WO2018196709A1 - 一种调控作物矮化及其产量的基因及其应用 - Google Patents

一种调控作物矮化及其产量的基因及其应用 Download PDF

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WO2018196709A1
WO2018196709A1 PCT/CN2018/084055 CN2018084055W WO2018196709A1 WO 2018196709 A1 WO2018196709 A1 WO 2018196709A1 CN 2018084055 W CN2018084055 W CN 2018084055W WO 2018196709 A1 WO2018196709 A1 WO 2018196709A1
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sbi
protein
gene
mutein
dwarf
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French (fr)
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李来庚
刘畅
杨远柱
符辰建
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中国科学院上海生命科学研究院
袁隆平农业高科技股份有限公司
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Publication of WO2018196709A1 publication Critical patent/WO2018196709A1/zh

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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    • 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
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    • 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)
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    • 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
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8226—Stem-specific, e.g. including tubers, beets
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    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
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    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
    • C12Y114/11013—Gibberellin 2-beta-dioxygenase (1.14.11.13)

Definitions

  • the present invention relates to the field of agronomy, and in particular to a gene for regulating crop dwarfing and its yield and application thereof.
  • Plant height is an important component of higher crop plant types and is closely related to crop yield.
  • the moderate dwarf height of the plant makes the crop's lodging resistance enhanced, suitable for high-density planting and easy mechanized management.
  • the plant height is too high and it is easy to cause lodging and yield reduction.
  • Moderate dwarfing of plant height is beneficial to crops suffering from fertilizer, lodging and high yield.
  • Dwarf breeding has greatly improved the yield per unit area of crops and has become one of the landmark achievements of the “Green Revolution”. It is of great significance to discover and identify dwarf genes for the genetic improvement of ideal plant types of crop varieties.
  • the sd-1 gene encodes a key enzyme GA20ox in the gibberellin synthesis pathway, which is strongly expressed in leaves, stems, and unopened flowers, so the plant height of rice carrying the sd-1 allele is reduced. And the output is not affected.
  • the sd-g gene is equivalent to the gibberellin receptor gene gid1, and the rice dwarf cultivar carrying the sd-g gene is not sensitive to exogenous gibberellin, resulting in cell elongation and dwarfing of the plant.
  • the wheat semi-dwarf genes Rht-B1b and Rht-D1b encode the DELLA protein, a negative regulator of the gibberellin signaling pathway. Mutations in this gene result in loss of DELLA protein function, downstream genes cannot be induced by gibberellin, and wheat plant height is low. Chemical.
  • japonica rice dwarf commonly used in China, namely, Dwarf Aberdeen, Dwarf France, Low Foot Wujian and Indonesian Paddy Valley. Its dwarfness is controlled by sd-1. The varieties derived therefrom accounted for 83.9% of the total varieties. Representative varieties include Plaza Dwarf, Guanglu Dwarf No. 4, Xiang Xiaozao No. 7, and Xiang Xiao Zao No. 9. Another semi-dwarf gene that has been used in large-scale production is sd-g. Its representative varieties are Guichao, Shuanggui, Teqing, Shengyou, etc. The annual promotion area has exceeded 667,000 hm.
  • the sterile lines and restorer lines in most hybrid rice combinations in China contain the semi-dwarf gene sd-1, and the sd-g gene is also utilized in hybrid rice.
  • the widespread use of a few dwarf genes has the potential to be brought about by genetic singularity. For example, in 1978, 76% of rice arable land in South Korea was planted with a uniform type of semi-dwarf varieties, most of which were affected by rice blast and low temperature. Indeed reduced production. Therefore, the discovery, identification and utilization of new dwarf source genes have become an important research content of crop breeding.
  • a dwarf related gene SBI mutein is provided, the mutein is a non-native protein, and the mutein has a catalytically active gibberellin or a precursor thereof GA 1 /GA 9 content Decreasing, catalytically inactive form of gibberellin GA 8 /GA 51 /GA 29 increased activity, and the mutant protein in the wild-type SBI protein corresponding to SEQ ID NO.: 1 selected from the group consisting of One or more core amino acid mutations associated with enzyme catalytic activity:
  • the aspartic acid (D) at position 308 is mutated to asparagine (N); and/or
  • the 338th glycine (G) is mutated to arginine (R).
  • amino acid sequence of the SBI mutein is set forth in SEQ ID NO.: 2.
  • the mutant protein has the same or substantially the same amino acid sequence as the sequence shown in SEQ ID NO.: 1 except for the mutation (e.g., positions 308, and/or 338).
  • the substantially identical is at most 1-5 amino acids different, wherein the different ones include substitutions, deletions or additions of amino acids, and the mutant protein still has catalytic activity.
  • the content of GA 1 /GA 9 is decreased in the presence of oxytetracycline or its precursor, and the activity of the gibberellin GA 8 /GA 51 /GA 29 content in the catalytically inactive form is increased.
  • sequence homology to the sequence of SEQ ID NO.: 1 is at least 80%, preferably at least 85% or 90%, more preferably at least 95%, optimally at least 98%.
  • the SBI mutein having catalytically active gibberellins (GA 1) reaction of gibberellin inactivation in the form of (GA 8).
  • the SBI muteins catalytically active gibberellin (GA 1) gibberellin (GA 29) generates an inactive form of a precursor.
  • the SBI mutein has one or more characteristics selected from the group consisting of:
  • the content of GA 1 /GA 9 is reduced by 60-100%, preferably 90-100%, compared to the wild-type SBI protein;
  • a second aspect of the invention provides a polynucleotide encoding the mutein of the first aspect of the invention.
  • polynucleotide is selected from the group consisting of:
  • the polynucleotide further comprises, in addition to the ORF of the SBI mutein, an auxiliary element selected from the group consisting of a signal peptide, a secreted peptide, a tag sequence (eg, 6His, and/or 3FLAG), Or a combination thereof.
  • an auxiliary element selected from the group consisting of a signal peptide, a secreted peptide, a tag sequence (eg, 6His, and/or 3FLAG), Or a combination thereof.
  • the polynucleotide is selected from the group consisting of a DNA sequence, an RNA sequence, or a combination thereof.
  • a third aspect of the invention provides a vector comprising the polynucleotide of the second aspect of the invention.
  • the vector comprises an expression vector, a shuttle vector, and/or an integration vector.
  • a fourth aspect of the invention provides a host cell comprising the vector of the third aspect of the invention, or the polynucleotide of the second aspect of the invention integrated in the genome.
  • the host cell is a eukaryotic cell, such as a yeast cell or a plant cell.
  • the host cell is a prokaryotic cell, such as E. coli.
  • a fifth aspect of the invention provides a method of producing the SBI mutein of the first aspect of the invention, comprising the steps of:
  • the host cell of the fourth aspect of the invention is cultured under conditions suitable for expression to thereby express an SBI mutein;
  • the SBI mutein is isolated.
  • a sixth aspect of the invention provides an enzyme preparation comprising the SBI mutein of the first aspect of the invention.
  • the enzyme preparation comprises an injection, and/or a lyophilized preparation.
  • a seventh aspect of the present invention provides the use of a substance selected from the group consisting of a dwarf related gene SBI or a protein encoded thereby, or a mutant protein thereof, or a promoter thereof, for improving agronomic traits of a crop,
  • the agronomic trait is selected from one or more of the group consisting of:
  • the dwarf-related gene SBI or its encoded protein, or a mutant protein thereof, or an enhancer thereof is also used to improve the lodging resistance of a crop.
  • An eighth aspect of the present invention provides the use of a substance selected from the group consisting of a dwarf related gene SBI or a protein encoded thereby, or a mutant protein thereof, or a promoter thereof, for use in one or more of the following use:
  • the promoter is a substance that promotes expression of the dwarf-associated gene SBI or its encoded protein, or a mutant protein thereof.
  • the promoter comprises a small molecule compound.
  • the crop is selected from the group consisting of grass crops, cruciferous crops, Solanaceae crops, legume crops, Malvaceae crops, Cucurbitaceae crops, or combinations thereof.
  • the crop is selected from the group consisting of rice (Oryza sativa), millia (Setaria italica), wheat (Triticum aestivum), sorghum bicolor, maize (Zea mays), Arabidopsis (Arabidopsis) Thaliana), Brassica rapa FPsc, Solanum Lycopersicum, Solanum tuberosum, Glycine max, Medicago truncatula, Gossypium raimondii, Cucumis sativas, or combinations thereof.
  • the rice comprises indica, japonica, or a combination thereof.
  • the dwarf-related gene comprises a cDNA sequence, a genomic sequence, or a combination thereof.
  • the dwarf related gene SBI is from a grass crop.
  • the dwarf-related gene SBI is derived from one or more crops selected from the group consisting of rice, wheat, or a combination thereof.
  • the dwarf-related gene is selected from the group consisting of the SBI gene of rice (LOC_Os05g43880), the SBI gene of wheat (Traes_3B_7ABEA6AAD Phytozome), or a combination thereof.
  • amino acid sequence of the dwarf-associated protein SBI is selected from the group consisting of:
  • nucleotide sequence of the dwarf-related gene SBI is selected from the group consisting of:
  • a ninth aspect of the invention provides a method of improving agronomic traits of a crop comprising the steps of:
  • the agronomic traits of the improved crop include:
  • the "reduced plant height” comprises the step of mutating the amino acid (aspartic acid) at position 308 of the dwarf-related protein SBI in the crop to asparagine, and/or 338
  • the amino acid (glycine) is mutated to arginine, thereby reducing the content of active gibberellin or its precursor GA 1 /GA 9 and increasing the content of the inactivated form of gibberellin GA 8 /GA 51 /GA 29 , thereby reducing Plant height.
  • a tenth aspect of the present invention provides the use of a promoter element of a dwarf-related gene SBI or a mutein thereof for spatiotemporal specific expression of a foreign protein, wherein said spatiotemporal specific expression refers to specificity at maturity Expressed at the base of the stem and stem.
  • Figure 1 shows a map clone of the SBI (shortened basal internodes) gene.
  • A strain 1S and SV14 mature plants
  • B strain 1S and SV14 mature plant height statistics
  • C SBI gene linkage region
  • D SBI gene map cloning strategy
  • E strain 1S and SV14SBI gene comparison
  • F SBI mutation site.
  • Figure 2 shows the results of verification of SBI candidate genes.
  • A gibberellin treatment of SV14 and strain 1S plants 6 weeks after germination
  • B gibberellin treatment plant height statistics
  • C strain 1S and SV14 base internode gibberellin
  • D H, Transgenic plant protein detection
  • E I, transgenic positive plants
  • Figure 3 shows the expression pattern analysis of SBI genes in various tissues of rice and the phylogenetic analysis of rice GA2ox family.
  • A rice OsGA2ox family gene expression level in rice tissue heatmap
  • B Realtime detection of SBI gene expression in rice tissue
  • C rice OsGA2ox family member system phylogenetic tree.
  • Figure 4 shows the results of staining observation of various tissues of SBI promoterSV14-GUS transgenic plants.
  • A rice leaves
  • B rice at the fourth internode
  • C rice at the fourth intersection
  • D rice inflorescence
  • E rice root
  • F rice second intersection
  • G rice second Intersection.
  • Figure 5 shows the statistical results of the length measurement of the stems of the transgenic plants. Among them, A, genetically modified rice stalk; B, transgenic rice stalk fourth inter-section slitting; C, transgenic rice stalk inter-section length statistics.
  • Figure 6 shows the results of in vitro enzyme activity assay of SBI. Wherein, A, GA chromatogram and mass spectrum of FIG. 9; B, the chromatogram and mass spectrum of the GA 51.
  • Figure 7 shows the comparison of SBI Zhu1S and SBI SV14 enzyme activity changes.
  • A SBI vitro expression of protein detection
  • B SBI SV14 comparison with the protease activity SBI Zhu1S
  • C transgenic plant endogenous gibberellin content detection.
  • Figure 8 shows the effect of single point mutations on SBI protease activity.
  • A F, transgenic plant protein detection
  • Figure 9 shows that stem-specific expression of SBI Zhu1S and SBI D308N enhances the lodging resistance of rice.
  • A B, transgenic plants and Zhonghua 11 lodging resistance difference
  • C transgenic plants and Zhonghua 11 mature spikes
  • DL transgenic plants yield traits statistics, from D to L are the lodging rate, ear length, Grain length, grain width, 100-grain weight, number per grain, heading date, number of tillers, grain weight per plant.
  • Figure 10 shows that SBI gene is involved in the domestication of plant height traits in indica and japonica rice; among them, A, indica rice and japonica rice plant height distribution; B, SBI gene region SNP in cultivated rice haplotype distribution statistics; C, SBI The haplotype distribution statistics of the gene region SNP in wild rice.
  • Figure 11 shows the results of comparison of strain 1S with the SV14SBI gene promoter.
  • A SBI gene promoter SNP and Indel distribution
  • B SNP and Indel ⁇ differentiation
  • C different source SBI promoter drives GUS gene in rice tissue activity statistics
  • D SBI gene in strain 1S and The amount expressed in the stem of SV14.
  • Figure 12 shows the results of alignment of rice OsGA2ox family protein sequences.
  • Figure 13 shows the SBI protein sequences and alignment results of higher plants and higher crops.
  • a crop such as rice
  • dwarf-related gene SBI or its encoded protein, or its mutant protein, or its molecular species by studying a large number of crop agronomic trait loci.
  • An accelerator for regulating agronomic traits of a crop being selected from the group consisting of one or more of (i) plant height; (ii) number of tillers; (iii) single plant weight; (iv) yield.
  • the present invention also finds for the first time that the amino acid (aspartic acid) at position 308 of the dwarf-related protein SBI in the crop is mutated to asparagine, and/or the amino acid (glycine) at position 338 is mutated to arginine.
  • the amino acid (aspartic acid) at position 308 of the dwarf-related protein SBI in the crop is mutated to asparagine, and/or the amino acid (glycine) at position 338 is mutated to arginine.
  • the present invention has been completed on this basis.
  • AxxB means that amino acid A at position xx is changed to amino acid B, for example "D308N” means that amino acid D at position 308 is mutated to N, and so on.
  • Mutant protein of the invention and nucleic acid encoding the same
  • mutein As used herein, the terms "mutein”, “mutein of the invention”, “SBI mutein of the invention”, “mutant protein of the dwarf-related gene SBI of the invention” are used interchangeably and refer to a non-naturally occurring SBI.
  • a mutein wherein the mutein is a protein artificially engineered based on the protein of SEQ ID NO.: 1, wherein the mutein contains a core amino acid associated with catalytic activity of the enzyme, and at least One is artificially engineered; and the mutein of the invention has a catalytically active gibberellin or a precursor thereof having a reduced content of GA 1 /GA 9 and a catalytically inactive form of gibberellin GA 8 /GA 51 /GA 29 active.
  • core amino acid refers to SEQ ID NO.: 1 and has a homology to SEQ ID NO.: 1 of at least 80%, such as 84%, 85%, 90%, 92%, 95%, 98%.
  • the corresponding site is a specific amino acid as described herein, such as the sequence based on SEQ ID NO.: 1, the core amino acid is:
  • mutant protein obtained by mutating the above core amino acid has a catalytically active gibberellin or a precursor thereof, wherein the content of GA 1 /GA 9 is decreased, and the catalytically inactive form of gibberellin GA 8 /GA 51 /GA 29 is increased. active.
  • the core amino acid of the present invention is mutated as follows:
  • the 338th glycine (G) is mutated to arginine (R); and/or
  • the aspartic acid (D) at position 308 was mutated to asparagine (N).
  • the amino acid numbering in the mutant protein of the present invention is based on SEQ ID NO.: 1.
  • the mutant protein may have a mismatch relative to the amino acid numbering of SEQ ID NO.: 1, such as a mismatch to the N-terminus or C-terminus of the amino acid, 1-5, using conventional sequence alignment techniques in the art, which are generally It is understood that such misalignment is within a reasonable range and should not result in 80% (eg 90%, 95%, 98%) homology with the same or similar catalytic activity of Gibberellium due to misalignment of amino acid numbering.
  • the amount of the GA 1 /GA 9 content of the hormone or its precursor is lowered, and the mutant protein which catalyzes the activity of the gibberellin GA 8 /GA 51 /GA 29 content in an inactivated form is not within the scope of the mutein of the present invention.
  • the muteins of the invention are synthetic or recombinant proteins, i.e., may be products of chemical synthesis, or produced by recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants).
  • the muteins of the invention may be glycosylated or may be non-glycosylated, depending on the host used in the recombinant production protocol.
  • the muteins of the invention may or may not include an initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of the muteins.
  • fragment refers to a protein that substantially retains the same biological function or activity of the mutein.
  • the mutein fragment, derivative or analog of the present invention may be (i) a mutein having one or more conserved or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acids
  • the residue may or may not be encoded by the genetic code, or (ii) a mutein having a substituent in one or more amino acid residues, or (iii) a mature mutein and another compound (such as an extended mutein) a half-life compound, such as polyethylene glycol), a fusion protein formed by fusion, or (iv) a mutant protein formed by fused an additional amino acid sequence to the mutant protein sequence (such as a leader or secretion sequence or used to purify the mutant protein) Sequence or proprotein sequence, or fusion protein with the formation of an antigenic IgG fragment).
  • conservatively substituted amino acids are preferably produced by amino acid
  • the active mutant protein of the present invention has a catalytically active gibberellin or a decrease in the content of the precursor GA 1 /GA 9 and a catalytically inactive form of the gibberellin GA 8 /GA 51 /GA 29 content.
  • the mutein is represented by SEQ ID NO.: 2. It is to be understood that the mutein of the present invention generally has higher homology (identity) than the sequence shown in SEQ ID NO.: 2, preferably, the mutein and SEQ ID NO.: 2 The sequence has a homology of at least 80%, preferably at least 85% to 90%, more preferably at least 95%, and most preferably at least 98%.
  • the muteins of the invention may also be modified. Modifications (usually without altering the primary structure) include: chemically derived forms of the mutant protein, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of muteins or in further processing steps. Such modification can be accomplished by exposing the mutein to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are muteins that have been modified to increase their resistance to proteolytic properties or to optimize solubility properties.
  • the invention also provides polynucleotide sequences encoding SBI polypeptides, proteins or variants thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include: DNA, genomic DNA, or synthetic DNA, which can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO.: 4 or a degenerate variant.
  • polynucleotide encoding a mutein may be a polynucleotide comprising a mutein of the invention, or a polynucleotide further comprising an additional coding and/or non-coding sequence.
  • a preferred polynucleotide sequence encoding a mutein is shown in SEQ ID NO.: 3.
  • the present invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or muteins having the same amino acid sequence as the present invention.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the encoded mutant protein thereof.
  • the invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions (or stringent conditions).
  • stringent conditions means: (1) hybridization and elution at a lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60 ° C; or (2) hybridization a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) at least 90% identity between the two sequences, more It is good that hybridization occurs more than 95%.
  • the muteins and polynucleotides of the invention are preferably provided in isolated form, and more preferably, purified to homogeneity.
  • SBI gene of the present invention is preferably derived from rice, other plants are highly homologous to the rice SBI gene (eg, having more than 80%, such as 85%, 90%, 95%, or 98% sequence identity). Genes are also within the scope of the present invention. Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.
  • the full length sequence of the polynucleotide of the present invention can usually be obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • a method of amplifying DNA/RNA using PCR technology is preferably used to obtain the polynucleotide of the present invention.
  • RACE method RACE-cDNA end rapid amplification method
  • primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein.
  • the amplified DNA/RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • wild-type SBI protein and “wild-type dwarf-related gene SBI protein” are used interchangeably to refer to a naturally occurring, unmodified SBI protein whose nucleotides can be genetically engineered. Obtained, such as genome sequencing, polymerase chain reaction (PCR), etc., whose amino acid sequence can be derived from a nucleotide sequence.
  • PCR polymerase chain reaction
  • amino acid sequence of a typical wild-type SBI protein of the present invention is shown in SEQ ID NO.: 1.
  • the invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered with the vectors of the invention or the mutant protein coding sequences of the invention, and methods of producing the polypeptides of the invention by recombinant techniques.
  • polynucleotide sequences of the present invention can be utilized to express or produce recombinant muteins by conventional recombinant DNA techniques. Generally there are the following steps:
  • the invention also provides a recombinant vector comprising the gene of the invention.
  • the promoter of the recombinant vector comprises a multiple cloning site or at least one cleavage site downstream.
  • the gene of interest is ligated into a suitable multiple cloning site or restriction site to operably link the gene of interest to the promoter.
  • the recombinant vector comprises (from the 5' to 3' direction): a promoter, a gene of interest, and a terminator.
  • the recombinant vector may further comprise an element selected from the group consisting of: a 3' polynucleotideization signal; a non-translated nucleic acid sequence; a transport and targeting nucleic acid sequence; a resistance selection marker (dihydrofolate reductase, Neomycin resistance, hygromycin resistance, and green fluorescent protein, etc.); enhancer; or operator.
  • a 3' polynucleotideization signal a non-translated nucleic acid sequence
  • a transport and targeting nucleic acid sequence a resistance selection marker (dihydrofolate reductase, Neomycin resistance, hygromycin resistance, and green fluorescent protein, etc.); enhancer; or operator.
  • a polynucleotide sequence encoding a mutein can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing the mutein encoding DNA sequences of the invention and suitable transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • expression vectors containing the genes of the present invention can construct expression vectors containing the genes of the present invention using well known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. When a recombinant expression vector is constructed using the gene of the present invention, any of the enhanced, constitutive, tissue-specific or inducible promoters can be added before the transcription initiation nucleotide.
  • a gene, expression cassette or vector comprising the invention can be used to transform a suitable host cell such that the host expresses the protein.
  • the host cell may be a prokaryotic cell such as Escherichia coli, Streptomyces, Agrobacterium; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. It will be apparent to one of ordinary skill in the art how to select an appropriate vector and host cell. Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote (such as E. coli), it can be treated with the CaCl 2 method or by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.).
  • the transformed plants can also be subjected to methods such as Agrobacterium transformation or gene gun transformation, such as leaf disc method, immature embryo transformation method, flower bud soaking method and the like.
  • Agrobacterium transformation or gene gun transformation such as leaf disc method, immature embryo transformation method, flower bud soaking method and the like.
  • plants can be regenerated by conventional methods to obtain transgenic plants.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, plant cells (such as rice cells).
  • an enhancer sequence is inserted into the vector.
  • An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, acting on a promoter to enhance transcription of the gene.
  • Usable examples include a 100 to 270 base pair SV40 enhancer on the late side of the replication initiation point, a polyoma enhancer on the late side of the replication initiation site, and an adenovirus enhancer.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the present invention finds for the first time that the dwarf-related gene SBI or its encoded protein, or its mutant protein, or its promoter can regulate the agronomic traits of crops (for example, plant height, number of tillers, yield, etc.).
  • the present inventors have found for the first time that the amino acid (aspartic acid) at position 308 of the dwarf-related protein SBI in the crop is mutated to asparagine, and/or the amino acid (glycine) at position 338 is mutated to arginine. , can significantly reduce the content of active gibberellin or its precursor GA 1 /GA 9 and significantly increase the content of gibberellin GA 8 /GA 51 /GA 29 in inactive form, thereby significantly reducing plant height and improving crops Resistance to lodging.
  • the present invention finds for the first time that the dwarf-related gene SBI or its encoded protein, or its mutant protein, is specifically expressed at the maturity of the stalk and the stem at the base of the stem, and the gene can be specifically shortened by overexpressing the gene in the stem.
  • the length of the base section is specifically expressed at the maturity of the stalk and the stem at the base of the stem, and the gene can be specifically shortened by overexpressing the gene in the stem. The length of the base section.
  • the present invention found for the first time that the transgenic plants with rice stem-specific overexpressing SBI gene were reasonably reduced in plant height, the lodging resistance was increased by 100%, the effective tiller number was increased by 30%, and the individual plant weight was increased by 29%.
  • the present inventors have found for the first time that the SBI gene can specifically reduce the gibberellin content at the base of the rice stem, thereby reasonably reducing the plant height, increasing the lodging resistance, increasing the number of tillers, increasing the yield, and the gene is in other
  • the homologous genes in the crop can be used to cultivate crop dwarf varieties and improve the lodging resistance of crops; at the same time, it can increase the effective tiller number of crops, especially gramineous crops, and increase crop yield.
  • the localization and cloning of SBI gene is extremely important for the storage of dwarf genes, and it is also an innovation in the creation and screening of new dwarf rice, and it has important application value and prospect for the cultivation of excellent traits of higher crops.
  • Rice male sterile lines 1S and SV14 were provided by Hunan Yahua Seed Industry Research Institute. 1000 F2 plants used for genetic analysis were produced by selfing of F1 plants orthogonally produced by strain 1S and SV14 and planted in Daejeon. The transgenic rice materials used in the phenotypic analysis and data statistics of this study were all transgenic T2 plants planted in Daejeon. The rice material used in the GUS staining experiment was planted in an artificial climate chamber (12-hour photoperiod, light intensity: 200-250 ⁇ mol ⁇ m-2 ⁇ s-1, temperature 28 ⁇ 1 °C), which was a transgenic T2 plant. Measurement of plant height phenotype: The plant height of mature plants was measured from the ground to the tip of the ear. The order between the nodes starts from the first section of the neck section, and the morphological upper end is between the second section and the fifth section.
  • the nuclear genomic DNA of rice was extracted by CTAB method. Secondly, the genomes of the 1S and F2 pools were sequenced by whole genome, and then the read lengths were compared with the Nipponbare genome, and the Nipponbare genome was modified according to the SNP and indel sites, and the corrected genome was used as the reference genome.
  • the F2DNA pool read length was compared with the reference genome, SNP extraction was performed, and sliding window calculation was performed based on the SNP index and plotted.
  • strain 1S and SV14SBI gene were cloned and subcloned into plasmid pCAMBIA1300 (commercially available plasmid), and then transferred to Agrobacterium tumefaciens strain EHA105 (obtained from Shanghai Institute of Biological Sciences, Chinese Academy of Sciences) and transferred.
  • plasmid pCAMBIA1300 commercially available plasmid
  • EHA105 Agrobacterium tumefaciens strain EHA105 (obtained from Shanghai Institute of Biological Sciences, Chinese Academy of Sciences) and transferred.
  • rice flower 11 obtained from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
  • strain 1S and SV14SBI gene was cloned and subcloned into plasmid pCAMBIA1301 (commercially available plasmid), and then transferred into Agrobacterium tumefaciens strain EHA105 and transferred into rice flower 11 to study strain 1S and SV14SBI genes. Promoter activity.
  • the mature tissue of the fifth internode of rice was placed in FAA fixative (70% ethanol, 5% acetic acid, 5% formaldehyde) at 4 ° C for more than 24 h. 70%, 85%, 95% ethanol dehydrated for 1h each time; anhydrous ethanol 30min, 2 times; anhydrous ethanol / xylene 1:1, 1h; xylene, 1h; xylene / paraffin 1:1, 2h; Paraffin I, 4h; paraffin II, overnight.
  • the paraffin wax was melted into the embedding frame, and the sample was placed in paraffin, cooled, and stored at 4 °C.
  • the slides were placed on a 42 °C roaster and covered with sterile water.
  • the repaired wax block was cut into 10 ⁇ m thick wax tape on a microtome, placed on a glass slide, and suspended in water. After the wax tape is flattened, absorb the moisture with absorbent paper and bake at 42 ° C overnight.
  • Toluidine blue staining The prepared sections were stained in 0.1% toluidine blue solution (0.01 M sodium acetate buffer, pH 4.4-4.6) for 30 min, washed with water, and dried in an oven at 37 ° C; xylene Dewaxing 2 times, each time 10 min; neutral gum seal. Microscopic observation.
  • the SBI recombinant protein of SV14 and strain 1S was subjected to in vitro induced purification.
  • a single colony was picked up in 5 ml of LB medium and cultured overnight at 37 ° C on a shaker at 210 rpm.
  • 1 ml of the bacterial solution was transferred to 100 ml of LB medium, and cultured at 37 ° C for 2 hours at 210 rpm, and the OD600 reading of the bacterial solution was measured to 0.6.
  • Induction was carried out by adding 1 M IPTG at a ratio of 1:2000, and was induced by shaking at 210 rpm overnight at 16 °C.
  • the cells were induced to express by centrifugation, and the cells were resuspended in cold lysate (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.4), sonicated (200 W, 10 s ultrasound, 20 s rest, 30 times). ). After centrifugation, the supernatant was added to 1 ml of equilibrated NEB Amylose Resin (E8021), and the column was passed at 4 ° C for about 2 hours, and then washed twice with 4 ml of the lysate and collected.
  • cold lysate 20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.4
  • sonicated 200 W, 10 s ultrasound, 20 s rest, 30 times.
  • Each component was collected by washing 4 times with 1 ml of eluate (10 mM maltose dissolved in lysate), each standing for 10 minutes.
  • the purified fraction was added to an ultrafiltration tube, 6000 g, 30 min to 200 ⁇ l. Transfer the storage buffer (100 mM Tris-Hcl pH 7.5) to a 4-5 ml storage buffer, centrifuge at 6000 g to 500 ⁇ l, and store at -80 °C. Thereafter, the purified protein was incubated with GA 9 and incubated at 30 ° C for 6 h or a corresponding time gradient.
  • GUS activity analysis was performed on the transgenic positive material.
  • the tissue was first treated with acetone (about 10 min, 4 ° C), and the residual acetone in the tissue was washed away with 100 mM NaPO4 buffer (pH 7.0) using GUS color developing solution [100 mM NaPO4 (pH 7. 0), 10 mM EDTA, 2 mM X-gluc, 5 mM K4Fe (CN) 6, 5 mM K3Fe (CN) 6, 0.2% Triton X-100] was incubated at 37 ° C for a suitable period of time. Finally, the reaction was terminated with 75% ethanol and decolorized.
  • GUS activity was quantitatively analyzed on the transgenic material, the tissue was ground in liquid nitrogen, and 1 ml of GUS extraction buffer [10 mM EDTA (pH 8.0), 0.1% SDS, 50 mM sodium phosphate (pH 7.0), 0.1% was added. Triton X-100, 100 mM ⁇ -mercaptoethanol, 25 ⁇ g/ml PMSF], vortexed for 5 min, centrifuged at 12000 rpm for 10 minutes, transferred the supernatant to a new centrifuge tube and placed on ice. 10 ⁇ l of the supernatant was added to 130 ⁇ l of GUS reaction solution (containing 1 mM 4-MUG in GUS buffer), and incubated at 37 ° C for 10 minutes. After the reaction was completed, 10 ⁇ l was added to 190 ⁇ l of 1 M sodium carbonate and mixed. The fluorescence value of 4-MU was detected by a microplate reader at 365 nm excitation light and 455 nm emission light.
  • RNA from different tissues was extracted using the Omega Plant Total RNA Extraction Kit (R6827).
  • the first strand cDNA was synthesized using Oligo dT primer using TransScript One-Step gDNA Removal and cDNA Synthesis Kit (AT311) (purchased from Beijing Quanjin Biotechnology Co., Ltd.) with 1 ⁇ g of total RNA as a template.
  • Use TransStart TipTop Green qPCR SuperMix reagent (AQ131) (purchased from Beijing Quanjin Biotechnology Co., Ltd.) was subjected to QRT-PCR analysis on MyiQ real-time PCR detection system (Bio-Rad) quantitative PCR instrument, and rice actin ( OsACT1, Os03g0718100) as an internal reference gene.
  • Plant samples were ground to a fine powder in liquid nitrogen and 1.5 volumes of protein extraction buffer (1M NaAc, pH 5.0,
  • the amino acid sequence of the rice GA2ox family was downloaded from the rice genome-wide database (TIGR Rice, http://rice.plantbiology.msu.edu/), and the corresponding amino acid sequences of other species were downloaded from the Pytozome database (https://phytozome.jgi.doe. Gov/).
  • the amino acid sequences were aligned and phylogenetically constructed using MEGA4.0. SNP information of different rice varieties was obtained from the RiceHAP3 database and statistically calculated.
  • Example 1 SBI (shortened basal internodes) gene encodes a gibberellin-2-beta dioxygenase
  • SV14 (selected by Yahua Seed Co., Ltd.) is a widely used thermo-sensitive male sterile line in breeding production. It is based on rice temperature-sensitive sterile line 1S (selected by Yahua Seed Co., Ltd.) as the chassis. Varieties, rice semi-dwarf varieties cultivated on the basis of somatic mutation combined with artificial breeding techniques. Compared with strain 1S, except for the plant height decreased by more than 20 cm, there was no significant change in other important agronomic traits of SV14 (Fig. 1A, B), and the lodging resistance of hybrid progeny was also enhanced.
  • the hybrid population of SV14 and strain 1S was constructed first, and the plant height traits of more than 1000 hybrid F2 plants were measured and counted. Extremely dwarfed F2 plants were further analyzed. Next, the genomic DNA of the above 65 extremely dwarfed F2 plants was extracted and mixed as an F2 dwarf gene pool. At the same time, the genomic DNA of strain 1S was extracted as a high-stalk gene pool. Then, the DNA of the two pools was sequenced by resequencing, and the single reference polymorphism (SNP) locus was searched by comparing with the rice reference genome IRGSP.
  • SNP single reference polymorphism
  • the dwarf gene pool genome was compared with the high stalk gene pool genome, and the SNP molecular markers were used for linkage analysis of dwarf traits (Fig. 1D).
  • a linkage region with a dwarf phenotype was found at chromosome 25.5-25.8Mb of rice (Fig. 1C).
  • annotated analysis of genes in a plurality of completely linked SNP loci in the target region is performed.
  • These fully linked SNP distributions contain three genes, one of which, LOC_Os05g43880, shares homology with the gibberellin-2-beta dioxygenase gene sequence.
  • the content of gibberellin GA 1 at the base of SV14 stem was decreased compared with strain 1S, and the corresponding inactivated form of gibberellin GA 8 was increased (Fig. 2C).
  • the application of gibberellin GA 3 exogenously to SV14 and strain 1S 6 weeks after germination restored the plant height of SV14 to a height consistent with strain 1S (Fig. 2A, B).
  • the plant height of transgenic positive strains with SBI zhu1S overexpressing stems was partially dwarfed, only about 10 cm (Fig.
  • LOC_Os05g43880 is the SBI gene which causes the semi-dwarf trait of SV14, and the gene was successfully cloned.
  • SV14 Compared with strain 1S, SV14 showed no significant changes in other important agronomic traits except plant height reduced by 20 cm. This suggests that the expression of the SBI gene may be tissue specific.
  • the rice gibberellin-2-beta dioxygenase family has 10 members. The expression pattern of all members of the family in various tissues of rice indicates that SBI is specifically expressed in rice stems and sheaths (Fig. 3A). The results of real-time quantitative PCR showed that the expression level of SBI gene in rice stalks was relatively high at maturity, and the expression level was the highest at the base of the stem, while the expression in other tissues was relatively low (Fig. 3B).
  • GUS staining of the tissues of transgenic positive plants driven by SBI promoter SV14 driving GUS reporter gene revealed that the enzyme activity of GUS in leaves, inflorescences and roots was weak, only in the wounds of leaves, vascular bundles of individual florets and new generation. The radicle in the vascular bundle is expressed (Fig. 4A, D, E). In the second and fourth internodes of the stem, GUS enzyme activity was significantly enhanced (Fig. 4B, G). Cross-slice staining of internodes showed that the GUS reporter gene was strongly expressed in various tissues in the stem (Fig. 4C, F).
  • the length of the stem of the SBIpromoter zhu1S- SBI Zhu1s transgenic plants was specifically shortened from the third internode to the fifth internode, and the length of the third internode was shortened by 50% and 70. %, the length of the fourth internode was shortened by 78% and 73%, the length of the fifth internode was shortened by 50% and 60%, and the length of parenchyma cells at the basal internode was also shortened; and the transgenic positive strain of SBIpromoter SV14 -SBI SV14 The stalks were extremely shortened at each node, and the shortening rate was over 90%. The elongation of the parenchyma cells at the base was severely blocked (Fig. 5A-C).
  • the SBI Zhu1S protein and the SBI SV14 protein were induced by E. coli (Fig. 7A), and they were co-incubated with the substrate C 19 gibberellin GA 9 under certain reaction conditions, respectively, and then the reaction was performed by LC-MS. GA 9 inactivated form gibberellin GA 51 detects the product.
  • the detection of gibberellin in the transgenic positive plants showed that the active red in the SBIPromoter zhu1S- SBI Zhu1S and SBIPromoter SV14- SBI SV14 transgenic positive plants was compared with the control Zhonghua 11 (obtained from the Shanghai Institute of Life Sciences, Chinese Academy of Sciences).
  • the content of GA 1 was decreased, and the content of inactivated gibberellin GA 29 was increased, but SBIpromoter SV14 -SBI SV14 was more severe than SBIpromoter zhu1S -SBI Zhu1S .
  • the content of GA 1 was reduced to 0, and the content of GA 29 was medium. Flower 4 is 4 times (Fig. 7C).
  • the yield-related traits of SBI zhu1S and SBI D308N transgenic plants and Zhonghua 11 were specifically investigated in the waxy maturity stage of rice, and it was found that overexpression of SBI zhu1S and SBI D308N in Zhonghua 11 stalk significantly improved.
  • the lodging resistance of Zhonghua 11 (Fig. 9A, B, D), in the wax maturity stage, 89% of the plant 11 plants had stem lodging, and the transgenic plants were almost 0; the ear length, grain length, grain Width, grain weight, number per grain,
  • Gibberellin plays a broad-spectrum regulation on plant cell elongation and plant height.
  • the elongation between the internodes and the second internodes of rice stems usually occurs in the late stage of rice reproductive growth, ie, jointing stage and heading stage.
  • the excessive shortening between the neck section and the second section may have adverse effects on rice jointing and heading.
  • the elongation of the internodes of rice stalks, including the internodes to the fifth internodes often occurs during vegetative growth and pre-growth, so the dwarfing of the internodes does not affect the development of rice flowers and seeds. Fine regulation of plant height can be achieved by using a gene that specifically shortens the base of the stem.
  • the present invention finds for the first time that the SBI gene is specifically expressed in rice stems and has the highest expression at the base of the stem, but is low in other tissues, especially in inflorescences and leaves. Excessive expression of this gene in rice stems can specifically shorten the internodes of the stem, and increase the number of effective tillers and yield per plant. Therefore, it is possible to specifically control the plant height by controlling the change in the expression level of SBI in rice stems under the premise of ensuring high yield and stable yield.
  • the present invention also demonstrates for the first time that a change in amino acid position 338 in the amino acid sequence of the SBI-encoded protein plays a key role in the change in SBI protease activity.
  • the alignment of the amino acid sequence of rice OsGA2ox family showed that the amino acid 338 was conserved in the C 19 GA2ox subfamily and was located in the conserved domain of C 19 GA2ox protein, while the site was relatively poorly conserved in the C 20 GA2ox subfamily. (Figure 12). This site is important for the specificity of the enzyme activity of C 19 GA2ox.
  • SBI gene is important in angiosperms, and SBI gene is also involved in the regulation of gibberellin on high traits of different higher crops.
  • the exploration of the function of the conserved locus of the present invention also provides genetic resources and molecular theoretical basis for the plant type breeding of lodging, fertilizer and high-yield varieties of other economic crops.
  • the SBI gene may be involved in the rice cultivar japonica rice subspecies and japonica rice. Subspecies plant height traits domestication process.
  • the polymorphic SNP locus in the SBI gene region can be used as a SNP for indica and japonica rice differentiation in the study of the source of chromosomal fragments in different rice cultivars.
  • the promoter of the SBI gene derived from indica is more potent than the SBI gene promoter derived from indica (Fig. 11C), but both function mainly between the stem segments.
  • the invention utilizes the SBI promoter SBIPromoter SV14 of indica rice source to drive SBI zhu1S to over-express in the stem 11 of the weak lodging resistance ability, which can significantly enhance the lodging resistance of Zhonghua 11 without affecting other yield traits (Fig. 9). Therefore, the SBI gene promoter can be developed and utilized, and the promoter of the SBI gene is used to drive the target gene of interest to artificially control the rice stem. It provides new ideas, methods and theoretical basis for the breeding of excellent traits of rice.

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Abstract

提供了一种调控作物矮化及产量的基因SBI和SBI突变蛋白，及其用于调控农作物的农艺性状的用途，所述农艺性状选自下组的一种或多种(i)株高；(ii)分蘖数；(iii)单株粒重；(iv)产量。将农作物中矮化相关蛋白SBI的第308位氨基酸(天冬氨酸)突变为天冬酰胺，和/或第338位氨基酸(甘氨酸)突变为精氨酸，可显著降低活性赤霉素GA 1/GA 9的含量，并显著提高失活形式的赤霉素GA 8/GA 51/GA 29的含量，从而显著降低株高，提高农作物的抗倒伏能力。

Description

一种调控作物矮化及其产量的基因及其应用 技术领域
本发明涉及农学领域，具体地，涉及一种调控作物矮化及其产量的基因及其应用。
背景技术
植株高度是高等作物株型的重要组成，与作物产量密切相关。植株高度的适度矮化使得作物抗倒伏能力增强，适宜高密度种植和便于机械化管理。例如，对于粮食作物如水稻、小麦，株高过高易引起倒伏而减产，株高适度矮化有利于作物耐肥、抗倒伏、高产。矮化育种使作物单位面积产量获得了极大的提高，并成为“绿色革命”的标志性成就之一，发掘和鉴定矮源基因对作物品种的理想株型的遗传改良具有十分重要的意义。
以水稻为例，目前人们发现的控制水稻矮化性状的基因已有130多个，主效基因已达70多个。株高既属于数量性状遗传，又表现为质量性状遗传。既由主效基因控制，同时也受到微效基因与环境因素的影响。对这些矮秆基因的分子机制研究表明，它们大多通过影响内源植物激素——赤霉素、生长素、油菜素内酯、独角金内酯、脱落酸及乙烯的生物合成及信号转导从而调节作物的株高，其中以赤霉素的调控最为重要。例如，sd-1基因编码一个赤霉素合成途径中的关键酶GA20ox，该基因主要在叶片、茎秆、未开放的花中表达强烈，因此携带sd-1等位突变基因的水稻株高降低，而产量不受影响。sd-g基因与赤霉素受体基因gid1相等位，携带sd-g基因的水稻矮秆品种对外源赤霉素不敏感，导致细胞伸长受到影响，植株发生矮化。小麦半矮秆基因Rht-B1b和Rht-D1b编码赤霉素信号通路中负调因子DELLA蛋白，该基因的突变导致DELLA蛋白功能缺失，下游基因无法被赤霉素诱导表达，小麦株高发生矮化。
中国普遍利用的籼稻矮源主要有4个，即矮仔占、矮脚南特、低脚乌尖和印尼水田谷。其矮生性均由sd-1控制。由其衍生的品种占总品种数的83.9％。代表性品种有广场矮、广陆矮四号、湘矮早7号、湘矮早9号等。另一个在生产上得到了大面积应用的半矮秆基因为sd-g，其代表性品种有桂朝、双桂、特青、胜优等，年推广面积都曾超过66.7万hm。除了常规稻品种，中国大多数杂交稻组合中的不育系和恢复系都含有半矮秆基因sd-1，而sd-g基因在杂交稻中也得到了利用。少数矮秆基因的广泛利用潜藏着由遗传单一带来的风险，例如1978年韩国有76％的水稻耕地面积种植统一类型的半矮秆品种，这些品种大多受到稻瘟病和低温的影响，造成了严重减产。因此，发掘、鉴定和利用新矮源基因成为作物育种的重要研究内容。迄今为止，国内外通过发掘和诱变已获得了70余个非sd-1等位矮生、半矮生基因，但这些矮秆材料大多农艺性状差、生长势弱、植株过矮、分蘖少或极多、穗型不理想等，在育种上很少得到应用。因此，在拓宽矮源的同时，改良种质、挖掘具有理想农艺性状的新型矮源极为重要。
发明内容
本发明的目的在于提供一种可改良农作物农艺性状的新的矮化相关基因及其突变蛋白。
在本发明第一方面，提供了一种矮化相关基因SBI突变蛋白，所述的突变蛋白为非天然蛋白，且所述突变蛋白具有催化活性赤霉素或其前体GA 1/GA 9含量降低，催化失活形式的赤霉素GA 8/GA 51/GA 29含量升高的活性，并且所述突变蛋白在野生型的SBI蛋白的对应于SEQ ID NO.:1的选自下组的一个或多个与酶催化活性相关的核心氨基酸发生突变：
第308位天冬氨酸(D)；和/或
第338位甘氨酸(G)。
在另一优选例中，所述第308位天冬氨酸(D)突变为天冬酰胺(N)；和/或
第338位甘氨酸(G)突变为精氨酸(R)。
在另一优选例中，所述SBI突变蛋白的氨基酸序列如SEQ ID NO.:2所示。
在另一优选例中，所述的突变蛋白除所述突变(如308、和/或338位)外，其余的氨基酸序列与SEQ ID NO.:1所示的序列相同或基本相同。
在另一优选例中，所述的基本相同是至多有1-5个氨基酸不相同，其中，所述的不相同包括氨基酸的取代、缺失或添加，且所述的突变蛋白仍具有催化活性赤霉素或其前体GA 1/GA 9含量降低，催化失活形式的赤霉素GA 8/GA 51/GA 29含量升高的活性。
在另一优选例中，与SEQ ID NO.:1所示序列的同源性至少为80％，较佳地至少为85％或90％，更佳地至少为95％，最佳地至少为98％。
在另一优选例中，所述SBI突变蛋白催化具有活性的赤霉素(GA 1)反应生成失活形式的赤霉素(GA 8)。
在另一优选例中，所述SBI突变蛋白催化具有活性的赤霉素(GA 1)的前体生成失活形式的赤霉素(GA 29)。
在另一优选例中，所述SBI突变蛋白催化具有活性的赤霉素前体(GA 9)反应生成失活形式的赤霉素(GA 51)。
在另一优选例中，所述SBI突变蛋白具有选自下组的一个或多个特征：
(a)与野生型SBI蛋白相比，在所述SBI突变蛋白的催化反应中，GA 1/GA 9的含量减少了60-100％，较佳地，90-100％；
(b)与野生型SBI蛋白相比，在所述SBI突变蛋白的催化反应中，GA 8/GA 51/GA 29的含量增加了50％－100％，较佳地，80％－100％。
本发明第二方面提供了一种多核苷酸，所述的多核苷酸编码本发明第一方面所述的突变蛋白。
在另一优选例中，所述多核苷酸选自下组：
(a)编码如SEQ ID NO.:2所示多肽的多核苷酸；
(b)序列如SEQ ID NO.:3所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:3所示序列的同源性≥95％(较佳地≥98％)，且编码SEQ ID NO.:1或2所示多肽的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述的多核苷酸在SBI突变蛋白的ORF的侧翼还额外含有 选自下组的辅助元件：信号肽、分泌肽、标签序列(如6His、和/或3FLAG)、或其组合。
在另一优选例中，所述的多核苷酸选自下组：DNA序列、RNA序列、或其组合。
本发明第三方面提供了一种载体，所述的载体含有本发明第二方面所述的多核苷酸。
在另一优选例中，所述载体包括表达载体、穿梭载体、和/或整合载体。
本发明第四方面提供了一种宿主细胞，所述的宿主细胞含有本发明第三方面所述的载体，或其基因组中整合有本发明第二方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞为真核细胞，如酵母细胞或植物细胞。
在另一优选例中，所述的宿主细胞为原核细胞，如大肠杆菌。
本发明第五方面提供了一种产生本发明第一方面所述SBI突变蛋白的方法，包括步骤：
在适合表达的条件下，培养本发明第四方面所述的宿主细胞，从而表达出SBI突变蛋白；和
分离所述SBI突变蛋白。
本发明第六方面提供了一种酶制剂，所述酶制剂包含本发明第一方面所述的SBI突变蛋白。
在另一优选例中，所述的酶制剂包括注射剂、和/或冻干制剂。
本发明第七方面提供了一种物质的用途，所述的物质选自下组：矮化相关基因SBI或其编码蛋白、或其突变蛋白、或其促进剂，用于改善农作物的农艺性状，所述农艺性状选自下组的一种或多种：
(i)株高；
(ii)分蘖数；
(iii)单株粒重；
(iv)产量；
(v)茎秆基部节间长度。
在另一优选例中，所述的矮化相关基因SBI或其编码蛋白、或其突变蛋白、或其促进剂还用于改善农作物的抗倒伏性。
本发明第八方面提供了一种物质的用途，所述的物质选自下组：矮化相关基因SBI或其编码蛋白、或其突变蛋白、或其促进剂，用于以下一种或多种用途：
(i-1)降低株高；
(ii-1)增加分蘖数；
(iii-1)提高产量；
(iv-1)缩短茎秆基部节间长度。
在另一优选例中，所述的促进剂为促进矮化相关基因SBI或其编码蛋白、或其突变蛋白表达的物质。
在另一优选例中，所述的促进剂包括小分子化合物。
在另一优选例中，所述的农作物选自下组：禾本科农作物、十字花科农作物、茄科农作物、豆科农作物、锦葵科农作物、葫芦科农作物、或其组合。
在另一优选例中，所述农作物选自下组：水稻(Oryza sativa)、谷子(Setaria italica)、小麦(Triticum aestivum)、高粱(Sorghum bicolor)、玉米 (Zea mays)，拟南芥(Arabidopsis thaliana)、油菜(Brassica rapa FPsc)、番茄(Solanum Lycopersicum)、马铃薯(Solanum tuberosum)、大豆(Glycine max)、苜蓿(Medicago truncatula)、棉花(Gossypium raimondii)、黄瓜(Cucumis sativas)、或其组合。
在另一优选例中，所述的水稻包括籼稻、粳稻、或其组合。
在另一优选例中，所述的矮化相关基因包括cDNA序列、基因组序列、或其组合。
在另一优选例中，所述矮化相关基因SBI来自禾本科作物。
在另一优选例中，所述的矮化相关基因SBI来自选自下组的一种或多种农作物：水稻、小麦、或其组合。
在另一优选例中，所述矮化相关基因选自下组：水稻的SBI基因(LOC_Os05g43880)、小麦的SBI基因(Traes_3B_7ABEA6AAD Phytozome)、或其组合。
在另一优选例中，所述矮化相关蛋白SBI的氨基酸序列选自下组：
(i)具有SEQ ID NO.:1所示氨基酸序列的多肽；
(ii)将如SEQ ID NO.:1所示的氨基酸序列经过一个或几个(如1-10个)氨基酸残基的取代、缺失或添加而形成的，具有所述调控农艺性状功能的、由(i)衍生的多肽；或(iii)氨基酸序列与SEQ ID NO.:1所示氨基酸序列的同源性≥90％(较佳地≥95％，更佳地≥98％)，具有所述调控农艺性状功能的多肽。
在另一优选例中，所述矮化相关基因SBI的核苷酸序列选自下组：
(a)编码如SEQ ID NO.:1所示多肽的多核苷酸；
(b)序列如SEQ ID NO.:4所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:4所示序列的同源性≥95％(较佳地≥98％，更佳地≥99％)的多核苷酸；
(d)在SEQ ID NO.:4所示多核苷酸的5’端和/或3’端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸；
(e)与(a)-(d)任一所述的多核苷酸互补的多核苷酸。
本发明第九方面提供了一种改良作物农艺性状的方法，包括步骤：
提高所述作物中矮化相关蛋白SBI或其突变蛋白的表达量或活性，从而改良作物的农艺性状。
在另一优选例中，所述的改良作物的农艺性状包括：
(i-1)降低株高；
(ii-1)增加分蘖数；
(iii-1)提高产量。
在另一优选例中，所述的“降低株高”包括步骤：将所述作物中矮化相关蛋白SBI的第308位氨基酸(天冬氨酸)突变为天冬酰胺，和/或第338位氨基酸(甘氨酸)突变为精氨酸，从而降低活性赤霉素或其前体GA 1/GA 9的含量，提高失活形式的赤霉素GA 8/GA 51/GA 29的含量，从而降低株高。
本发明第十方面提供了一种矮化相关基因SBI或其突变蛋白的启动子元件的用途，用于时空特异性表达外源蛋白，其中所述的时空特异性表达指在成熟期特异性地表达于茎杆、茎杆基部节间。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了SBI(shortened basal internodes)基因的图位克隆。其中，A，株1S与SV14成熟植株；B，株1S与SV14成熟植株株高统计；C，SBI基因连锁区域；D，SBI基因图位克隆策略；E，株1S与SV14SBI基因比较；F，SBI突变位点。
图2显示了SBI候选基因的验证结果。其中，A，外施赤霉素处理萌发后6周SV14与株1S植株；B，赤霉素处理后植株株高统计；C，株1S与SV14基部节间赤霉素含量；D、H，转基因植株蛋白检测；E、I，转基因阳性植株；F、J，转基因植株基因表达量检测；G、K，转基因植株株高统计。
图3显示了SBI基因在水稻各组织表达模式分析及水稻GA2ox家族系统发生分析。其中，A，水稻OsGA2ox家族基因在水稻个组织表达量heatmap；B，Realtime检测SBI基因在水稻个组织表达量；C，水稻OsGA2ox家族成员系统发生树。
图4显示了SBI promoterSV14-GUS转基因植株各组织的染色观察结果。其中，A，水稻叶片；B，水稻第四节间；C，水稻第四节间横切；D，水稻花序；E，水稻根；F，水稻第二节间横切；G，水稻第二节间。
图5显示了转基因植株茎秆各节间长度测量统计结果。其中，A，转基因水稻茎秆；B，转基因水稻茎秆第四节间纵切；C，转基因水稻茎秆各节间长度统计。
图6显示了SBI体外酶活性检测结果。其中，A，GA 9的色谱图及质谱图；B，GA 51的色谱图及质谱图。
图7显示了SBI Zhu1S与SBI SV14酶活性变化结果比较。其中，A，体外表达SBI蛋白检测；B，SBI SV14与SBI Zhu1S蛋白酶活比较；C，转基因植株内源赤霉素含量检测。
图8显示了单点突变对SBI蛋白酶活的影响。其中，A、F，转基因植株蛋白检测；B、G，转基因植株；C、H，转基因植株表达量检测；D、I，转基因植株株高统计；E、J，转基因植株赤霉素含量检测。
图9显示了茎秆特异表达SBI Zhu1S及SBI D308N增强了水稻的抗倒伏能力。其中，A、B，转基因植株与中花11抗倒伏能力差别；C，转基因植株与中花11成熟穗子对比；D-L，转基因植株产量性状调查统计，从D到L分别是倒伏率、穗长、粒长、粒宽、百粒重、每穗粒数、抽穗期、分蘖数、单株粒重。
图10显示了SBI基因参与了籼稻与粳稻株高性状的驯化；其中，A，籼稻群体与粳稻群体株高分布；B，SBI基因区域SNP在栽培稻中的单体型分布统计；C，SBI基因区域SNP在野生稻中的单体型分布统计。
图11显示了株1S与SV14SBI基因启动子比较结果。其中，A，SBI基因启动子SNP与Indel的分布；B，SNP与Indel的籼粳分化；C，不同来源SBI启动子驱动GUS基因在水稻各组织酶活统计；D，SBI基因在株1S与SV14茎秆中表达量。
图12显示了水稻OsGA2ox家族蛋白序列比对结果。
图13显示了高等植物及高等作物SBI蛋白序列及比对结果。
具体实施方式
经过广泛而深入的研究，本发明人通过对大量的农作物农艺性状位点的研究，首次意外发现了一种农作物(如水稻)矮化相关基因SBI或其编码蛋白、或其突变蛋白、或其促进剂，用于调控农作物的农艺性状，所述农艺性状选自下组的一种或多种(i)株高；(ii)分蘖数；(iii)单株粒重；(iv)产量。此外，本发明还首次发现，将农作物中矮化相关蛋白SBI的第308位氨基酸(天冬氨酸)突变为天冬酰胺，和/或第338位氨基酸(甘氨酸)突变为精氨酸，可显著降低活性赤霉素或其前体GA 1/GA 9的含量，并显著提高失活形式的赤霉素GA 8/GA 51/GA 29的含量，从而显著降低株高，提高农作物的抗倒伏能力。在此基础上完成了本发明。
术语
如本文所用，术语“AxxB”表示第xx位的氨基酸A变为氨基酸B，例如“D308N”表示第308位的氨基酸D突变为N，以此类推。
本发明突变蛋白及其编码核酸
如本文所用，术语“突变蛋白”、“本发明突变蛋白”、“本发明SBI突变蛋白”、“本发明的矮化相关基因SBI的突变蛋白”可互换使用，均指非天然存在的SBI突变蛋白，且所述突变蛋白为基于SEQ ID NO.:1所示蛋白进行人工改造的蛋白，其中，所述的突变蛋白含有与酶催化活性相关的核心氨基酸，且所述核心氨基酸中至少有一个是经过人工改造的；并且本发明突变蛋白具有催化活性赤霉素或其前体GA 1/GA 9含量降低，催化失活形式的赤霉素GA 8/GA 51/GA 29含量升高的活性。
术语“核心氨基酸”指的是基于SEQ ID NO.:1，且与SEQ ID NO.:1同源性达至少80％，如84％、85％、90％、92％、95％、98％的序列中，相应位点是本文所述的特定氨基酸，如基于SEQ ID NO.:1所示的序列，核心氨基酸为：
第338位甘氨酸(G)；和/或
第308位天冬氨酸(D)；
且对上述核心氨基酸进行突变所得到的突变蛋白具有催化活性赤霉素或其前体GA 1/GA 9含量降低，催化失活形式的赤霉素GA 8/GA 51/GA 29含量升高的活性。
优选地，在本发明中，对本发明的所述核心氨基酸进行如下突变：
第338位甘氨酸(G)突变为精氨酸(R)；和/或
第308位天冬氨酸(D)突变为天冬酰胺(N)。
应理解，本发明突变蛋白中的氨基酸编号基于SEQ ID NO.:1作出，当某一具体突变蛋白与SEQ ID NO.:1所示序列的同源性达到80％或以上时，突变蛋白的氨基酸编号可能会有相对于SEQ ID NO.:1的氨基酸编号的错位，如向氨基酸的N末端或C末端错位1-5位，而采用本领域常规的序列比对技术，本领域技术 人员通常可以理解这样的错位是在合理范围内的，且不应当由于氨基酸编号的错位而使同源性达80％(如90％、95％、98％)的、具有相同或相似的催化活性赤霉素或其前体GA 1/GA 9含量降低，催化失活形式的赤霉素GA 8/GA 51/GA 29含量升高的活性的突变蛋白不在本发明突变蛋白的范围内。
本发明突变蛋白是合成蛋白或重组蛋白，即可以是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、植物)中产生。根据重组生产方案所用的宿主，本发明的突变蛋白可以是糖基化的，或可以是非糖基化的。本发明的突变蛋白还可包括或不包括起始的甲硫氨酸残基。
本发明还包括所述突变蛋白的片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持所述突变蛋白相同的生物学功能或活性的蛋白。
本发明的突变蛋白片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的突变蛋白，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的突变蛋白，或(iii)成熟突变蛋白与另一个化合物(比如延长突变蛋白半衰期的化合物，例如聚乙二醇)融合所形成的突变蛋白，或(iv)附加的氨基酸序列融合到此突变蛋白序列而形成的突变蛋白(如前导序列或分泌序列或用来纯化此突变蛋白的序列或蛋白原序列，或与抗原IgG片段的形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。本发明中，保守性替换的氨基酸最好根据表I进行氨基酸替换而产生。
表I
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明的活性突变蛋白具有催化活性赤霉素或前体GA 1/GA 9含量降低，催化失活形式的赤霉素GA 8/GA 51/GA 29含量升高的活性。
优选地，所述的突变蛋白如SEQ ID NO.:2所示。应理解，本发明突变蛋白与SEQ ID NO.:2所示的序列相比，通常具有较高的同源性(相同性)，优选地，所述的突变蛋白与SEQ ID NO.:2所示序列的同源性至少为80％，较佳地至少为85％-90％，更佳地至少为95％，最佳地至少为98％。
此外，还可以对本发明突变蛋白进行修饰。修饰(通常不改变一级结构)形式包括：体内或体外的突变蛋白的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在突变蛋白的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的突变蛋白。这种修饰可以通过将突变蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的突变蛋白。
本发明还提供了编码SBI多肽、蛋白或其变体的多核苷酸序列。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括：DNA、基因组DNA或人工合成的DNA，DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NO.:4所示的编码区序列相同或者是简并的变异体。
术语“编码突变蛋白的多核苷酸”可以是包括编码本发明突变蛋白的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。在本发明中，一种优选的编码突变蛋白的多核苷酸序列如SEQ ID NO.:3所示。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或突变蛋白的片段、类似物和衍生物。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码的突变蛋白的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件(或严紧条件)下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。
本发明的突变蛋白和多核苷酸优选以分离的形式提供，更佳地，被纯化至均质。
应理解，虽然本发明的SBI基因优选来自水稻，但是来自其它植物的与水稻SBI基因高度同源(如具有80％以上，如85％,90％,95％甚至98％序列相同性)的其它基因也在本发明考虑的范围之内。比对序列相同性的方法和工具也是本领域周知的，例如BLAST。
本发明多核苷酸全长序列通常可以通过PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅 读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的多核苷酸。特别是很难从文库中得到全长的cDNA时，可优选使用RACE法(RACE-cDNA末端快速扩增法)，用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
野生型SBI蛋白
如本文所用，“野生型SBI蛋白”、“野生型矮化相关基因SBI蛋白”可互换使用，是指天然存在的、未经过人工改造的SBI蛋白，其核苷酸可以通过基因工程技术来获得，如基因组测序、聚合酶链式反应(PCR)等，其氨基酸序列可由核苷酸序列推导而得到。本发明的一种典型的野生型SBI蛋白的氨基酸序列如SEQ ID NO.:1所示。
表达载体
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或本发明突变蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述多肽的方法。
通过常规的重组DNA技术，可利用本发明的多聚核苷酸序列可用来表达或生产重组的突变蛋白。一般来说有以下步骤：
(1).用本发明的编码本发明突变蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本发明还提供了一种包括本发明的基因的重组载体。作为一种优选的方式，重组载体的启动子下游包含多克隆位点或至少一个酶切位点。当需要表达本发明目的基因时，将目的基因连接入适合的多克隆位点或酶切位点内，从而将目的基因与启动子可操作地连接。作为另一种优选方式，所述的重组载体包括(从5’到3’方向)：启动子，目的基因，和终止子。如果需要，所述的重组载体还可以包括选自下组的元件：3’多聚核苷酸化信号；非翻译核酸序列；转运和靶向核酸序列；抗性选择标记(二氢叶酸还原酶、新霉素抗性、潮霉素抗性以及绿色荧光蛋白等)；增强子；或操作子。
在本发明中，编码突变蛋白的多核苷酸序列可插入到重组表达载体中。术语“重组表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒或其他载体。只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含本发明突变蛋白编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTRs和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
本领域普通技术人员可以使用熟知的方法构建含有本发明所述的基因的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。使用本发明的基因构建重组表达载体时，可在其转录起始核苷酸前加上任何一种增强型、组成型、组织特异型或诱导型启动子。
包括本发明基因、表达盒或载体可以用于转化适当的宿主细胞，以使宿主表达蛋白质。宿主细胞可以是原核细胞，如大肠杆菌，链霉菌属、农杆菌；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物细胞。本领域一般技术人员都清楚如何选择适当的载体和宿主细胞。用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物(如大肠杆菌)时，可以用CaCl 2法处理，也可用电穿孔法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法(如显微注射、电穿孔、脂质体包装等)。转化植物也可使用农杆菌转化或基因枪转化等方法，例如叶盘法、幼胚转化法、花芽浸泡法等。对于转化的植物细胞、组织或器官可以用常规方法再生成植株，从而获得转基因的植物。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母、植物细胞(如水稻细胞)。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期一侧的100到270个碱基对的SV40增强子、在复制起始点晚期一侧的多瘤增强子以及腺病毒增强子等。
本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的主要优点包括：
(1)本发明首次发现矮化相关基因SBI或其编码蛋白、或其突变蛋白、或其促进剂可调控农作物的农艺性状(比如，株高、分蘖数、产量等)。
(2)本发明首次发现，将所述作物中矮化相关蛋白SBI的第308位氨基酸(天冬氨酸)突变为天冬酰胺，和/或第338位氨基酸(甘氨酸)突变为精氨酸，可显著降低活性赤霉素或其前体GA 1/GA 9的含量，并显著提高失活形式的赤霉素GA 8/GA 51/GA 29的含量，从而显著降低株高，提高农作物的抗倒伏能力。
(3)本发明首次发现矮化相关基因SBI或其编码蛋白、或其突变蛋白在成熟期特异性地表达于茎杆、茎杆基部节间，并且在茎杆中过表达该基因可以特异缩短基部节间的长度。
(4)本发明首次发现，水稻茎秆特异过表达SBI基因的转基因植株株高合理降低，抗倒伏能力增强100％，有效分蘖数增多30％，单株粒重增加29％。
(5)本发明首次发现，SBI基因可以通过特异降低水稻茎秆基部节间活性赤霉素含量从而使水稻株高合理降低、抗倒伏能力增强、分蘖数增多、产量增加，同时该基因在其他作物中的同源基因可用于培育作物矮化品种，提高作物的抗倒伏能力；同时还能增加作物，尤其是禾本科作物的有效分蘖数，提高作物产量。SBI基因的定位克隆对于矮秆基因的储备极为重要，同时在创建、筛选新矮源水稻上也是一次创新，并且对于高等作物优良性状的培育具有十分重要的应用价值与前景。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非有特别说明，否则实施例中所用的材料和试剂均为市售产品。
实验方法
1.植物材料的培养条件及测量方法
水稻雄性不育系株1S与SV14由湖南亚华种业研究所提供。用于遗传分析的1000株F2代植株由株1S与SV14正交产生的F1代植株自交而产生并种植于大田。本研究表型分析及数据统计所用转基因水稻材料均为种植于大田的转基因T2代植株。GUS染色实验中所用水稻材料为人工气候室(12小时光周期、光强照度：200-250μmol·m-2·s-1，温度28±1℃)种植，为转基因T2代植株。株高表型的测量：成熟植株株高测量，从地面量至穗尖。节间的次序由穗颈节开始为第一节间，以此从形态学上端往下为第二节间至第五节间。
2.SBI基因的图位克隆
利用CTAB法对水稻的核基因组DNA进行提取。其次将株1S与F2混池的基因组分别进行全基因组测序，然后将测序得到的读长与日本晴基因组比对，根据SNP与indel位点对日本晴基因组进行修正，并以修正后基因组为参考基因组。将F2DNA混池读长与参考基因组比对，进行SNP提取，在SNP指数基础上进行滑窗计算并绘图。
3.载体的构建与转化
将株1S与SV14SBI基因的启动子序列及基因组序列克隆，并亚克隆到质粒pCAMBIA1300(市售质粒)上，验证后转入农杆菌EHA105菌株(获自中国科学院上海生命科学研究院)，并转入水稻中花11(获自中国科学院上海生命科学研究院)中，以研究株1S与SV14SBI基因的功能。
以pCAMBIA1300-SBIpromoter SV14-SBI SV14载体序列为模板，利用YEASEN公司Hieff Mut TMSite-Directed Mutagenesis Kit(11003ES10)对SBI氨基酸序列第308位点及第338位点进行单点突变，验证后转入农杆菌EHA105菌株，并转入水稻中花11中，以研究单点突变对蛋白质功能的影响。
将株1S与SV14SBI基因CDS序列全长克隆，并亚克隆到NEB公司蛋白表达质粒pMAL-c5x(N8108)上，验证后转入Transgen公司蛋白表达菌株Transetta(CD801)，用于体外表达株1S与SV14SBI重组蛋白。
将株1S与SV14SBI基因的启动子序列克隆，并亚克隆到质粒pCAMBIA1301(市售质粒)上，验证后转入农杆菌EHA105菌株，并转入水稻中花11中，以研究株1S与SV14SBI基因启动子的活性。
4.石蜡切片
取水稻第五节间成熟组织部分置于FAA固定液(70％乙醇，5％乙酸，5％甲醛)中，4℃，24h以上。70％、85％、95％乙醇逐级脱水各1h；无水乙醇30min,2次；无水乙醇/二甲苯1:1，1h；二甲苯，1h；二甲苯/石蜡1:1，2h；石蜡I，4h；石蜡II，过夜。将石蜡融于包埋框，置样品于石蜡中，冷却，保存于4℃。将载玻片置于42℃烤片机上，加无菌水均匀覆盖。修好的蜡块在切片机切好10μm厚蜡带，置于载玻片上，悬浮于水中。待蜡带展平，用吸水纸吸干水分，42℃烤片过夜。
甲苯胺蓝染色：将制备好的切片于0.1％的甲苯胺蓝溶液(0.01M乙酸乙酸钠缓冲液，pH 4.4～4.6)中染色30min，清水洗去浮色，37℃烘箱烘干；二甲苯中脱蜡2次，每次10min；中性树胶封片。显微观察。
5.植物体内赤霉素含量测定
取生长在田间的八周的株1S与SV14的第四节间与第五节间伸长区以及生长在人工气候室中的六周的转基因植株的小苗进行测定，每份待测样品包含均匀混合的独立生长的三份水稻样品。赤霉素的测定方法参考(Lu et al.,2016)
6.SBI体外酶活测定
首先，将SV14与株1S的SBI重组蛋白进行体外诱导纯化。挑取单菌落于5ml LB培养基，37℃摇床210rpm过夜培养。取1ml菌液移入100ml LB培养基，37℃摇床210rpm培养2小时，测量菌液OD600读数至0.6。以1：2000的比例加入1M的IPTG诱导，16℃摇床210rpm过夜诱导。离心收集诱导表达菌体，用遇冷的裂解液(20mM Tris-HCl,200mM NaCl,1mM EDTA,1mM DTT,pH7.4)重悬菌体，超声波破碎(200W，10s超声，20s休息，30次)。离心，将上清加入平衡过的NEB公司Amylose Resin(E8021)1ml，4℃过柱，约2h，之后用4ml裂解液洗两次，收集。用1ml洗脱液(10mM麦芽糖溶于裂解液)洗4次，每次静置10分钟，收集各个组分。将纯化好的组分加入到超滤管中，6000g，30min至200μl。换储存buffer(100mM Tris-Hcl pH 7.5)加入4-5ml储存buffer，6000g离心至500μl，-80℃保存。之后，将纯化好的蛋白与GA 9进行孵育，30℃孵育6h或相应的时间梯度。之后用液相色谱质谱联用仪(LC1200-MS/QTOF6520)进行GA 51的检测与定量分析，具体测定方法参考(Lee and Zeevaart,2002；Lo et al.,2008)。
7.启动子活性分析
对转基因阳性材料进行GUS活性分析，组织首先用丙酮处理(约10min，4℃)，用100mMNaPO4缓冲液(pH7.0)洗掉组织中残留的丙酮，用GUS显色液[100mM NaPO4(pH7.0)，10mM EDTA，2mM X-gluc，5mM K4Fe(CN)6，5mM K3Fe(CN)6，0.2％Triton X-100]于37℃保温适当时间。最后用75％乙醇终止反应及脱色。
对转基因材料进行GUS活性定量分析，组织在液氮中研磨，加入1ml GUS提取缓冲液[10mM EDTA(pH8.0)，0.1％SDS，50mM磷酸钠(sodium phosphate)(pH7.0)，0.1％Triton X-100，100mMβ－巯基乙醇(β-mercaptoethanol)，25μg/ml PMSF]，振荡混匀5min，12000rpm离心10分钟，转移上清至新离心管，置于冰上。取10μl上清液加入到130μl GUS反应液[GUS缓冲液中含1mM4-MUG]中，37℃保温10分钟。反应结束后，取10μl加入到190μl的1M碳酸钠中，混匀。用酶标仪在365nm激发光和455nm发射光下检测4-MU的荧光值。
8.基因表达分析
将不同组织的总RNA用Omega植物总RNA提取试剂盒(R6827)进行提取。利用TransScript One-Step gDNA Removal and cDNA Synthesis Kit(AT311)(购自北京全式金生物科技有限公司)，并以1μg总RNA为模板，采用Oligo dT引物合成第一链cDNA。使用TransStart
Figure PCTCN2018084055-appb-000001
TipTop Green qPCR SuperMix试剂(AQ131)(购自北京全式金生物科技有限公司)在MyiQ real-time PCR detection system(Bio-Rad)定量PCR仪上进行QRT-PCR分析，并以水稻肌动蛋白(OsACT1，Os03g0718100)作为内参基因。
植物样品在液氮中研磨成细粉，加入1.5体积的蛋白提取缓冲液(1M NaAc，pH 5.0，
10mM EDTA，10mM NaN3，3mM PMSF冰上放置1h，中间翻转几次。4 ℃，10000g，30min。上清过10000Mr超滤管纯化浓缩。用BCA试剂测定酶蛋白浓度，以牛血清白蛋白(BSA)为对照。取等浓度的蛋白用western检测蛋白表达水平，并以水稻肌动蛋白(OsACT1，Os03g0718100)作为内参蛋白。
9.系统发生与进化分析
水稻GA2ox家族氨基酸序列从水稻全基因组数据库下载(TIGR Rice，http://rice.plantbiology.msu.edu/),其他物种相应的氨基酸序列从Pytozome数据库下载(https://phytozome.jgi.doe.gov/)。用MEGA4.0对其氨基酸序列进行比对和进化树构建。不同水稻品种SNP信息从RiceHAP3数据库获取并进行统计。
实施例1SBI(shortened basal internodes)基因编码一个赤霉素-2-β双加氧酶
SV14(由亚华种业有限公司选育)是一个在育种生产中广泛应用温敏不育系，它是以水稻温敏不育系株1S(由亚华种业有限公司选育)为底盘品种，在体细胞诱变基础之上结合人工选育技术培育而来的水稻半矮秆品种。与株1S相比，除株高降低了20cm以上，SV14其他重要农艺性状均没有明显改变(图1A，B)，其杂交后代抗倒伏性也有所增强。为了对SV14的半矮秆基因进行图位克隆，首先构建了SV14与株1S的杂交群体，并对1000多株杂交F2代植株的株高性状进行了测量统计，选定了65株株高发生极端矮化的F2代植株做进一步分析。其次，提取了上述65株极端矮化的F2代植株的基因组DNA，并进行等量混合作为F2矮秆基因池；同时，提取了株1S的基因组DNA作为高秆基因池。之后，利用重测序技术对两个池的DNA进行全基因组测序，并分别与水稻参考基因组IRGSP做比对寻找单核苷酸多态性(SNP)位点。最后，将矮秆基因池基因组与高秆基因池基因组相比对，利用SNP分子标记对矮秆性状进行连锁分析(图1D)。利用该方法，在水稻第5号染色体25.5-25.8Mb处找到了与矮秆表型连锁区域(图1C)。进一步，对目标区域内连续多个完全连锁的SNP位点所处位置的基因进行注释分析。这些完全连锁的SNP分布的范围内包含三个基因，其中一个基因LOC_Os05g43880与赤霉素-2-β双加氧酶基因序列具有同源性。通过对株1S与SV14的LOC_Os05g43880基因序列分析，发现在外显子上有6处SNP差别，其中有两处发生了错义突变，分别导致了氨基酸序列中第308位氨基酸由天冬氨酸(D；株1S)替换为天冬酰胺(N；SV14)以及第338位氨基酸由甘氨酸(G；株1S)替换为精氨酸(R，SV14)(图1E)。发明人将该基因命名为SBI(shortened basal internodes)基因。通过对不同植物SBI蛋白氨基酸序列比对分析发现，第308位氨基酸替换发生在SBI蛋白氨基酸序列高变区，而第338位氨基酸替换发生在SBI蛋白氨基酸序列保守区(图1F)。
根据发明人的检测，与株1S相比，SV14茎秆基部节间活性赤霉素GA 1含量降低，其相应失活形式的赤霉素GA 8含量升高(图2C)。同时，对萌发后6周的SV14与株1S外源施加有活性的赤霉素GA 3可以使SV14的株高恢复到与株1S一致的高度(图2A，B)。为了对候选基因进一步验证，我们将株1S与SV14的SBI基因在其自身启动子的驱动下转化进入粳稻栽培品种中花11。茎秆过量表达SBI  zhu1S的转基因阳性株系植株株高发生了部分矮化，仅减少了大约10cm(图2H-K)，而茎秆过量表达SBI  SV14的转基因阳性株系植株在与SBI  zhu1S转基 因阳性株系相比SBI基因表达量相同或更少的情况下株高发生了严重矮化，降低了大约70cm(图2D-G)。该结果提示，SBI  zhu1S是SBI的功能较弱的等位基因，SBI  SV14则是一个功能较强的等位基因。
综合上述实验结果，证实了LOC_Os05g43880为导致SV14半矮秆性状的SBI基因，并成功克隆了该基因。
实施例2SBI基因在水稻茎秆特异表达
SV14与株1S相比，除株高降低了20cm以外其他重要农艺性状均没有明显改变。这暗示SBI基因的表达可能存在组织特异性。水稻赤霉素-2-β双加氧酶家族共有10个成员，通过对该家族所有成员在水稻各个组织的表达模式分析表明SBI在水稻茎秆与叶鞘中特异表达(图3A)。实时定量PCR结果表明，SBI基因在成熟期水稻茎秆表达量相对较高，且在茎秆基部节间的表达量最高，而在其他组织中的表达量相对较低(图3B)。
对SBI promoter SV14驱动GUS报告基因的转基因阳性植株的各个组织进行GUS染色观察，发现在叶片、花序及根中GUS的酶活性微弱，仅在叶片伤口处、个别小花颖壳的维管束以及新生成的幼根维管束中有表达(图4A，D，E)。而在茎秆第二节间及第四节间中，GUS酶活性明显增强(图4B，G)。对节间进行横切染色观察，GUS报告基因在茎秆中各组织内均有较强表达(图4C，F)。
通过对转基因阳性植株组织水平及细胞水平的观察统计，发现SBIpromoter zhu1S-SBI Zhu1s转基因阳性植株茎秆基部第三节间至第五节间长度特异缩短，第三节间长度缩短了50％和70％，第四节间长度缩短了78％和73％，第五节间长度缩短了50％和60％，基部节间薄壁细胞长度也变短；而SBIpromoter SV14-SBI SV14的转基因阳性株系茎秆各节间均发生极端缩短，缩短率达到90％以上，基部节间薄壁细胞伸长严重受阻(图5A-C)。
以上研究结果表明，SBI基因在水稻茎秆中特异表达，并且在茎秆基部节间表达量最高，在茎秆中过表达该基因可以特异缩短基部节间的长度。
实施例3SV14中的SBI蛋白酶活性增强
利用大肠杆菌诱导表达出SBI Zhu1S蛋白与SBI SV14蛋白(图7A)，并分别将它们与底物C 19赤霉素GA 9在一定的反应条件下共孵育，之后利用液质联用技术对反应产物中GA 9失活形式的赤霉素GA 51进行检测。
结果显示，GA 9与SBI SV14反应产物及GA 9与SBI Zhu1S反应产物中，在与GA 51标准品相对应的保留时间及核质比处均检测到了色谱峰及质谱峰；而不含蛋白的对照组中则没有检测到相应的色谱峰及质谱峰。该结果表明，SBI蛋白质具有与C 19赤霉素发生反应并将其失活的酶活性(图6A，B)。
为了进一步探讨SV14与株1S的突变对SBI蛋白功能造成的影响，对SBI Zhu1S及SBI SV14与GA 9的体外酶活反应在不同反应时间条件下的反应产物GA 51含量进行检测。在SBI SV14与GA 9的反应体系中，随着反应时间的延长，产物GA 51的丰度也逐渐增加，在8小时到10小时之间反应速率最快。而在SBI Zhu1S与GA 9的反应体系中，所调查的每一时间点GA 51丰度都小于SBI SV14，且反应速率迟缓(图7B)。该结果表明，SBI SV14在体外具有更高的酶活性。
对转基因阳性植株幼苗体内赤霉素含量进行检测发现，与对照中花11(获 自中国科学院上海生命科学研究院)相比，SBIPromoter zhu1S-SBI Zhu1S及SBIPromoter SV14-SBI SV14转基因阳性植株中活性赤霉素GA 1含量均降低，失活赤霉素GA 29含量均升高，但SBIpromoter SV14-SBI SV14比SBIpromoter zhu1S-SBI Zhu1S变化更剧烈，GA 1的含量减少至0，GA 29的含量是中花11的4倍(图7C)。
SV14与株1S的SBI蛋白氨基酸序列中有两处氨基酸发生了替换，分别是第308位氨基酸由天冬氨酸(D；株1S)替换为天冬酰胺(N；SV14)以及第338位氨基酸由甘氨酸(G；株1S)替换为精氨酸(R；SV14)(图1E)。为了分别验证这两处氨基酸替换对SBI SV14蛋白酶活性增强所起到的作用，构建了由SBIpromoter SV14驱动的SBI D308N及SBI G338R的单点突变双源载体并转化粳稻品种中花11。茎秆过量表达SBI D308N的转基因阳性植株株高发生了部分矮化，株高降低了30％(图8A-D)，而茎秆过量表达SBI G338R的转基因阳性株系植株在与SBI D308N转基因阳性株系相比SBI基因表达量相同的情况下株高发生了严重矮化，降低了88％(图8F-I)。SBI G338R的转基因阳性植株体内活性赤霉素GA 1的含量降低为0，失活赤霉素GA 29的含量上升为中花11的7倍以及SBI D308N转基因阳性植株的3.5倍(图8E，J)。由此可见，第338位的氨基酸变化对SBI SV14蛋白质酶活的增强及SBI Zhu1S蛋白质酶活的减弱提供了关键作用。
以上实验结果证明了，株1S的SBI蛋白酶活性较弱，而SV14中的SBI蛋白酶活性显著增强，且SBI氨基酸序列中第338位氨基酸的变化对SBI酶活性的改变起主要作用。
虽然SBI SV14酶活性显著增强，但茎秆过量表达SBI SV14的转基因阳性植株由于株高发生了极端矮化，使得转基因植株的产量性状也受到了影响(图2E；图8G)。由于SBI zhu1S及SBI D308N的酶活性较弱，在茎秆过量表达的SBI zhu1S及SBI D308N的转基因阳性植株中，株高只发生了部分矮化(图2I；图8B)。在水稻蜡熟期对茎秆特异高表达的SBI zhu1S及SBI D308N的转基因植株及中花11的产量相关性状进行了调查，发现在中花11茎秆中过量表达SBI zhu1S及SBI D308N显著提高了中花11的抗倒伏能力(图9A，B，D)，在蜡熟期，89％的中花11植株都发生了茎倒伏，转基因植株发生倒伏植株几乎为0；穗长、粒长、粒宽、粒重、每穗粒数、
以及抽穗期都没有收到影响(图9E-J)；过量表达SBI zhu1S及SBI D308N转基因植株的有效分蘖数与中花11相比分别增多了30％和14％(图9K)，单株粒重分别增多了16％和29％(图9L)。在水稻茎秆中过表达SBI zhu1S及SBI D308N使得水稻植株抗倒伏能力显著增强、有效分蘖数增多、产量增高，可以作为抗倒伏高产稳产水稻品种选育的一种遗传资源储备与方法选择。
实施例4SBI基因及启动子在籼稻亚种与粳稻亚种的差别
由于SV14与株1S的单点突变可以通过改变SBI蛋白酶活性从而对水稻株高进行调控，为了探究SBI基因等位分化对SBI蛋白功能的影响，将研究范围从只针对SV14与株1S扩大到对更多水稻品种的调查。对SBI基因所在染色体区域内7个具有多态性的SNP在栽培稻籼稻亚种与粳稻亚种及野生稻群体中的多态型进行分析，发现这7个SNP与非参考序列一致的单倍型频率随着籼稻向粳稻的转变从100％逐渐变为0％(图10B)，而在野生稻群体内部这些SNP的频率则没有规律性变化(图10C)。同时，对134个籼稻品种及102个粳稻品种 的株高进行了测量统计，在粳稻群体中，株高分布趋于标准正态分布；而在籼稻群体中株高分布呈偏态分布，偏向株高较高的一边(图10A)。这些结果提示，栽培稻的籼稻亚种与粳稻亚种在进化过程中对株高性状的选择产生了差异，且SBI基因很可能参与了栽培稻籼稻亚种与粳稻亚种株高性状的驯化过程中。
接着，对株1S与SV14SBI基因的启动子序列进行了比较，发现了多处SNP以及Indel的变化(图11A)，且这些变化也具有籼粳分化特征(图11B)。为了探讨这些突变是否导致SBI启动子功能的改变，我们构建了SBIPromoter indica及SBIPromoter japonica驱动GUS报告基因的转基因植株，并对转基因阳性植株各个组织中的GUS酶活进行检测(图4)。SBIPromoter indica及SBIPromoter japonica驱动的GUS酶活均在茎秆节间最高，而SBIPromoter japonica在基部节间具有更高的特异性(图11C)。同时，SBI基因在SV14的茎秆基部节间的表达量高于株1S(图11D)。以上数据说明，SBI基因启动子的差别对水稻株高的变化也会造成一定的影响。
讨论
赤霉素对植物细胞伸长及植株高度起到广谱调控作用。传统水稻矮秆品种中茎秆各节间都发生了同等幅度的矮化。水稻茎秆穗颈节间及第二节间的伸长通常发生在水稻生殖生长后期即拔节期与抽穗期，穗颈节和第二节间的过度缩短会对水稻拔节与抽穗造成不良影响。水稻茎秆基部节间包括第三节间至第五节间的伸长往往发生在营养生长时期和生殖生长前期，因此基部节间的矮化不会影响水稻花与种子的发育。利用能够使茎秆基部特异缩短的基因可以实现对株高的精细调控。
本发明首次发现SBI基因在水稻茎秆中特异表达且在茎秆基部节间表达量最高，而在其他组织中尤其是在花序及叶片中表达量很低。在水稻茎秆中过量表达该基因可以使茎秆基部节间特异缩短，同时还能增加水稻有效分蘖数及单株产量。因此，可以在保证高产稳产的前提下通过控制SBI在水稻茎秆中的表达量的变化从而特异的对株高进行精细调控。
本发明还首次证明了SBI编码蛋白的氨基酸序列中第338位氨基酸的改变对SBI蛋白酶活性的变化起到关键作用。水稻OsGA2ox家族氨基酸序列的比对结果显示，第338位氨基酸在C 19GA2ox亚家族中很保守且位于C 19GA2ox蛋白保守结构域内，而在C 20GA2ox亚家族中该位点保守性相对较差(图12)。说明该位点对于C 19GA2ox的酶活的特异性很重要。株1S与SV14的SBI蛋白序列中该位点的氨基酸由甘氨酸(G；株1S)向精氨酸(R，SV14)的替换使SBI SV14酶活显著增强。该位点氨基酸不仅对C 19GA2ox的酶活特异性起作用，同时也参与C 19GA2ox蛋白酶活性强弱的调控。在水稻茎秆中过量表达SBI Zhu1s可以在保证高产稳产的前提下特异降低茎秆高度而增强水稻品种的抗倒伏能力，本发明首次发现的这一位点可以进一步被开发为筛选水稻抗倒伏品种的分子标记，同时也为水稻抗倒伏品种选育途径提供了新的选择。
通过对不同植物中SBI蛋白序列的比对，发现水稻SBI蛋白第338位氨基酸在其他单子叶植物如谷子(Setaria italica)、小麦(Triticum aestivum)、高粱(Sorghum bicolor)、玉米(Zea mays)，及双子叶植物如拟南芥 (Arabidopsis thaliana)、油菜(Brassica rapa FPsc)、番茄(Solanum Lycopersicum)、马铃薯(Solanum tuberosum)、大豆(Glycine max)、苜蓿(Medicago truncatula)、棉花(Gossypium raimondii)、黄瓜(Cucumis sativas)的SBI蛋白序列中十分保守(图13)。说明SBI基因在被子植物中的功能重要，SBI基因也参与了赤霉素对不同高等作物株高性状的调控过程。本发明对这一保守位点功能的探索也为其他经济作物的抗倒伏、耐肥、高产品种的株型育种提供了遗传基因资源与分子理论依据。
SBI基因及其启动子区域中的多态性SNP位点存在显著的籼粳分化差异，籼稻群体与粳稻群体株高分布也存在差异；因此，SBI基因可能参与了水稻栽培品种籼稻亚种与粳稻亚种的株高性状驯化过程。同时，SBI基因区域中的多态性SNP位点可以作为籼稻与粳稻特异分化的SNP在对不同水稻品种染色体片段籼粳来源的研究中被利用。粳稻来源的SBI基因的启动子的功能比籼稻来源的SBI基因启动子的功能更强(图11C)，但二者均主要在茎秆节间发挥功能。本发明利用粳稻来源的SBI启动子SBIPromoter SV14驱动SBI zhu1S在抗倒伏能力弱品种中花11茎秆中过表达可以显著增强中花11的抗倒伏能力，且不影响其他产量性状(图9)。因此，可以对SBI基因启动子进行开发利用，用SBI基因的启动子驱动感兴趣的目的基因特异对水稻茎秆进行人工控制。为水稻优良性状品种的选育提供新的思路、方法及理论依据。
参考文献
1.Lee,D.J.,and Zeevaart,J.A.(2002).Differential regulation of RNA levels of gibberellin dioxygenases by photoperiod in spinach.Plant Physiol 130,2085-2094.
2.Lo,S.F.,Yang,S.Y.,Chen,K.T.,Hsing,Y.I.,Zeevaart,J.A.,Chen,L.J.,and Yu,S.M.(2008).A novel class of gibberellin 2-oxidases control semidwarfism,tillering,and root development in rice.Plant Cell 20,2603-2618.
3.Lu,Y.H.,Cao,Y.M.,Guo,X.F.,Wang,H.,and Zhang,H.S.(2016).Determination of gibberellins using HPLC coupled with fluorescence detection.Anal Methods-Uk 8,1520-1526.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种矮化相关基因SBI突变蛋白，其特征在于，所述的突变蛋白为非天然蛋白，且所述突变蛋白具有催化活性赤霉素或其前体GA 1/GA 9含量降低，催化失活形式的赤霉素GA 8/GA 51/GA 29含量升高的活性，并且所述突变蛋白在野生型的SBI蛋白的对应于SEQ ID NO.:1的选自下组的一个或多个与酶催化活性相关的核心氨基酸发生突变：
    第308位天冬氨酸(D)；和/或
    第338位甘氨酸(G)。
  2. 一种多核苷酸，其特征在于，所述的多核苷酸编码权利要求1所述的突变蛋白。
  3. 一种载体，其特征在于，所述的载体含有权利要求2所述的多核苷酸。
  4. 一种宿主细胞，其特征在于，所述的宿主细胞含有权利要求3所述的载体，或其基因组中整合有权利要求2所述的多核苷酸。
  5. 一种产生权利要求1所述SBI突变蛋白的方法，其特征在于，包括步骤：
    在适合表达的条件下，培养权利要求4所述的宿主细胞，从而表达出SBI突变蛋白；和
    分离所述SBI突变蛋白。
  6. 一种酶制剂，其特征在于，所述酶制剂包含权利要求1所述的SBI突变蛋白。
  7. 一种物质的用途，所述的物质选自下组：矮化相关基因SBI或其编码蛋白、或其突变蛋白、或其促进剂，其特征在于，用于改善农作物的农艺性状，所述农艺性状选自下组的一种或多种：
    (i)株高；
    (ii)分蘖数；
    (iii)单株粒重；
    (iv)产量；
    (v)茎秆基部节间长度。
  8. 一种物质的用途，所述的物质选自下组：矮化相关基因SBI或其编码蛋白、或其突变蛋白、或其促进剂，其特征在于，用于以下一种或多种用途：
    (i-1)降低株高；
    (ii-1)增加分蘖数；
    (iii-1)提高产量；
    (iv-1)缩短茎秆基部节间长度。
  9. 一种改良作物农艺性状的方法，其特征在于，包括步骤：
    提高所述作物中矮化相关蛋白SBI或其突变蛋白的表达量或活性，从而改良作物的农艺性状。
  10. 一种矮化相关基因SBI或其突变蛋白的启动子元件的用途，其特征在于，用于时空特异性表达外源蛋白，其中所述的时空特异性表达指在成熟期特异性地表达于茎杆、茎杆基部节间。
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CN110444253B (zh) * 2019-08-30 2023-09-29 上海美吉生物医药科技有限公司 一种适用于混池基因定位的方法及系统
CN113004382A (zh) * 2019-12-20 2021-06-22 中国科学院分子植物科学卓越创新中心 一种EmBP1基因或其蛋白的应用
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CN111848765B (zh) * 2020-07-22 2021-10-08 中国水稻研究所 水稻基因OsFBK4及其突变体与应用
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CN113151551A (zh) * 2021-04-22 2021-07-23 山西农业大学 Caps分子标记在鉴定谷子株高性状的用途及引物和检测试剂盒
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CN116769792B (zh) * 2023-06-15 2024-03-22 安徽农业大学 一种毛竹茎秆伸长相关基因PheLBD12及其应用

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