12 hours light/12 hours dark.
  • the heading dates of wild-type and transgenic wheat were counted, and the photosynthesis rate of the flag leaves of the tested wheat at the heading stage was measured with a LICOR-6400XT portable photosynthesis instrument (LI-COR, USA), and the light intensity was set to 1000 ⁇ mol m -2 s - 1 . After the wheat grains were mature, the number of grains per ear, 1000-grain weight and grain yield per plant were determined.
  • the materials to be tested were wild-type Arabidopsis (Col-0), overexpressing OsDREB1C Arabidopsis (AtOE-10, AtOE-11 and AtOE-12). Cultures were cultured under short-day (8 hours light/16 hours dark) for 2 weeks and then transferred to long day (16 hours light/8 hours dark) cultures.
  • Bolting time is the number of days from sowing to stem elongation and flowering.
  • the tested materials were 709 rice core germplasm materials, including 299 indica, 355 temperate japonica, 14 tropical japonica, and 41 intermediate.
  • the heading period is the number of days from sowing to heading.
  • the nucleotides of these 10 positions of Hap.1 are A, A, G, C, A, G, G, C, T and G in sequence
  • the nucleotides of these 10 positions of Hap.2 are in order A, G, G, C, T, G, G, C, T and G
  • the nucleotides of these 10 positions of Hap.3 are G, G, A, G, T, A, A, T in order , G and T
  • the haplotypes of each rice material are shown in Tables 13 and 14.
  • the heading date of Hap.1 haplotype rice is 93.6 ⁇ 0.9 days, that of Hap2 haplotype rice is 85.9 ⁇ 2.8 days, and that of Hap.3 haplotype rice is 85.9 ⁇ 2.8 days.
  • the heading date was 82.8 ⁇ 0.7 days.
  • the heading date of Hap1 haplotype rice was significantly greater than that of Hap.2 haplotype and Hap.3 haplotype. There was no significant difference in rice (Fig. 9), indicating that the three haplotypes of rice are related to the heading date of a single rice plant and can be used for rice breeding.
  • the OsDREB1C and related biological materials of the present invention can improve the photosynthetic efficiency of plants, can promote nitrogen absorption and transport, increase nitrogen content in plants and grains, can promote early heading, and can also increase yield.
  • the invention starts from the synergistic improvement of crop photosynthetic efficiency, nitrogen utilization efficiency and heading stage, realizes the synergistic improvement of nitrogen utilization efficiency while realizing a substantial increase in crop yield, and also provides a solution for the contradiction between high crop yield and early maturity. Therefore, the present invention can further greatly improve the crop yield potential and nitrogen fertilizer utilization efficiency, and realize "high yield and high efficiency";
  • the problems of early maturity and high yield and the "superparent late maturity" of inter-subspecies hybrid rice have important application potential.
  • haplotypes 2 and 3 have excellent traits of advancing the heading date of rice, It has broad application prospects in rice genetics breeding and variety improvement.

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Abstract

La présente invention concerne une protéine OsDREB1C pour raccourcir le stade d'épiaison du riz, ainsi qu'un gène codant et une application associée. La séquence de OsDREB1C est la séquence 1 dans une table de séquence, et la séquence d'un gène codant de celle-ci est la séquence 2 dans la table de séquence. L'OsDREB1C et un biomatériau apparenté peuvent améliorer l'efficacité photosynthétique des plantes, accélérer l'absorption et le transport de l'azote et augmenter la teneur en azote des plantes et des grains, accélérer l'épiaison précoce et améliorer le rendement. La présente invention concerne également les haplotypes Hap.2 et Hap.3 basés sur le OsDREB1C, et les haplotypes présentent d'excellentes caractéristiques d'avancement du stade d'épiaison du riz.
PCT/CN2021/100543 2021-03-10 2021-06-17 Protéine pour raccourcir le stade d'épiaison du riz, et gène codant pour cette protéine et son application WO2022188287A1 (fr)

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CN202110359893.9 2021-04-02
CN202110359893.9A CN113073142B (zh) 2021-04-02 2021-04-02 利用三种单倍型检测水稻抽穗期性状的方法

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