WO2018184333A1 - 蛋白质nog1在调控植物产量和穗粒数中的应用 - Google Patents

蛋白质nog1在调控植物产量和穗粒数中的应用 Download PDF

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WO2018184333A1
WO2018184333A1 PCT/CN2017/097608 CN2017097608W WO2018184333A1 WO 2018184333 A1 WO2018184333 A1 WO 2018184333A1 CN 2017097608 W CN2017097608 W CN 2017097608W WO 2018184333 A1 WO2018184333 A1 WO 2018184333A1
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plant
protein
nog1
dna molecule
sequence
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PCT/CN2017/097608
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French (fr)
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孙传清
霍兴
谭禄宾
刘凤霞
付永彩
朱作峰
顾凭
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中国农业大学
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Priority to US16/603,199 priority Critical patent/US20210079414A1/en
Priority to JP2019555031A priority patent/JP7023979B2/ja
Priority to KR1020197031047A priority patent/KR102539626B1/ko
Publication of WO2018184333A1 publication Critical patent/WO2018184333A1/zh

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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
    • A01H5/10—Seeds
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • 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
    • 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 protein nog1 in regulating plant yield and kernel number.
  • Rice is one of the most important food crops in the world. Rice is grown in more than 120 countries around the world. The cultivated area is maintained at more than 150 million hectares per year, and the population of 50% of the world's population is mainly rice. Today, as the population continues to increase and the area of cultivated arable land decreases year by year, increasing rice yield is one of the powerful measures to ensure world food security. Looking back at the rice breeding history for more than half a century, China's rice yield has experienced two leapfrogs. The first is the green revolution marked by dwarf breeding, and the second is the utilization of rice heterosis. But in the past 20 years, rice yields have been stagnant.
  • Oryza rufipogon Griff. is a wild ancestor of cultivated rice in Asia. It has richer genetic diversity and genetic resources than cultivated rice after artificial domestication. Oryza sativa has more genetic differentiation types than cultivated rice, and contains abundant genes that can increase rice yield. Therefore, it is of great theoretical significance and practical value to excavate and utilize the excellent domesticated genes that have been lost or weakened in cultivated rice from the common wild rice genome, and apply them to rice breeding production. It is also a solution to the current rice breeding problem. An effective way.
  • the technical problem to be solved by the present invention is how to regulate plant yield and kernel number.
  • the present invention first provides the use of protein nog1 for regulating plant yield and/or kernel number; the protein nog1 may be a1) or a2) or a3) or a4):
  • amino acid sequence is the protein shown in SEQ ID NO: 2 in the Sequence Listing;
  • A2 a fusion protein obtained by ligating the N-terminus or/and C-terminus of the protein shown in SEQ ID NO: 2 in the Sequence Listing;
  • A3 a protein related to plant yield and/or kernel number obtained by subjecting the amino acid sequence shown in SEQ ID NO: 2 of the sequence listing to substitution and/or deletion and/or addition of one or several amino acid residues;
  • A4 A protein having 80% or more identity with the amino acid sequence defined by a1).
  • sequence 2 in the sequence listing consists of 389 amino acid residues.
  • a label as shown in Table 1 may be attached to the amino terminus or carboxy terminus of the protein shown in SEQ ID NO: 2 in the Sequence Listing.
  • substitution and/or deletion and/or addition of the one or several amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues.
  • the protein in the above a3) can be artificially synthesized, or the encoded gene can be synthesized first, and then obtained by biological expression.
  • the gene encoding the protein in a3) above may be obtained by deleting a codon of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 1 in the sequence listing, and/or performing one or several base pair missense mutations. And/or the coding sequence of the tag shown in Table 1 is attached at its 5' end and/or 3' end.
  • identity refers to the sequence similarity to the amino acid sequence of the protein shown by SEQ ID NO: 2 in the Sequence Listing. "Identity” includes amino acids having 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the amino acid sequence shown in SEQ ID NO: 2 of the Sequence Listing of the present invention. sequence. Identity can be evaluated using the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.
  • nucleic acid molecule encoding the protein nog1 for regulating plant yield and/or kernel number is also within the scope of the invention.
  • the nucleic acid molecule encoding the protein nog1 may be a DNA molecule as shown in b1) or b2) or b3) or b4):
  • the b2) nucleotide sequence is the DNA molecule shown in SEQ ID NO: 1 in the Sequence Listing;
  • B3 having 75% or more of the identity of the nucleotide sequence defined by b1) or b2), and encoding the DNA molecule of the protein nog1;
  • B4 a DNA molecule which hybridizes under stringent conditions to a nucleotide sequence defined by b1) or b2) and which encodes the protein nog1.
  • the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.
  • sequence 1 in the sequence listing is composed of 1170 nucleotides, and the nucleotide of the sequence 1 in the sequence listing encodes the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
  • nucleotide sequence encoding the protein nog1 of the present invention can readily mutate the nucleotide sequence encoding the protein nog1 of the present invention using known methods, such as directed evolution and point mutation methods. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of the protein nog1 isolated from the present invention, as long as the protein nog1 is encoded, are derived from the nucleoside of the present invention.
  • the acid sequence is identical to the sequence of the invention.
  • identity refers to sequence similarity to a native nucleic acid sequence. “Identity” includes a core of protein nog1 consisting of the amino acid sequence shown in SEQ ID NO: 2 of the coding sequence listing of the present invention.
  • the nucleotide sequence has a nucleotide sequence of 75% or higher, or 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher.
  • Identity can be evaluated using the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.
  • the regulated plant yield may be a regulation of plant yield per plant.
  • the number of spikes in the regulated plant may be the number of spikes of the main stem of the plant and/or the number of kernels per ear.
  • the plant may be any one of the following c1) to c7): c1) dicotyledon; c2) monocotyledon; c3) gramineous plant; c4) rice; c5) japonica; c6) rice Variety Guichao 2; c7) Dongxiang common wild rice infiltration system SIL176.
  • the present invention also provides a method 1 for cultivating a transgenic plant A or a method 2 for cultivating a transgenic plant B.
  • the method for cultivating a transgenic plant A according to the present invention may comprise the step of introducing a nucleic acid molecule encoding the protein nog1 into a recipient plant A to obtain a transgenic plant A; compared with the recipient plant A, The yield of the transgenic plant A is increased and/or the number of kernels is increased.
  • the "introducing the nucleic acid molecule encoding the protein nog1 into the recipient plant A” can be achieved by introducing the recombinant vector A into the recipient plant A; the recombinant vector A can be inserted into the expression vector.
  • the recombinant vector A may specifically be the recombinant plasmid pCAMBIA1300-NOG1.
  • the recombinant plasmid pCAMBIA1300-NOG1 may specifically replace the DNA fragment between the restriction enzyme BglII recognition sequence of the engineered plant expression vector pCAMBIA1300 and the MluI recognition sequence into a nucleotide sequence as shown in sequence 3 of the sequence listing. DNA molecule.
  • the receptor plant A may be any one of d1)-d6): d1) monocotyledon; d2) dicotyledon; d3) gramineous plant; d4) rice; d5) japonica; D6) Dongxiang common wild rice infiltration system SIL176.
  • the method for cultivating the transgenic plant A provided by the present invention may include the step of introducing a substance which inhibits the expression of the protein nog1 into the recipient plant B to obtain the transgenic plant B; compared with the recipient plant B, The yield of transgenic plant B is reduced and/or the number of kernels is reduced.
  • the "substance that inhibits expression of protein nog1" may be a specific DNA molecule, an expression cassette containing the specific DNA molecule, or a recombinant plasmid containing the specific DNA molecule;
  • the specific DNA molecule includes a sense fragment, an antisense fragment, and a spacer fragment located therebetween; the sense fragment is the inverse of the DNA molecule shown in sequence 1 to position 522 of the sequence 1 of the sequence listing from the 5' end.
  • the complementary sequence; the antisense fragment is the DNA molecule shown in sequence 1 to position 522 of the sequence 1 of the sequence listing from the 5' end.
  • the recombinant plasmid containing the specific DNA molecule may specifically be the recombinant plasmid pRNAi-nog1.
  • the recombinant plasmid pRNAi-nog1 may specifically replace the small DNA fragment between the BamHI recognition sequence and the KpnI recognition sequence of the vector pTCK303/JL1460 with a nucleotide sequence, and the sequence 1 of the sequence table is from the 5th end to the 155th position.
  • the recipient plant B may be any one of e1)-e6): e1) monocotyledon; e2) dicotyledon; e3) gramineous plant; e4) rice; e5) japonica; E6) Rice variety Gui Chao No. 2.
  • the present invention also provides a method 3 for cultivating a transgenic plant C.
  • the method for cultivating the transgenic plant C according to the present invention may comprise the step of introducing a substance which increases the expression and/or activity of the protein nog1 into the receptor plant C, to obtain a transgenic plant C; and the receptor plant C In comparison, the yield of the transgenic plant C is increased and/or the number of kernels is increased.
  • the "substance for increasing the expression and/or activity of the protein nog1" may specifically be the recombinant vector A.
  • the receptor plant C may be any one of d1)-d6): d1) monocotyledon; d2) dicotyledon; d3) gramineous plant; d4) rice; d5) japonica; D6) Dongxiang common wild rice infiltration system SIL176.
  • the nucleic acid molecule encoding the protein nog1 may be a DNA molecule as shown in b1) or b2) or b3) or b4):
  • the b2) nucleotide sequence is the DNA molecule shown in SEQ ID NO: 1 in the Sequence Listing;
  • B3 having 75% or more of the identity of the nucleotide sequence defined by b1) or b2), and encoding the DNA molecule of the protein nog1;
  • B4 a DNA molecule which hybridizes under stringent conditions to a nucleotide sequence defined by b1) or b2) and which encodes the protein nog1.
  • the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.
  • sequence 1 in the sequence listing is composed of 1170 nucleotides, and the nucleotide of the sequence 1 in the sequence listing encodes the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
  • the present invention also provides a plant breeding method 1 or a plant breeding method 2.
  • the plant breeding method 1 provided by the present invention may comprise the steps of increasing the content and/or activity of the protein nog1 in the plant, thereby increasing the plant yield and/or the number of kernels.
  • the "increasing the content and/or activity of the protein nog1 in the plant” can be achieved by increasing the number of copies, a promoter, a regulatory factor, a transgene, etc., as is well known in the art.
  • the plant breeding method 2 provided by the present invention may comprise the steps of reducing the content and/or activity of the protein nog1 in the plant, thereby reducing plant yield and/or kernel number.
  • the "reducing the content and/or activity of the protein nog1 in the plant” can achieve the reduction of the protein in the plant by methods well known in the art such as RNA interference, homologous recombination, gene-site editing, and the like.
  • the plant may be any one of f1) to f4): f1) monocotyledon; f2) dicotyledon; f3) gramineous plant; f4) rice.
  • the yield can be a single plant yield.
  • the number of kernels per spike may be the number of spikes in the main stem and/or the number of kernels in the average.
  • the "substance which inhibits the expression of the protein nog1" is also within the scope of the present invention.
  • the substance for inhibiting the expression of the protein nog1 may specifically be a specific DNA molecule, an expression cassette containing the specific DNA molecule, or a recombinant plasmid containing the specific DNA molecule.
  • the specific DNA molecule includes a sense fragment, an antisense fragment, and a spacer fragment located therebetween.
  • the sense fragment is the reverse complement of the DNA molecule shown in 155th to 522th position from the 5' end of the sequence 1 of the sequence listing; the antisense fragment is the sequence 1 of the sequence listing from the 5' end DNA molecules shown in positions 161 to 522.
  • the recombinant plasmid containing the specific DNA molecule may specifically be the recombinant plasmid pRNAi-nog1.
  • the recombinant plasmid pRNAi-nog1 may specifically replace the small DNA fragment between the BamHI recognition sequence and the KpnI recognition sequence of the vector pTCK303/JL1460 with a nucleotide sequence, and the sequence 1 of the sequence table is from the 5th end to the 155th position.
  • the reverse complement of the DNA molecule shown in position 522, the small fragment of DNA between the SpeI recognition sequence and the SacI recognition sequence is replaced by the nucleotide sequence.
  • Sequence 1 of the sequence listing is from position 161 to position 522 from the 5' end. The DNA molecule shown.
  • Figure 1 shows the morphology of the main stem ear of SIL176 and Guichao 2, the number of spikes in the main stem and the yield per plant.
  • FIG 2 is a T 2 generation of homozygous lines and RNAi of the main spike 2 Guichao morphology, comparing gene expression nog1, main stem and grain yield per plant.
  • the vector pTCK303/JL1460 is described in Wang Z, Chen CG, Xu YY, Jiang RX, Han Y, Xu ZH and Chong KA Practical Vector for Efficient Knockdown of Gene Expression in Rice (Oryza sativa L.). Plant Molecular Biology Reporter, 2004, 22: 409–417.
  • Gui Chao No. 2 is recorded in the following documents: Zhang X, Zhou S X, Fu Y C, et al. Identification of a drought tolerant introgression line derived from Dongxiang common wild rice (O.rufipogon Griff.). Plant Mol Biol, 2006 62:247 ⁇ 259, the public can get it from China Agricultural University.
  • Guichao 2 is referred to as Guichao 2 in the following.
  • Guichao 2 belongs to japonica rice.
  • Jiangxi Dongxiang wild rice is described in the following literature: Tian F, Li D J, Fu Q, Zhu Z F, Fu Y C, Wang X K, Sun C Q. 2006. Construction of introgression lines carrying wild rice (Oryza rufipogon Griff. Segments in cultivated rice (O. sativa L.) background and characterization of introgressed segments associated with yield-related traits. Theoretical and Applied Genetics, 112, 570-80. The public is available from China Agricultural University.
  • Agrobacterium tumefaciens EHA105 (named Agrobacterium tumefaciens strain EHA105 in the literature) is described in the following literature: GLUTELIN PRECURSOR ACCUMULATION3 encodes a regulator of post-Golgi vesicular traffic essential for vacuolar protein sorting in rice endosperm.Plant Cell.2014 Jan;26 (1): 410-25. The public can obtain from the China Agricultural University to repeat the experiment of this application.
  • the Dongxiang Oryza sativa infiltration line SIL176 is the progeny of multiple crosses and backcrosses between Guichao 2 and Dongxiang wild rice in Jiangxi Republic. It is recorded in the following literature: Tian F, Li D J, Fu Q, Zhu Z F, Fu Y C, Wang X K,Sun C Q.2006.Construction of introgression lines carrying wild rice(Oryza rufipogon Griff.)segments in cultivated rice(O.sativa L.)background and characterization of introgressed segments associated with yield-related traits.Theoretical and Applied Genetics , 112, 570-80. The public can get it from China Agricultural University.
  • the Dongxiang common wild rice infiltration system SIL176 is hereinafter referred to as SIL176.
  • the DNA content of Guichao 2 cDNA is approximately 200 ng/ ⁇ L.
  • SuperScript II reverse transcriptase is a product of Invitrogen, catalog number 18064-014.
  • the engineered plant expression vector pCAMBIA1300 increased the restriction endonuclease BglII recognition sequence at the 5' end of the recognition sequence of the restriction enzyme KpnI of the vector pCAMBIA1300, and increased the restriction of the 5' end of the recognition sequence of the restriction enzyme BamHI
  • the recognition sequence of the restriction enzyme MluI, and the other nucleotide sequences remain unchanged.
  • Example 2 Acquisition and phenotypic identification of homozygous RNAi interference lines of T 2 generation
  • Primers 860-rnai-320F, 860-rnai-681R, 860-rnai-681F and 860-rnai-314R were designed and synthesized according to the sequence of the nog1 gene shown in SEQ ID NO:1 in the sequence listing; the primer sequences are as follows:
  • 860-rnai-320F 5'-GG ACTAGT GGGAGAAAGATGAGGA-3' (underlined as the recognition site for restriction endonuclease SpeI);
  • 860-rnai-681R 5'-TCC GAGCTC GGTCAAAGCCAGGTAC-3' (underlined as restriction endonuclease SacI recognition site);
  • 860-rnai-681F 5'- CG GGATCC GGTCAAAGCCAGGTAC-3 '( underlined BamHI restriction endonuclease recognition site);
  • 860-rnai-314R 5'-GG GGTACC AGAGCTGGGAGAAAGA-3' (underlined as restriction endonuclease KpnI recognition site).
  • the DNA fragment A was digested with restriction endonucleases SpeI and SacI, and the digested product 1 was recovered.
  • the vector pTCK303/JL1460 was digested with restriction endonucleases SpeI and SacI to recover a carrier backbone 1 of about 14.6 kb.
  • the digested product 1 is ligated to the vector backbone 1 to obtain an intermediate plasmid.
  • the DNA fragment B was digested with restriction endonucleases BamHI and KpnI, and the digested product 2 was recovered.
  • the intermediate plasmid was digested with restriction endonucleases BamHI and KpnI to recover a carrier backbone 2 of about 14.9 kb.
  • the digested product 2 was ligated to the vector backbone 2 to obtain a recombinant plasmid pRNAi-nog1.
  • the recombinant plasmid pRNAi-nog1 was structurally described as follows: the small DNA fragment between the BamHI recognition sequence and the KpnI recognition sequence of the vector pTCK303/JL1460 was replaced with the nucleotide sequence. Sequence 1 of the sequence listing was from the 5' end. The reverse complement of the DNA molecule shown in positions 155 to 522, the small DNA fragment between the SpeI recognition sequence and the SacI recognition sequence is replaced with the nucleotide sequence, and the sequence 1 of the sequence listing is the 161th position from the 5' end. To the DNA molecule shown at position 522.
  • the recombinant plasmid pRNAi-nog1 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium EHA105/pRNAi-nog1.
  • Hiei et al. Hiei Y, Ohta S, Komari T&Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 1994, 6:271– 282) the recombinant Agrobacterium EHA105 / pRNAi-nog1 conversion Guichao 2, T 0 generation of RNAi, is obtained strains.
  • RNAi-1-T 0 to RNAi-3-T 0 Three T 0 generation RNAi interference strains (named RNAi-1-T 0 to RNAi-3-T 0 ) were randomly selected for real-time quantitative PCR detection. The specific steps are as follows:
  • the two-week seedlings of three T 0 generation RNAi interference strains were used to extract total RNA with TRIZOL reagent, and then reverse-transcribed with SuperScript II reverse transcriptase to obtain the cDNA of each T 0 generation silenced strain.
  • the DNA content of the cDNA of the three T 0 generation RNAi interference strains was about 200 ng/ ⁇ L.
  • the primer for detecting the nog1 gene was forward primer 1:5'-TCCGACTTACAATGAACAC-3' and reverse primer 1:5'-GGTAGCAGGACTCCACTT-3'.
  • the primers for detecting the UBI gene were forward primer 2: 5'-CTGTCAACTGCCGCAAGAAG-3' and reverse primer 2: 5'-GGCGAGTGACGCTCTAGTTC-3'.
  • the T 0 generation RNAi interference strain was replaced with Guichao 2, and the other steps were unchanged, and the relative expression level of the nog1 gene in Guichao 2 was obtained.
  • the relative expression level of the nog1 gene in Guichao 2 was taken as 1, and the relative expression level of the nog1 gene in other rice plants was counted. The results showed that compared with Guichao 2, the relative expression levels of nog1 gene in the three T 0 generation RNAi interference strains were significantly decreased.
  • RNAi-1-T 0 , RNAi-2-T 0 and RNAi-3-T 0 are both T 0 generation RNAi interference strains.
  • RNAi-1-T 0 to RNAi-3-T 0 were self-crossed for two consecutive generations to obtain T 2 generation homozygous RNAi interference lines, designated as RNAi-1 to RNAi-3, respectively.
  • step four real-time quantitative PCR detection was performed on RNAi-1 to RNAi-3 and Guichao 2, respectively.
  • the seeds of the tested rice (Guichao 2, RNAi-1, RNAi-2 or RNAi-3) were planted in pots containing nutrient soil and vermiculite (the ratio of nutrient soil to vermiculite was 1:1), 25 °C and light were alternately cultured, and the morphology of the main stem ear, the number of spikes in the main stem, the average kernel number per spike and the yield per plant were compared and counted during the growth and development. The experiment was repeated three times, with 30 strains repeated each time.
  • Example 3 Acquisition and phenotypic identification of homozygous complementary lines of T 2 generation
  • the PCR amplification product was digested with restriction endonucleases BglII and MluI, and the digested product was recovered.
  • the plant expression vector pCAMBIA1300 was digested with restriction endonucleases BglII and MluI to recover a vector backbone of about 9 kb.
  • the restriction enzyme product was ligated to the vector backbone to obtain a recombinant plasmid pCAMBIA1300-NOG1.
  • the recombinant plasmid pCAMBIA1300-NOG1 was structurally described as follows: the restriction fragment enzyme BglII recognition sequence of the engineered plant expression vector pCAMBIA1300 and the DNA fragment between the MluI recognition sequence were replaced with nucleotide sequences which are sequence listings.
  • the recombinant plasmid pCAMBIA1300-NOG1 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium EHA105/pCAMBIA1300-NOG1.
  • Hiei et al. Hiei Y, Ohta S, Komari T&Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 1994, 6:271– 282) the recombinant Agrobacterium EHA105 / pCAMBIA1300-NOG1 conversion SIL176, T 0 is obtained substituting the complementary strain.
  • T 0 generation complementary strains (named CTP-1-T 0 to CTP-3-T 0 ) were randomly selected for real-time quantitative PCR detection. The specific steps are as follows:
  • RNA was extracted with TRIZOL reagent from the two-week seedlings of three T 0 complementary strains, and then reverse-transcribed with SuperScript II reverse transcriptase to obtain the cDNA of each T 0 complementary strain.
  • the DNA content of the cDNA of the three T 0 complementary strains was about 200 ng/ ⁇ L.
  • the primer for detecting the nog1 gene was forward primer 1:5'-TCCGACTTACAATGAACAC-3' and reverse primer 1:5'-GGTAGCAGGACTCCACTT-3'.
  • the primers for detecting the UBI gene were forward primer 2: 5'-CTGTCAACTGCCGCAAGAAG-3' and reverse primer 2: 5'-GGCGAGTGACGCTCTAGTTC-3'.
  • the T 0 generation complementary strain was replaced with SIL176, and the other steps were unchanged, and the relative expression amount of the nog1 gene in SIL176 was obtained.
  • the relative expression level of the nog1 gene in SIL176 was used as 1, and the relative expression level of the nog1 gene in other rice plants was counted. The results showed that the relative expression levels of the nog1 gene in the three T 0 generation complement strains were significantly increased compared with SIL176.
  • CTP-1-T 0 , CTP-2-T 0 and CTP-3-T 0 are T 0 generation complementary transgenic rice.
  • CTP-1-T 0 to CTP-3-T 0 were self-crossed for two consecutive generations to obtain T 2 generation homozygous complementary lines, which were named CTP-1 to CTP-3, respectively.
  • step four real-time quantitative PCR detection was performed on CTP-1 to CTP-3 and SIL176, respectively.
  • Seeds of rice to be tested (SIL176, CTP-1, CTP-2 or CTP-3) were planted in pots containing nutrient soil and vermiculite (nutrient soil and vermiculite volume ratio was 1:1), 25 °C The light was alternately cultured, and the morphology of the main stem ear, the number of spikes in the main stem, the average kernel number per spike, and the yield per plant were compared and counted during the growth and development. The experiment was repeated three times, with 30 strains repeated each time.
  • a transgenic rice which inhibits the expression of the protein nog1 is introduced into the starting rice (e.g., Guichao 2) to obtain a transgenic rice having a reduced yield per plant and/or a reduced number of main stems.
  • the nucleic acid molecule encoding the protein nog1 is introduced into the starting rice (such as SIL176) to obtain an increase in the yield per plant and/or the number of spikes in the main stem and/or Or transgenic rice with an increased average kernel number per spike. Therefore, protein nog1 can regulate rice yield and kernel number, which has important application value.

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Abstract

本发明提供了蛋白质nog1在调控植物产量和/或穗粒数中的应用，所述蛋白质nog1的氨基酸序列如序列2所示，所述产量为单株产量，所述穗粒数为主茎穗粒数。与未处理的桂朝2号相比，蛋白质nog1的表达受到抑制的转基因处理的桂朝2号的单株产量和/或主茎穗粒数减少。将编码蛋白质nog1的核酸分子导入SIL176中，得到的转基因植株较未转化的SIL176的单株产量和/或主茎穗粒数增加。

Description

蛋白质nog1在调控植物产量和穗粒数中的应用 技术领域
本发明涉及生物技术领域，具体涉及蛋白质nog1在调控植物产量和穗粒数中的应用。
背景技术
水稻是世界上最重要的粮食作物之一，全球有120多个国家种植水稻，栽培面积常年保持在1.5亿公顷以上，并有占世界50％比例的人口以稻米为主食。在人口持续增加和可种植耕地面积逐年减少的今天，提高水稻单产是保障世界粮食安全的有力措施之一。回顾半个多世纪以来的水稻育种史，我国水稻单产经历了两次飞跃，第一次是以矮化育种为标志的绿色革命，第二次是水稻杂种优势的利用。但近20年来，水稻单产一直停滞不前。研究者认为，目前生产上利用的许多栽培品种具有相同或类似的遗传来源，对水稻栽培种遗传资源的利用趋于饱和，水稻品种间狭窄的遗传多样性造成了水稻品种的遗传基础和基因型相近，这已成为制约水稻产量潜力进一步提高的瓶颈。
普通野生稻(Oryza rufipogon Griff.)是亚洲栽培稻的野生祖先种，相比于经过人工驯化后的栽培稻，具有更丰富的遗传多样性和基因资源。普通野生稻的遗传分化类型远多于栽培稻，蕴含着丰富的可以提高水稻产量的基因。因此从普通野生稻基因组中发掘和利用在栽培稻中已丢失或削弱的优异驯化基因，并把它们应用于水稻育种生产中具有十分重要的理论意义和实践价值，也是解决当前水稻育种难题的一条行之有效的途径。
发明公开
本发明所要解决的技术问题是如何调控植物产量和穗粒数。
为解决上述技术问题，本发明首先提供了蛋白质nog1在调控植物产量和/或穗粒数中的应用；所述蛋白质nog1可为a1)或a2)或a3)或a4)：
a1)氨基酸序列是序列表中序列2所示的蛋白质；
a2)在序列表中序列2所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
a3)将序列表中序列2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与植物产量和/或穗粒数相关的蛋白质；
a4)与a1)限定的氨基酸序列具有80％或80％以上同一性的蛋白质。
其中，序列表中序列2由389个氨基酸残基组成。
为了使a1)中的蛋白质便于纯化，可在序列表中序列2所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。
表1.标签的序列
标签 残基 序列
Poly-Arg 5-6(通常为5个) RRRRR
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
上述a3)中的蛋白质，所述一个或几个氨基酸残基的取代和/或缺失和/或添加为不超过10个氨基酸残基的取代和/或缺失和/或添加。
上述a3)中的蛋白质可人工合成，也可先合成其编码基因，再进行生物表达得到。
上述a3)中的蛋白质的编码基因可通过将序列表中序列1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。
上述a4)中使用的术语“同一性”指与序列表中序列2所示的蛋白质的氨基酸序列的序列相似性。“同一性”包括与本发明的序列表的序列2所示的氨基酸序列具有80％或更高，或85％或更高，或90％或更高，或95％或更高同一性的氨基酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
编码所述蛋白质nog1的核酸分子在调控植物产量和/或穗粒数中的应用也属于本发明的保护范围。
编码所述蛋白质nog1的核酸分子可为如下b1)或b2)或b3)或b4)所示的DNA分子：
b1)编码区如序列表中序列1所示的DNA分子；
b2)核苷酸序列是序列表中序列1所示的DNA分子；
b3)与b1)或b2)限定的核苷酸序列具有75％或75％以上同一性，且编码所述蛋白质nog1的DNA分子；
b4)在严格条件下与b1)或b2)限定的核苷酸序列杂交，且编码所述蛋白质nog1的DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
其中，序列表中序列1由1170个核苷酸组成，序列表中序列1的核苷酸编码序列表中序列2所示的氨基酸序列。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码所述蛋白质nog1的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明分离得到的所述蛋白质nog1的核苷酸序列75％或者更高同一性的核苷酸，只要编码所述蛋白质nog1，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码序列表的序列2所示的氨基酸序列组成的蛋白质nog1的核 苷酸序列具有75％或更高，或80％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
上述应用中，所述调控植物产量可为调控植物单株产量。所述调控植物穗粒数可为调控植物主茎穗粒数和/或平均穗粒数。
上述应用中，所述植物可为如下c1)至c7)中的任一种：c1)双子叶植物；c2)单子叶植物；c3)禾本科植物；c4)水稻；c5)籼稻；c6)水稻品种桂朝2号；c7)东乡普通野生稻渗入系SIL176。
为解决上述技术问题，本发明还提供了培育转基因植物甲的方法一或培育转基因植物乙的方法二。
本发明所提供的培育转基因植物甲的方法一，可包括将编码所述蛋白质nog1的核酸分子导入受体植物甲中，得到转基因植物甲的步骤；与所述受体植物甲相比，所述转基因植物甲的产量增加和/或穗粒数增加。
上述方法一中，所述“将编码蛋白质nog1的核酸分子导入受体植物甲中”可通过向受体植物甲中导入重组载体甲实现；所述重组载体甲可为向表达载体插入编码所述蛋白质nog1的核酸分子得到的重组质粒。
所述重组载体甲具体可为重组质粒pCAMBIA1300-NOG1。所述重组质粒pCAMBIA1300-NOG1具体可为将改造的植物表达载体pCAMBIA1300的限制性酶切酶BglII识别序列和MluI识别序列间的DNA小片段替换为核苷酸序列是序列表的序列3所示的DNA分子。
上述方法一中，所述受体植物甲可为d1)-d6)中的任一种：d1)单子叶植物；d2)双子叶植物；d3)禾本科植物；d4)水稻；d5)籼稻；d6)东乡普通野生稻渗入系SIL176。
本发明所提供的培育转基因植物甲的方法二，可包括向受体植物乙中导入抑制所述蛋白质nog1表达的物质，得到转基因植物乙的步骤；与所述受体植物乙相比，所述转基因植物乙的产量减少和/或穗粒数减少。
上述方法二中，所述“抑制蛋白质nog1表达的物质”可为特异DNA分子、含有所述特异DNA分子的表达盒或含有所述特异DNA分子重组质粒；
所述特异DNA分子包括正义片段、反义片段以及位于它们之间的间隔片段；所述正义片段为序列表的序列1自5′末端起第155位至第522位所示的DNA分子的反向互补序列；所述反义片段为序列表的序列1自5′末端起第161位至第522位所示的DNA分子。
上述方法二中，所述含有特异DNA分子重组质粒具体可为重组质粒pRNAi-nog1。所述重组质粒pRNAi-nog1具体可为将载体pTCK303/JL1460的BamHI识别序列和KpnI识别序列间的DNA小片段替换为核苷酸序列是序列表的序列1自5′末端起第155位至第522位所示的DNA分子的反向互补序列，SpeI识别 序列和SacI识别序列间的DNA小片段替换为核苷酸序列是序列表的序列1自5′末端起第161位至第522位所示的DNA分子。
上述方法二中，所述受体植物乙可为e1)-e6)中的任一种：e1)单子叶植物；e2)双子叶植物；e3)禾本科植物；e4)水稻；e5)籼稻；e6)水稻品种桂朝2号。
为解决上述技术问题，本发明还提供了培育转基因植物丙的方法三。
本发明所提供的培育转基因植物丙的方法三，可包括向受体植物丙中导入提高所述蛋白质nog1表达和/或活性的物质，得到转基因植物丙的步骤；与所述受体植物丙相比，所述转基因植物丙的产量增加和/或穗粒数增加。
上述方法三中，所述“提高所述蛋白质nog1表达和/或活性的物质”具体可为所述重组载体甲。
上述方法三中，所述受体植物丙可为d1)-d6)中的任一种：d1)单子叶植物；d2)双子叶植物；d3)禾本科植物；d4)水稻；d5)籼稻；d6)东乡普通野生稻渗入系SIL176。
上述方法中，编码所述蛋白质nog1的核酸分子可为如下b1)或b2)或b3)或b4)所示的DNA分子：
b1)编码区如序列表中序列1所示的DNA分子；
b2)核苷酸序列是序列表中序列1所示的DNA分子；
b3)与b1)或b2)限定的核苷酸序列具有75％或75％以上同一性，且编码所述蛋白质nog1的DNA分子；
b4)在严格条件下与b1)或b2)限定的核苷酸序列杂交，且编码所述蛋白质nog1的DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
其中，序列表中序列1由1170个核苷酸组成，序列表中序列1的核苷酸编码序列表中序列2所示的氨基酸序列。
为解决上述技术问题，本发明还提供了植物育种方法一或植物育种方法二。
本发明所提供的植物育种方法一，可包括如下步骤：增加植物中所述蛋白质nog1的含量和/或活性，从而增加植物产量和/或穗粒数。
上述植物育种方法一中，所述“增加植物中所述蛋白质nog1的含量和/或活性”可通过多拷贝、改变启动子、调控因子、转基因等本领域熟知的方法，达到增加植物中所述蛋白质nog1的含量和/或活性的效果。
本发明所提供的植物育种方法二，可包括如下步骤：降低植物中所述蛋白质nog1的含量和/或活性，从而减少植物产量和/或穗粒数。
上述植物育种方法二中，所述“降低植物中所述蛋白质nog1的含量和/或活性”可通过RNA干扰、同源重组、基因定点编辑等本领域熟知的方法，达到降低植物中所述蛋白质nog1的含量和/或活性的目的。
上述方法中，所述植物可为f1)-f4)中的任一种：f1)单子叶植物；f2)双子叶植物；f3)禾本科植物；f4)水稻。
上述任一所述方法中，所述产量可为单株产量。所述穗粒数可为主茎穗粒数和/或平均穗粒数。
所述“抑制所述蛋白质nog1表达的物质”也属于本发明的保护范围。
上述任一所述抑制所述蛋白质nog1表达的物质具体可为特异DNA分子、含有所述特异DNA分子的表达盒或含有所述特异DNA分子重组质粒。
所述特异DNA分子包括正义片段、反义片段以及位于它们之间的间隔片段。
所述正义片段为序列表的序列1自5′末端起第155位至第522位所示的DNA分子的反向互补序列；所述反义片段为序列表的序列1自5′末端起第161位至第522位所示的DNA分子。
含有所述特异DNA分子重组质粒具体可为重组质粒pRNAi-nog1。所述重组质粒pRNAi-nog1具体可为将载体pTCK303/JL1460的BamHI识别序列和KpnI识别序列间的DNA小片段替换为核苷酸序列是序列表的序列1自5′末端起第155位至第522位所示的DNA分子的反向互补序列，SpeI识别序列和SacI识别序列间的DNA小片段替换为核苷酸序列是序列表的序列1自5′末端起第161位至第522位所示的DNA分子。
实验证明，向Guichao 2中导入抑制所述蛋白质nog1表达的物质(即重组质粒pRNAi-nog1)，得到转基因植物乙；与Guichao 2相比，转基因植物乙的单株产量减少和/或主茎穗粒数减少和/或平均穗粒数减少。将编码蛋白质nog1的核酸分子导入SIL176中，得到转基因植物甲；与SIL176相比，转基因植物甲的单株产量增加和/或主茎穗粒数增加和/或平均穗粒数增加。结果表明，蛋白质nog1对调控水稻产量和穗粒数具有非常重要的作用。
附图说明
图1为SIL176与Guichao 2的主茎穗的形态、主茎穗粒数和单株产量的比较。
图2为T2代纯合RNAi干扰株系与Guichao 2的主茎穗的形态、nog1基因表达量、主茎穗粒数和单株产量的比较。
图3为T2代纯合互补株系与SIL176的主茎穗的形态、nog1基因表达量、主茎穗粒数和单株产量的比较。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
载体pTCK303/JL1460记载于如下文献中：Wang Z，Chen CG，Xu YY，Jiang RX，Han Y，Xu ZH and Chong K.A Practical Vector for Efficient Knockdown of Gene Expression in Rice(Oryza sativa L.).Plant Molecular Biology Reporter，2004，22:409–417.
桂朝2号记载于如下文献中：Zhang X，Zhou S X，Fu Y C,et al.Identification of a drought tolerant introgression line derived from Dongxiang common wild rice(O.rufipogon Griff.).Plant Mol Biol，2006，62:247～259，公众可以从中国农业大学获得。桂朝2号在下文中简称Guichao 2。桂朝2号属于籼稻。
江西东乡野生稻记载于如下文献中：Tian F，Li D J，Fu Q，Zhu Z F，Fu Y C，Wang X K，Sun C Q.2006.Construction of introgression lines carrying wild rice(Oryza rufipogon Griff.)segments in cultivated rice(O.sativa L.)background and characterization of introgressed segments associated with yield-related traits.Theoretical and Applied Genetics，112，570-80.公众可以从中国农业大学获得。
根癌农杆菌EHA105(文献中的名称为Agrobacterium tumefaciens strain EHA105)记载于如下文献中：GLUTELIN PRECURSOR ACCUMULATION3 encodes a regulator of post-Golgi vesicular traffic essential for vacuolar protein sorting in rice endosperm.Plant Cell.2014 Jan；26(1):410-25.公众可从中国农业大学获得，以重复本申请实验。
东乡普通野生稻渗入系SIL176为Guichao 2与江西东乡野生稻多次杂交和回交的后代，其记载于如下文献中：Tian F，Li D J，Fu Q，Zhu Z F，Fu Y C，Wang X K，Sun C Q.2006.Construction of introgression lines carrying wild rice(Oryza rufipogon Griff.)segments in cultivated rice(O.sativa L.)background and characterization of introgressed segments associated with yield-related traits.Theoretical and Applied Genetics，112，570-80.公众可以从中国农业大学获得。东乡普通野生稻渗入系SIL176在下文中简称SIL176。
Guichao 2的cDNA：以Guichao 2的2周苗实验材料，先用TRIZOL试剂提取总RNA，然后用SuperScriptII反转录酶进行反转录得到。Guichao 2的cDNA中DNA含量约为200ng/μL。SuperScriptII反转录酶为Invitrogen公司的产品，产品目录号为18064-014。
改造的植物表达载体pCAMBIA1300：将载体pCAMBIA1300的限制性酶切酶KpnI的识别序列的5’末端增加限制性酶切酶BglII的识别序列，限制性酶切酶BamHI的识别序列的5’末端增加限制性酶切酶MluI的识别序列，其它的核苷酸序列均保持不变得到的载体。
实施例1、nog1基因的发现
利用江西东乡野生稻为供体亲本，与Guichao 2为轮回亲本通过杂交、回交构建了一套含有265个系的渗入系群体(其中一个系为SIL176)。该套群体野生稻基因组的覆盖率达79.4％，其中有15个系(其中一个系为SIL176)的单株产量比Guichao 2减少35％以上。将Guichao 2与SIL176的主茎穗的形态、主茎穗粒数和单株产量进行比较及统计。实验重复三次，每次重复30株。
实验结果见图1(A为主茎穗的形态，bar＝5cm；B为主茎穗粒数；C为单株产量；**表示P<0.01差异极显著)。结果表明，与Guichao 2相比，SIL176的主茎穗粒数和单株产量显著减少。
对SIL176进行图位克隆和功能分析。结果在第1染色体长臂发现了一个与水稻产量相关的QTL，命名为nog1基因。nog1基因的开放阅读框如序列表中序列1所示，编码的蛋白命名为nog1，其氨基酸序列如序列表中序列2所示，由389个氨基酸残基组成。
实施例2、T2代纯合RNAi干扰株系的获得和表型鉴定
一、重组质粒pRNAi-nog1的构建
重组质粒pRNAi-nog1的构建步骤如下：
1、合成引物
根据序列表中序列1所示的nog1基因的序列，设计并合成引物860-rnai-320F、860-rnai-681R、860-rnai-681F和860-rnai-314R；引物序列具体如下：
860-rnai-320F：5′-GGACTAGTGGGAGAAAGATGAGGA-3′(下划线为限制性内切酶SpeI的识别位点)；
860-rnai-681R：5′-TCCGAGCTCGGTCAAAGCCAGGTAC-3′(下划线为限制性内切酶SacI识别位点)；
860-rnai-681F：5′-CGGGATCCGGTCAAAGCCAGGTAC-3′(下划线为限制性内切酶BamHI识别位点)；
860-rnai-314R：5′-GGGGTACCAGAGCTGGGAGAAAGA-3′(下划线为限制性内切酶KpnI识别位点)。
2、以Guichao 2的cDNA为模板，以860-rnai-320F和860-rnai-681R为引物，进行PCR扩增，得到约360bp的DNA片段A。
3、以Guichao 2的cDNA为模板，以860-rnai-681F和860-rnai-314R为引物，进行PCR扩增，得到约360bp的DNA片段B。
4、用限制性内切酶SpeI和SacI酶切DNA片段A，回收酶切产物1。
5、用限制性内切酶SpeI和SacI酶切载体pTCK303/JL1460，回收约14.6kb的载体骨架1。
6、将酶切产物1与载体骨架1连接，得到中间质粒。
7、用限制性内切酶BamHI和KpnI酶切DNA片段B，回收酶切产物2。
8、用限制性内切酶BamHI和KpnI酶切中间质粒，回收约14.9kb的载体骨架2。
9、将酶切产物2与载体骨架2连接，得到重组质粒pRNAi-nog1。
根据测序结果，对重组质粒pRNAi-nog1进行结构描述如下：将载体pTCK303/JL1460的BamHI识别序列和KpnI识别序列间的DNA小片段替换为核苷酸序列是序列表的序列1自5′末端起第155位至第522位所示的DNA分子的反向互补序列，SpeI识别序列和SacI识别序列间的DNA小片段替换为核苷酸序列是序列表的序列1自5′末端起第161位至第522位所示的DNA分子。
二、重组农杆菌的获得
将重组质粒pRNAi-nog1导入根癌农杆菌EHA105中，得到重组农杆菌EHA105/pRNAi-nog1。
三、T0代RNAi干扰株的获得
采用Hiei等的方法(Hiei Y,Ohta S,Komari T&Kumashiro T.Efficient transformation of rice(Oryza sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.Plant J.1994，6:271–282)将重组农杆菌EHA105/pRNAi-nog1转化Guichao 2，获得T0代RNAi干扰株。
四、T0代RNAi干扰株的实时定量PCR检测
随机选取3个T0代RNAi干扰株(分别命名为RNAi-1-T0至RNAi-3-T0)进行实时定量PCR检测，具体步骤如下：
1、分别以3个T0代RNAi干扰株的2周苗实验材料，先用TRIZOL试剂提取总RNA，然后用SuperScriptII反转录酶进行反转录，得到各个T0代拟沉默株的cDNA。3个T0代RNAi干扰株的cDNA中DNA含量约为200ng/μL。
2、使用RT-qPCR技术分别检测3个T0代RNAi干扰株中nog1基因的相对表达量(以UBI基因作为内参基因)。
检测nog1基因的引物为正向引物1：5’-TCCGACTTACAATGAACAC-3’和反向引物1：5’-GGTAGCAGGACTCCACTT-3’。检测UBI基因的引物为正向引物2：5’-CTGTCAACTGCCGCAAGAAG-3’和反向引物2：5’-GGCGAGTGACGCTCTAGTTC-3’。
按照上述方法，将T0代RNAi干扰株替换为Guichao 2，其它步骤均不变，得到Guichao 2中nog1基因的相对表达量。
以Guichao 2中nog1基因的相对表达量作为1，统计其它水稻植株中nog1基因的相对表达量。结果表明，与Guichao 2相比，3个T0代RNAi干扰株中nog1基因的相对表达量均显著降低。
上述结果表明，RNAi-1-T0、RNAi-2-T0和RNAi-3-T0均为T0代RNAi干扰株。
五、T2代纯合RNAi干扰株系的获得及实时定量PCR检测
将RNAi-1-T0至RNAi-3-T0经连续两代自交，获得T2代纯合RNAi干扰株系，分别命名为RNAi-1至RNAi-3。
按照步骤四的方法，对RNAi-1至RNAi-3和Guichao 2分别进行实时定量PCR检测。
部分检测结果见图2中B(**表示P<0.01差异极显著)。结果表明，与Guichao 2相比，RNAi-1至RNAi-3中nog1基因的相对表达量均显著降低。
六、T2代纯合RNAi干扰株系的表型鉴定
将待测水稻(Guichao 2、RNAi-1、RNAi-2或RNAi-3)的种子分别种植在装有营养土和蛭石的盆中(营养土和蛭石体积比为1:1)，25℃、光照交替培养，在生长发育过程中对待测水稻的主茎穗的形态、主茎穗粒数、平均穗粒数和单株产量进行比较及统计。实验重复三次，每次重复30株。
部分实验结果见图2中A、C和D(A为主茎穗的形态，bar＝5cm；C为主茎穗粒数；D为单株产量；**表示P<0.01差异极显著)。结果表明，与Guichao2相比，RNAi-1、RNAi-2和RNAi-3的主茎穗粒数、平均穗粒数和单株产量均显著降低。
实施例3、T2代纯合互补株系的获得和表型鉴定
一、重组质粒pCAMBIA1300-NOG1的构建
重组质粒pCAMBIA1300-NOG1的构建步骤如下：
1、以Guichao 2的2周苗实验材料，提取基因组DNA并以其为模板，以860HBF：5'-GAAGATCTCATCTGATGCCTCATACTGA-3'(下划线为限制性内切酶BglII识别位点)和860HBR：5'-CCGACGCGTCATGCTTAGGCTGTTGAT-3'(下划线为限制性内切酶MluI识别位点)为引物，进行PCR扩增，得到约7kb的PCR扩增产物。
2、用限制性内切酶BglII和MluI酶切PCR扩增产物，回收酶切产物。
3、用限制性内切酶BglII和MluI酶切改造的植物表达载体pCAMBIA1300，回收约9kb的载体骨架。
4、将酶切产物与载体骨架连接，得到重组质粒pCAMBIA1300-NOG1。
根据测序结果，对重组质粒pCAMBIA1300-NOG1进行结构描述如下：将改造的植物表达载体pCAMBIA1300的限制性酶切酶BglII识别序列和MluI识别序列间的DNA小片段替换为核苷酸序列是序列表的序列3所示的DNA分子。
二、重组农杆菌的获得
将重组质粒pCAMBIA1300-NOG1导入根癌农杆菌EHA105中，得到重组农杆菌EHA105/pCAMBIA1300-NOG1。
三、T0代互补株的获得
采用Hiei等的方法(Hiei Y,Ohta S,Komari T&Kumashiro T.Efficient transformation of rice(Oryza sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.Plant J.1994，6:271–282)将重组农杆菌EHA105/pCAMBIA1300-NOG1转化SIL176，获得T0代互补株。
四、T0代互补株的实时定量PCR检测
随机选取3个T0代互补株(分别命名为CTP-1-T0至CTP-3-T0)进行实时定量PCR检测，具体步骤如下：
1、分别以3个T0代互补株的2周苗实验材料，先用TRIZOL试剂提取总RNA，然后用SuperScriptII反转录酶进行反转录，得到各个T0代互补株的cDNA。3个T0代互补株的cDNA中DNA含量约为200ng/μL。
2、使用RT-qPCR技术分别检测3个T0代互补株中nog1基因的相对表达量(以UBI基因作为内参基因)。
检测nog1基因的引物为正向引物1：5’-TCCGACTTACAATGAACAC-3’和反向引物1：5’-GGTAGCAGGACTCCACTT-3’。检测UBI基因的引物为正向引物2：5’-CTGTCAACTGCCGCAAGAAG-3’和反向引物2：5’-GGCGAGTGACGCTCTAGTTC-3’。
按照上述方法，将T0代互补株替换为SIL176，其它步骤均不变，得到SIL176中nog1基因的相对表达量。
以SIL176中nog1基因的相对表达量作为1，统计其它水稻植株中nog1基因的相对表达量。结果表明，与SIL176相比，3个T0代互补株中nog1基因的相对表达量均显著增加。
上述结果表明，CTP-1-T0、CTP-2-T0和CTP-3-T0均为T0代互补转基因水稻。
五、T2代纯合互补株系的获得及实时定量PCR检测
将CTP-1-T0至CTP-3-T0经连续两代自交，获得T2代纯合互补株系，分别命名为CTP-1至CTP-3。
按照步骤四的方法，对CTP-1至CTP-3和SIL176分别进行实时定量PCR检测。
部分检测结果见图3中B(**表示P<0.01差异极显著)。结果表明，与SIL176相比，CTP-1至CTP-3中nog1基因的相对表达量均显著增加。
六、T2代纯合互补株系的表型鉴定
将待测水稻(SIL176、CTP-1、CTP-2或CTP-3)的种子分别种植在装有营养土和蛭石的盆中(营养土和蛭石体积比为1:1)，25℃、光照交替培养，在生长发育过程中对待测水稻的主茎穗的形态、主茎穗粒数、平均穗粒数和单株产量进行比较及统计。实验重复三次，每次重复30株。
部分实验结果见图3中A、C和D(A为主茎穗的形态，bar＝5cm；C为主茎穗粒数；D为单株产量；**表示P<0.01差异极显著)。结果表明，与SIL176相比，CTP-1、CTP-2和CTP-3的主茎穗粒数、平均穗粒数和单株产量均显著增加。
工业应用
向出发水稻(如Guichao 2)中导入抑制所述蛋白质nog1表达的物质，得到单株产量减少和/或主茎穗粒数减少的转基因水稻。将编码蛋白质nog1的核酸分子导入出发水稻(如SIL176)中，得到单株产量增加和/或主茎穗粒数和/ 或平均穗粒数增加的转基因水稻。因此，蛋白质nog1可以调控水稻产量和穗粒数，具有重要的应用价值。

Claims (15)

  1. 蛋白质nog1在调控植物产量和/或穗粒数中的应用；所述蛋白质nog1为a1)或a2)或a3)或a4)：
    a1)氨基酸序列是序列表中序列2所示的蛋白质；
    a2)在序列表中序列2所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
    a3)将序列表中序列2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与植物产量和/或穗粒数相关的蛋白质；
    a4)与a1)限定的氨基酸序列具有80％或80％以上同一性的蛋白质。
  2. 编码权利要求1中所述蛋白质nog1的核酸分子在调控植物产量和/或穗粒数中的应用。
  3. 如权利要求2所述的应用，其特征在于：所述核酸分子为如下b1)或b2)或b3)或b4)所示的DNA分子：
    b1)编码区如序列表中序列1所示的DNA分子；
    b2)核苷酸序列是序列表中序列1所示的DNA分子；
    b3)与b1)或b2)限定的核苷酸序列具有75％或75％以上同一性，且编码权利要求1中所述蛋白质nog1的DNA分子；
    b4)在严格条件下与b1)或b2)限定的核苷酸序列杂交，且编码权利要求1中所述蛋白质nog1的DNA分子。
  4. 如权利要求1至3任一所述的应用，其特征在于：所述调控植物产量为调控植物单株产量。
  5. 如权利要求1至3任一所述的应用，其特征在于：所述调控植物穗粒数为调控植物主茎穗粒数和/或平均穗粒数。
  6. 如权利要求1至5任一所述的应用，其特征在于：所述植物为如下c1)至c7)中的任一种：c1)双子叶植物；c2)单子叶植物；c3)禾本科植物；c4)水稻；c5)籼稻；c6)水稻品种桂朝2号；c7)东乡普通野生稻渗入系SIL176。
  7. 培育转基因植物甲的方法一或培育转基因植物乙的方法二：
    所述培育转基因植物甲的方法一，包括将编码权利要求1中所述蛋白质nog1的核酸分子导入受体植物甲中，得到转基因植物甲的步骤；与所述受体植物甲相比，所述转基因植物甲的产量增加和/或穗粒数增加；
    所述培育转基因植物乙的方法二，包括向受体植物乙中导入抑制权利要求1中所述蛋白质nog1表达的物质，得到转基因植物乙的步骤；与所述受体植物乙相比，所述转基因植物乙的产量减少和/或穗粒数减少。
  8. 如权利要求7所述的培育转基因植物甲的方法一，其特征在于：所述“将编码蛋白质nog1的核酸分子导入受体植物甲中”可通过向受体植物甲中导入重组载体甲实现；所述重组载体甲可为向表达载体插入编码所述蛋白质nog1的核酸分子得到的重组质粒。
  9. 如权利要求7所述的培育转基因植物乙的方法二，其特征在于：所述“抑制蛋白质nog1表达的物质”为特异DNA分子、含有所述特异DNA分子的表达盒或含有所述特异DNA分子重组质粒；
    所述特异DNA分子包括正义片段、反义片段以及位于它们之间的间隔片段；
    所述正义片段为序列表的序列1自5′末端起第155位至第522位所示的DNA分子的反向互补序列；
    所述反义片段为序列表的序列1自5′末端起第161位至第522位所示的DNA分子。
  10. 一种培育转基因植物丙的方法三，包括向受体植物丙中导入提高权利要求1中所述蛋白质nog1表达和/或活性的物质，得到转基因植物丙的步骤；与所述受体植物丙相比，所述转基因植物丙的产量增加和/或穗粒数增加。
  11. 植物育种方法一或植物育种方法二：
    所述植物育种方法一，包括如下步骤：增加植物中权利要求1中所述蛋白质nog1的含量和/或活性，从而增加植物产量和/或穗粒数；
    所述植物育种方法二，包括如下步骤：降低植物中权利要求1中所述蛋白质nog1的含量和/或活性，从而减少植物产量和/或穗粒数。
  12. 如权利要求7至11任一所述的方法，其特征在于：所述植物为f1)-f4)中的任一种：f1)单子叶植物；f2)双子叶植物；f3)禾本科植物；f4)水稻。
  13. 如权利要7至12任一所述的方法，其特征在于：所述产量为单株产量。
  14. 如权利要7至12任一所述的方法，其特征在于：所述穗粒数为主茎穗粒数和/或平均穗粒数。
  15. 特异DNA分子、含有所述特异DNA分子的表达盒或含有所述特异DNA分子重组质粒；
    所述特异DNA分子包括正义片段、反义片段以及位于它们之间的间隔片段；
    所述正义片段为序列表的序列1自5′末端起第155位至第522位所示的DNA分子的反向互补序列；
    所述反义片段为序列表的序列1自5′末端起第161位至第522位所示的DNA分子。
PCT/CN2017/097608 2017-04-06 2017-08-16 蛋白质nog1在调控植物产量和穗粒数中的应用 WO2018184333A1 (zh)

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KR1020197031047A KR102539626B1 (ko) 2017-04-06 2017-08-16 식물 수확량 및 이삭 당 낟알 갯수의 조절에 있어서 단백질 nog1의 용도

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