WO2021047377A1 - Tpst基因在调控植物性状中的应用 - Google Patents

Tpst基因在调控植物性状中的应用 Download PDF

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WO2021047377A1
WO2021047377A1 PCT/CN2020/110574 CN2020110574W WO2021047377A1 WO 2021047377 A1 WO2021047377 A1 WO 2021047377A1 CN 2020110574 W CN2020110574 W CN 2020110574W WO 2021047377 A1 WO2021047377 A1 WO 2021047377A1
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plant
traits
tpst
gene
sequence
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PCT/CN2020/110574
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French (fr)
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山东舜丰生物科技有限公司
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Priority to CN202080003113.0A priority Critical patent/CN113242906B/zh
Priority to CN202211497019.2A priority patent/CN116042563A/zh
Publication of WO2021047377A1 publication Critical patent/WO2021047377A1/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
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    • A—HUMAN NECESSITIES
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    • 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
    • A01H5/08—Fruits
    • 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
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    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
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    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
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Definitions

  • the present invention relates to the field of agronomy, in particular, to the application of TPST gene in regulating plant traits, and more specifically, to the application of TPST gene in regulating plant agronomic traits, especially rice stress resistance, yield and the like.
  • Rice is one of the most important food crops in the world and the main source of human energy and protein. Its output and consumption have always been the top food crops. Due to the lack of arable land, the increase in population, and the problems of soil erosion, the selection and production of new varieties of high-yield, high-quality, and more resistant rice has always been the theme of rice breeding in China. Therefore, mining relevant functional genes and using them to cultivate good varieties with target traits and opening up new breeding paths is of great significance for promoting my country's transformation from a large seed industry to a strong seed industry.
  • the purpose of the present invention is to provide the use of TPST gene or its encoded protein or its promoter in the regulation of plant (such as rice) traits.
  • one or more traits in plants can be improved by increasing the expression or activity of the TPST gene or its encoded protein, including enhancing plant stress resistance, increasing thousand-seed weight, increasing yield and/or biomass, Increase the size, weight and/or number of fruits and/or seeds, increase root length or root weight.
  • the invention provides a new technical means for the improvement of plant traits and molecular breeding.
  • the first aspect of the present invention provides the use of a substance, the substance being the TPST gene or its encoded protein, or its promoter, for regulating plant traits or preparing a preparation or composition regulating plant traits, wherein
  • the traits of the plant include one or more traits selected from the following group:
  • the stress resistance is selected from the group consisting of salt resistance, drought resistance, disease and insect pest resistance, or a combination thereof.
  • the trait further includes one or more selected from the following group:
  • the traits of the regulated plant include:
  • the traits of the regulated plant further include:
  • the composition includes an agricultural composition.
  • the formulation includes an agricultural formulation.
  • the composition comprises (a) the TPST gene or its encoded protein, or its promoter; and (b) an agronomically acceptable carrier.
  • the composition contains 0.0001-99wt%, preferably 0.1-90wt% of component (a), based on the total weight of the composition.
  • the dosage form of the composition or preparation is selected from the following group: solution, emulsion, suspension, powder, foam, paste, granule, aerosol, or a combination thereof.
  • composition further includes other substances that regulate plant traits.
  • the other substances that regulate plant traits include osmotic regulators, brassinoids, seaweeds, fertilizers with high potassium or nitrogen or phosphorus content, trace elements (such as boron zinc, calcium, silicon), three Azole fungicides (such as difenoconazole, propiconazole, tebuconazole), high potassium foliar fertilizer, plant hormones (such as abscisic acid, ethylene, cytokinin, polyamines), rare earths, PP 333 , benzene Formic acid, salicylic acid, uniconazole.
  • trace elements such as boron zinc, calcium, silicon
  • three Azole fungicides such as difenoconazole, propiconazole, tebuconazole
  • high potassium foliar fertilizer such as abscisic acid, ethylene, cytokinin, polyamines
  • plant hormones such as abscisic acid, ethylene, cytokinin, polyamines
  • rare earths such
  • the osmotic regulator is selected from the group consisting of inorganic regulators, organic regulators, growth regulators, or combinations thereof.
  • the inorganic regulator includes Ca 2+ and salicylic acid.
  • the organic regulator includes betaine, proline, and sodium nitroprusside (SNP).
  • the growth regulator includes abscisic acid (ABA).
  • the promoter includes a small molecule compound that promotes the expression of the TPST gene or its encoded protein.
  • the promoter is selected from the following group: small molecule compounds, nucleic acid molecules, or a combination thereof.
  • the plants include monocots, dicots, and/or gymnosperms.
  • the plants include crops, forestry plants, vegetables, fruits, flowers, and pastures (including lawn grasses).
  • the plant is selected from the following group: Cruciferae, Gramineae, Leguminosae, Solanaceae, Umbelliferae, Chenopodiaceae, or a combination thereof.
  • the plant is selected from the group consisting of Arabidopsis, rice, soybean, tomato, corn, sorghum, tobacco, wheat, sorghum, millet, quinoa, potato, sweet potato, rape, cabbage, spinach, Lettuce, cucumber, chrysanthemum, water spinach, celery, lettuce, or a combination thereof.
  • the plants include: rice, wheat, corn, and/or sorghum.
  • the rice is selected from the group consisting of indica rice, japonica rice, or a combination thereof.
  • the TPST gene is selected from the group consisting of cDNA sequence, genomic sequence, or a combination thereof.
  • the TPST gene is derived from one or more plants selected from the group consisting of cruciferous plants, gramineous plants, solanaceae, legumes, and Chenopodiaceae.
  • the TPST gene is derived from one or more plants selected from the group consisting of Arabidopsis, rice, corn, sorghum, wheat, millet, Brachypodium dilatum, and quinoa.
  • the TPST gene is selected from the group consisting of: Arabidopsis TPST gene (AtTPST, AT1G08030), rice TPST gene (OsTPST, accession number LOC9267276), maize TPST gene (maize ZmTPST, accession number LOC100280275), sugarcane TPST gene (sugarcane SbTPST, accession number LOC8071351), camelina TPST gene (camelina CsTPST, accession number LOC104754980), rapeseed TPST gene (rape BrTPST, accession number LOC103871547), radish TPST gene (radish RsTPST, accession number LOC108862166), or a combination thereof.
  • the TPST gene includes a wild-type TPST gene and a mutant TPST gene.
  • the mutant type includes a mutant form in which the function of the encoded protein remains unchanged (that is, the function is the same as or substantially the same as the wild-type encoded protein) and the function is enhanced after the mutation.
  • polypeptide encoded by the mutant TPST gene is the same or substantially the same as the polypeptide encoded by the wild-type TPST gene.
  • the mutant TPST gene includes homology of ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95%, more preferably, ⁇ 98% compared with the wild-type TPST gene). % Or 99%) polynucleotides.
  • the mutant TPST gene is included in the 5'end and/or 3'end of the wild-type TPST gene, truncated or added 1-60 (preferably 1-30, more preferably 1 -10) nucleotide polynucleotides.
  • amino acid sequence of the TPST protein is selected from the following group:
  • amino acid sequence shown in SEQ ID NO.: 3 is formed by the substitution, deletion or addition of one or several (such as 1-10) amino acid residues, which has the function of regulating plant traits (i) Derived polypeptide; or
  • the homology between the amino acid sequence and the amino acid sequence shown in SEQ ID NO.: 3 is greater than or equal to 80% (preferably greater than or equal to 90%, more preferably greater than or equal to 95% or greater than or equal to 98%), a polypeptide having the TPST activity .
  • nucleotide sequence of the TPST gene is selected from the following group:
  • the nucleotide sequence has a homology of ⁇ 75% (preferably ⁇ 85%, more preferably ⁇ 90% or ⁇ 95%) with the sequence shown in SEQ ID NO.: 1, 2 or 5 Polynucleotide
  • the second aspect of the present invention provides a composition comprising:
  • the composition includes an agricultural composition.
  • the dosage form of the composition is selected from the following group: solution, emulsion, suspension, powder, foam, paste, granule, aerosol, or a combination thereof.
  • the composition contains 0.0001-99wt%, preferably 0.1-90wt% of component (a), based on the total weight of the composition.
  • the content (wt%) of the promoter of the TPST gene or its encoded protein in the composition is 0.05%-10%, preferably 0.1%-8%, more preferably , 0.5%-6%.
  • the promoter is selected from the following group: small molecule compounds, nucleic acid molecules, or a combination thereof.
  • composition further includes other substances that regulate plant traits.
  • the other substances that regulate plant traits include osmotic regulators, brassinolide, seaweed, fertilizers with high potassium or nitrogen or phosphorus content, trace elements (such as boron zinc, calcium, silicon), three Azole fungicides (such as difenoconazole, propiconazole, tebuconazole), high potassium foliar fertilizer, plant hormones (such as abscisic acid, ethylene, cytokinin, polyamines), rare earths, paclobutrazol (PP 333 ), benzoic acid, salicylic acid, uniconazole.
  • the osmotic regulator is selected from the group consisting of inorganic regulators, organic regulators, growth regulators, or combinations thereof.
  • the inorganic regulator includes Ca 2+ and salicylic acid.
  • the organic regulator includes betaine, proline, and sodium nitroprusside (SNP).
  • the growth regulator includes abscisic acid (ABA).
  • the third aspect of the present invention provides a use of the composition according to the second aspect of the present invention for improving plant properties.
  • the fourth aspect of the present invention provides a method for improving plant traits, including the steps:
  • the method includes administering a plant TPST gene or a promoter of its encoded protein.
  • the promoter is a substance that promotes the expression of the TPST gene or its encoded protein.
  • the promoter is selected from the following group: small molecule compounds, nucleic acid molecules, or a combination thereof.
  • the method includes introducing an exogenous TPST gene into the plant.
  • the method includes introducing into the plant a substance that promotes the expression of the endogenous TPST gene or its encoded protein.
  • the method includes promoting the expression of endogenous TPST gene or its encoded protein in the plant.
  • the method includes the steps:
  • the TPST gene sequence is introduced into the plant or plant cell, thereby obtaining a transgenic plant or plant cell.
  • the method includes the steps:
  • step (b) Contacting the plant cell or tissue or organ with the Agrobacterium in step (a), so that the TPST gene sequence is transferred into the plant cell and integrated into the chromosome of the plant cell;
  • step (d) Regenerating the plant cells or tissues or organs in step (c) into plants.
  • the expression or activity of the TPST gene or its encoded protein in the plant tissue or plant cell is increased by ⁇ 5%, ⁇ 10%, ⁇ 20%, and more preferably, ⁇ 50%.
  • the "increased" means that the expression or activity of the TPST gene or its encoded protein is increased to meet the following conditions:
  • the ratio of A1/A0 is ⁇ 5%, more preferably, ⁇ 10%, more preferably ⁇ 20%, and most preferably 50-200%; among them, A1 is the TPST gene or its coded protein in plant tissues or plant cells Expression or activity; A0 is the expression or activity of the same TPST gene or its encoded protein in wild-type plant tissues or plant cells of the same type.
  • the ratio of the activity E1 of TPST in the plant to the background activity E0 of the same TPST in wild-type plants of the same type is ⁇ 2 times, preferably ⁇ 5 times, more The best place is ⁇ 10 times.
  • the fifth aspect of the present invention provides a method for preparing genetically engineered plant tissues or plant cells, including the steps:
  • the genetic engineering includes transgene.
  • the method further includes introducing the TPST gene or the promoter of the encoded protein thereof into the plant tissue or plant cell.
  • the sixth aspect of the present invention provides a method for preparing plants with improved traits, including the steps:
  • the genetically engineered plant tissue or plant cell prepared by the method of the fifth aspect of the present invention is regenerated into a plant body to obtain a plant with improved traits.
  • the traits include one or more traits selected from the following group:
  • the traits further include one or more selected from the following group:
  • the trait improvement includes:
  • the improvement of the traits further includes:
  • the seventh aspect of the present invention provides a genetically engineered plant into which the TPST gene or its encoded protein, or its promoter, or the plant is prepared by the method described in the sixth aspect of the present invention.
  • Figure 1 shows the AtTPST gene expression detection of AtTPST transgenic T1 rice lines.
  • control is the Nipponbare negative control transferred into the empty vector pCambia1305.
  • P35S-3, 46, 51, 52 are TPST CDs transgenic rice lines driven by the 35S promoter;
  • PTPST-31, 40, 46, 47 are pseudonan The mustard TPST endogenous promoter drives the transgenic rice line of AtTPST genome sequence.
  • Figure 2 shows the comparison of metal ion content in transgenic and wild-type rice seeds.
  • Figure 2A potassium content
  • control is the Nipponbare negative control transferred to the empty vector pCambia1305
  • P35S is the 35S promoter driven TPST CDs transgenic rice
  • PTPST is the Arabidopsis TPST endogenous promoter driven AtTPST Transgenic rice with genomic sequence.
  • Figure 3 shows the comparison of the grain weight of transgenic and wild-type rice seeds.
  • control is the Nipponbare negative control transferred into the empty vector pCambia1305
  • P35S is the 35S promoter-driven TPST CDs transgenic rice
  • PTPST is the Arabidopsis TPST endogenous promoter.
  • Transgenic rice with AtTPST genome sequence is the Nipponbare negative control transferred into the empty vector pCambia1305
  • P35S is the 35S promoter-driven TPST CDs transgenic rice
  • PTPST is the Arabidopsis TPST endogenous promoter.
  • Figure 4 shows AtTPST transgenic rice hydroponic seedlings with more developed root systems
  • Figure 4A Arabidopsis TPST endogenous promoter drives AtTPST genome sequence transgenic lines No. 7 and No. 31 and Japonica, empty vector control transformation Nipponbare negative control (control) has a more developed root system
  • Figure 4B Arabidopsis TPST endogenous promoter drives AtTPST genome sequence transgenic line 7 and line 31 and Nipponbare (Japonica), empty vector control transformed Nipponbare Compared with the negative control (control), the root length is increased
  • Fig. 4C The Arabidopsis TPST endogenous promoter drives the AtTPST genome sequence transgenic lines No. 7 and No.
  • the empty vector control transforms the Nipponbare negative control (control) ) Compared with the increase in fresh root weight, the aboveground part also increased; Japonica refers to Nipponbare; control refers to the empty vector control transformed Nipponbare negative control; PTPST-7 refers to the Arabidopsis TPST endogenous promoter driving AtTPST genome sequence transgenic No. 7 strain Line; PTPST-31 refers to the No. 31 line of AtTPST genome sequence transgene driven by the Arabidopsis TPST endogenous promoter.
  • Figure 5 shows the drought resistance of AtTPST transgenic rice, where A: normal-growing rice seedlings; B: rice seedlings that have germinated for 2 weeks without watering for two weeks of drought treatment; C: 1 week after resuming watering.
  • Figure 6 shows that increasing the expression level of endogenous OsTPST in rice can improve the traits of rice plants.
  • Figure 6A shows the results of root growth of AMV-enhanced OsTPST expression plants (AMV-OsTPST-1) and control plants (AMV Control). Increasing the expression details of OsTPST in rice can promote root growth;
  • Figure 6B shows AMV enhancement The development results of OsTPST-expressing plants (AMV Inserted Lines), control groups (Control), and other non-AMV sequence-inserted plants (DNA Fragment Inserted Line), as shown in Figure 6B, increase the expression of endogenous OsTPST in rice , Can make the rice plant more robust at the seedling stage.
  • the inventors After extensive and in-depth research, the inventors through the research and screening of a large number of plant trait sites, and found for the first time that increasing the expression or activity of the TPST gene or its encoded protein in the plant (such as rice) can significantly improve The traits of plants. On this basis, the inventor completed the present invention.
  • the expression or activity of the TPST gene or its encoded protein in the plant when the expression or activity of the TPST gene or its encoded protein in the plant is increased, it can (i) enhance plant stress resistance; and/or (ii) increase thousand-grain weight; and/or (iii) increase yield and /Or biomass; and/or (iv) increase the size, weight and/or number of fruits and/or seeds; and/or (v) root length; and/or (vi) root weight.
  • TPST stands for tyrosyl-protein sulfo-transferase, which is called tyrosyl sulfo-transferase in Chinese. It participates in the sulfation modification of protein translation. Transferred to the tyrosine residues of the protein, this modification makes the secreted protein or membrane protein have mature biological functions [1] .
  • TPST gene of the present invention and “TPST gene” are used interchangeably, and both refer to TPST genes derived from plants (such as rice, Arabidopsis) or variants thereof.
  • the nucleotide sequence of the TPST gene of the present invention is shown in SEQ ID NO.: 2.
  • the gene variants can be obtained by inserting or deleting regulatory regions, performing random or site-directed mutations, and the like.
  • the present invention also includes 50% or more of the preferred gene sequence (SEQ ID NO.: 2) of the present invention (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, The most preferred is 98% or more, such as 99%) homologous nucleic acid, which can also effectively regulate the traits of plants (such as rice).
  • “Homology” refers to the level of similarity (ie sequence similarity or identity) between two or more nucleic acids according to the percentage of positional identity.
  • the nucleotide sequence in SEQ ID NO.: 2 may be substituted, deleted or added one or more (usually 1-90, preferably 1-60, more preferably 1-20 Numbers, preferably 1-10), and add a few at the 5'and/or 3'end (usually within 60, preferably within 30, more preferably within 10, optimally Within 5 nucleotides), the derived sequence of SEQ ID NO.: 2 is generated. Due to the degeneracy of the codon, even if the homology with SEQ ID NO.: 2 is low, it can basically encode as SEQ ID NO.: 2 ID NO.: The amino acid sequence shown in 3.
  • nucleotide sequence in SEQ ID NO.: 2 has been substituted, deleted or added at least one nucleotide-derived sequence
  • it can be used under moderately stringent conditions, and more preferably under highly stringent conditions.
  • These variants include (but are not limited to): deletion of several (usually 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) nucleotides , Insertion and/or substitution, and adding several at the 5'and/or 3'end (usually within 60, preferably within 30, more preferably within 10, most preferably within 5 ) Nucleotide.
  • genes provided in the examples of the present invention are derived from Arabidopsis thaliana, they are derived from other similar plants (especially those belonging to the same family or genus with Arabidopsis thaliana, or their homology to Arabidopsis thaliana is relatively high Plants of other families or genera), and the sequence of the present invention (preferably, the sequence is shown in SEQ ID NO.: 2) have certain homology (conservative, such as 80% or more, such as 85%, 90 %, 95% or even 98% sequence identity) of the TPST gene sequence is also included in the scope of the present invention, as long as those skilled in the art can easily obtain information from other plants based on the information provided in this application after reading this application.
  • the sequence is isolated, and methods and tools for aligning sequence identity are also well known in the art, such as BLAST.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include: DNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • 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.: 2 or a degenerate variant.
  • a polynucleotide encoding a mature polypeptide includes: a coding sequence that only encodes the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequences) of the mature polypeptide and non-coding sequences.
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs and derivatives of polyglycosides or polypeptides having the same amino acid sequence as the present invention.
  • the variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants.
  • allelic variant is an alternative form of a polynucleotide. It may be a substitution, deletion or insertion of one or more nucleotides, but it will not substantially change the function of the encoded polypeptide. .
  • the present invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) methylphthalamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, More preferably, the hybridization occurs when 95% or more occurs.
  • the full-length TPST nucleotide sequence of the present invention or its fragments can usually be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available DNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used.
  • 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 splice the amplified fragments together in the correct order.
  • the recombination method can be used to obtain the relevant sequence in large quantities. It is usually cloned into a vector, and then transferred into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
  • artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • polypeptide of the present invention and “encoded protein of TPST gene” can be used interchangeably, and both refer to polypeptides derived from plant TPST and variants thereof.
  • a typical amino acid sequence of the polypeptide of the present invention is shown in SEQ ID NO.:3.
  • the present invention relates to a TPST polypeptide and variants thereof that regulate plant traits.
  • the amino acid sequence of the polypeptide is shown in SEQ ID NO.: 3.
  • the polypeptide of the present invention can effectively regulate the traits of plants (such as rice).
  • the present invention also includes 50% or more of the sequence shown in SEQ ID NO.: 3 of the present invention (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, such as 99%) homologous polypeptides or proteins with the same or similar functions.
  • the "same or similar function” mainly refers to: "regulate the traits of plants or crops (such as rice)”.
  • the polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide.
  • the polypeptide of the present invention can be a natural purified product, or a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plant, insect, and mammalian cells) using recombinant technology.
  • a prokaryotic or eukaryotic host for example, bacteria, yeast, higher plant, insect, and mammalian cells
  • the polypeptide of the present invention may be glycosylated or non-glycosylated.
  • the polypeptide of the present invention may also include or not include the initial methionine residue.
  • the present invention also includes TPST protein fragments and analogs having TPST protein activity.
  • fragment and analogs having TPST protein activity refer to polypeptides that substantially retain the same biological function or activity as the natural TPST protein of the present invention.
  • polypeptide fragments, derivatives or analogues of the present invention may be: (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues
  • the base may or may not be encoded by the genetic code; or (ii) a polypeptide with a substitution group in one or more amino acid residues; or (iii) the mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, For example, polyethylene glycol) fused to form a polypeptide; or (iv) additional amino acid sequence is fused to the polypeptide sequence to form a polypeptide (such as a leader sequence or secretory sequence, or a sequence or proprotein sequence used to purify the polypeptide, or Fusion protein).
  • these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.
  • the polypeptide variant is the amino acid sequence shown in SEQ ID NO.: 3, after several (usually 1-60, preferably 1-30, and more preferably 1-20) , Preferably 1-10) substitution, deletion or addition of at least one amino acid derived sequence, and adding one or several (usually within 20, preferably within 10) at the C-terminal and/or N-terminal , More preferably within 5) amino acids.
  • amino acids with similar or similar properties are substituted, the function of the protein is usually not changed, and the addition of one or several amino acids to the C-terminal and/or ⁇ terminal usually does not change the function of the protein.
  • the present invention also includes analogs of the claimed protein.
  • the difference between these analogs and the natural SEQ ID NO.: 3 may be the difference in the amino acid sequence, the difference in the modified form that does not affect the sequence, or both.
  • Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, site-directed mutagenesis or other known biological techniques. Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as ⁇ , ⁇ -amino acids). It should be understood that the protein of the present invention is not limited to the representative proteins exemplified above.
  • Modified (usually not changing the primary structure) forms include: chemically derived forms of proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those that undergo glycosylation modifications during protein synthesis and processing. This modification can be accomplished by exposing the protein to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, and phosphothreonine).
  • the present invention also relates to a vector containing the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or a mutant protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention through recombinant technology.
  • the polynucleotide sequence of the present invention can be used to express or produce the protein of the present invention or its variants. Generally speaking, there are the following steps:
  • the present invention also provides a recombinant vector including the gene of the present invention.
  • the downstream of the promoter of the recombinant vector contains a multiple cloning site or at least one restriction site.
  • the target gene is ligated into a suitable multiple cloning site or restriction site, so that the target gene and the promoter are operably linked.
  • the recombinant vector includes (from 5'to 3'direction): a promoter, a target gene, and a terminator.
  • the recombinant vector may also include elements selected from the following group: 3'polynucleotideization signal; untranslated nucleic acid sequence; transport and targeting nucleic acid sequence; resistance selection marker (dihydrofolate reductase, Neomycin resistance, hygromycin resistance, fluorescent protein, etc.); enhancer; or operator.
  • the polynucleotide sequence encoding the protein can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus 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 an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.
  • Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence encoding the protein of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. When constructing a recombinant expression vector using the gene of the present invention, any enhanced, constitutive, tissue-specific or inducible promoter can be added before the transcription initiation nucleotide.
  • the DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis.
  • promoters are: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti Transcriptional virus LTRs and some other known promoters that can control gene expression in prokaryotic or eukaryotic cells or viruses.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the vector including the gene, expression cassette or of the present invention can be used to transform an appropriate host cell so that the host expresses the protein.
  • the host cell can 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.
  • a prokaryotic cell such as Escherichia coli, Streptomyces, Agrobacterium
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a plant cell.
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method.
  • the steps used are well known in the art.
  • Another method is to use MgCl 2 .
  • transformation can also be carried out by electroporation.
  • the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • Agrobacterium transformation or gene gun transformation can also be used to transform plants, such as leaf disc method, immature embryo transformation method, flower bud soaking method, etc.
  • the transformed plant cells, tissues or organs can be regenerated by conventional methods to obtain transgenic plants.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting 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 selecting 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.
  • a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express 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 include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast and plant cells (such as rice cells).
  • Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, acting on promoters to enhance gene transcription. Examples include the 100 to 270 base pair SV40 enhancer on the late side of the replication initiation point, the polyoma enhancer on the late side of the replication initiation point, and adenovirus enhancers.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the protein of the present invention can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, 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.
  • conventional renaturation treatment treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange
  • a method for improving plant traits specifically, promoting or increasing the expression of TPST gene or its encoded protein, thereby improving plant traits, the traits being selected from one or more of the following group :
  • the trait further includes one or more selected from the following group:
  • the traits of the improved plant include:
  • the traits of the improved plant further include:
  • the present invention found for the first time that increasing the content of the TPST gene in a plant or up-regulating the expression of the gene can improve the agronomic traits of the plant, such as stress resistance, thousand-grain weight, yield, biomass, fruit or seed size, weight, quantity, root length , Root weight, etc.
  • the present invention finds for the first time that using an endogenous promoter of Arabidopsis to drive the expression of the Arabidopsis AtTPST gene in rice or to increase the expression of the endogenous OsTPST gene of rice can promote the root development of rice at the seedling stage. Under natural cultivation conditions It is bound to be conducive to the growth of the above-ground parts of rice and play a strong seedling effect. The well-developed and strong root system can also make rice have excellent growth characteristics of drought resistance and barren resistance, and promote the strength of late rice plants in many aspects. Moreover, this gene-transformed rice can increase the thousand-grain weight of grains, directly promote rice yield, and make plants have excellent yield traits. The most direct and effective solution to the problems of food shortages and efficient use of land.
  • the total volume of the reaction system is 25 ⁇ L
  • the template is Arabidopsis genomic DNA for gene cloning to obtain the full-length sequence of the TPST gene (SEQ ID NO:1)
  • the promoter sequence is shown in SEQ ID NO: 4
  • the genome sequence is shown in SEQ ID NO: shown in 5.
  • the primers used are as follows:
  • TPST-F GTAAGCTTCATGGGAGCTCCA (SEQ ID NO.: 6);
  • TPST-R AATCTTAACTTTGGAGGTTCTTCT (SEQ ID NO.: 7).
  • the total volume of the reaction system is 25 ⁇ L, and the template is Arabidopsis cDNA for gene cloning to obtain the TPST CDS sequence (sequence is shown in SEQ ID NO: 2), and the amino acid sequence encoded by the gene is shown in SEQ ID NO: 3;
  • the primers used are as follows:
  • TPST-CDS-F ATGCAAATGAACTCTGTTTGGA (SEQ ID NO.: 8);
  • TPST-CDS-R AATCTTAACTTTGGAGGTTCTTCT (SEQ ID NO.: 9).
  • SEQ ID NO: 1 The sequence shown in SEQ ID NO: 1 was cloned into the pCambia1305 vector, and HA (influenza hemagglutinin epitope: YPYDVPDYA (SEQ ID NO.: 10)) tag was fused and expressed to construct a TPST gene transgenic vector.
  • HA influenza hemagglutinin epitope: YPYDVPDYA (SEQ ID NO.: 10)
  • SEQ ID NO: 2 The sequence shown in SEQ ID NO: 2 was cloned into the pCambia1305 vector, and expressed by fusion with the HA tag to construct a 35S-TPST gene transgenic vector.
  • the genetic transformation of rice adopts the genetic transformation method mediated by Agrobacterium EHA105, and the details are as follows:
  • the obtained resistant callus is transferred to a differentiation medium and differentiated in a light culture room for about 40 days to produce regenerated plants; the regenerated plants are transferred to a rooting medium and cultivated for 10 days, and seedlings are refined for 3-5 days and then transplanted.
  • the rooted transgenic plants are each genetically constructed into lines, transplanted into a greenhouse, and leaves are taken for realtime qRT-PCR expression identification.
  • the primers used are:
  • Amplification process 98°C 2min; (98°C 20sec; 60°C 30sec) for 45 cycles;
  • the total RNA of the T1 transgenic rice line was extracted after germination and RT-qPCR was performed.
  • AtTPST gene transgenic rice lines PTPST-31/40/46/47 driven by the AtTPST endogenous promoter all have AtTPST gene transcripts detected, and the AtTPST endogenous promoter activity in the tested lines It is significantly higher than the CMV35S promoter strain, and the transcription activity in T1 generation rice is 3.5 times higher on average.
  • the metal ion content of AtTPST transgenic rice seeds is significantly different, especially the higher K content.
  • the K content of the transgenic AtTPST plants is higher than 130% of the control group ( Figure 2A).
  • the lower Na content is about It is 50% of the control group ( Figure 2B).
  • the seeds of the transgenic plants are heavier than the control, with an average weight gain of about 15% (Figure 3).
  • AtTPST transgenic rice seedlings have a more developed root system, with root length increased by more than 14%, root weight increased by more than 60%, and a significantly more developed root system (Figure 4A-4C).
  • the Arabidopsis TPST endogenous promoter drives AtTPST Genome sequence transgenic No. 7 and No. 31 strains have more developed root systems than Japonica, the empty vector control transformed Nipponbare negative control (Figure 4A), and the root length is significantly increased (Figure 4B).
  • the root fresh weight increased significantly, and the above-ground part also increased (Figure 4C).
  • the seeds were soaked in water to promote germination for three days. Weigh dry soil of equal quality into the flower pot, place the flower pot on the same tray, and irrigate it with water until the upper soil is moist. Plant the germinated seeds in the soil. Greenhouse cultivation. Ensure that the upper soil is kept moist before the drought treatment. Stop watering the rice seedlings after two weeks of growth, and observe phenotypic changes after two weeks of growth. Then resume watering and grow for 1 week, and observe phenotypic changes.
  • TPST content can reduce the Na/K ratio in the plant, thereby enhancing the salt resistance of the plant; at the same time, it significantly increases the thousand-grain weight of the seed, thereby increasing the yield of crops; significantly increasing the root length and root weight of the plant, thereby improving the drought resistance of the plant Resistance, resistance to diseases and insect pests, lodging resistance, and enhance the ability to adapt to the environment. It shows that TPST has important scientific application value in cultivating new varieties of high-yield, stress-tolerant plants.
  • Insert AHD and/or AMV upstream or downstream of the OsTPST promoter to promote the expression of OsTPST and increase its expression level.
  • construct a CRISPR vector AMVKI sgRNA: CCGCCTCGAACCGGGGCCG
  • AMV enhancer sequence AMV enhancer sequence targeting the 5′UTR position of OsTPST (accession number LOC9267276)
  • transfer it into Nipponbare rice callus by gene gun along with the AMV enhancer sequence which can be detected by detecting T1 generation plants Homozygous plants with insertion of AMV and homozygous lines with insertion of other non-AMV sequences.
  • the insertion of AMV can enhance the expression of OsTPST.
  • Figure 6 A-B
  • increasing the expression of endogenous OsTPST in rice can make rice plants stronger in seedling stage and longer and stronger root systems.
  • the expression of endogenous TPST in rice can be significantly increased, and some rice traits can be improved, such as increasing the thousand-grain weight of rice, enhancing stress resistance, increasing root length and root weight, etc.
  • This method provides a new way for the improvement of plant traits. means.

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Abstract

提供了酪氨酰蛋白质磺基转移酶（TPST）基因或其编码蛋白、或其促进剂在调控植物的性状或制备调控植物性状的制剂或组合物中的应用，其中，提高所述植物中TPST基因或其编码蛋白的表达量或活性，可改良植物的性状，所述植物的性状包括选自下组的一种或多种性状：(i)抗逆性；(ii)千粒重；(iii)产量和/或生物量；(iv)果实和/或种子的大小、重量和/或数量。

Description

TPST基因在调控植物性状中的应用 技术领域
本发明涉及农学领域，具体地，涉及TPST基因在调控植物性状中的应用，更具体地，涉及TPST基因在调控植物农艺性状，尤其是水稻抗逆性、产量等方面中的应用。
背景技术
生物体内的基因数目众多、功能多样，他们相互协同作用共同完成生命过程。如模式植物拟南芥的基因组约有2.5万个基因、水稻的基因组包含3-5万个基因。随着拟南芥、大豆、水稻、玉米等植物全基因组序列图谱的完成，许多重要基因相继被克隆，对其功能也有了较深入的研究。近年来，随着世界粮食问题日益严重，为提高粮食作物产量，改善品种，提高抗性、达到高产、稳产、优质的目的，科学家们正逐渐从单纯的基因功能研究转移到更多地关注所研究的基因功能域重要农艺性状的关系。粮食作物中主要围绕作物产量、抗性、品种等新装展开研究，以期发掘调控目标性状的重要基因，并通过基因工程、分子标记辅助育种等手段培育优良品种。部分研究成果已经成功地应用到作物改良上，在粮食生产中显现出巨大的应用前景，凸显了植物功能基因研究的重要意义。
水稻是世界上最重要的粮食作物之一，是人类能量和蛋白质的主要来源，其产量和消费量一直居粮食作物之首。由于现在耕地的缺乏、人口的增多、水土流失问题，高产、优质、多抗水稻新种类的选育与出产使用一直是中国水稻育种的主题。因此挖掘相关功能性基因，并利用其培育目标性状优良品种，开辟育种新路径，对于促进我国从种业大国迈向种业强国具有重要意义。
发明内容
本发明的目的在于提供TPST基因或其编码蛋白或其促进剂在植物(如水稻)性状调控中的用途。
具体地，在本发明中，通过增加TPST基因或其编码蛋白表达量或活性可改良植物中一种或多种性状，包括增强植物的抗逆性、增加千粒重、增加产量 和/或生物量、增加果实和/或种子的大小、重量和/或数量、增加根长或根重。本发明为植物性状改良和分子育种提供了新的技术手段。
本发明的第一方面提供了一种物质的用途，所述物质为TPST基因或其编码蛋白、或其促进剂，用于调控植物的性状或制备调控植物性状的制剂或组合物，其中，所述植物的性状包括选自下组的一种或多种性状：
(i)抗逆性；
(ii)千粒重；
(iii)产量和/或生物量；
(iv)果实和/或种子的大小、重量和/或数量。
在另一优选例中，所述抗逆性选自下组：抗盐性、抗旱性、抗病虫害、或其组合。
在另一优选例中，所述性状还包括选自下组的一种或多种：
(v)根长；
(vi)根重。
在另一优选例中，所述调控植物的性状包括：
(i)增强植物抗逆性；和/或
(ii)增加千粒重；和/或
(iii)增加产量和/或生物量；和/或
(iv)增加果实和/或种子的大小、重量和/或数量。
在另一优选例中，所述调控植物的性状还包括：
(v)增加根长；和/或
(vi)增加根重。
在另一优选例中，所述组合物包括农用组合物。
在另一优选例中，所述制剂包括农用制剂。
在另一优选例中，所述组合物包含(a)TPST基因或其编码蛋白、或其促进剂；和(b)农学上可接受的载体。
在另一优选例中，所述组合物中，含有0.0001-99wt％，较佳地0.1-90wt％的组分(a)，以所述组合物的总重量计。
在另一优选例中，所述组合物或制剂的剂型选自下组：溶液剂、乳剂、混悬剂、粉剂、泡沫剂、糊剂、颗粒剂、气雾剂、或其组合。
在另一优选例中，所述组合物还包括其他调控植物性状的物质。
在另一优选例中，所述其他调控植物性状的物质包括渗透调节剂、芸苔素，海藻素、高钾或氮或磷含量的肥料、微量元素(如硼锌、钙、硅)、***类杀菌剂(如苯醚甲环唑、丙环唑、戊唑醇)、高钾叶面肥、植物激素(如脱落酸、乙烯、细胞***素、多胺)、稀土、PP 333、苯甲酸、水杨酸、烯效唑。
在另一优选例中，所述渗透调节剂选自下组：无机调节剂、有机调节剂、生长调节剂、或其组合。
在另一优选例中，所述的无机调节剂包括Ca 2+、水杨酸。
在另一优选例中，所述有机调节剂包括甜菜碱、脯氨酸、硝普钠(sodium nitroprusside,SNP)。
在另一优选例中，所述生长调节剂包括：脱落酸(ABA)。
在另一优选例中，所述促进剂包括促进TPST基因或其编码蛋白表达的小分子化合物。
在另一优选例中，所述的促进剂选自下组：小分子化合物、核酸分子、或其组合。
在另一优选例中，所述的植物包括单子叶植物、双子叶植物、和/或裸子植物。
在另一优选例中，所述植物包括农作物、林业植物、蔬菜、瓜果、花卉、牧草(包括草坪草)。
在另一优选例中，所述植物选自下组：十字花科、禾本科、豆科、茄科、伞形科、藜科、或其组合。
在另一优选例中，所述植物选自下组：拟南芥、水稻、大豆、番茄、玉米、高粱、烟草、小麦、高粱、谷子、藜麦、马铃薯、红薯、油菜、白菜、菠菜、生菜、黄瓜、茼蒿、空心菜、芹菜、油麦菜、或其组合。
另一优选例中，所述的植物包括：水稻、小麦、玉米、和/或高粱。
在另一优选例中，所述的水稻选自下组：籼稻、粳稻、或其组合。
在另一优选例中，所述的TPST基因选自下组：cDNA序列、基因组序列、或其组合。
在另一优选例中，所述TPST基因来自选自下组的一种或多种植物：十字花科植物、禾本科植物、茄科、豆科、藜科。
在另一优选例中，所述的TPST基因来自选自下组的一种或多种植物：拟南芥、 水稻、玉米、高粱、小麦、谷子、二穗短柄草、藜麦。
在另一优选例中，所述TPST基因选自下组：拟南芥的TPST基因(AtTPST，AT1G08030)、水稻的TPST基因(OsTPST，登录号LOC9267276)、玉米的TPST基因(玉米ZmTPST，登录号LOC100280275)、甘蔗的TPST基因(甘蔗SbTPST，登录号LOC8071351)、亚麻荠的TPST基因(亚麻荠CsTPST，登录号LOC104754980)、油菜的TPST基因(油菜BrTPST，登录号LOC103871547)、萝卜的TPST基因(萝卜RsTPST，登录号LOC108862166)、或其组合。
在另一优选例中，所述TPST基因包括野生型TPST基因和突变型TPST基因。
在另一优选例中，所述的突变型包括突变后编码蛋白的功能未发生改变(即功能与野生型编码蛋白相同或基本相同)和功能增强的突变形式。
在另一优选例中，所述的突变型TPST基因编码的多肽与野生型TPST基因所编码的多肽相同或基本相同。
在另一优选例中，所述的突变型TPST基因包括与野生型TPST基因相比，同源性≥80％(较佳地≥90％，更佳地≥95％，更佳地，≥98％或99％)的多核苷酸。
在另一优选例中，所述的突变型TPST基因包括在野生型TPST基因的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。
在另一优选例中，所述TPST蛋白的氨基酸序列选自下组：
(i)具有SEQ ID NO.:3所示氨基酸序列的多肽；
(ii)将如SEQ ID NO.:3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，具有所述调控植物性状功能的由(i)衍生的多肽；或
(iii)氨基酸序列与SEQ ID NO.:3所示氨基酸序列的同源性≥80％(较佳地≥90％，更佳地≥95％或≥98％)，具有所述TPST活性的多肽。
在另一优选例中，所述TPST基因的核苷酸序列选自下组：
(a)编码如SEQ ID NO.:3所示多肽的多核苷酸；
(b)序列如SEQ ID NO.:1、2或5任一所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.:1、2或5任一所示序列的同源性≥75％(较佳地≥85％，更佳地≥90％或≥95％)的多核苷酸；
(d)在SEQ ID NO.:1、2或5任一所示多核苷酸的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。
本发明第二方面提供了一种组合物，包括：
(a)TPST基因或其编码蛋白的促进剂；和
(b)农学上可接受的载体。
在另一优选例中，所述组合物包括农用组合物。
在另一优选例中，所述组合物的剂型选自下组：溶液剂、乳剂、混悬剂、粉剂、泡沫剂、糊剂、颗粒剂、气雾剂、或其组合。
在另一优选例中，所述组合物中，含有0.0001-99wt％，较佳地0.1-90wt％的组分(a)，以所述组合物的总重量计。
在另一优选例中，所述组合物中，所述TPST基因或其编码蛋白的促进剂的含量(wt％)为0.05％-10％，较佳地，0.1％－8％，更佳地，0.5％－6％。
在另一优选例中，所述的促进剂选自下组：小分子化合物、核酸分子、或其组合。
在另一优选例中，所述组合物还包括其他调控植物的性状的物质。
在另一优选例中，所述其他调控植物性状的物质包括渗透调节剂、芸苔素、海藻素、高钾或氮或磷含量的肥料、微量元素(比如硼锌、钙、硅)、***类杀菌剂(如苯醚甲环唑、丙环唑、戊唑醇)、高钾叶面肥、植物激素(如脱落酸、乙烯、细胞***素、多胺)、稀土、多效唑(PP 333)、苯甲酸、水杨酸、烯效唑。在另一优选例中，所述渗透调节剂选自下组：无机调节剂、有机调节剂、生长调节剂、或其组合。
在另一优选例中，所述的无机调节剂包括Ca 2+、水杨酸。
在另一优选例中，所述有机调节剂包括甜菜碱、脯氨酸、硝普钠(sodium nitroprusside,SNP)。
在另一优选例中，所述生长调节剂包括：脱落酸(ABA)。
本发明第三方面提供了一种本发明第二方面所述的组合物的用途，用于改良植物性状。
本发明第四方面提供了一种改良植物性状的方法，包括步骤：
提高所述植物中TPST基因或其编码蛋白的表达量和/或活性，从而改良植物的性状。
在另一优选例中，所述方法包括给予植物TPST基因或其编码蛋白的促进剂。
在另一优选例中，所述促进剂为促进TPST基因或其编码蛋白表达的物质。
在另一优选例中，所述的促进剂选自下组：小分子化合物、核酸分子、或其组合。
在另一优选例中，所述方法包括向植物中导入外源的TPST基因。
在另一优选例中，所述方法包括向植物中导入促进内源的TPST基因或其编码蛋白表达的物质。
在另一优选例中，所述方法包括促进植物中内源的TPST基因或其编码蛋白表达。
在另一优选例中，所述方法包括步骤：
(i)提供一植物或植物细胞；和
(ii)将TPST基因序列导入所述植物或植物细胞，从而获得转基因的植物或植物细胞。
在另一优选例中，所述方法包括步骤：
(a)提供携带TPST基因序列的表达载体的农杆菌；
(b)将植物细胞或组织或器官与步骤(a)中的农杆菌接触，从而使TPST的基因序列转入植物细胞，并且整合到植物细胞的染色体上；
(c)选择已转入TPST基因序列的植物细胞或组织或器官；和
(d)将步骤(c)中的植物细胞或组织或器官再生为植株。
在另一优选例中，所述植物组织或植物细胞中TPST基因或其编码蛋白的表达量或活性提高了≥5％，≥10％，≥20％，更佳地，≥50％。
在另一优选例中，所述“提高”是指TPST基因或其编码蛋白的表达或活性提高满足以下条件：
A1/A0的比值≥5％，更佳地，≥10％，更佳地≥20％，最佳地为50-200％；其中，A1为植物组织或植物细胞中TPST基因或其编码蛋白的表达或活性；A0为野生型同种类型植物组织或植物细胞中相同TPST基因或其编码蛋白的表达或活性。
在另一优选例中，所述植物中TPST的活性E1与野生型同种类型的植物中的相同TPST本底活性E0之比(E1/E0)≥2倍，较佳地≥5倍，更佳地≥10倍。
本发明第五方面提供了一种制备基因工程的植物组织或植物细胞的方法，包括步骤：
提高植物组织或植物细胞中的TPST基因或其编码蛋白的表达量和/或活性，从而获得基因工程的植物组织或植物细胞。
在另一优选例中，所述的基因工程包括转基因。
在另一优选例中，所述方法还包括向植物组织或植物细胞中导入TPST基因或其编码蛋白的促进剂。
本发明第六方面提供了一种制备性状改良的植物的方法，包括步骤：
将本发明第五方面所述方法制备的基因工程的植物组织或植物细胞再生为植物体，从而获得性状改良的植物。
在另一优选例中，所述的性状包括选自下组的一种或多种性状：
(i)抗逆性；
(ii)千粒重；
(iii)产量和/或生物量；
(iv)果实和/或种子的大小、重量和/或数量。
在另一优选例中，所述的性状还包括选自下组的一种或多种：
(iii)根长；
(iv)根重。
在另一优选例中，所述性状改良包括：
(i)增强植物抗逆性；和/或
(ii)增加千粒重；和/或
(iii)增加产量和/或生物量；和/或
(iv)增加果实和/或种子的大小、重量和/或数量。
在另一优选例中，所述性状改良还包括：
(iii)增加根长；和/或
(iv)增加根重。
本发明第七方面提供了一种基因工程植株，所述植株中导入TPST基因或其编码蛋白、或其促进剂或所述的植株是用本发明第六方面所述的方法制备的。
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为AtTPST转基因T1代水稻株系AtTPST基因表达检测。其中，control是转入空载体pCambia1305的日本晴阴性对照，其中，P35S-3，46，51，52是35S启动子驱动的TPST CDs转基因水稻株系；PTPST-31，40，46，47是拟南芥TPST内源启动子驱动AtTPST基因组序列的转基因水稻株系。
图2为转基因及野生型水稻种子金属离子含量比较。其中，图2A钾元素 含量，图2B钠元素含量；control是转入空载体pCambia1305的日本晴阴性对照，P35S是35S启动子驱动的TPST CDs转基因水稻，PTPST是拟南芥TPST内源启动子驱动AtTPST基因组序列的转基因水稻。
图3为转基因及野生型水稻种子籽粒重量比较，其中，control是转入空载体pCambia1305的日本晴阴性对照，P35S是35S启动子驱动的TPST CDs转基因水稻，PTPST是拟南芥TPST内源启动子驱动AtTPST基因组序列的转基因水稻。
图4为为AtTPST转基因水稻水培苗具有更发达的根系，图4A拟南芥TPST内源启动子驱动AtTPST基因组序列转基因7号株系与31号株系与日本晴(Japonica)，空载体对照转化日本晴阴性对照(control)相比均有更发达的根系；图4B拟南芥TPST内源启动子驱动AtTPST基因组序列转基因7号株系与31号株系与日本晴(Japonica)，空载体对照转化日本晴阴性对照(control)相比根长度增加；图4C拟南芥TPST内源启动子驱动AtTPST基因组序列转基因7号株系与31号株系与日本晴(Japonica)，空载体对照转化日本晴阴性对照(control)相比根鲜重增加，地上部分也有增加；Japonica是指日本晴；control是指空载体对照转化日本晴阴性对照；PTPST-7是指拟南芥TPST内源启动子驱动AtTPST基因组序列转基因7号株系；PTPST-31是指拟南芥TPST内源启动子驱动AtTPST基因组序列转基因31号株系。
图5显示了AtTPST转基因水稻抗旱性，其中A：正常生长的水稻苗；B：萌发2周的水稻苗停止浇水做干旱处理两周；C:恢复浇水后1周。
图6显示了在水稻中提高内源性的OsTPST的表达量，可以提高水稻植株的性状。其中图6A显示了AMV增强OsTPST表达的植株(AMV-OsTPST-1)与对照植株(AMV Control)的根系生长的结果，提高水稻中OsTPST的表达明细可以促进根系的生长；图6B显示了AMV增强OsTPST表达的植株(AMV Inserted Lines)与对照组(Control)以及其他非AMV序列***的植株(DNA Fragment Inserted Line)的发育结果，由图6B可明显看出，提高水稻内源性OsTPST的表达量，可以使得水稻株系苗期更加强壮。
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植物性状位点的研究和筛选，首次发现，提高所述植物(如水稻)中TPST基因或其编码蛋白的表达量或活 性，可显著改良植物的性状。在此基础上，发明人完成了本发明。
具体地，当提高所述植物中TPST基因或其编码蛋白的表达量或活性时，可以(i)增强植物抗逆性；和/或(ii)增加千粒重；和/或(iii)增加产量和/或生物量；和/或(iv)增加果实和/或种子的大小、重量和/或数量；和/或(v)根长；和/或(vi)根重。
TPST基因
TPST即tyrosyl-protein sulfo-transferase，中文名为酪氨酰磺基转移酶，参与蛋白翻译后的硫酸化修饰，它将底物3-磷酸腺苷-5磷酸硫酸(PAPS)的磺酸基团转移到蛋白质的酪氨酸残基上，该修饰使分泌蛋白或膜蛋白具有成熟的生物学功能 [1]。
如本文所用，术语“本发明的TPST基因”、“TPST基因”可互换使用，均指来源于植物(如水稻、拟南芥)的TPST基因或其变体。在一优选实施方式中，本发明的TPST基因的核苷酸序列如SEQ ID NO.:2所示。所述基因的变体可以通过***或删除调控区域，进行随机或定点突变等来获得。
本发明还包括与本发明的优选基因序列(SEQ ID NO.:2)具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％)同源性的核酸，所述核酸也能有效地调控植物(如水稻)的性状。“同源性”是指按照位置相同的百分比，两条或多条核酸之间的相似水平(即序列相似性或同一性)。
在本发明中，SEQ ID NO.:2中的核苷酸序列可以经过取代、缺失或添加一个或多个(通常为1-90个，较佳地1-60个，更佳地1-20个，最佳地1-10个)，以及在5’和/或3’端添加数个(通常为60个以内，较佳地为30个以内，更佳地为10个以内，最佳地为5个以内)核苷酸，生成SEQ ID NO.:2的衍生序列，由于密码子的简并性，即使与SEQ ID NO.:2的同源性较低，也能基本编码出如SEQ ID NO.:3所示的氨基酸序列。
另外，“在SEQ ID NO.:2中的核苷酸序列经过取代、缺失或添加至少一个核苷酸衍生序列”的含义还包括能在中度严谨条件下，更佳的在高度严谨条件下与SEQ ID NO.:2所示的核苷酸序列杂交的核苷酸序列。这些变异形式包括(但并小限于)：若干个(通常为1-90个，较佳地1-60个，更佳地1-20个，最佳地1-10个)核苷酸的缺失、***和/或取代，以及在5’和/或3’端添加数个(通常为 60个以内，较佳地为30个以内，更佳地为10个以内，最佳地为5个以内)核苷酸。
应理解，尽管本发明的实例中提供的基因来源于拟南芥，但是来源于其它类似的植物(尤其是与拟南芥属于同一科或属的植物或于拟南芥的同源性比较高的其他科或属的植物)的、与本发明的序列(优选地，序列如SEQ ID NO.:2所示)具有一定同源性(保守性，如具有80％以上，如85％,90％,95％甚至98％序列相同性)的TPST的基因序列，也包括在本发明的范围内，只要本领域技术人员在阅读了本申请后根据本申请提供的信息可以方便地从其它植物中分离得到该序列，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.:2所示的编码区序列相同或者是简并的变异体。
编码成熟多肽的多核苷酸包括:只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。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苷或多肽的片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和***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***，但不会从实质上改变其编码的多肽的功能。
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)甲酞胺，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TPST核苷酸全长序列或其片段通常可以用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DNA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。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。
TPST基因编码的多肽
如本文所用，术语“本发明多肽”、“TPST基因的编码蛋白”、可以互换使用，都是指来源于植物的TPST的多肽及其变体。在一优选实施方式中，本发明多肽的一种典型的氨基酸序列如SEQ ID NO.:3所示。
本发明涉及一种调控植物性状的TPST多肽及其变体，在本发明的一个优选例中，所述多肽的氨基酸序列如SEQ ID NO.:3所示。本发明的多肽能够有效调控植物(如水稻)的性状。
本发明还包括与本发明的SEQ ID NO.:3所示序列具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％)同源性的具有相同或相似功能的多肽或蛋白。
所述“相同或相似功能”主要是指：“调控植物或农作物(如水稻)的性状”。
本发明的多肽可以是重组多肽、天然多肽、合成多肽。本发明的多肽可以是天然纯化的产物，或是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主，本发明的多肽可以是糖基化的，或可以是非糖基化的。本发明的多肽还可包括或不包括起始的甲硫氨酸残基。
本发明还包括具有TPST蛋白活性的TPST蛋白片段和类似物。如本文所用，术语“片段”和“类似物”是指基本上保持本发明的天然TPST蛋白相同的生物学功 能或活性的多肽。
本发明的多肽片段、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)被取代的多肽，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多肽；或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽；或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或融合蛋白)。根据本文的定义这些片段、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，所述的多肽变体是如SEQ ID NO.:3所示的氨基酸序列，经过若干个(通常为1-60个，较佳地1-30个，更佳地1-20个，最佳地1-10个)取代、缺失或添加至少一个氨基酸所得的衍生序列，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在所述蛋白中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能，在C末端和/或\末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。这些保守性变异最好根据表1进行替换而产生。
表1
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
本发明还包括所要求保护的蛋白的类似物。这些类似物与天然SEQ ID NO.:3差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些蛋白的类似物包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分了生物学的技术。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的蛋白并不限于上述例举的代表性的蛋白。
修饰(通常不改变一级结构)形式包括：体内或体外蛋白的化学衍生形式如乙酸化或羧基化。修饰还包括糖基化，如那些在蛋白质合成和加工中进行糖基化修饰。这种修饰可以通过将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。
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本发明所述的蛋白或其变异体。一般来说有以下步骤：
(1).用编码本发明蛋白或其变异体的多核苷酸，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、潮霉素抗性以及荧光蛋白等)；增强子；或操作子。
在本发明中，编码蛋白的多核苷酸序列可***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。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。
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。
包括本发明基因、表达盒或的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，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。
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***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。
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。
本发明所述的蛋白可在细胞内、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。
改良植物的性状
在本发明中，还提供了一种改良植物性状的方法，具体地，促进或提高TPST基因或其编码蛋白的表达，从而改良植物的性状，所述性状选自下组的一种或多种：
(i)抗逆性；
(ii)千粒重。
(iii)产量和/或生物量；
(iv)果实和/或种子的大小、重量和/或数量。
在一优选实施方式中，所述性状还包括选自下组的一种或多种：
(v)根长；
(vi)根重。
在一优选实施方式中，所述改良植物的性状包括：
(i)增强植物抗逆性；和/或
(ii)增加千粒重；和/或
(iii)增加产量和/或生物量；和/或
(iv)增加果实和/或种子的大小、重量和/或数量。
在一优选实施方式中，所述改良植物的性状还包括：
(v)增加根长；和/或
(vi)增加根重。
本发明的主要优点包括：
(1)本发明首次发现，增加植物中TPST基因的含量或上调表达该基因可改善植物的农艺性状，如抗逆性、千粒重、产量、生物量、果实或种子大小、重量、数量、根长、根重等。
(2)本发明首次发现，采用拟南芥内源启动子驱动拟南芥AtTPST基因在水稻中表达或者提高水稻内源性OsTPST基因的表达可以促进幼苗期水稻的根系发育，在自然栽培条件下必然有利于水稻地上部分的生长，起到壮苗的效果。发达强壮的根系还可以使水稻具有抗旱和抗贫瘠的优良生长性状，从多方面促进后期水稻植株的强壮。而且这种基因转化水稻可以提高籽粒千粒重，直接促进水稻产量，使植株具有优良的产量性状。最直接有效的解决粮食短缺和土地高效利用的问题。
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。
实施例1TPST基因的获得：
1)TPST基因启动子及基因组序列克隆
反应体系的总体积为25μL，模板为拟南芥基因组DNA进行基因克隆，得到TPST基因全长序列(SEQ ID NO:1),启动子序列如SEQ ID NO:4所示，基因组序列如SEQ ID NO:5所示.
所用引物(本发明人设计)如下：
TPST-F:GTAAGCTTCATGGGAGCTCCA(SEQ ID NO.:6)；
TPST-R:AATCTTAACTTTGGAGGTTCTTCT(SEQ ID NO.:7)。
反应体系：
Figure PCTCN2020110574-appb-000001
加水至50μL。
扩增过程：98℃ 2min；
(98℃ 20sec；58℃ 30min；72℃ 90sec)for 30 cycles；
72℃ 5min。
2)TPST CDS基因序列的克隆
反应体系的总体积为25μL，模板为拟南芥cDNA进行基因克隆，得到TPST CDS序列(序列如SEQ ID NO:2所示)，该基因编码的氨基酸序列如SEQ ID NO:3所示；
所用引物(本发明人设计)如下：
TPST-CDS-F:ATGCAAATGAACTCTGTTTGGA(SEQ ID NO.:8)；
TPST-CDS-R:AATCTTAACTTTGGAGGTTCTTCT(SEQ ID NO.:9)。
反应体系：
Figure PCTCN2020110574-appb-000002
加水至50μL
扩增过程：98℃ 2min；
(98℃ 20sec；58℃ 30min；72℃ 90sec)for 30 cycles；
72℃ 5min。
实施例2：转基因植株的构建及性状分析
1.转基因载体构建
1)TPST基因转基因载体的构建
将SEQ ID NO:1所示序列克隆至pCambia1305载体，与HA(流感病毒血凝素，influenza hemagglutinin epitope:YPYDVPDYA(SEQ ID NO.:10))标签融合表达，构建TPST基因转基因载体。
2)35S-TPST转基因载体的构建
将SEQ ID NO:2所示序列克隆至pCambia1305载体，与HA标签融合表达，构建35S-TPST基因转基因载体。
2.水稻遗传转化
水稻遗传转化均采用农杆菌EHA105介导的遗传转化方法，具体如下：
将成熟的水稻种子去壳用升汞消毒后，在愈伤诱导培养基上26℃黑暗培养30d；将诱导愈伤在继代培养基上培养15d；挑选鲜黄的愈伤用携带目标载体的农杆菌菌液浸泡30min后，吹干于共培养基上18℃培养2d；无菌水清洗愈伤后吹干置于抗性的筛选培养基上，经过2轮抗性筛选，每次15d；将获得的抗性愈伤转移到分化培养基上，在光照培养室分化约40d产生再生植株；将再生植株转移到生根培养基上培养10d，炼苗3-5d后移栽。
3.移栽、表达量鉴定和表型分析
将生根的转基因植株每个遗传构建成系，移栽温室，取叶片进行realtime qRT-PCR表达量鉴定。
所用引物(本发明人设计)为：
qTPST-1F TTACTTCTTAGCTCAGTTATTGGC(SEQ ID NO.:11)
qTPST-1R CAATGAAAATATGTTCTGCCTCCA(SEQ ID NO.:12)
反应体系：
Figure PCTCN2020110574-appb-000003
加水至15μL
扩增过程：98℃ 2min；(98℃ 20sec；60℃ 30sec)for 45 cycles；
添加融合曲线。
4.结果
1)AtTPST转基因T1代水稻中AtTPST基因表达检测
如图1，T1代水稻转基因株系种子萌发后提取总RNA并进行RT-qPCR检测，与转化空载体的阴性对照相比较，PCMV35S启动子驱动的AtTPST表达株系P35-3/46/51/52，与AtTPST内源性启动子驱动的AtTPST基因转基因水稻株系PTPST-31/40/46/47都有AtTPST基因转录本被检测到，并且在检测的株系中AtTPST内源性启动子活性明显高于CMV35S启动子株系，在T1代水稻中转录活性平均高出3.5倍。
2)AtTPST转基因T1代水稻的表型鉴定
鉴定结果请参见图2-4。AtTPST转基因水稻种子金属离子含量与对照相比有显著差异，特别是有较高的K含量，转AtTPST植物K含量均高于对照组的130％(图2A)，同时较低的Na含量，约为对照组的50％(图2B)。
在籽粒千粒重的重量上，转基因植株的种子要较对照更重，平均增重约为15％(图3)。
AtTPST转基因水稻苗具有更加发达的根系，根长增加14％以上，根重增加60％以上，具有明显更加发达的根系(图4A-图4C)，其中，拟南芥TPST内源启动子驱动AtTPST基因组序列转基因7号株系与31号株系与日本晴(Japonica)，空载体对照转化日本晴阴性对照(control)相比均有更发达的根系(图4A)，根长度明显增加(图4B)，根鲜重明显增加，地上部分也有增加(图4C)。
3)AtTPST转基因T1代水稻抗旱性测试
种子浸水催芽萌发三天。称取等质量的干土于花盆中，将花盆置于同一个托盘，浸水浇灌至上层土湿润。将萌发的种子种植于土中。温室培养。干旱处理前确保上层土壤保持湿润。水稻苗生长两周后停止浇水，生长两周后观察表型变化。然后恢复浇水后生长1周，观察表型变化。
结果显示：正常条件下，野生型组和载体对照组无明显差异，见图5A。干旱处理两周后的野生型组和载体对照组的幼苗，生长受到明显抑制，相比转基因植株的株高显著变矮、叶片较为萎蔫，见图5B。复水后，野生型组和载体对照组的幼苗无明显改善，转基因植株的得到一定程度改善，见图5C。
5.结论
TPST含量的增加可以降低植物中Na/K比，从而增强植物的抗盐性；同时显著的增加种子的千粒重，从而提高农作物的产量；显著的增加植物根长、根重，从而提高植物的抗旱性、抗病虫害、抗倒伏性，增强环境适应能力。表明 TPST在培育高产、耐逆的植物新品种中具有重要的科学应用价值。
实施例3增强水稻内源性TPST(OsTPST)基因相关表达量实验
进一步的，发明人还将通过以下方法增加水稻中OsTPST表达量：
(1)通过向水稻中转入OsTPST相关基因，增加其内源性含量；
(2)在内源性强启动子如UBQ1,UBQ2,UBI9或ACT2之后***OsTPST增加其表达量；
(3)在OsTPST启动子上游或下游***AHD和/或AMV促进OsTPST表达，增加其表达量。例如，构建靶向OsTPST(登录号LOC9267276)5′UTR位置的CRISPR载体(AMVKI sgRNA:CCGCCTCGAACCGGGGCCG)，与AMV增强子序列一同用基因枪方式转入日本晴水稻愈伤，通过检测T1代植株可检测到AMV***的纯合植株，以及其他非AMV序列***的纯合株系。AMV的***可以增强OsTPST的表达。如图6(A-B)所示，提高水稻内源性OsTPST的表达量，可以使得水稻株系苗期更加强壮、根系更长更壮。
此外，通过对水稻内源OsTPST进行基因敲除，发现基因敲除的植株表现出显著发育缺陷或根发育缺陷，这也提示了OsTPST基因对水稻发育有不可或缺的重要功能。
通过以上方法可以显著增强水稻内源TPST的表达量，改良水稻的部分性状，如增加水稻的千粒重、增强抗逆性、增加根长根重等，该方法为植物性状改良提供了一种新的手段。
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。
参考文献
[1]Moore K L(2003).The biology and enzymology of protein tyrosine Osulfation.J Biol Chem，278(27):24243-24246.

Claims (10)

  1. 一种物质的用途，其特征在于，所述物质为TPST基因或其编码蛋白、或其促进剂，用于调控植物的性状或制备调控植物性状的制剂或组合物，其中，所述植物的性状包括选自下组的一种或多种性状：
    (i)抗逆性；
    (ii)千粒重；
    (iii)产量和/或生物量；
    (iv)果实和/或种子的大小、重量和/或数量。
  2. 如权利要求1所述的用途，其特征在于，所述抗逆性选自下组：抗盐性、抗旱性、抗病虫害、或其组合。
  3. 如权利要求1所述的用途，其特征在于，所述性状还包括选自下组的一种或多种：
    (v)根长；
    (vi)根重。
  4. 如权利要求1所述的用途，其特征在于，所述调控植物的性状包括：
    (i)增强植物抗逆性；和/或
    (ii)增加千粒重；和/或
    (iii)增加产量和/或生物量；和/或
    (iv)增加果实和/或种子的大小、重量和/或数量。
  5. 一种组合物，其特征在于，包括：
    (a)TPST基因或其编码蛋白的促进剂；和
    (b)农学上可接受的载体。
  6. 如权利要求5所述的组合物，其特征在于，所述组合物还包括其他调控植物的性状的物质。
  7. 一种权利要求5所述的组合物的用途，其特征在于，用于改良植物性状。
  8. 一种改良植物性状的方法，其特征在于，包括步骤：
    提高所述植物中TPST基因或其编码蛋白的表达量和/或活性，从而改良植物的性状。
  9. 一种制备基因工程的植物组织或植物细胞的方法，其特征在于，包括步骤：
    提高植物组织或植物细胞中的TPST基因或其编码蛋白的表达量和/或活性，从而获得基因工程的植物组织或植物细胞。
  10. 一种制备性状改良的植物的方法，其特征在于，包括步骤：
    将权利要求9所述方法制备的基因工程的植物组织或植物细胞再生为植物体，从而获得性状改良的植物。
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