WO2017067214A1 - 蛋白质VdAL在提高植物产量、产品品质和抗旱性及促进植物果实着色中的应用 - Google Patents

蛋白质VdAL在提高植物产量、产品品质和抗旱性及促进植物果实着色中的应用 Download PDF

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WO2017067214A1
WO2017067214A1 PCT/CN2016/088074 CN2016088074W WO2017067214A1 WO 2017067214 A1 WO2017067214 A1 WO 2017067214A1 CN 2016088074 W CN2016088074 W CN 2016088074W WO 2017067214 A1 WO2017067214 A1 WO 2017067214A1
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vdal
plant
diluted
growth
protein
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PCT/CN2016/088074
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English (en)
French (fr)
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齐俊生
巩志忠
洪旭辉
张定朋
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中国农业大学
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Priority claimed from CN201510691295.6A external-priority patent/CN105198967B/zh
Priority claimed from CN201510691515.5A external-priority patent/CN105218649B/zh
Application filed by 中国农业大学 filed Critical 中国农业大学
Priority to US15/769,746 priority Critical patent/US10806145B2/en
Priority to CA3002881A priority patent/CA3002881A1/en
Priority to EP16856654.5A priority patent/EP3351555A4/en
Publication of WO2017067214A1 publication Critical patent/WO2017067214A1/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
    • A01H3/00—Processes for modifying phenotypes, e.g. symbiosis with bacteria
    • A01H3/04—Processes for modifying phenotypes, e.g. symbiosis with bacteria by treatment with chemicals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
    • A01N47/44—Guanidine; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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Definitions

  • the invention relates to the application of protein VdAL in the field of biotechnology for improving plant yield, product quality and drought resistance and promoting plant fruit coloration.
  • Excessive fertilization will increase the antagonism between ions when the nutrient elements are absorbed by the crop. If the high concentration of a certain nutrient segregant can inhibit the activity of another or more nutrient segregants, it will affect the crop to another nutrient ion. absorb. If the application of nitrogen fertilizer is too much in acidic soil, it is difficult for crops to absorb calcium ions. Excessive application of calcium fertilizer can induce the lack of trace elements such as zinc, boron, iron, magnesium and manganese in crops. Excessive potassium use will also affect the absorption of calcium and magnesium ions by crops; and excessive fertilization may cause crop poisoning.
  • the first technical problem to be solved by the present invention is how to promote plant growth and how to increase plant yield.
  • the present invention first provides the use of proteins for increasing plant yield and/or regulating plant growth.
  • VdAL protein provided by the present invention for increasing plant yield and/or regulating plant growth
  • the protein is named VdAL and is as follows A1) or A2) or A3):
  • amino acid sequence is the protein of sequence 1;
  • A2 a protein derived from A1) having the same function after substitution and/or deletion and/or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO:1;
  • A3 A fusion protein obtained by ligating the N-terminus or/and C-terminus of A1) or A2).
  • sequence 1 consists of 297 amino acids.
  • the label shown in Table 1 may be attached to the amino terminus or the carboxy terminus of the protein represented by the amino acid of SEQ ID NO: 1 or SEQ ID NO: 1 in the Sequence Listing.
  • the VdAL in the above A2) can be artificially synthesized, or the coding gene can be synthesized first, and then obtained by biological expression.
  • the gene encoding the VdAL in the above A2) can be obtained by deleting a codon of one or several amino acid residues in the DNA sequence shown in the sequence 2 in the sequence listing, and/or performing one or several base pair missense mutations. And/or the coding sequence of the tag shown in Table 1 is attached at its 5' end and/or 3' end.
  • the regulating plant growth may be to promote plant growth.
  • the present invention also provides an application of a biological material related to VdAL in increasing plant yield and/or regulating plant growth.
  • VdAL-related biological material provided by the present invention for increasing plant yield and/or regulating plant growth
  • the biological material is any one of the following B1) to B20):
  • B2 an expression cassette comprising the nucleic acid molecule of B1);
  • B3 a recombinant vector comprising the nucleic acid molecule of B1);
  • B4 a recombinant vector comprising the expression cassette of B2)
  • B5 a recombinant microorganism comprising the nucleic acid molecule of B1);
  • B7 a recombinant microorganism comprising the recombinant vector of B3);
  • B9 a transgenic plant cell line comprising said nucleic acid molecule of B1);
  • B11 a transgenic plant cell line comprising the recombinant vector of B3);
  • B12 a transgenic plant cell line comprising the recombinant vector of B4)
  • B13 a transgenic plant tissue comprising the nucleic acid molecule of B1);
  • B14 a transgenic plant tissue comprising the expression cassette of B2)
  • B15 a transgenic plant tissue comprising the recombinant vector of B3);
  • B16 a transgenic plant tissue comprising the recombinant vector of B4)
  • B17 a transgenic plant organ comprising said nucleic acid molecule of B1);
  • B18 a transgenic plant organ comprising the expression cassette of B2)
  • B19 a transgenic plant organ comprising the recombinant vector of B3);
  • B20 A transgenic plant organ comprising the recombinant vector of B4).
  • the nucleic acid molecule may be a gene of b1) or b2) or b3) as follows:
  • nucleotide sequence is a cDNA molecule or a DNA molecule of SEQ ID NO: 2 in the Sequence Listing;
  • B2 having 75% or more of the identity of the nucleotide sequence defined by b1), and a cDNA molecule or genomic DNA molecule encoding VdAL;
  • B3 a cDNA molecule or genomic DNA molecule which hybridizes under stringent conditions to a nucleotide sequence defined by b1) and which encodes VdAL.
  • the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.
  • sequence 2 consists of 894 nucleotides and encodes the protein shown in SEQ ID NO: 1.
  • nucleotide sequence encoding a VdAL of the present invention can readily mutate a nucleotide sequence encoding a VdAL of the present invention using known methods, such as directed evolution and point mutation.
  • Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of VdAL isolated from the present invention are nucleotide sequences derived from the present invention as long as they encode VdAL and have VdAL function. And equivalent to the sequence of the invention.
  • identity refers to sequence similarity to a native nucleic acid sequence. “Identity” includes 75% or more, or 85% or more, or 90% or more, or 95% or more of the nucleotide sequence of a protein consisting of the amino acid sequence of the present invention encoding VdAL. Highly identical nucleotide sequence. Identity can be evaluated using the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.
  • VdAL-related biomaterials are used in the process of increasing plant yield and/or regulating plant growth, the stringent conditions being hybridization and washing at 2 times in a solution of 2 x SSC, 0.1% SDS at 68 ° C, Each time 5 min, in a solution of 0.5 ⁇ SSC, 0.1% SDS, hybridize at 68 ° C and wash the membrane twice, each time 15 min; or, 0.1 ⁇ SSPE (or 0.1 ⁇ SSC), 0.1% SDS solution Hybridization and washing at 65 °C.
  • the above 75% or more of the identity may be 80%, 85%, 90% or 95% or more.
  • the expression cassette (VdAL gene expression cassette) containing the nucleic acid molecule encoding VdAL described in B2) means capable of being in a host cell.
  • a DNA expressing VdAL which may include not only a promoter that initiates transcription of the VdAL gene, but also a terminator that terminates transcription of the VdAL gene.
  • the expression cassette may further comprise an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development specific promoters, and inducible promoters.
  • promoters include, but are not limited to, constitutive promoter of cauliflower mosaic virus 35S: a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester); tomatoes Protease inhibitor II promoter (PIN2) or LAP promoter (both induced by methyl jasmonate); heat shock promoter (U.S. Patent 5,187,267); tetracycline-inducible promoter (U.S.
  • Patent 5,057,422 seed-specific promoter
  • the millet seed-specific promoter pF128 CN101063139B (Chinese Patent 200710099169.7)
  • the seed storage protein-specific promoter for example, the promoters of Bean globulin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO) J.4: 3047-3053). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety.
  • Suitable transcription terminators include, but are not limited to, Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine Synthase terminator (see, for example, Odell et al. (I 985 ) Nature 313: 810; Rosenberg et al. (1987) Gene, 56: 125; Guerineau et al. (1991) Mol. Gen. Genet, 262: 141; 1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5: 141; Mogen et al.
  • NOS terminator Agrobacterium nopaline synthase terminator
  • CaMV 35S terminator cauliflower mosaic virus CaMV 35S terminator
  • tml terminator tml terminator
  • pea rbcS E9 terminator nopaline and octopine Synth
  • a recombinant vector containing the VdAL gene expression cassette can be constructed using an existing expression vector.
  • the plant expression vector includes a dual Agrobacterium vector and a vector which can be used for plant microprojectile bombardment and the like. For example, pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA).
  • the plant expression vector may further comprise a 3' untranslated region of the foreign gene, ie, comprising a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression.
  • the polyadenylation signal can direct polyadenylation to the 3' end of the mRNA precursor, such as Agrobacterium tumefaciens-induced (Ti) plasmid genes (such as nopaline synthase gene Nos), plant genes (such as soybean).
  • the untranslated regions transcribed at the 3' end of the storage protein gene all have similar functions.
  • an enhancer including a translation enhancer or a transcription enhancer, may be used, and these enhancer regions may be an ATG start codon or a contiguous region start codon, etc., but are required to be encoded.
  • the reading frames of the sequence are identical to ensure proper translation of the entire sequence.
  • the sources of the translational control signals and initiation codons are broad and may be natural or synthetic.
  • the translation initiation region can be from a transcription initiation region or a structural gene.
  • the plant expression vector used can be processed, such as a gene encoding a color-changing enzyme or luminescent compound (GUS gene, luciferase) which can be expressed in plants.
  • marker genes for antibiotics such as the nptII gene conferring resistance to kanamycin and related antibiotics, the bar gene conferring resistance to the herbicide phosphinothricin, and the hph gene conferring antibiotic resistance to hygromycin
  • the dhfr gene that confers resistance to methotrexate, confers glyphosate-resistant EPSPS gene) or chemical-resistant marker gene (such as anti-tuberant gene) and provides mannose-6-capable mannose-capable mannose-- Phosphoric acid Enzyme gene. From the safety of transgenic plants, the transformed plants can be directly screened by adversity without any selectable marker genes.
  • the vector may be a plasmid, cosmid, phage or viral vector.
  • the plasmid may be the vector pET42a(+).
  • VdAL-related biomaterials in increasing plant yield and/or regulating plant growth may be yeast, bacteria, algae or fungi, such as E. coli.
  • the E. coli may be Escherichia coli JM109.
  • the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ may neither include the propagation material nor the propagation material.
  • the transgenic plant organ may also be a seed of a transgenic plant.
  • the transgenic plants can include seeds, callus, whole plants, and cells.
  • the coding gene of VdAL is introduced into Escherichia coli JM109 by a recombinant vector containing an expression cassette for the gene encoding VdAL to obtain a recombinant microorganism.
  • the recombinant vector is obtained by replacing the sequence between the NdeI and KpnI recognition sites of the vector pET42a(+) with the DNA molecule represented by nucleotides 1-894 of the sequence 2 in the sequence table (ie, the VdAL gene).
  • Recombinant vector expresses VdAL shown in SEQ ID NO: 1.
  • the recombinant microorganism expresses VdAL shown in SEQ ID NO: 1.
  • the regulation of plant growth may be to promote plant growth.
  • the present invention also provides a method of increasing plant yield and/or promoting plant growth.
  • a method for increasing plant yield and/or promoting plant growth comprising applying VdAL, the biological material or biological agent to a plant of interest, increasing yield of the plant of interest and/or promoting growth of the plant of interest;
  • the active ingredient of the biological preparation is VdAL or the biological material.
  • the biological preparation can be prepared according to the method 1), wherein the recombinant microorganism is cultured to express a gene encoding the VdAL to obtain the biological preparation.
  • the method 1) may specifically be as follows 11) and 12):
  • the method 1) may further comprise drying the biological preparation.
  • the coding gene may be B1) the nucleic acid molecule.
  • the recombinant microorganism is a recombinant microorganism obtained by introducing a recombinant vector containing an expression cassette of a gene encoding a VdAL into Escherichia coli JM109.
  • the recombinant vector is a sequence in which the sequence between the NdeI and KpnI recognition sites of the vector pET42a(+) is replaced with the sequence 1-894 of the sequence 2.
  • a DNA molecule represented by a nucleotide ie, a VdAL gene
  • the recombinant vector expresses VdAL shown in SEQ ID NO: 1.
  • the recombinant microorganism expresses VdAL shown in SEQ ID NO: 1.
  • the applying the VdAL, the biological material or the biological preparation to the plant of interest may specifically be to apply VdAL, the biological material or the biological preparation to the leaf of the plant of interest, such as spraying VdAL on the plant of interest.
  • the biological material or the biological preparation, the seed of the plant of interest may also be treated with VdAL, the biological material or the biological preparation, such as with VdAL, the biological material or a liquid preparation of the biological preparation for the purpose Seeds of plants are soaked.
  • the liquid preparation of VdAL, the biological material or the biological preparation may be a liquid obtained by dissolving or suspending VdAL, the biological material or the biological preparation directly with water.
  • the amount of VdAL, the biological material or biological agent applied to the plant of interest may be determined according to the species and/or growth period of the plant of interest.
  • the present invention also provides the use of the biological preparation for increasing plant yield and/or regulating plant growth.
  • the present invention also provides the biological preparation.
  • the name of the biological preparation is Vidalian.
  • the plant is a dicot or a monocot.
  • the dicotyledonous plant may be a Malvaceae plant, a Cucurbitaceae plant, a Solanaceae plant, a cruciferous plant, a Rosaceae plant, a grape plant, a Liaaceae plant, a Polygonaceae plant or a legume plant.
  • the mallow plant may be cotton.
  • the Cucurbitaceae plant can be watermelon, cucumber or melon.
  • the Solanaceae plant may be tomato, succulent or Solanaceae.
  • the cruciferous plant may be a green stalk or a radish, such as a can of radish.
  • the Rosaceae plant may be a strawberry.
  • the legume may be soybean.
  • the grape plant can be a grape.
  • the subfamily plant can be a cherry.
  • the plant may be spinach.
  • the monocot may be a gramineous plant such as rice or wheat.
  • the yield may be an economic yield, that is, a product yield required for cultivation purposes.
  • a product yield required for cultivation purposes Such as cereal seeds, cotton seed cotton or lint, green stem or spinach plants, watermelon, tomato, cucumber, sacred fruit, strawberry, melon, sweet pepper, cherry, grape fruit, radish roots.
  • the yield can be embodied by weight.
  • the growth of the promoted plant can ultimately be manifested as an increase in crop yield.
  • Increases in yields such as cotton, watermelon, wheat, tomato, green stalk, cucumber, sacred fruit, strawberry, melon, rice, grape, cherry, radish, spinach, sweet pepper or soybean.
  • the promoting the growth of the plant may be to promote vegetative growth and/or reproductive growth of the plant, such as promoting the growth of the plant fruit.
  • the vegetative growth can be embodied in promoting seed germination, promoting plant growth, promoting fruit growth, increasing the number of fruit sets, and/or delaying plant senescence.
  • the promoting vegetative growth of the plant is embodied in the following aspects: increasing the germination rate of the cotton, the number of bolls per plant, the total number of bolls per mu and the weight of the single boll; and increasing the number of fruits per plant; Improve the germination rate of wheat, promote the growth of wheat plants; increase the number of tomato per plant; promote the growth of green stalk, spinach or radish plants; increase the number of cucumber fruit, promote the expansion of cucumber fruit, promote the growth of cucumber plants, delay the senescence of cucumber plants; Female fruit, cherry, grape fruit number; promote strawberry plant growth; promote melon plant growth; promote sweet pepper plant growth; promote soybean emergence and plant growth. Promoting plant reproduction The growth is embodied by increasing the number of fruits in cotton, watermelon, tomato, cucumber and sacred fruit.
  • the effect of VdAL or the biological agent on different plants may result in different results depending on the concentration of VdAL or the biological agent.
  • the concentration of VdAL or the biological preparation may be adjusted according to the crop or its variety and the purpose of administering VdAL or the biological preparation (such as promoting seed germination or increasing biological yield, etc.) The concentration of the biological agent.
  • the liquid obtained by diluting the biological preparation by 7500 times can promote the germination of the cotton; the liquid obtained by diluting the biological preparation by 3000-5000 times can increase the pre-frost flower yield and total yield of the cotton and per mu.
  • the liquid obtained by diluting the biological preparation by 2000 times can promote the watermelon result and increase the watermelon yield.
  • the liquid obtained by diluting the biological preparation by 5000-10000 times can promote the germination of the wheat seed; the liquid obtained by diluting the biological preparation by 5000-20000 times can increase the yield of the wheat.
  • the liquid obtained by diluting the biological preparation by 1000 times can promote the tomato result and increase the tomato yield.
  • the liquid obtained by diluting the biological preparation by 2000-4000 times can increase the yield of the green stem.
  • the liquid obtained by diluting the biological preparation by 300-2000 times can increase the fruit setting rate of the cucumber, promote the expansion of the cucumber, increase the yield of the cucumber, promote the growth of the top of the cucumber plant, and delay the senescence of the cucumber plant.
  • the liquid obtained by diluting the biological preparation by 2000 times can increase the fruit setting rate of the virgin fruit.
  • the liquid obtained by diluting the biological preparation by 2000 times can promote the growth of strawberry and melon plants.
  • the liquid obtained by diluting the biological preparation 1000 times can promote the growth of the sweet pepper plant.
  • the liquid obtained by diluting the biological preparation by 3000-5000 times can promote the emergence and growth of soybean.
  • the liquid obtained by diluting the biological preparation by 10,000 to 20,000 times can increase the yield of rice.
  • the liquid obtained by diluting the biological preparation by 3000 times can promote the ripening of the grapes.
  • the liquid obtained by diluting the biological preparation by 5000 times can promote the ripening of the cherry and the growth of the radish.
  • the liquid obtained by diluting the biological preparation by 6000 times can promote the growth of spinach.
  • the second technical problem to be solved by the present invention is how to improve the quality of plant products.
  • the plant product is a product required by humans produced by human cultivated plants.
  • the evaluation criteria for the quality vary depending on the use of the product.
  • the quality of the product used as food can be mentioned in terms of the nutritional quality and the eating quality of the product; the quality of the product used as the raw material for the clothing can be mentioned in the fiber quality of the product.
  • To evaluate the quality of products generally use two indicators, one is the content of biochemical components and harmful substances, such as nutrient content and trace element content; the second is physical indicators, such as product shape, size, taste, aroma, color, seed coat thickness , uniformity, fiber length, fiber strength, etc., such as cotton clothing.
  • the present invention first provides the use of proteins in improving the quality of plant products.
  • VdAL In the application of the protein provided by the invention in improving the quality of a plant product, the protein is named VdAL and is as follows A1) or A2) or A3):
  • amino acid sequence is the protein of sequence 1;
  • A2 a protein derived from A1) having the same function after substitution and/or deletion and/or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO:1;
  • A3 A fusion protein obtained by ligating the N-terminus or/and C-terminus of A1) or A2).
  • sequence 1 consists of 297 amino acids.
  • a label as shown in Table 1 may be attached to the amino terminus or carboxy terminus of the protein shown in SEQ ID NO: 1 in the Sequence Listing.
  • the VdAL in the above A2) can be artificially synthesized, or the coding gene can be synthesized first, and then obtained by biological expression.
  • the gene encoding the VdAL in the above A2) can be obtained by deleting a codon of one or several amino acid residues in the DNA sequence shown in the sequence 2 in the sequence listing, and/or performing one or several base pair missense mutations. And/or the coding sequence of the tag shown in Table 1 is attached at its 5' end and/or 3' end.
  • the improving the quality of the plant product may be to increase the content of the nutrient component of the plant product and/or increase the content of trace elements in the plant product and/or increase the plant product.
  • the nutrient component of the plant product may specifically be vitamin C, soluble sugar and/or protein.
  • the trace elements may specifically be Cu, Fe, K, Mn and/or Zn.
  • the palatability can be manifested in a decrease in dry matter rate and/or crude fiber content, or in the brittleness of the edible portion of the plant.
  • the improving the quality of the plant product may improve the quality of the plant product by promoting the ripening of the plant product.
  • the application of VdAL promotes the maturation of the cotton product, and the cotton pre-frost flower yield is increased, thereby improving the quality of the cotton.
  • the plant may be a dicot or a monocot.
  • the dicot may be a), b), c), d), e), f), g) or h):
  • the present invention also provides an application of a biological material related to VdAL in improving the quality of a plant product.
  • VdAL-related biological material provided by the present invention for improving the quality of a plant product
  • the biological material is any one of the following B1) to B20):
  • B2 an expression cassette comprising the nucleic acid molecule of B1);
  • B3 a recombinant vector comprising the nucleic acid molecule of B1);
  • B4 a recombinant vector comprising the expression cassette of B2)
  • B5 a recombinant microorganism comprising the nucleic acid molecule of B1);
  • B7 a recombinant microorganism comprising the recombinant vector of B3);
  • B9 a transgenic plant cell line comprising said nucleic acid molecule of B1);
  • B11 a transgenic plant cell line comprising the recombinant vector of B3);
  • B12 a transgenic plant cell line comprising the recombinant vector of B4)
  • B13 a transgenic plant tissue comprising the nucleic acid molecule of B1);
  • B14 a transgenic plant tissue comprising the expression cassette of B2)
  • B15 a transgenic plant tissue comprising the recombinant vector of B3);
  • B16 a transgenic plant tissue comprising the recombinant vector of B4)
  • B17 a transgenic plant organ comprising said nucleic acid molecule of B1);
  • B18 a transgenic plant organ comprising the expression cassette of B2)
  • B19 a transgenic plant organ comprising the recombinant vector of B3);
  • B20 A transgenic plant organ comprising the recombinant vector of B4).
  • the nucleic acid molecule of B1) may be a gene of b1) or b2) or b3) as follows:
  • nucleotide sequence is a cDNA molecule or a DNA molecule of SEQ ID NO: 2 in the Sequence Listing;
  • B2 having 75% or more of the identity of the nucleotide sequence defined by b1), and a cDNA molecule or genomic DNA molecule encoding VdAL;
  • B3 a cDNA molecule or genomic DNA molecule which hybridizes under stringent conditions to a nucleotide sequence defined by b1) and which encodes VdAL.
  • the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.
  • sequence 2 consists of 894 nucleotides and encodes the protein shown in SEQ ID NO: 1.
  • nucleotide sequence encoding a VdAL of the present invention can readily mutate a nucleotide sequence encoding a VdAL of the present invention using known methods, such as directed evolution and point mutation.
  • Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of VdAL isolated from the present invention are nucleotide sequences derived from the present invention as long as they encode VdAL and have VdAL function. And equivalent to the sequence of the invention.
  • identity refers to sequence similarity to a native nucleic acid sequence. “Identity” includes 75% or more, or 85% or more, or 90% or more, or 95% or more of the nucleotide sequence of a protein consisting of the amino acid sequence of the present invention encoding VdAL. Highly identical nucleotide sequence. Identity can be evaluated using the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.
  • the stringent condition is that the solution is hybridized and washed twice at a temperature of 68 ° C in a solution of 2 ⁇ SSC, 0.1% SDS for 5 minutes each time. Hybridization and washing at 68 ° C for 2 times in a solution of 0.5 ⁇ SSC, 0.1% SDS for 15 min; or 0.1 ⁇ SSPE (or 0.1 ⁇ SSC), 0.1% SDS solution at 65 ° C Hybridize and wash the membrane.
  • the above 75% or more of the identity may be 80%, 85%, 90% or 95% or more.
  • the expression cassette (VdAL gene expression cassette) containing the nucleic acid molecule encoding VdAL described in B2) refers to a DNA capable of expressing VdAL in a host cell,
  • the DNA may include not only a promoter that initiates transcription of the VdAL gene, but also a terminator that terminates transcription of the VdAL gene.
  • the expression cassette may further comprise an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development specific promoters, and inducible promoters.
  • promoters include, but are not limited to, constitutive promoter of cauliflower mosaic virus 35S: a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester); tomatoes Protease inhibitor II promoter (PIN2) or LAP promoter (both induced by methyl jasmonate); heat shock promoter (U.S. Patent 5,187,267); tetracycline-inducible promoter (U.S.
  • Patent 5,057,422 seed-specific promoter
  • the millet seed-specific promoter pF128 CN101063139B (Chinese Patent 200710099169.7)
  • the seed storage protein-specific promoter for example, the promoters of Bean globulin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO) J.4: 3047-3053). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety.
  • Suitable transcription terminators include, but are not limited to, Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine Synthase terminator (see, for example, Odell et al. (I 985 ) Nature 313: 810; Rosenberg et al. (1987) Gene, 56: 125; Guerineau et al. (1991) Mol. Gen. Genet, 262: 141; (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5: 141; Mogen et al.
  • NOS terminator Agrobacterium nopaline synthase terminator
  • cauliflower mosaic virus CaMV 35S terminator tml terminator
  • pea rbcS E9 terminator nopaline and octopine Synthase terminator
  • a recombinant vector containing the VdAL gene expression cassette can be constructed using an existing expression vector.
  • the plant expression vector includes a dual Agrobacterium vector and a plant expression vector which can be used for microprojectile bombardment and the like.
  • the plant expression vector may further comprise a 3' untranslated region of the foreign gene, ie, comprising a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression.
  • the polyadenylation signal can direct polyadenylation to the 3' end of the mRNA precursor, such as Agrobacterium tumefaciens-induced (Ti) plasmid genes (such as nopaline synthase gene Nos), plant genes (such as soybean).
  • the untranslated regions transcribed at the 3' end of the storage protein gene all have similar functions.
  • an enhancer including a translation enhancer or a transcription enhancer, may be used, and these enhancer regions may be an ATG start codon or a contiguous region start codon, etc., but are required to be encoded.
  • the reading frames of the sequence are identical to ensure proper translation of the entire sequence.
  • the sources of the translational control signals and initiation codons are broad and may be natural or synthetic.
  • the translation initiation region can be from a transcription initiation region or a structural gene.
  • the plant expression vector used can be processed, such as a gene encoding a color-changing enzyme or luminescent compound (GUS gene, luciferase) which can be expressed in plants.
  • a marker gene for antibiotics such as the nptII gene conferring resistance to kanamycin and related antibiotics, conferring The herbicide phosphinothricin-resistant bar gene, the hph gene conferring resistance to the antibiotic hygromycin, and the dhfr gene conferring resistance to methotrexate, conferring glyphosate-resistant EPSPS gene) or chemical resistance
  • a marker gene or the like such as a herbicide-resistant gene
  • a mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose. From the safety of transgenic plants, the transformed plants can be directly screened by adversity without any selectable marker genes.
  • the vector may be a plasmid, a cosmid, a phage or a viral vector.
  • the plasmid may be the vector pET42a(+).
  • the microorganism may be a yeast, a bacterium, an alga or a fungus such as Escherichia coli.
  • the E. coli may be Escherichia coli JM109.
  • the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ may neither include the propagation material nor the propagation material.
  • the transgenic plant organ may also be a seed of the transgenic plant.
  • the transgenic plants can include seeds, callus, whole plants, and cells.
  • the coding gene of VdAL is introduced into Escherichia coli JM109 by a recombinant vector containing an expression cassette for the gene encoding VdAL to obtain a recombinant microorganism.
  • the recombinant vector is obtained by replacing the sequence between the NdeI and KpnI recognition sites of the vector pET42a(+) with the DNA molecule represented by nucleotides 1-894 of the sequence 2 in the sequence table (ie, the VdAL gene).
  • Recombinant vector expresses VdAL shown in SEQ ID NO: 1.
  • the recombinant microorganism expresses VdAL shown in SEQ ID NO: 1.
  • the improving the quality of the plant product may be to increase the content of the nutrient component of the plant product and/or increase the content of trace elements in the plant product and/or Improve the palatability of the plant product.
  • the nutrient component of the plant product may specifically be vitamin C, soluble sugar and/or protein.
  • the trace elements may specifically be Cu, Fe, K, Mn and/or Zn. The palatability can be manifested in a decrease in the dry matter rate and/or the content of the crude fiber.
  • the improving the quality of the plant product may improve the quality of the plant product by promoting the ripening of the plant product.
  • the application of VdAL promotes the maturation of the cotton product, and the cotton pre-frost flower yield is increased, thereby improving the quality of the cotton.
  • the plant may be a dicot or a monocot.
  • the dicot may be a), b), c), d), e), f), g) or h):
  • the present invention also provides a method for improving the quality of a plant product.
  • the method for improving the quality of a plant product provided by the invention comprises applying VdAL to a plant of interest A biological material or biological preparation for improving the quality of the plant product of interest; the active ingredient of the biological preparation is VdAL or the biological material.
  • the biological preparation can be prepared according to the method 1), wherein the recombinant microorganism is cultured to express a gene encoding the VdAL to obtain the biological preparation.
  • the method 1) may specifically be as follows 11) and 12):
  • the method 1) may further comprise drying the biological preparation.
  • the coding gene may be the nucleic acid molecule of B1).
  • the recombinant microorganism is a recombinant microorganism obtained by introducing a recombinant vector containing an expression cassette of a gene encoding a VdAL into Escherichia coli JM109.
  • the recombinant vector is obtained by replacing the sequence between the NdeI and KpnI recognition sites of the vector pET42a(+) with the DNA molecule represented by nucleotides 1-894 of the sequence 2 in the sequence table (ie, the VdAL gene).
  • Recombinant vector expresses VdAL shown in SEQ ID NO: 1.
  • the recombinant microorganism expresses VdAL shown in SEQ ID NO: 1.
  • the applying the VdAL to the plant of interest, the biological material or the biological preparation may specifically be applying VdAL, the biological material or the biological preparation to the leaf of the plant of interest, such as a plant of interest.
  • Spraying VdAL, the biological material or the biological preparation, the seed of the plant of interest may also be treated with VdAL, the biological material or the biological preparation, such as a liquid using VdAL, the biological material or the biological preparation
  • the preparation is soaked in seeds of the plant of interest.
  • the liquid preparation of VdAL, the biological material or the biological preparation may be a liquid obtained by dissolving or suspending VdAL, the biological material or the biological preparation directly with water.
  • the amount of VdAL, the biological material or biological agent applied to the plant of interest may be determined according to the species and/or growth period of the plant of interest.
  • the improving the quality of the plant product may be to increase the content of the nutrient component of the plant product and/or increase the content of trace elements in the plant product and/or improve the palatability of the plant product.
  • the nutrient component of the plant product may specifically be vitamin C, soluble sugar and/or protein.
  • the trace elements may specifically be Cu, Fe, K, Mn and/or Zn. The palatability can be manifested in a decrease in the dry matter rate and/or the content of the crude fiber.
  • the improving the quality of the plant product may improve the quality of the plant product by promoting the ripening of the plant product.
  • the application of VdAL promotes the maturation of the cotton product, and the cotton pre-frost flower yield is increased, thereby improving the quality of the cotton.
  • the plant may be a dicot or a monocot.
  • the dicot may be a), b), c), d), e), f), g) or h):
  • the present invention also provides the use of the biological preparation for improving the quality of a plant product.
  • the improving the quality of the plant product may be to increase the content of the nutrient component of the plant product and/or increase the content of trace elements in the plant product and/or increase the plant.
  • the palatability of the product may specifically be vitamin C, soluble sugar and/or protein.
  • the trace elements may specifically be Cu, Fe, K, Mn and/or Zn.
  • the palatability can be manifested in a decrease in dry matter rate and/or crude fiber content, or in the brittleness of the edible portion of the plant.
  • the improving the quality of the plant product may improve the quality of the plant product by promoting the ripening of the plant product.
  • the application of VdAL promotes the maturation of the cotton product, and the cotton pre-frost flower yield is increased, thereby improving the quality of the cotton.
  • the plant may be a dicot or a monocot.
  • the dicot may be a), b), c), d), e), f), g) or h):
  • VdAL or the biological agent may result in different results depending on the concentration of VdAL or the biological agent.
  • the concentration of VdAL or the biological preparation may adjust VdAL according to the crop or its variety and the purpose of administering VdAL or the biological preparation (such as increasing the content of nutrients in the plant product of interest) Or the concentration of the biological agent.
  • the liquid obtained by diluting the biological preparation by 2000 times can increase the content of nutrients and trace elements in the watermelon.
  • the liquid obtained by diluting the biological preparation by 3000-5000 times can increase the clothing fraction of the cotton.
  • the liquid obtained by diluting the biological preparation by 3000 times can increase the vitamin C content of the grape.
  • the liquid obtained by diluting the biological preparation by 5000 times can increase the brittleness and sweetness of the canned radish.
  • the radish may be a pot of radish.
  • a third technical problem to be solved by the present invention is how to improve the drought resistance of plants or to promote the coloration of plant fruits.
  • the present invention first provides the use of proteins in regulating plant drought resistance or regulating plant fruit coloration.
  • VdAL which is as follows: A1) or A2) or A3):
  • the protein is as follows A1) or A2) or A3):
  • amino acid sequence is the protein of sequence 1;
  • A2 a protein derived from A1) having the same function after substitution and/or deletion and/or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NO:1;
  • A3 A fusion protein obtained by ligating the N-terminus or/and C-terminus of A1) or A2).
  • sequence 1 consists of 297 amino acids.
  • a label as shown in Table 1 may be attached to the amino terminus or carboxy terminus of the protein shown in SEQ ID NO: 1 in the Sequence Listing.
  • the VdAL in the above A2) can be artificially synthesized, or the coding gene can be synthesized first, and then obtained by biological expression.
  • the gene encoding the VdAL in the above A2) can be obtained by deleting a codon of one or several amino acid residues in the DNA sequence shown in the sequence 2 in the sequence listing, and/or performing one or several base pair missense mutations. And/or the coding sequence of the tag shown in Table 1 is attached at its 5' end and/or 3' end.
  • VdAL may be a dicot or a monocot in the application of controlling drought resistance of a plant or regulating the coloration of a plant fruit.
  • the present invention also provides an application of a biological material related to VdAL in regulating drought resistance of plants or regulating coloration of plant fruits.
  • VdAL-related biological material provided by the present invention for regulating drought resistance of plants or regulating coloration of plant fruits
  • the biological material is any one of the following B1) to B20):
  • B2 an expression cassette comprising the nucleic acid molecule of B1);
  • B3 a recombinant vector comprising the nucleic acid molecule of B1);
  • B4 a recombinant vector comprising the expression cassette of B2)
  • B5 a recombinant microorganism comprising the nucleic acid molecule of B1);
  • B7 a recombinant microorganism comprising the recombinant vector of B3);
  • B9 a transgenic plant cell line comprising said nucleic acid molecule of B1);
  • B11 a transgenic plant cell line comprising the recombinant vector of B3);
  • B12 a transgenic plant cell line comprising the recombinant vector of B4)
  • B13 a transgenic plant tissue comprising the nucleic acid molecule of B1);
  • B14 a transgenic plant tissue comprising the expression cassette of B2)
  • B15 a transgenic plant tissue comprising the recombinant vector of B3);
  • B16 a transgenic plant tissue comprising the recombinant vector of B4)
  • B17 a transgenic plant organ comprising said nucleic acid molecule of B1);
  • B18 a transgenic plant organ comprising the expression cassette of B2)
  • B19 a transgenic plant organ comprising the recombinant vector of B3);
  • B20 A transgenic plant organ comprising the recombinant vector of B4).
  • the nucleic acid molecule is a gene of b1) or b2) or b3):
  • nucleotide sequence is a cDNA molecule or a DNA molecule of SEQ ID NO: 2 in the Sequence Listing;
  • B2 having 75% or more of the identity of the nucleotide sequence defined by b1), and a cDNA molecule or genomic DNA molecule encoding VdAL;
  • B3 a cDNA molecule or genomic DNA molecule which hybridizes under stringent conditions to a nucleotide sequence defined by b1) and which encodes VdAL.
  • the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.
  • sequence 2 consists of 894 nucleotides and encodes the protein shown in SEQ ID NO: 1.
  • nucleotide sequence encoding a VdAL of the present invention can readily mutate a nucleotide sequence encoding a VdAL of the present invention using known methods, such as directed evolution and point mutation.
  • Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of VdAL isolated from the present invention are nucleotide sequences derived from the present invention as long as they encode VdAL and have VdAL function. And equivalent to the sequence of the invention.
  • identity refers to sequence similarity to a native nucleic acid sequence. “Identity” includes 75% or more, or 85% or more, or 90% or more, or 95% or more of the nucleotide sequence of a protein consisting of the amino acid sequence of the present invention encoding VdAL. Highly identical nucleotide sequence. Identity can be evaluated using the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed in percentage (%), which can be used to evaluate the identity between related sequences.
  • the above-mentioned biological materials related to VdAL are used in the regulation of drought resistance of plants or the regulation of plant fruit coloration, and the stringent conditions are: hybridization and washing of the membrane at 68 ° C for 2 times in a solution of 2 ⁇ SSC, 0.1% SDS, Each time 5 min, in a solution of 0.5 ⁇ SSC, 0.1% SDS, hybridize at 68 ° C and wash the membrane twice, each time 15 min; or, 0.1 ⁇ SSPE (or 0.1 ⁇ SSC), 0.1% SDS solution Hybridization and washing at 65 °C.
  • the above 75% or more of the identity may be 80%, 85%, 90% or 95% or more.
  • the expression cassette (VdAL gene expression cassette) containing the nucleic acid molecule encoding VdAL described in B2) means capable of being in a host cell.
  • a DNA expressing VdAL which may include not only a promoter that initiates transcription of the VdAL gene, but also a terminator that terminates transcription of the VdAL gene.
  • the expression cassette may further comprise an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters, tissue, organ and development specific promoters, and inducible promoters.
  • promoters include, but are not limited to, constitutive promoter of cauliflower mosaic virus 35S: a wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester); tomatoes Protease inhibitor II promoter (PIN2) or LAP promoter (both induced by methyl jasmonate); heat shock promoter (U.S. Patent 5,187,267); tetracycline-inducible promoter (U.S.
  • Patent 5,057,422 seed-specific promoter
  • the millet seed-specific promoter pF128 CN101063139B (Chinese Patent 200710099169.7)
  • the seed storage protein-specific promoter for example, the promoters of Bean globulin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO) J.4: 3047-3053). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety.
  • Suitable transcription terminators include, but are not limited to, Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine Synthase terminator (see, for example, Odell et al. (I 985 ) Nature 313: 810; Rosenberg et al. (1987) Gene, 56: 125; Guerineau et al. (1991) Mol. Gen. Genet, 262: 141; 1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5: 141; Mogen et al.
  • NOS terminator Agrobacterium nopaline synthase terminator
  • CaMV 35S terminator cauliflower mosaic virus CaMV 35S terminator
  • tml terminator tml terminator
  • pea rbcS E9 terminator nopaline and octopine Synth
  • a recombinant vector containing the VdAL gene expression cassette can be constructed using an existing expression vector.
  • the plant expression vector includes a dual Agrobacterium vector and a plant expression vector which can be used for microprojectile bombardment and the like.
  • the plant expression vector may further comprise a 3' untranslated region of the foreign gene, ie, comprising a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression.
  • the polyadenylation signal can direct polyadenylation to the 3' end of the mRNA precursor, such as Agrobacterium tumefaciens-induced (Ti) plasmid genes (such as nopaline synthase gene Nos), plant genes (such as soybean).
  • the untranslated regions transcribed at the 3' end of the storage protein gene all have similar functions.
  • an enhancer including a translation enhancer or a transcription enhancer, may be used, and these enhancer regions may be an ATG start codon or a contiguous region start codon, etc., but are required to be encoded.
  • the reading frames of the sequence are identical to ensure proper translation of the entire sequence.
  • the sources of the translational control signals and initiation codons are broad and may be natural or synthetic.
  • the translation initiation region can be from a transcription initiation region or a structural gene.
  • the plant expression vector used can be processed, such as a gene encoding a color-changing enzyme or luminescent compound (GUS gene, luciferase) which can be expressed in plants.
  • marker genes for antibiotics such as the nptII gene conferring resistance to kanamycin and related antibiotics, the bar gene conferring resistance to the herbicide phosphinothricin, and the hph gene conferring antibiotic resistance to hygromycin
  • the dhfr gene that confers resistance to methotrexate, confers glyphosate-resistant EPSPS gene) or chemical-resistant marker gene (such as anti-tuberant gene) and provides mannose-6-capable mannose-capable mannose-- Phosphoisomerase gene. From the safety of transgenic plants, the transformed plants can be directly screened by adversity without any selectable marker genes.
  • the above-mentioned biological material related to VdAL may be a plasmid, a cosmid, a phage or a viral vector in the application of regulating drought resistance of a plant or regulating the coloration of a plant fruit.
  • the plasmid may be the vector pET42a(+).
  • the above-mentioned biomaterials related to VdAL may be yeast, bacteria, algae or fungi, such as Escherichia coli, in the application of controlling drought resistance of plants or regulating the coloration of plant fruits.
  • the E. coli can For E. coli JM109.
  • the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ may neither include the propagation material nor the propagation material.
  • the above-mentioned VdAL-related biological material may be a seed of a transgenic plant in the application of regulating plant drought resistance or regulating plant fruit coloration.
  • the transgenic plants can include seeds, callus, whole plants, and cells.
  • the coding gene of VdAL is introduced into Escherichia coli JM109 by a recombinant vector containing an expression cassette for the gene encoding VdAL to obtain a recombinant microorganism.
  • the recombinant vector is obtained by replacing the sequence between the NdeI and KpnI recognition sites of the vector pET42a(+) with the DNA molecule represented by nucleotides 1-894 of the sequence 2 in the sequence table (ie, the VdAL gene).
  • Recombinant vector expresses VdAL shown in SEQ ID NO: 1.
  • the recombinant microorganism expresses VdAL shown in SEQ ID NO: 1.
  • the above-mentioned biological material related to VdAL is used in the regulation of drought resistance of plants or the regulation of coloration of plant fruits, which are monocotyledons or dicotyledons.
  • the present invention also provides a method for improving drought resistance of a plant or promoting coloration of a plant fruit.
  • the invention provides a method for improving drought resistance of a plant or promoting coloration of a plant fruit, comprising applying VdAL, the biological material or a biological preparation to a plant of interest to improve the quality of the plant product of interest; the active ingredient of the biological agent is VdAL or the biological material.
  • the biological preparation can be prepared according to the method 1), wherein the method 1) is: cultivating the recombinant microorganism, expressing a gene encoding VdAL, and obtaining the biological preparation. .
  • the method 1) may specifically be as follows 11) and 12):
  • the method 1) may further comprise drying the biological preparation.
  • the coding gene may be the nucleic acid molecule of B1).
  • the recombinant microorganism is a recombinant microorganism obtained by introducing a recombinant vector containing an expression cassette of a gene encoding a VdAL into Escherichia coli JM109.
  • the recombinant vector is obtained by replacing the sequence between the NdeI and KpnI recognition sites of the vector pET42a(+) with the DNA molecule represented by nucleotides 1-894 of the sequence 2 in the sequence table (ie, the VdAL gene).
  • Recombinant vector expresses VdAL shown in SEQ ID NO: 1.
  • the recombinant microorganism expresses VdAL shown in SEQ ID NO: 1.
  • the VdAL, the biological material or the biological preparation may be a VdAL, the biological material or the biological preparation applied to the leaf of the plant of interest, such as spraying VdAL, the biological material or the biological preparation on the plant of interest
  • the seed of the plant of interest may be treated with VdAL, the biological material or the biological agent, such as seeding the seed of the plant of interest with a liquid formulation of VdAL, the biological material or the biological agent.
  • the liquid preparation of VdAL, the biological material or the biological preparation may be a liquid obtained by dissolving or suspending VdAL, the biological material or the biological preparation directly with water.
  • the amount of VdAL, the biological material or biological agent applied to the plant of interest may be determined according to the species and/or growth period of the plant of interest.
  • the plant is a monocot or a dicot.
  • the present invention also provides the use of the biological preparation for improving drought resistance of a plant or promoting fruit coloration.
  • the plant is a monocot or a dicot.
  • VdAL or the biological agent on different plants above may result in different results depending on the concentration of VdAL or the biological agent.
  • concentration of VdAL or the biological agent at the time of administration of VdAL or the biological preparation may adjust the concentration of VdAL or the biological preparation depending on the crop or its variety and the purpose of administering VdAL or the biological preparation.
  • the biological preparation can increase the drought resistance of the wheat at a mass percentage of 0.2 to 4%.
  • the liquid obtained by diluting the biological preparation by 3000-5000 times can promote the coloration of cherries or grapes.
  • Figure 1 shows the results of the Vidlian appearance and the western-blot test of VdAL in Vidalian.
  • A is the appearance of Vadalian
  • B is the western-blot test result of VdAL in Vadalian
  • the fan shows the fine powder entering the air outlet part during the drying process of the sample
  • the main tower shows the normal powder in the drying process of the sample.
  • FIG. 2 shows that Vadalian can promote the vegetative growth of watermelon.
  • CK represents untreated watermelon
  • VdAL represents Vadalian treated watermelon.
  • FIG. 3 shows that Vadalian can promote cucumber fruit and fruit enlargement.
  • CK represents untreated cucumber
  • 300 represents diluted 300-fold VdAL-treated cucumber
  • 600 represents 600-fold diluted VdAL-treated cucumber
  • 900 represents 900-fold diluted VdAL-treated cucumber.
  • Figure 4 shows the growth of cucumbers treated differently. Among them, CK represents untreated cucumber, and 300 represents 300 times diluted VdAL-treated cucumber.
  • Figure 5 shows the senescence of cucumbers treated differently. Among them, CK represents untreated cucumber, and 2000 ⁇ represents 2000 times diluted VdAL-treated cucumber.
  • Figure 6 shows the fruit setting of the differently processed cherries.
  • CK represents the untreated virgin fruit
  • 2000 ⁇ represents a VdAL-treated virgin fruit diluted 2000 times.
  • Figure 7 shows differently treated strawberries. Among them, CK represents untreated strawberries, and 2000 ⁇ represents 2000 times diluted VdAL-treated strawberries.
  • Figure 8 shows different processed melons. Among them, CK represents untreated melon, and 2000 ⁇ represents 2000 times diluted VdAL-treated melon.
  • Figure 9 shows differently treated sweet peppers. Among them, CK represents untreated sweet pepper, and 1000 ⁇ represents 1000 times diluted VdAL-treated sweet pepper.
  • Figure 10 shows the germination of differently treated soybean seeds.
  • CK represents untreated soybean seeds
  • 1000 represents 1000 times diluted VdAL-treated soybean seeds
  • 3000 represents diluted 3000 times of VdAL-treated soybean seeds
  • 5000 represents diluted 5000 times of VdAL-treated soybean seeds
  • 10000 represents dilution of 10,000 seeds.
  • the doubling of VdAL treated soybean seeds, 20000, represents 20,000 times diluted VdAL treated soybean seeds.
  • Figure 11 shows the growth status of soybean seedlings treated differently. Among them, CK represents untreated soybean seedlings, and 5000 ⁇ represents 5000 times diluted VdAL-treated soybean seedlings.
  • Figure 12 shows the yield per mu of Vidalantan after treatment of rice.
  • Figure 13 shows the growth status of wheat in each group on the 7th day of water control.
  • Figure 14 shows the chlorophyll content in each group of wheat leaves.
  • Figure 15 shows the rehydration index of each group of wheat.
  • Figure 16 shows two differently treated grapes.
  • Figure 17 shows two differently treated cherries.
  • Figure 18 is the appearance of two treated canned radishes.
  • 1/300, 1/600, 1/900, 1/1000, 1/200, 1/250, 1/3000, 1/4000, 1/5000, 1/6000, 1/7500, 1/10000, 1/20000, 1/40000 times VdAL means 300 times diluted VdAL, diluted 600 times VdAL, diluted 900 times VdAL, diluted 1000 times VdAL, diluted 2000 times VdAL, diluted 2500 times VdAL, diluted 3000 VdAL multiple times, VdAL diluted 4000 times, VdAL diluted 5000 times, VdAL diluted 6000 times, VdAL diluted 7500 times, VdAL diluted 10000 times, VdAL diluted 20,000 times and VdAL diluted 40,000 times.
  • the Escherichia coli JM109 in the following examples is a product of Beijing Suo Laibao Technology Co., Ltd., and the catalog number is C1300.
  • the carrier pET42a(+) in the following examples is a product of Beijing Bio Orange Biotech Co., Ltd., catalog number is S18-16.
  • Xinluzao No. 58 is the product of Xinjiang Jinmian Seed Industry Technology Co., Ltd.
  • Lu 30, 616, Lu 25 and Luyan Cotton No. 24 are products of Shandong Nongxing Seed Industry Co., Ltd.
  • the watermelon variety Jinlidu in the following examples is the product of Shouguang Jishan Trading Co., Ltd.
  • the Zhongmai 816 in the following examples is the product of Beijing Longshengyuan Technology Development Co., Ltd.
  • the tomato variety Fukes in the following examples is the product of Shouguang Jinpeng Seed Co., Ltd.
  • the gorgeous green stalk F1 in the following examples is a product of Shanghai Hongqiao Tianlong Seed Industry Co., Ltd.
  • the cucumber variety Jinza No. 1 in the following examples is a product of Shandong Nongxing Seed Industry Co., Ltd.
  • the sacred fruit variety Jingdan 2 in the following examples is the product of Beijing Bei Nong Luheng Technology Development Co., Ltd.
  • the strawberry variety Fengxiang in the following examples is the product of Beijing Bei Nong Luheng Technology Development Co., Ltd.
  • the Super Cuibao sweet melon variety in the following examples is the product of Jinan Ruihao Agricultural Technology Development Co., Ltd.
  • the sweet pepper variety Ruiyou 816 in the following examples is the product of Jinan Ruihao Agricultural Technology Development Co., Ltd.
  • soybean variety Zhonghua Soybean in the following examples is the product of Jinan Ruihao Agricultural Technology Development Co., Ltd.
  • Nongda 502 in the following examples is a product of Beijing Bei Nong Luheng Technology Co., Ltd.
  • Vidalian was developed by China Agricultural University and developed and produced by Shandong Huimin Huhao Biotechnology Co., Ltd.
  • the recombinant vector pET42a-VdAL expresses the protein VdAL shown in SEQ ID NO: 1 in the Sequence Listing.
  • sequence 2 is composed of 894 nucleotides, and encodes the amino acid sequence shown in the sequence 1.
  • JM109-pET42a-VdAL was introduced into Escherichia coli JM109 to obtain a recombinant strain, and the obtained recombinant strain was named as JM109-pET42a-VdAL, and the protein represented by JM109-pET42a-VdAL expression sequence 1.
  • the JM109-pET42a-VdAL was fermented at 37 ° C to an OD value of 0.6 to obtain a pre-fermentation solution, and IPTG was added to the pre-fermentation solution to make the concentration of IPTG 1 mM to obtain an induction solution, and the induction solution was at 25 ° C.
  • the fermentation broth was obtained by fermentation for 6 hours. The fermentation broth was centrifuged, the supernatant was discarded, and the obtained bacterial body was crushed, and then spray-dried at 150 ° C to obtain a dry powder preparation (A in Fig. 1), which was named Vidalyan.
  • VdAL in Vadalian was detected by western-blot method.
  • the primary antibody was VdAL antibody (VdAL antibody is a polyclonal antibody prepared by the protein shown in sequence 2), and the results showed that Vadalian contained VdAL (B in Fig. 1), the content of VdAL in Vidalian was 7.28 mg/g.
  • the vitamins were dissolved in water to obtain Vidalian concentrations of 10 g/3 kg, 10 g/6 kg, 10 g/9 kg, 1 g/kg, 1 g/2 kg, 1 g/2.5 kg, 1 g/3 kg, 1 g/4 kg, 1 g, respectively.
  • these liquids are respectively named 300 times diluted VdAL, Diluted 600 times VdAL, diluted 900 times VdAL, diluted 1000 times VdAL, diluted 2000 times VdAL, diluted 2500 times VdAL, diluted 3000 times VdAL, diluted 4000 times VdAL, diluted 5000 times VdAL, diluted 6000 VdAL, diluted 7500 times VdAL, diluted 10000 times VdAL, diluted 20,000 times VdAL and diluted 40,000 times VdAL.
  • the field experiment design is as follows: The experiment adopts random block design, and sets three repeating zones. Each repeating zone is randomly set with nine processing zones, namely, Lud 30 VdAL Processing Zone 1, Lu 30 VdAL Processing Zone 2, Lu 30 Control treatment area, 616VdAL treatment 1 area, 616VdAL treatment 2 area, 616 control treatment area, Lu 25 VdAL treatment 1 area, Lu 25 VdAL treatment 2 area, and Lu 25 control treatment area, the area of each treatment area It is 0.2 mu.
  • the seedling stage and the flowering bell stage of Lu 30 in the VdAL treatment area 1 of Lu 30 were treated as follows: the top of the cotton spray was sprayed with the dilution of Example 1 by the top spray (mechanical spray). VdAL, spray 45-50kg per acre, and get 3000 times diluted VdAL treated Lu 30. Spraying once in the seedling stage and flowering bell; spraying time in the morning or evening, it is best to avoid the noon high temperature period; it is strictly prohibited to mix with drugs and fertilizers during spraying; if spraying within 2 hours after spraying, it needs to be sprayed Apply once.
  • VdAL treatment zone 1 According to the treatment method of Lud 30 VdAL treatment zone 1, the VdAL diluted 3000 times was replaced with VdAL diluted 5000 times, and the VdAL treatment zone 2 of Lu 30 was treated. The other steps were unchanged, and the VdAL treatment was diluted 5000 times. Lu 30.
  • the diluted 3000 times VdAL was replaced with Qingshui to treat the Lu 30 control treatment zone, and the other steps were unchanged, and the untreated Lu 30 was obtained.
  • VdAL treatment zone 1 According to the treatment method of No.30 VdAL treatment zone 1, the 616VdAL treatment zone 1 and the Lu 25 VdAL treatment zone 1 were treated separately, and the other steps were unchanged.
  • the VdAL treated 616 and Lu 25 were diluted 3000 times respectively.
  • VdAL diluted 3000 times was replaced with VdAL diluted 5000 times, and the 616VdAL treatment zone 2 and Lu 25 VdAL treatment zone 2 were treated separately, and the other steps were unchanged.
  • a 5,000-fold diluted VdAL-treated 616, Lu 25 was obtained.
  • VdAL treatment zone 1 According to the treatment method of No. 30 VdAL treatment zone 1, the diluted 3000 times VdAL was replaced with clean water, and the 616 control treatment zone and Lu 25 control treatment zone were respectively treated, and the other steps were unchanged, respectively, and untreated 616 was obtained respectively. Lu, No. 25.
  • VdAL treated Lu 30 diluted 5000 times VdAL treated Lu 30, untreated Lu 30, diluted 3000 times VdAL treated 616, diluted 5000 times VdAL treated 616, not counted separately Treated 616, diluted 3000 times VdAL treated Lu 25, diluted 5000 times VdAL treated Lu 25 and untreated Lu 25 before frost production, after frost yield and total yield, the results are shown in the table 2 is shown.
  • Process 1 represents a process of diluting 3000 times VdAL
  • Process 2 represents a process of diluting 5000 times VdAL
  • CK means untreated (ie, treatment with water).
  • the field experiment design is as follows: The experiment adopts a random block design, and three repeating zones are set, and four processing zones are randomly set in each repeating zone, which are VdAL processing zone 1, VdAL treatment zone 2, VdAL treatment zone 3 and control treatment zone, respectively. Each treatment area has an area of 10 acres.
  • the seedling stage and the flowering bell stage of Luyan Cotton No. 24 in the VdAL treatment area 1 were carried out as follows. Rationale: Spraying 3000d VdAL of Example 1 on cotton leaves by top spray (mechanical spraying), spraying 45-50kg per acre, and controlling the speed of locomotive operation at a speed of about 6 kilometers per hour, which is diluted 3000 times. Luyan Cotton No. 24 treated by VdAL. Spraying once in the seedling stage and flowering bell; spraying time in the morning or evening, it is best to avoid the noon high temperature period; it is strictly prohibited to mix with drugs and fertilizers during spraying; if spraying within 2 hours after spraying, it needs to be sprayed Apply once.
  • the VdAL diluted 3000 times was replaced with the VdAL diluted 5000 times, and the VdAL treatment zone 2 was treated, and the other steps were unchanged, and the VdAL-treated Luyan cotton No. 24 which was diluted 5000 times was obtained.
  • the VdAL diluted 3000 times was replaced with the VdAL diluted 10,000 times, and the VdAL treatment zone 3 was treated, and the other steps were unchanged, and the VdAL-treated Luyan cotton No. 24 diluted 10,000 times was obtained.
  • the diluted treatment 3,000 times of VdAL was replaced with clean water to treat the control treatment zone, and the other steps were unchanged, and the untreated Luyan cotton No. 24 was obtained.
  • Process 1 represents a treatment performed by diluting 3000 times VdAL
  • Process 2 represents a process of diluting 5000 times VdAL
  • Process 3 represents a process of diluting 10000 times VdAL
  • CK means untreated (ie, treatment with water) .
  • Process 1 represents a treatment performed by diluting 3000 times VdAL
  • Process 2 represents a process of diluting 5000 times VdAL
  • Process 3 represents a process of diluting 10000 times VdAL
  • CK means untreated (ie, treatment with water) .
  • VdAL diluted 5000 times When treated with VdAL diluted 5000 times, the number of plants per acre, single plant The number of bells, the total number of bolls, the weight of single bolls, the number of litters and the yield per mu increased, and the increase rates were 1.4%, 3.41%, 4.41%, 1.9%, 0.49%, and 5.22%, respectively; VdAL diluted 10,000 times.
  • the increase amount of each item was not increased by the treatment of VdAL diluted 3000 times and VdAL diluted by 5000 times. It shows that the application of the appropriate concentration of Vidalian can improve the quality and yield of cotton.
  • the field experiment design is as follows: The experiment adopts random block design, and sets three repeating zones. Each repeating zone is randomly set with two processing zones, namely VdLA processing zone and control processing zone, each of which has an area of 0.2 mu.
  • the watermelon variety Jinlidu in the VdLA treatment area was treated as follows: the watermelon leaf was sprayed with 2000 times of VdLA diluted in Example 1 by means of top spray (mechanical spraying), sprayed 30 kg per acre, and diluted. 2000 times VdAL treated watermelon. Spraying from the seedling stage of watermelon, and spraying it once after 15 days of the first spraying, a total of 2 sprays; spraying time in the morning or evening, preferably avoiding the noon high temperature period; spraying is best not Mix with drugs and fertilizers; if it rains within 2 hours after spraying, it needs to be sprayed once.
  • the diluted treatment 2000 times of VdAL was replaced with clean water to treat the control treatment zone, and the other steps were unchanged, and untreated watermelon was obtained.
  • the water content, dry matter rate, Vc content, soluble sugar content, titratable acid content, soluble solid content, melon skin thickness, protein content, crude fiber content (Table 5) and trace elements of different treated watermelons were determined at the maturity stage of watermelon.
  • the contents of Ca, Cu, Fe, K, Mg, Mn, Na, P and Zn (Table 6).
  • the water content and the dry matter rate (the percentage of dry matter in watermelon accounted for the fresh weight of watermelon) were determined after drying the watermelon, and the Vc content was determined by the 2,6-dichloroindophenol titration method.
  • the soluble sugar content was determined according to the oxime ratio. The color method is used for the determination.
  • the titratable acid content is determined by the colorimetric method.
  • the soluble solid content and protein content are determined by spectrophotometry.
  • the crude fiber content is measured by the Winder method.
  • the content of trace elements Ca, Cu, Fe, K, Mg, Mn, Na, P and Zn was measured by a trace element analyzer.
  • each trace element in lines 3, 5, 7, 9, 11, 13, 15, 17 and 19 refers to the mass of each trace element in fresh watermelon, 2, 4, 6, 8, 10, 12
  • the content of each trace element in lines 14, 16 and 18 refers to the content of each trace element in fresh watermelon.
  • Example 4 Vadalian can promote wheat growth and increase wheat yield
  • the seeds of Zhongmai 816 were separately soaked in 1000 times of VdAL diluted in Example 1, VdAL diluted 2000 times, VdAL diluted 5000 times, VdAL diluted 10,000 times and fresh water for 12 hours, and then laid in a germination box (germination box). There are two layers of sterile filter paper soaked in sterile water for 96 hours at 25 ° C. The germination rate of wheat seeds is counted. The germination rate of wheat seeds is: the seed buds are half or more than the length of the seeds, and the root length is Or more than the length of the seed). 100 seeds were treated for each treatment and the experiment was repeated three times.
  • the seeds of Zhongmai 816 were soaked in VdAL diluted 5,000 times, VdAL diluted 10000 times and water in Example 1 for 24 hours, respectively, and then sown in the soil, and the day of sowing was recorded as the first day of sowing, on the third day of sowing.
  • the wheat seedlings were taken out of the soil and the fresh weight of the wheat seedlings was weighed.
  • the field experiment design is as follows: The experiment adopts random block design, and sets three repeating zones. Each repeating zone is randomly set with five processing zones, namely VdAL processing zone 1, VdAL processing zone 2, VdAL processing zone 3, and VdAL processing zone 4. And the control treatment area, each treatment area has an area of 0.5 mu.
  • the wheat was treated once as follows: the wheat leaves were sprayed with the VdAL diluted by 2500 times in Example 1 by means of top spray (manual spray), per acre. 45-50 kg was sprayed to obtain 2,500-fold diluted VdAL-treated wheat. Spraying time in the morning or evening, it is best to avoid the noon high temperature period; it is strictly prohibited to mix with drugs and fertilizers during spraying; if it rains within 2 hours after spraying, it needs to be sprayed once.
  • the VdAL diluted 2500 times was replaced with the VdAL diluted 5000 times, and the VdAL treatment zone 2 was treated, and the other steps were unchanged, and the VdAL-treated wheat diluted 5000 times was obtained.
  • the VdAL diluted 2500 times was replaced with the VdAL diluted 10000 times, and the VdAL treatment zone 3 was treated, and the other steps were unchanged, and the VdAL-treated wheat diluted 10,000 times was obtained.
  • the VdAL diluted 2500 times was replaced with the VdAL diluted 20,000 times, and the VdAL treatment zone 4 was treated, and the other steps were unchanged, and the VdAL-treated wheat diluted 20,000 times was obtained.
  • the VdAL diluted 2500 times was replaced with clean water to treat the control treatment zone, and the other steps were unchanged, and untreated wheat was obtained.
  • the wheat yield of each treatment was counted. The results showed that the yield of wheat was not increased after 2,500 times dilution of VdAL, and the yield was 8679 kg/ha, diluted 5000 times of VdAL, diluted 10,000 times of VdAL, diluted 20,000 times of VdAL and treated with water. The average yield of wheat was 8942kg/ha, 9485kg/ha, 9231kg/ha and 8772kg/ha, respectively. After diluting 5000 times of VdAL, diluting 10,000 times of VdAL, and diluting 20,000 times of VdAL to treat wheat, the yield increased by 1.94%. , 8.13%, 5.23%.
  • the 1000-grain weight, the number of effective panicles, the number of panicles and the panicle length of the differently treated wheat were counted, and the results are shown in Table 7.
  • the results showed that after 5,000 times of VdAL treatment, the number of effective panicles increased by 1.5%, the number of panicles increased by 0.5%; the dilution of 10000 times of VdAL increased the number of effective panicles by 7%, and the number of panicles increased by 3.5%; Diluted 20,000 times VdAL, the effective panicle number increased by 2.3%, and the number of panicles increased by 3.7%.
  • Table 7 The average value of 1000-grain weight, effective panicle number, panicle number and panicle length of differently treated wheat
  • Process 1 represents a process of diluting 20,000 times VdAL
  • Process 2 represents a process of diluting 10,000 times VdAL
  • Process 3 represents a process of diluting 5000 times VdAL
  • Process 4 represents a process of diluting 2500 times VdAL.
  • CK indicates untreated (ie, treatment with clean water).
  • Example 5 Vadalian can increase the number of tomato plants per plant
  • the field experiment design is as follows: The experiment adopts random block design, and sets three repeating zones. Each repeating zone randomly sets three processing zones, which are VdAL processing zone 1, VdAL processing zone 2 and control treatment zone, and each treatment zone The area is 0.1 mu.
  • VdAL was sprayed in the early stage of flowering of the tomato variety F. in the VdAL treatment zone 1 and on the 15th day after the first treatment: Spraying the tomato leaves by top spray (manual spray)
  • Example 1 Dilute 1000 times VdAL, spray 45-50kg per acre to obtain 1000 times diluted VdAL-treated tomato, and record the first day of spraying as the 0th day after spraying. Spraying time in the morning or evening, it is best to avoid the noon high temperature period; it is strictly prohibited to mix with drugs and fertilizers during spraying; if it rains within 2 hours after spraying, it needs to be sprayed once.
  • the VdAL diluted 1000 times was replaced with the VdAL diluted 3000 times, and the VdAL treatment zone 2 was treated, and the other steps were unchanged, and the VdAL-treated tomato was diluted 3000 times.
  • the 1000-fold diluted VdAL was replaced with clean water to treat the control treatment zone, and the other steps were unchanged, and the untreated tomato was obtained.
  • the number of individual results of tomato was counted on the 20th day after spraying.
  • the results showed that the average number of single plants of tomato after dilution of 1000 times VdAL, 3000 times dilution of VdAL and water treatment of tomato were 34.9 plants/40.4, respectively.
  • /Strain and 33.3 plants/plants after 1000-fold dilution of VdAL and 3000-fold dilution of VdAL, the number of tomato plants increased by 4.8% and 21.3%, respectively. It shows that Vadalian can promote tomato results.
  • the field experiment design is as follows: The experiment adopts random block design, and sets three repeating zones. Each repeating zone is randomly set with five processing zones, namely VdAL processing zone 1, VdAL processing zone 2, VdAL processing zone 3, and VdAL processing zone 4. And the control treatment area, each treatment area has an area of 0.2 mu.
  • VdAL was sprayed once as follows: the tomato leaves were sprayed 2000 times by the top spray (manual spray) method. VdAL, spray 45-50kg per acre to obtain 2000 times diluted VdAL-treated green stalks, and record the day of Vadalian as the first day of treatment. Spraying time in the morning or evening, it is best to avoid the noon high temperature period; it is strictly prohibited to mix with drugs and fertilizers during spraying; if it rains within 2 hours after spraying, it needs to be sprayed once.
  • the VdAL diluted 2000 times was replaced with the VdAL diluted 2500 times, and the VdAL treatment zone 2 was treated, and the other steps were unchanged, and the VdAL-treated stalks were diluted 2500 times.
  • the VdAL diluted 2000 times was replaced with the VdAL diluted 3000 times, and the VdAL treatment zone 3 was treated, and the other steps were unchanged, and the VdAL-treated blue stem vegetables diluted 3000 times were obtained.
  • the VdAL diluted 2000 times was replaced with the VdAL diluted 4000 times, and the VdAL treatment zone 4 was treated, and the other steps were unchanged, and the VdAL-treated green stem vegetables diluted 4000 times were obtained.
  • the 2000d dilution of VdAL was replaced with clean water to treat the control treatment zone, and the other steps were unchanged, and the untreated green stem vegetables were obtained.
  • Vadalian can promote cucumber growth, increase yield, delay aging
  • the field experiment design is as follows: The experiment adopts random block design, and sets three repeating zones. Each repeating zone is randomly set with five processing zones, namely VdAL processing zone 1, VdAL processing zone 2, VdAL processing zone 3, and VdAL processing zone 4. And the control treatment area, each treatment area has an area of 0.1 mu.
  • the VdAL diluted 300 times was replaced with the VdAL diluted 600 times, and the VdAL treatment zone 2 was treated, and the other steps were unchanged, and the VdAL-treated cucumber diluted 600 times was obtained.
  • VdAL treatment zone 1 According to the treatment method of VdAL treatment zone 1, the VdAL diluted 300 times was replaced with VdAL diluted 900 times, and the VdAL treatment zone 3 was treated. The other steps were unchanged, and the VdAL was diluted 900 times. The cucumber.
  • the VdAL diluted 300 times was replaced with the VdAL diluted 2000 times, and the VdAL treatment zone 4 was treated, and the other steps were unchanged, and the VdAL-treated cucumber was diluted 2000 times.
  • the 300-fold diluted VdAL was replaced with clean water to treat the control treatment zone, and the other steps were unchanged, and the untreated cucumber was obtained.
  • Vadalian can increase the fruit setting rate and increase the yield of the virgin fruit.
  • the field experiment design is as follows: The experiment adopts random block design, and sets three repeating zones. Each repeating zone randomly sets two processing zones, which are VdAL processing zone and control processing zone, and each processing zone has an area of 1 mu.
  • the flowering period of the sacred fruit variety Jingdan 2 in the VdAL treatment area was sprayed once in the following manner by VdAL: the top dilution (manual spray) was applied to the leaves of the sacred fruit fruit by the dilution of Example 1 2000 times.
  • the VdAL was sprayed 45-50 kg per acre, and the VdAL-treated virgin fruit was diluted 2000 times, and the day of the Vadalian spray was recorded as the 0th day of treatment. Spraying time in the morning or evening, it is best to avoid the noon high temperature period; it is strictly prohibited to mix with drugs and fertilizers during spraying; if it rains within 2 hours after spraying, it needs to be sprayed once.
  • the diluted treatment 2000 times of VdAL was replaced with clean water to treat the control treatment zone, and the other steps were unchanged, and the untreated virgin fruit was obtained.
  • the field experiment design is as follows: the variety is Fengxiang, the experiment adopts random block design, and three repeating zones are set. Two processing zones are randomly set in each repeating zone, which are VdAL processing zone and control processing zone, and the area of each processing zone. Both are 1 mu.
  • the Fengxiang strawberry variety in the VdAL treatment area was sprayed once in the flowering stage according to the following method: sprayed on the strawberry leaves by the top spray (manual spray) method to dilute 2000 times of VdAL of Example 1 and spray per acre. 45-50 kg was applied, and a VdAL-treated strawberry diluted 2000 times was obtained, and the day of the Vadalian spray was recorded as the 0th day of the treatment. Spraying time in the morning or evening, it is best to avoid the noon high temperature period; it is strictly prohibited to mix with drugs and fertilizers during spraying; if it rains within 2 hours after spraying, it needs to be sprayed once.
  • VdAL treatment area replace the diluted 2000 times VdAL with clear water to the control area.
  • the area is treated and the other steps are unchanged to obtain untreated strawberries.
  • Vadalian can promote the growth of melon
  • the field experiment design is as follows: the melon variety is Super Cuibaoxiang.
  • the experiment adopts a random block design, and three repeating zones are set. Two processing zones are randomly set in each repeating zone, which are VdAL processing zone and control processing zone, respectively.
  • the area of the area is 1 mu.
  • spray VdAL once in the seedling stage according to the following method: spray the 2000d VdAL of the melon leaves by the top spray (manual spray) method. 45-50 kg per mu was sprayed, and a VdAL-treated melon diluted 2000 times was obtained, and the day of the Vadalian spray was recorded as the 0th day of the treatment. Spraying time in the morning or evening, it is best to avoid the noon high temperature period; it is strictly prohibited to mix with drugs and fertilizers during spraying; if it rains within 2 hours after spraying, it needs to be sprayed once.
  • the diluted treatment 2000 times of VdAL was replaced with clean water to treat the control treatment zone, and the other steps were unchanged, and untreated melon was obtained.
  • Vadalian can promote the growth of sweet pepper
  • the field experiment design is as follows: The experiment adopts random block design, and sets three repeating zones. Each repeating zone randomly sets two processing zones, which are VdAL processing zone and control processing zone, and each processing zone has an area of 1 mu.
  • the flowering period of the sweet pepper variety Ruiyou 816 in the VdAL treatment area was sprayed once in the following manner by VdAL: the sweet pepper leaves were sprayed with the VdAL diluted 1000 times in Example 1 by means of top spray (manual spray). Spray 45-50 kg per acre to obtain a VdAL-treated sweet pepper diluted 1000 times, and record the day of the Vadalian spray as the 0th day of treatment. Spraying time in the morning or evening, it is best to avoid the noon high temperature period; it is strictly prohibited to mix with drugs and fertilizers during spraying; if it rains within 2 hours after spraying, it needs to be sprayed once.
  • the 1000-fold diluted VdAL was replaced with clean water to treat the control treatment zone, and the other steps were unchanged, and the untreated sweet pepper was obtained.
  • Soybean variety Zhonghua Soybean seed was soaked in 1000 times of VdAL diluted in Example 1, VdAL diluted 3000 times, VdAL diluted 5000 times, VdAL diluted 10000 times, VdAL diluted 20,000 times and fresh water for 24 hours, then The germination rate of soybean seeds was counted by germination at 25 ° C for 72 hours in a tray (two layers of sterile filter paper soaked with sterile water). 100 seeds were treated for each treatment and the experiment was repeated three times.
  • Soybean variety Zhonghua Soybean seed was soaked in VdAL diluted with 5000 times of Example 1 and clear water for 24 hours, then sown in the soil, the day of sowing was recorded as the first day of sowing, and the soybean seedlings were taken from the 20th day of sowing. The soil was taken out and the fresh weight of soybean seedlings was weighed (Fig. 11).
  • Example 8 Vidalian can promote rice growth and increase rice yield
  • the field experiment design is as follows: The experiment adopts a random block design, and three repeating zones are set, and four processing zones are randomly set in each repeating zone, which are VdAL processing zone 1, VdAL treatment zone 2, VdAL treatment zone 3 and control treatment zone, respectively. Each treatment area has an area of 0.1 mu.
  • the rice leaf was sprayed with the 10000-fold diluted VdAL of the first embodiment by means of top spray (manual spray). 45-50 kg per mu was sprayed to obtain 10,000 times diluted VdAL-treated rice (1/10000). Spraying time in the morning or evening, it is best to avoid the noon high temperature period; it is strictly prohibited to mix with drugs and fertilizers during spraying; if it rains within 2 hours after spraying, it needs to be sprayed once.
  • VdAL treatment zone 1 According to the treatment method of VdAL treatment zone 1, the VdAL diluted by 10000 times was replaced with VdAL diluted by 20,000 times, and the VdAL treatment zone 2 was treated. The other steps were unchanged, and the diluted 20,000-fold VdAL-treated rice (1/20000) was obtained. .
  • VdAL treatment zone 1 According to the treatment method of VdAL treatment zone 1, the VdAL diluted by 10,000 times was replaced with VdAL diluted 40,000 times, and the VdAL treatment zone 3 was treated. The other steps were unchanged, and the diluted 40000 times of VdAL-treated rice (1/40000) was obtained. .
  • the 10000-fold diluted VdAL was replaced with clean water to treat the control treatment zone, and the other steps were unchanged, and untreated rice (CK) was obtained.
  • the Zhongmai 816 seeds were divided into 4 groups, namely group 1, group 2, group 3 and group 4, with 50 seeds per group.
  • Example 1 The fudali of Example 1 was added to Fuwu (Beinong (Haili) Luzhou Seed Coating Co., Ltd.) with a mass percentage concentration of 6% to obtain a liquid having a Vydarian mass percentage concentration of 2%, and the liquid was obtained. Named 2.0% + Fu Wu.
  • the group 1 wheat seeds were treated with 2.0% + Fufu as follows: 2.0% + Fufu 100-200 g, 1.5-2 kg of water, stirred evenly, mixed with 100 kg of seeds, thoroughly mixed and dried in the dry, and then sown. The wheat seeds of Group 1 were then sown in the soil, and at the three-leaf stage of the wheat, the wheat was stopped from watering (ie, water treatment).
  • the Vedaliment of Example 1 was added to a 6% by weight concentration of effluent to obtain a liquid having a Vadalian mass percent concentration of 1%, and the liquid was named 1.0% + fufu.
  • the group 2 wheat seeds were treated with 1.0% + Fufu as follows: 1.0% + Fufu 100-200g, mixed with water 1.5-2kg, evenly mixed, 100kg of seeded, thoroughly mixed and dried in the dry and then sown.
  • the wheat seeds of Group 2 were then sown in the soil, and at the three-leaf stage of the wheat, the wheat was stopped from watering (ie, water treatment).
  • the group 3 wheat seeds were treated with 1.0% + Fufu as follows: the mass percentage concentration of Beinong (Haili) Luzhou Seed Coating Co., Ltd. was 6% Fufu 100-200g, and the water was mixed 1.5-2kg evenly, mixed Plant 100kg, mix well and dry in the summer and sow. The wheat seeds of Group 3 were then sown in the soil, and at the three-leaf stage of the wheat, the wheat was stopped from watering (ie, water treatment).
  • Group 4 wheat seeds were treated with water as follows: as a control (CK): 100 kg of rice was mixed with 1.5-2 kg of water, thoroughly mixed, and then dried and then sown. The wheat seeds of Group 4 were then sown in the soil, and at the three-leaf stage of the wheat, the wheat was stopped from watering (ie, water treatment).
  • CK a control
  • 100 kg of rice was mixed with 1.5-2 kg of water, thoroughly mixed, and then dried and then sown.
  • the wheat seeds of Group 4 were then sown in the soil, and at the three-leaf stage of the wheat, the wheat was stopped from watering (ie, water treatment).
  • the first day when the watering was stopped was recorded as water control for 1 day.
  • the results showed that the treatment of wheat seeds with Vadalian added to the seed coating could significantly improve the drought tolerance of wheat: Group 1 wheat all died within 20 days of controlling water, and group 2 wheat all died within 15 days of water control, group 3 The wheat was all killed in the water control for 10 days, the group 4 wheat all died in the 12 days of water control, and the time of all the dead of the group 1 wheat was significantly greater than that of the group 3 and group 4, and the time of the group 2 all died. Significantly greater than the time when all of the groups 3 and 4 were dead.
  • the growth status of wheat in each group on the 7th day of water control is shown in Fig. 13.
  • the Zhongmai 816 seeds were divided into 6 groups, namely group 1, group 2, group 3, group 4, group 5 and group 6, with 50 seeds per group.
  • the wheat seeds of the group 1 were treated with water as follows: as a control (CK1): 100 kg of seed dressing was carried out with 1.5-2 kg of water, thoroughly mixed and dried, and then sown. The wheat seeds of Group 1 were then sown in the soil, and at the 3-leaf stage, the wheat was stopped from watering, and after the watering 7 was stopped, the wheat was rehydrated.
  • CK1 100 kg of seed dressing was carried out with 1.5-2 kg of water, thoroughly mixed and dried, and then sown.
  • the wheat seeds of Group 1 were then sown in the soil, and at the 3-leaf stage, the wheat was stopped from watering, and after the watering 7 was stopped, the wheat was rehydrated.
  • Example 1 The Vadalian of Example 1 was added to a 6% by weight concentration of effluent to obtain a liquid having a Vddalan mass percent concentration of 0.2%, and the liquid was named 0.2% + fufu.
  • Group 2 wheat seeds were treated with 0.2% + Fufu as follows: 0.2% + Fufu 100-200g, 1.5-2kg with water, stirred evenly, 100kg of seeded, thoroughly mixed and dried, and then sown. The wheat seeds of Group 2 were then sown in the soil, and at the 3-leaf stage, the wheat was stopped from watering, and after the watering 7 was stopped, the wheat was rehydrated.
  • the Vedaliment of Example 1 was added to a 6% by weight concentration of effluent to obtain a liquid having a Vadalian mass percent concentration of 1%, and the liquid was named 1.0% + fufu.
  • Use 1.0% + Fu Fu according to The following method treatment group 3 wheat seeds: 1.0% + Fu-Ping 100-200g, mixed with water 1.5-2kg evenly, 100kg of seed dressing, fully mixed and dried, and then sown.
  • the wheat seeds of Group 3 were then sown in the soil, and at the 3-leaf stage, the wheat was stopped from watering, and after the watering was stopped, the wheat was rehydrated.
  • Example 1 The Vadalian of Example 1 was added to a 6% by weight concentration of effluent to obtain a liquid having a Vadalian mass percent concentration of 2%, and the liquid was named 2.0% + fufu.
  • the group 4 wheat seeds were treated with 2.0% + Fufu as follows: 2.0% + Fufu 100-200g, mixed with water 1.5-2kg, mixed with 100kg, thoroughly mixed and dried, and then sown.
  • the wheat seeds of Group 4 were then sown in the soil, and at the 3-leaf stage, the wheat was stopped from watering, and after the watering was stopped, the wheat was rehydrated.
  • Example 1 The Vadalian of Example 1 was added to a 6% by weight concentration of effluent to obtain a liquid having a Vadalian mass percent concentration of 4%, and the liquid was named 4.0% + fufu.
  • the group 5 wheat seeds were treated with 4.0% + Fufu as follows: 4.0% + Fufu 100-200 g, mixed with water 1.5-2 kg, mixed with 100 kg, thoroughly mixed, dried and then sown.
  • the wheat seeds of Group 5 were then sown in the soil, and at the 3-leaf stage, the wheat was stopped from watering, and after the watering was stopped, the wheat was rehydrated.
  • Group 6 wheat seeds were treated with 6% Fufu as follows (CK2): the mass concentration of Beinong (Haili) Zhangzhou Seed Coating Co., Ltd. was 6% Fufu 100-200g, 1.5-water Stir well with 2kg, mix 100kg, mix thoroughly and dry and sown. The wheat seeds of Group 6 were then sown in the soil, and at the 3-leaf stage, the wheat was stopped from watering, and after the watering was stopped, the wheat was rehydrated.
  • CK2 the mass concentration of Beinong (Haili) Zhangzhou Seed Coating Co., Ltd. was 6% Fufu 100-200g, 1.5-water Stir well with 2kg, mix 100kg, mix thoroughly and dry and sown.
  • the wheat seeds of Group 6 were then sown in the soil, and at the 3-leaf stage, the wheat was stopped from watering, and after the watering was stopped, the wheat was rehydrated.
  • Fig. 14 On the day when the water treatment was stopped in each group, SPAD-502Plus (Beijing Boren Jingwei Technology Development Co., Ltd.) was used to determine the chlorophyll content in each group of wheat leaves. The results are shown in Fig. 14.
  • 1 indicates group 1
  • 2 Indicates group 2
  • 3 represents group 3
  • 4 represents group 4
  • 5 represents group 5
  • 6 represents group 6, and the unit of ordinate is SPAD.
  • the average chlorophyll content of Group 1 Group 2, Group 3, Group 4, Group 5 and Group 6 wheat were 34.1 SPAD, 34.5 SPAD, 34.5 SPAD, 34.4 SPAD, 33.6 SPAD, 33.9 SPAD, Group 2, Group 3 and Group 4, respectively.
  • the chlorophyll content of wheat was significantly higher than that of group 1 and group 6, while the chlorophyll content of group 5 was lower than that of group 1 and group 6, indicating that the appropriate concentration of vedaliam could increase the chlorophyll content of wheat.
  • Grade II After reconstitution of watering, the area of curl of a single plant blade is greater than or equal to 50% less than 100%;
  • Grade III After reconstitution of watering, the area of curl of individual leaves is greater than 0% and less than 50%;
  • Fig. 15 The results are shown in Fig. 15.
  • 1 indicates group 1
  • 2 indicates group 2
  • 3 indicates group 3
  • 4 indicates group 4
  • 5 indicates group 5
  • 6 indicates group 6.
  • the average rehydration indices of group 1 (CK1), group 2, group 3, group 4, group 5 and group 6 (CK2) wheat were 0.348, 0.516, 0.523, 0.43, 0.219, 0.323, group 2, group 3 and group, respectively. 4
  • the rehydration index of wheat was significantly higher than that of group 1 and group 6, while the rehydration index of group 5 was lower than that of group 1 and group 6, indicating that the appropriate concentration of vedaliam could improve the drought resistance of wheat.
  • the field experiment design is as follows: The experimental site is Dunhuang, Gansu. The experiment uses a randomized block design, and three repeating zones are set. Each repeating zone is randomly set with two processing zones, namely VdAL processing zone and control processing zone, and each processing zone The area is 0.1 mu.
  • the red grape (Yongdeng Yongsheng Red Grape Professional Cooperative of Gansu province) was treated once in the flowering period according to the following method: spraying the grape leaves by means of top spray (manual spraying)
  • the diluted 3000-fold VdAL of Example 1 was sprayed with 45-50 kg per acre to obtain a 3000-fold diluted VdAL-treated grape (1/3000).
  • Spraying time in the morning or evening it is best to avoid the noon high temperature period; it is strictly prohibited to mix with drugs and fertilizers during spraying; if it rains within 2 hours after spraying, it needs to be sprayed once.
  • the diluted treatment 3,000 times of VdAL was replaced with clean water to treat the control treatment zone, and the other steps were unchanged, and untreated grapes (control, CK) were obtained.
  • the ripening time of each treated grape was counted.
  • the results showed that the average ripening time of the grapes diluted with 3000 times of VdAL and water treatment was September 15 (85 days from flowering period, 75 days from VdAL treatment) and September 20 (After 90 days of flowering, or 80 days from clear water treatment), after 3,000 times of VdAL treatment of grapes, the ripening time of the grapes was 5 days earlier.
  • the Vc content of the grapes at the time of ripening was determined. It was found that the average Vc content of the grapes diluted with 3000 times of VdAL and water treatment was 18.20 mg/100 g fresh weight and 12.10 mg/100 g fresh, respectively. After weighing 3000 times with VdAL, the grape Vc content increased by 50.4%.
  • Example 11 Vidalian can promote the ripening and coloration of cherries and increase the yield of cherries
  • the field experiment design is as follows: The experimental site is Fushan District, Yantai City. The experiment adopts a random block design, and three repeating zones are set. Two processing zones are randomly set in each repeating zone, which are VdAL processing zone and control processing zone, respectively. The area of the area is 0.1 mu.
  • the diluted treatment 5,000 times of VdAL was replaced with clean water to treat the control treatment zone, and the other steps were unchanged, and untreated cherries (control, CK) were obtained.
  • the ripening time of each treated cherry was counted.
  • the results showed that the average ripening time of 5,000-fold diluted VdAL and water-treated cherries was May 1 (52 days from the flowering period VdAL treatment) and May 6 (distance from VdAL treatment). 57 days), after 5,000 times dilution of VdAL treated cherries, the ripening time of the cherries was 5 days earlier.
  • Example 12 Vadalian can promote the growth of canned radish and improve its palatability
  • the field experiment design is as follows: The experiment adopts a random block design, and three repeating zones are set. Two processing zones are randomly set in each repeating zone, which are respectively VdAL processing zone and control processing zone, and the area of each processing zone is 0.1 mu.
  • the root swell period of the canned radish variety 791 (Zhengzhou Sanhe Agricultural Science and Technology Development Co., Ltd.) in the VdAL treatment area was treated once in the following manner: the top radish (manual spray) method
  • the diluted 5000-fold VdAL of Example 1 was sprayed, and 45-50 kg per mu was sprayed to obtain a VdAL-treated canned radish (1/5000) diluted 5000 times.
  • the diluted treatment 5,000 times of VdAL was replaced with clean water to treat the control treatment zone, and the other steps were unchanged, and untreated canned radish (control, CK) was obtained.
  • the canned radish yield of each treatment was counted (Fig. 18).
  • the results showed that the average root weight of the VdAL diluted with 5000 times of water and the treated radish of the water was 0.90 kg and 0.85 kg, respectively, diluted 5000 times.
  • the average root weight of the canned radish increased by 5.88%; the average fresh weight of the radish diluted with 5000 times of VdAL and the treated water was 1.20kg and 1.09kg, respectively, and the VdAL treatment tank diluted 5000 times.
  • the average root weight of the canned radish increased by 10.09%.
  • Vadalian can improve the crispness and sweetness of canned radish, and the canned radish with Vadalian treatment Have a strong palatability.
  • the field experiment design is as follows: The experiment adopts random block design, and sets three repeating zones. Each repeating zone randomly sets three processing zones, which are VdAL processing zone 1, VdAL processing zone 2 and control treatment zone, and each treatment zone The area is 0.1 mu.
  • VdAL treatment zone 1 According to the treatment method of VdAL treatment zone 1, the VdAL diluted 4000 times was replaced with VdAL diluted 6000 times, and the VdAL treatment zone 2 was treated. The other steps were unchanged, and the VdAL-treated spinach (1/6000) diluted 6000 times was obtained. .
  • the Dd diluted 4000 times of VdAL was replaced with clean water to treat the control treatment zone, and the other steps were unchanged, and untreated spinach (CK) was obtained.
  • the yield of spinach in each treatment was counted in VdAL/water treatment for 20 days.
  • the results showed that the average yield of VdAL diluted 4000 times, VdAL diluted 6000 times and spinach treated with water was 0.58 kg/0.12 m 2 and 0.98 kg/0.12, respectively.
  • m 2 and 0.63kg/0.12m 2 diluted 4000 times VdAL, diluted 6000 times VdAL treatment of spinach, the yield increased by -7.45%, 56.38%, respectively, indicating that the appropriate concentration of VdAL treatment of spinach can increase its yield.
  • VdAL Vadal biological preparation
  • the average germination rate of cotton after VdAL treatment of cotton seeds diluted 7500 times was 1.08 times of the average germination rate of cotton after water treatment.
  • Treatment of Lu 30 with diluted 3000 times VdAL the flower yield before frost increased by 19.48%, the total yield increased by 17.04%; when treated with VdAL diluted 5000 times, the flower yield of Lu 30 was increased by 21.25%, and the total yield increased by 20.00. %, 616 pre-frost flower yield increased by 19.06%, total output increased by 19.69%, Lu 25 before frost production increased by 16.37%, total output increased by 17.60%.
  • the germination rate is about 2 times, 9 times, 12 times and respectively. 11 times.
  • the germination rate of wheat seeds diluted with 5000 times of VdAL and 10,000 times diluted with VdAL was increased by 2.48% and 4.40%, respectively.
  • the fresh weight per plant of wheat increased by 15.32% and 27.03%, respectively.
  • the yield increased by 1.94%, 8.13%, and 5.23%, respectively.
  • the effective panicle number increased by 1.5%, the number of panicles increased by 0.5%; the diluted 10000-fold VdAL, the effective panicle number increased by 7%, the panicle number increased by 3.5%; diluted 20,000 times For VdAL, the effective panicle number increased by 2.3% and the panicle number increased by 3.7%.
  • VdAL can also increase the fruit setting rate of cucumber, promote the expansion of cucumber, increase the yield of cucumber, promote the growth of cucumber plants, delay the senescence of cucumber plants; VdAL can also improve the fruit setting rate of the fruit; promote strawberry, melon, The growth of sweet pepper plants; promote the emergence and growth of soybeans. After 5,000 times of VdAL soaking, the fresh weight of soybeans increased by 16.91%, and the fresh weight of soybeans increased by 28.30%.
  • the average root weight of the radish treated with VdAL diluted with 5000 times and treated with water is 0.90kg and 0.85kg respectively. After 5,000 times of VdAL treated cherry, the average root weight of the canned radish is increased by 5.88%; diluted by 5000 times. The average fresh weight of VdAL and water-treated canned radish was 1.20kg and 1.09kg, respectively. After 5,000-fold dilution of VdAL-treated cherries, the average root weight of canned radish increased by 10.09%.
  • VDLA Vadalan biological preparation
  • VdAL was diluted 3000 times, the Vc content of the grapes was increased by 50.4%, and the color was more beautiful, the ears were compact and not easy to be granulated, and the ripening time of the grapes was 5 days earlier.
  • VDLA Vadalan biological preparation
  • the solution treated plants with a concentration percentage of 1%-2% of Vidalian are all dead in 15-20 days after water control.
  • the plants not treated with Vidalian are all dead in water for 10-12 days, and the mass percentage of Vidalian
  • the dead time of the plants treated with the solution at a concentration of 1% to 2% was significantly greater than that of the wheat treated without the Vadalian treatment.
  • the lycopene content of the Vadalian treated plants was determined to be 34.5 SPAD-34.4 SPAD, which was significantly higher than that of the plants treated with Vodaline in a concentration of 0.2% to 2%.
  • Plants treated with Vadalian; Vadalian treated plants have a rehydration index of 0.43-0.52, which is significantly higher than plants that have not been treated with Vidalan. It shows that the appropriate concentration of vedaliam can improve the drought resistance of wheat.
  • VDLA Vadalan biological preparation
  • VdAL-treated cherries diluted 5000 times is significantly higher than that of untreated cherry red, indicating that VdAL can promote the coloration of cherries.
  • the experiment proves that the protein VdAL of the invention and the biological preparation thereof can promote plant growth, increase plant yield, improve plant product quality and palatability of plant fruits, can improve drought resistance of plants, and can promote coloration of plant fruits and promote fruit ripening. .

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Abstract

提供了蛋白质VdAL在提高植物产量、产品品质、抗旱性及促进植物果实着色中的应用，所述蛋白质VdAL选自A1）-A3）之一：A1）氨基酸序列为序列1的蛋白质；A2）序列1经过取代和/或缺失和/或添加一个或几个氨基酸残基得到的具有相同功能的由A1）衍生的蛋白质；A3）在A1）或A2）的N端或/和C端连接标签得到的融合蛋白质。

Description

蛋白质VdAL在提高植物产量、产品品质和抗旱性及促进植物果实着色中的应用 技术领域
本发明涉及生物技术领域中蛋白质VdAL在提高植物产量、产品品质和抗旱性及促进植物果实着色中的应用。
背景技术
目前我国大面积粮食及经济作物的持续高产主要依赖大量使用化肥、防治病虫害主要依赖大量喷施化学农药。中国农业科学院农业经济与发展研究所研究员在谈及农业污染问题时他指出，我们国家现在还是相当严重的，化肥和农药的过量使用，以及养殖业中大量使用的抗生素和重金属添加剂对农业生产都将造成严重的污染(胡定寰，2013)。
仅据辽宁一个省的统计：目前化肥年使用量在3万吨以上，单位面积化肥施用强度约340千克每公顷，国家生态区建设要求每公顷限值则为每公顷250千克，重氮肥轻磷肥钾肥现象普遍不规范施肥问题也较为突出，辽宁省农药年用量超过1.4万吨且呈逐年上升趋势。残留农药通过大气沉降和雨水冲刷的形式进入环境和农产品中极易造成环境污染事件。近年来随着农村环境污染问题日渐突出环境信访案件呈高发态势。前几年该省的该省环境信访案件每年平均4310件，其中涉农案件近2795件占信访案件的65％左右。
过量施肥会造成农作物吸收营养元素时离子之间增强拮抗作用，若某种养分离子高浓度的存在能够抑制另一种或多种养分离子的活性，从而影响农作物对另一种营养离子的吸收。如在酸性土壤里氮肥的施入量过多，作物吸收钙离子就很困难。如果过量地施用钙肥会诱发农作物的锌、硼、铁、镁、锰等微量元素缺乏。钾肥用量过多也会影响农作物对钙离子、镁离子的吸收；而且过量施肥易引起农作物中毒，因施入大量肥料，增加了土壤溶液的浓度，使农作物根系吸收水分困难，造成地上部萎蔫，植株枯死。此外，过量施肥还影响农产品的品质，农作物生长中、后期，如果大量施入化肥，会使农产品器官含糖量降低，不耐贮藏，从而影响商品价值，降低农作物的经济效益。
发明公开
本发明所要解决的第一个技术问题是如何促进植物生长和如何提高植物产量。
为解决上述技术问题，本发明首先提供了蛋白质在提高植物产量和/或调控植物生长中的应用。
本发明所提供的蛋白质在提高植物产量和/或调控植物生长中的应用中，所述蛋白质的名称为VdAL，为如下A1)或A2)或A3)：
A1)氨基酸序列为序列1的蛋白质；
A2)在序列1的氨基酸序列中经过取代和/或缺失和/或添加一个或几个氨基酸残基得到的具有相同功能的由A1)衍生的蛋白质；
A3)在A1)或A2)的N端或/和C端连接标签得到的融合蛋白质。
其中，序列1由297个氨基酸组成。
为了使A1)中的蛋白质便于纯化，可在序列表中序列1或序列1的氨基酸所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。
表1、标签的序列
标签 残基 序列
Poly-Arg 5-6(通常为5个) RRRRR
Poly-His 2-10(通常为6个) HHHHHH
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
上述A2)中的VdAL可人工合成，也可先合成其编码基因，再进行生物表达得到。上述A2)中的VdAL的编码基因可通过将序列表中序列2所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
上述VdAL在提高植物产量和/或调控植物生长中的应用中，所述调控植物生长可为促进植物生长。
为解决上述技术问题，本发明还提供了与VdAL相关的生物材料在提高植物产量和/或调控植物生长中的应用。
本发明所提供的与VdAL相关的生物材料在提高植物产量和/或调控植物生长中的应用；
所述生物材料，为下述B1)至B20)中的任一种：
B1)编码VdAL的核酸分子；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体；
B4)含有B2)所述表达盒的重组载体；
B5)含有B1)所述核酸分子的重组微生物；
B6)含有B2)所述表达盒的重组微生物；
B7)含有B3)所述重组载体的重组微生物；
B8)含有B4)所述重组载体的重组微生物；
B9)含有B1)所述核酸分子的转基因植物细胞系；
B10)含有B2)所述表达盒的转基因植物细胞系；
B11)含有B3)所述重组载体的转基因植物细胞系；
B12)含有B4)所述重组载体的转基因植物细胞系；
B13)含有B1)所述核酸分子的转基因植物组织；
B14)含有B2)所述表达盒的转基因植物组织；
B15)含有B3)所述重组载体的转基因植物组织；
B16)含有B4)所述重组载体的转基因植物组织；
B17)含有B1)所述核酸分子的转基因植物器官；
B18)含有B2)所述表达盒的转基因植物器官；
B19)含有B3)所述重组载体的转基因植物器官；
B20)含有B4)所述重组载体的转基因植物器官。
上述与VdAL相关的生物材料在提高植物产量和/或调控植物生长中的应用中，B1)所述核酸分子可为如下b1)或b2)或b3)的基因：
b1)核苷酸序列是序列表中序列2的cDNA分子或DNA分子；
b2)与b1)限定的核苷酸序列具有75％或75％以上同一性，且编码VdAL的cDNA分子或基因组DNA分子；
b3)在严格条件下与b1)限定的核苷酸序列杂交，且编码VdAL的cDNA分子或基因组DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
其中，序列2由894个核苷酸组成，编码序列1所示的蛋白质。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码VdAL的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明分离得到的VdAL的核苷酸序列75％或者更高同一性的核苷酸，只要编码VdAL且具有VdAL功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码VdAL所示的氨基酸序列组成的蛋白质的核苷酸序列具有75％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
上述与VdAL相关的生物材料在提高植物产量和/或调控植物生长中的应用中，所述严格条件是在2×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次5min，又于0.5×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次15min；或，0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。
上述75％或75％以上同一性，可为80％、85％、90％或95％以上的同一性。
上述与VdAL相关的生物材料在提高植物产量和/或调控植物生长中的应用中，B2)所述的含有编码VdAL的核酸分子的表达盒(VdAL基因表达盒)，是指能够在宿主细胞中表达VdAL的DNA，该DNA不但可包括启动VdAL基因转录的启动子，还可包括终止VdAL基因转录的终止子。进一步，所述表达盒还可包括增强子序列。可用于本发明的启动子包括但不限于：组成型启动子，组织、器官和发育特异的启动子，和诱导型启动子。启动子的例子包括但不限于：花椰菜 花叶病毒的组成型启动子35S：来自西红柿的创伤诱导型启动子，亮氨酸氨基肽酶("LAP",Chao等人(1999)Plant Physiol 120：979-992)；来自烟草的化学诱导型启动子，发病机理相关1(PR1)(由水杨酸和BTH(苯并噻二唑-7-硫代羟酸S-甲酯)诱导)；西红柿蛋白酶抑制剂II启动子(PIN2)或LAP启动子(均可用茉莉酮酸甲酯诱导)；热休克启动子(美国专利5,187,267)；四环素诱导型启动子(美国专利5,057,422)；种子特异性启动子，如谷子种子特异性启动子pF128(CN101063139B(中国专利200710099169.7))，种子贮存蛋白质特异的启动子(例如，菜豆球蛋白、napin,oleosin和大豆beta conglycin的启动子(Beachy等人(1985)EMBO J.4：3047-3053))。它们可单独使用或与其它的植物启动子结合使用。此处引用的所有参考文献均全文引用。合适的转录终止子包括但不限于：农杆菌胭脂碱合成酶终止子(NOS终止子)、花椰菜花叶病毒CaMV 35S终止子、tml终止子、豌豆rbcS E9终止子和胭脂氨酸和章鱼氨酸合酶终止子(参见，例如：Odell等人(I985)Nature313:810；Rosenberg等人(1987)Gene,56:125；Guerineau等人(1991)Mol.Gen.Genet,262:141；Proudfoot(1991)Cell,64:671；Sanfacon等人Genes Dev.,5:141；Mogen等人(1990)Plant Cell,2:1261；Munroe等人(1990)Gene,91:151；Ballad等人(1989)Nucleic Acids Res.17:7891；Joshi等人(1987)Nucleic Acid Res.,15:9627)。
可用现有的表达载体构建含有所述VdAL基因表达盒的重组载体。所述植物表达载体包括双元农杆菌载体和可用于植物微弹轰击的载体等。如pAHC25、pBin438、pCAMBIA1302、pCAMBIA2301、pCAMBIA1301、pCAMBIA1300、pBI121、pCAMBIA1391-Xa或pCAMBIA1391-Xb(CAMBIA公司)等。所述植物表达载体还可包含外源基因的3′端非翻译区域，即包含聚腺苷酸信号和任何其它参与mRNA加工或基因表达的DNA片段。所述聚腺苷酸信号可引导聚腺苷酸加入到mRNA前体的3′端，如农杆菌冠瘿瘤诱导(Ti)质粒基因(如胭脂碱合成酶基因Nos)、植物基因(如大豆贮存蛋白基因)3′端转录的非翻译区均具有类似功能。使用本发明的基因构建植物表达载体时，还可使用增强子，包括翻译增强子或转录增强子，这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的，可以是天然的，也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。为了便于对转基因植物细胞或植物进行鉴定及筛选，可对所用植物表达载体进行加工，如加入可在植物中表达的编码可产生颜色变化的酶或发光化合物的基因(GUS基因、萤光素酶基因等)、抗生素的标记基因(如赋予对卡那霉素和相关抗生素抗性的nptII基因，赋予对除草剂膦丝菌素抗性的bar基因，赋予对抗生素潮霉素抗性的hph基因，和赋予对氨甲喋呤抗性的dhfr基因，赋予对草甘磷抗性的EPSPS基因)或是抗化学试剂标记基因等(如抗除莠剂基因)、提供代谢甘露糖能力的甘露糖-6-磷酸异 构酶基因。从转基因植物的安全性考虑，可不加任何选择性标记基因，直接以逆境筛选转化植株。
上述与VdAL相关的生物材料在提高植物产量和/或调控植物生长中的应用中，所述载体可为质粒、黏粒、噬菌体或病毒载体。所述质粒可为载体pET42a(+)。
上述与VdAL相关的生物材料在提高植物产量和/或调控植物生长中的应用中，所述微生物可为酵母、细菌、藻或真菌，如大肠杆菌。所述大肠杆菌可为大肠杆菌JM109。
上述与VdAL相关的生物材料在提高植物产量和/或调控植物生长中的应用中，所述转基因植物细胞系、转基因植物组织和转基因植物器官既可均不包括繁殖材料，也可均包括繁殖材料。
上述与VdAL相关的生物材料在提高植物产量和/或调控植物生长中的应用中，所述转基因植物器官也可为转基因植物的种子。所述转基因植物可包括种子、愈伤组织、完整植株和细胞。
在本发明的一个实施方式中，VdAL的编码基因通过含有VdAL的编码基因的表达盒的重组载体导入大肠杆菌JM109中得到重组微生物。所述重组载体为将载体pET42a(+)的NdeI和KpnI识别位点间的序列替换为序列表中序列2的第1-894位核苷酸所示的DNA分子(即VdAL基因)，得到的重组载体。所述重组载体表达序列1所示的VdAL。所述重组微生物表达序列1所示的VdAL。
上述与VdAL相关的生物材料在提高植物产量和/或调控植物生长中的应用中，所述调控植物生长可为促进植物生长。
为解决上述技术问题，本发明还提供了一种提高植物产量和/或促进植物生长的方法。
本发明所提供的提高植物产量和/或促进植物生长的方法，包括对目的植物施用VdAL、所述生物材料或生物制剂，提高所述目的植物产量和/或促进所述目的植物生长；所述生物制剂的活性成分为VdAL或所述生物材料。
上述方法中，所述生物制剂可按照方法1)制备，所述方法1)为：培养所述重组微生物，使VdAL的编码基因表达，得到所述生物制剂。
上述方法中，所述方法1)具体可为如下11)和12)：
11)培养所述重组微生物，使所述编码基因表达，得到表达所述蛋白质的重组微生物培养物；
12)破碎所述微生物培养物中的菌体，得到所述生物制剂。
上述方法中，所述方法1)还可包括对所述生物制剂进行干燥。
上述方法中，所述编码基因可为B1)所述核酸分子。
在本发明的一个实施方式中，所述重组微生物为将含有VdAL的编码基因的表达盒的重组载体导入大肠杆菌JM109中得到的重组微生物。所述重组载体为将载体pET42a(+)的NdeI和KpnI识别位点间的序列替换为序列2的第1-894 位核苷酸所示的DNA分子(即VdAL基因)，得到的重组载体。所述重组载体表达序列1所示的VdAL。所述重组微生物表达序列1所示的VdAL。
上述方法中，所述对目的植物施用VdAL、所述生物材料或生物制剂具体可为对所述目的植物叶片施用VdAL、所述生物材料或所述生物制剂，如对目的植物喷施VdAL、所述生物材料或所述生物制剂，也可用VdAL、所述生物材料或所述生物制剂处理所述目的植物的种子，如用VdAL、所述生物材料或所述生物制剂的液体制剂对所述目的植物的种子进行浸种。VdAL、所述生物材料或所述生物制剂的液体制剂可为直接用清水溶解或悬浮VdAL、所述生物材料或所述生物制剂得到的液体。对目的植物施用VdAL、所述生物材料或生物制剂的量可以根据所述目的植物的种类和/或生长时期确定。
为解决上述技术问题，本发明还提供了所述生物制剂在提高植物产量和/或调控植物生长中的应用。
为解决上述技术问题，本发明还提供了所述生物制剂。在本发明中，所述生物制剂的名称为维达利安。
上文中，所述植物为双子叶植物或单子叶植物。所述双子叶植物可为锦葵科植物、葫芦科植物、茄科植物、十字花科植物、蔷薇科植物、葡萄科植物、李亚科植物、藜科植物或豆科植物。所述锦葵科植物可为棉花。所述葫芦科植物可为西瓜、黄瓜或甜瓜。所述茄科植物可为番茄、圣女果或茄科。所述十字花科植物可为青梗菜或萝卜，如罐萝卜。所述蔷薇科植物可为草莓。所述豆科植物可为大豆。所述葡萄科植物可为葡萄。所述李亚科植物可为樱桃。所述藜科植物可为菠菜。所述单子叶植物可为禾本科植物，如水稻或小麦。
上文中，所述产量可为经济产量，即栽培目的所需要的产品收获量。如禾谷类的种子，棉花的籽棉或皮棉，青梗菜或菠菜的植株，西瓜、番茄、黄瓜、圣女果、草莓、甜瓜、甜椒、樱桃、葡萄的果实，萝卜的块根。所述产量可以重量来体现。
在生产中，所述促进植物的生长最终可表现为作物产量的增加。如棉花、西瓜、小麦、番茄、青梗菜、黄瓜、圣女果、草莓、甜瓜、水稻、葡萄、樱桃、萝卜、菠菜、甜椒或大豆产量的增加。
上文中，所述促进植物的生长可为促进所述植物的营养生长和/或生殖生长，如促进所述植物果实的生长。所述营养生长具体可体现在促进种子发芽、促进植株生长、促进果实生长、提高座果数目和/或延缓植株衰老。在本发明的实施例中，所述促进植物的营养生长具体体现在以下几个方面：提高棉花发芽率、单株成铃数、亩总铃数和单铃重；提高西瓜单株结果数；提高小麦发芽率，促进小麦植株生长；提高番茄单株结果数；促进青梗菜、菠菜或萝卜植株生长；提高黄瓜座果数目，促进黄瓜果实膨大，促进黄瓜植株生长，延缓黄瓜植株衰老；提高圣女果、樱桃、葡萄座果数目；促进草莓植株生长；促进甜瓜植株生长；促进甜椒植株生长；促进大豆的出苗和植株的生长。所述促进植物的生殖 生长具体体现为提高棉花、西瓜、番茄、黄瓜和圣女果的座果数目。
上文中，VdAL或所述生物制剂对不同植物的作用可因VdAL或所述生物制剂浓度的不同而出现不同的结果。在施用VdAL或所述生物制剂时，VdAL或所述生物制剂的浓度可以根据作物或其品种以及施用VdAL或所述生物制剂的目的(如促进种子发芽或提高生物产量等)调节VdAL或所述生物制剂的浓度。
在本发明的实施例中，所述生物制剂稀释7500倍得到的液体可以促进棉花的发芽；所述生物制剂稀释3000-5000倍得到的液体可以提高棉花的霜前花产量和总产量以及每亩植株数、单株成铃数、亩总铃数、衣分和亩产量。所述生物制剂稀释2000倍得到的液体可以促进西瓜结果，提高西瓜产量。所述生物制剂稀释5000-10000倍得到的液体可以促进小麦种子的发芽；所述生物制剂稀释5000-20000倍得到的液体可以提高小麦产量。所述生物制剂稀释1000倍得到的液体可以促进番茄结果，提高番茄产量。所述生物制剂稀释2000-4000倍得到的液体可以提高青梗菜的产量。所述生物制剂稀释300-2000倍得到的液体可以提高黄瓜的座果率、促进黄瓜的膨大，提高黄瓜产量，促进黄瓜植株的顶端生长，延缓黄瓜植株的衰老。所述生物制剂稀释2000倍得到的液体可以提高圣女果的座果率。所述生物制剂稀释2000倍得到的液体可以促进草莓、甜瓜植株的生长。所述生物制剂稀释1000倍得到的液体可以促进甜椒植株的生长。所述生物制剂稀释3000-5000倍得到的液体可以促进大豆的出苗与生长。所述生物制剂稀释10000-20000倍得到的液体可以提高水稻的产量。所述生物制剂稀释3000倍得到的液体可以促进葡萄的成熟。所述生物制剂稀释5000倍得到的液体可以促进樱桃的成熟与萝卜的生长。所述生物制剂稀释6000倍得到的液体可以促进菠菜的生长。
本发明所要解决的第二个技术问题是如何提高植物产品品质。所述植物产品为人类栽培植物生产的人类所需要的产品。所述品质的评价标准因产品的用途不同而异。用作食物的产品的品质可提现在产品的营养品质和食用品质上；用作衣着原料的产品的品质可提现在产品的纤维品质上。评价产品品质，一般采用两种指标，一是生化成分以及有害物质的含量，如营养物质含量和微量元素含量；二是物理指标，如产品的形状、大小、滋味、香气、色泽、种皮厚薄、整齐度、纤维长度、纤维强度等，如棉花的衣分。
为解决上述技术问题，本发明首先提供了蛋白质在提高植物产品品质中的应用。
本发明所提供的蛋白质在提高植物产品品质中的应用中，所述蛋白质的名称为VdAL，为如下A1)或A2)或A3)：
A1)氨基酸序列为序列1的蛋白质；
A2)在序列1的氨基酸序列中经过取代和/或缺失和/或添加一个或几个氨基酸残基得到的具有相同功能的由A1)衍生的蛋白质；
A3)在A1)或A2)的N端或/和C端连接标签得到的融合蛋白质。
其中，序列1由297个氨基酸组成。
为了使A1)中的蛋白质便于纯化，可在序列表中序列1所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。
上述A2)中的VdAL可人工合成，也可先合成其编码基因，再进行生物表达得到。上述A2)中的VdAL的编码基因可通过将序列表中序列2所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
上述VdAL在提高植物产品品质中的应用中，所述提高植物产品品质可为提高所述植物产品营养成分的含量和/或提高所述植物产品中微量元素的含量和/或提高所述植物产品的适口性。所述植物产品营养成分具体可为维生素C、可溶性糖和/或蛋白质。所述微量元素具体可为Cu、Fe、K、Mn和/或Zn。所述适口性可体现在干物率和/或粗纤维的含量下降上，或植物可食用部分的脆度上。
上述VdAL在提高植物产品品质中的应用中，所述提高植物产品品质可为通过促进所述植物产品的成熟而提高所述植物产品的品质。在本发明的一个实施例中，VdAL的施用促进棉花产品的成熟，棉花的霜前花产量增加，进而提高棉花品质。
上述VdAL在提高植物产品品质中的应用中，所述植物可为双子叶植物或单子叶植物。
上述VdAL在提高植物产品品质中的应用中，所述双子叶植物可为下述a)、b)、c)、d)、e)、f)、g)或h)：
a)葫芦科植物；b)西瓜；c)锦葵科植物；d)棉花；e)葡萄科植物；f)葡萄；g)十字花科植物；h)萝卜。
为解决上述技术问题，本发明还提供了与VdAL相关的生物材料在提高植物产品品质中的应用。
本发明所提供的与VdAL相关的生物材料在提高植物产品品质中的应用；
所述生物材料，为下述B1)至B20)中的任一种：
B1)编码VdAL的核酸分子；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体；
B4)含有B2)所述表达盒的重组载体；
B5)含有B1)所述核酸分子的重组微生物；
B6)含有B2)所述表达盒的重组微生物；
B7)含有B3)所述重组载体的重组微生物；
B8)含有B4)所述重组载体的重组微生物；
B9)含有B1)所述核酸分子的转基因植物细胞系；
B10)含有B2)所述表达盒的转基因植物细胞系；
B11)含有B3)所述重组载体的转基因植物细胞系；
B12)含有B4)所述重组载体的转基因植物细胞系；
B13)含有B1)所述核酸分子的转基因植物组织；
B14)含有B2)所述表达盒的转基因植物组织；
B15)含有B3)所述重组载体的转基因植物组织；
B16)含有B4)所述重组载体的转基因植物组织；
B17)含有B1)所述核酸分子的转基因植物器官；
B18)含有B2)所述表达盒的转基因植物器官；
B19)含有B3)所述重组载体的转基因植物器官；
B20)含有B4)所述重组载体的转基因植物器官。
上述与VdAL相关的生物材料在提高植物产品品质中的应用中，B1)所述核酸分子可为如下b1)或b2)或b3)的基因：
b1)核苷酸序列是序列表中序列2的cDNA分子或DNA分子；
b2)与b1)限定的核苷酸序列具有75％或75％以上同一性，且编码VdAL的cDNA分子或基因组DNA分子；
b3)在严格条件下与b1)限定的核苷酸序列杂交，且编码VdAL的cDNA分子或基因组DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
其中，序列2由894个核苷酸组成，编码序列1所示的蛋白质。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码VdAL的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明分离得到的VdAL的核苷酸序列75％或者更高同一性的核苷酸，只要编码VdAL且具有VdAL功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码VdAL所示的氨基酸序列组成的蛋白质的核苷酸序列具有75％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
上述与VdAL相关的生物材料在提高植物产品品质中的应用中，所述严格条件是在2×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次5min，又于0.5×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次15min；或，0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。
上述75％或75％以上同一性，可为80％、85％、90％或95％以上的同一性。
上述与VdAL相关的生物材料在提高植物产品品质中的应用中，B2)所述的含有编码VdAL的核酸分子的表达盒(VdAL基因表达盒)，是指能够在宿主细胞中表达VdAL的DNA，该DNA不但可包括启动VdAL基因转录的启动子，还可包括 终止VdAL基因转录的终止子。进一步，所述表达盒还可包括增强子序列。可用于本发明的启动子包括但不限于：组成型启动子，组织、器官和发育特异的启动子，和诱导型启动子。启动子的例子包括但不限于：花椰菜花叶病毒的组成型启动子35S：来自西红柿的创伤诱导型启动子，亮氨酸氨基肽酶("LAP",Chao等人(1999)Plant Physiol 120：979-992)；来自烟草的化学诱导型启动子，发病机理相关1(PR1)(由水杨酸和BTH(苯并噻二唑-7-硫代羟酸S-甲酯)诱导)；西红柿蛋白酶抑制剂II启动子(PIN2)或LAP启动子(均可用茉莉酮酸甲酯诱导)；热休克启动子(美国专利5,187,267)；四环素诱导型启动子(美国专利5,057,422)；种子特异性启动子，如谷子种子特异性启动子pF128(CN101063139B(中国专利200710099169.7))，种子贮存蛋白质特异的启动子(例如，菜豆球蛋白、napin,oleosin和大豆beta conglycin的启动子(Beachy等人(1985)EMBO J.4：3047-3053))。它们可单独使用或与其它的植物启动子结合使用。此处引用的所有参考文献均全文引用。合适的转录终止子包括但不限于：农杆菌胭脂碱合成酶终止子(NOS终止子)、花椰菜花叶病毒CaMV 35S终止子、tml终止子、豌豆rbcS E9终止子和胭脂氨酸和章鱼氨酸合酶终止子(参见，例如：Odell等人(I985)Nature 313:810；Rosenberg等人(1987)Gene,56:125；Guerineau等人(1991)Mol.Gen.Genet,262:141；Proudfoot(1991)Cell,64:671；Sanfacon等人Genes Dev.,5:141；Mogen等人(1990)Plant Cell,2:1261；Munroe等人(1990)Gene,91:151；Ballad等人(1989)Nucleic Acids Res.17:7891；Joshi等人(1987)Nucleic Acid Res.,15:9627)。
可用现有的表达载体构建含有所述VdAL基因表达盒的重组载体。所述植物表达载体包括双元农杆菌载体和可用于微弹轰击的植物表达载体等。如pAHC25、pBin438、pCAMBIA1302、pCAMBIA2301、pCAMBIA1301、pCAMBIA1300、pBI121、pCAMBIA1391-Xa或pCAMBIA1391-Xb(CAMBIA公司)等。所述植物表达载体还可包含外源基因的3′端非翻译区域，即包含聚腺苷酸信号和任何其它参与mRNA加工或基因表达的DNA片段。所述聚腺苷酸信号可引导聚腺苷酸加入到mRNA前体的3′端，如农杆菌冠瘿瘤诱导(Ti)质粒基因(如胭脂碱合成酶基因Nos)、植物基因(如大豆贮存蛋白基因)3′端转录的非翻译区均具有类似功能。使用本发明的基因构建植物表达载体时，还可使用增强子，包括翻译增强子或转录增强子，这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的，可以是天然的，也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。为了便于对转基因植物细胞或植物进行鉴定及筛选，可对所用植物表达载体进行加工，如加入可在植物中表达的编码可产生颜色变化的酶或发光化合物的基因(GUS基因、萤光素酶基因等)、抗生素的标记基因(如赋予对卡那霉素和相关抗生素抗性的nptII基因，赋予对 除草剂膦丝菌素抗性的bar基因，赋予对抗生素潮霉素抗性的hph基因，和赋予对氨甲喋呤抗性的dhfr基因，赋予对草甘磷抗性的EPSPS基因)或是抗化学试剂标记基因等(如抗除莠剂基因)、提供代谢甘露糖能力的甘露糖-6-磷酸异构酶基因。从转基因植物的安全性考虑，可不加任何选择性标记基因，直接以逆境筛选转化植株。
上述与VdAL相关的生物材料在提高植物产品品质中的应用中，所述载体可为质粒、黏粒、噬菌体或病毒载体。所述质粒可为载体pET42a(+)。
上述与VdAL相关的生物材料在提高植物产品品质中的应用中，所述微生物可为酵母、细菌、藻或真菌，如大肠杆菌。所述大肠杆菌可为大肠杆菌JM109。
上述与VdAL相关的生物材料在提高植物产品品质中的应用中，所述转基因植物细胞系、转基因植物组织和转基因植物器官既可均不包括繁殖材料，也可均包括繁殖材料。
上述与VdAL相关的生物材料在提高植物产品品质中的应用中，所述转基因植物器官也可为转基因植物的种子。所述转基因植物可包括种子、愈伤组织、完整植株和细胞。
在本发明的一个实施方式中，VdAL的编码基因通过含有VdAL的编码基因的表达盒的重组载体导入大肠杆菌JM109中得到重组微生物。所述重组载体为将载体pET42a(+)的NdeI和KpnI识别位点间的序列替换为序列表中序列2的第1-894位核苷酸所示的DNA分子(即VdAL基因)，得到的重组载体。所述重组载体表达序列1所示的VdAL。所述重组微生物表达序列1所示的VdAL。
上述与VdAL相关的生物材料在提高植物产品品质中的应用中，所述提高植物产品品质可为提高所述植物产品营养成分的含量和/或提高所述植物产品中微量元素的含量和/或提高所述植物产品的适口性。所述植物产品营养成分具体可为维生素C、可溶性糖和/或蛋白质。所述微量元素具体可为Cu、Fe、K、Mn和/或Zn。所述适口性可体现在干物率和/或粗纤维的含量下降上。
上述与VdAL相关的生物材料在提高植物产品品质中的应用中，所述提高植物产品品质可为通过促进所述植物产品的成熟而提高所述植物产品的品质。在本发明的一个实施例中，VdAL的施用促进棉花产品的成熟，棉花的霜前花产量增加，进而提高棉花品质。
上述与VdAL相关的生物材料在提高植物产品品质中的应用中，所述植物可为双子叶植物或单子叶植物。
上述与VdAL相关的生物材料在提高植物产品品质中的应用中，所述双子叶植物可为下述a)、b)、c)、d)、e)、f)、g)或h)：
a)葫芦科植物；b)西瓜；c)锦葵科植物；d)棉花；e)葡萄科植物；f)葡萄；g)十字花科植物；h)萝卜。
为解决上述技术问题，本发明还提供了一种提高植物产品品质的方法。
本发明所提供的提高植物产品品质的方法，包括对目的植物施用VdAL、所 述生物材料或生物制剂，提高所述目的植物产品品质；所述生物制剂的活性成分为VdAL或所述生物材料。
上述提高植物产品品质的方法中，所述生物制剂可按照方法1)制备，所述方法1)为：培养所述重组微生物，使VdAL的编码基因表达，得到所述生物制剂。
上述提高植物产品品质的方法中，所述方法1)具体可为如下11)和12)：
11)培养所述重组微生物，使所述编码基因表达，得到表达所述蛋白质的重组微生物培养物；
12)破碎所述微生物培养物中的菌体，得到所述生物制剂。
上述提高植物产品品质的方法中，所述方法1)还可包括对所述生物制剂进行干燥。
上述提高植物产品品质的方法中，所述编码基因可为B1)所述核酸分子。
在本发明的一个实施方式中，所述重组微生物为将含有VdAL的编码基因的表达盒的重组载体导入大肠杆菌JM109中得到的重组微生物。所述重组载体为将载体pET42a(+)的NdeI和KpnI识别位点间的序列替换为序列表中序列2的第1-894位核苷酸所示的DNA分子(即VdAL基因)，得到的重组载体。所述重组载体表达序列1所示的VdAL。所述重组微生物表达序列1所示的VdAL。
上述提高植物产品品质的方法中，所述对目的植物施用VdAL、所述生物材料或生物制剂具体可为对所述目的植物叶片施用VdAL、所述生物材料或所述生物制剂，如对目的植物喷施VdAL、所述生物材料或所述生物制剂，也可用VdAL、所述生物材料或所述生物制剂处理所述目的植物的种子，如用VdAL、所述生物材料或所述生物制剂的液体制剂对所述目的植物的种子进行浸种。VdAL、所述生物材料或所述生物制剂的液体制剂可为直接用清水溶解或悬浮VdAL、所述生物材料或所述生物制剂得到的液体。对目的植物施用VdAL、所述生物材料或生物制剂的量可以根据所述目的植物的种类和/或生长时期确定。
上述提高植物产品品质的方法中，所述提高植物产品品质可为提高所述植物产品营养成分的含量和/或提高所述植物产品中微量元素的含量和/或提高所述植物产品的适口性。所述植物产品营养成分具体可为维生素C、可溶性糖和/或蛋白质。所述微量元素具体可为Cu、Fe、K、Mn和/或Zn。所述适口性可体现在干物率和/或粗纤维的含量下降上。
上述提高植物产品品质的方法中，所述提高植物产品品质可为通过促进所述植物产品的成熟而提高所述植物产品的品质。在本发明的一个实施例中，VdAL的施用促进棉花产品的成熟，棉花的霜前花产量增加，进而提高棉花品质。
上述提高植物产品品质的方法中，所述植物可为双子叶植物或单子叶植物。
上述提高植物产品品质的方法中，所述双子叶植物可为下述a)、b)、c)、d)、e)、f)、g)或h)：
a)葫芦科植物；b)西瓜；c)锦葵科植物；d)棉花；e)葡萄科植物；f) 葡萄；g)十字花科植物；h)萝卜。
为解决上述技术问题，本发明还提供了所述生物制剂在提高植物产品品质中的应用。
上述生物制剂在提高植物产品品质中的应用中，所述提高植物产品品质可为提高所述植物产品营养成分的含量和/或提高所述植物产品中微量元素的含量和/或提高所述植物产品的适口性。所述植物产品营养成分具体可为维生素C、可溶性糖和/或蛋白质。所述微量元素具体可为Cu、Fe、K、Mn和/或Zn。所述适口性可体现在干物率和/或粗纤维的含量下降上，或植物可食用部分的脆度上。
上述生物制剂在提高植物产品品质中的应用中，所述提高植物产品品质可为通过促进所述植物产品的成熟而提高所述植物产品的品质。在本发明的一个实施例中，VdAL的施用促进棉花产品的成熟，棉花的霜前花产量增加，进而提高棉花品质。
上述生物制剂在提高植物产品品质中的应用中，所述植物可为双子叶植物或单子叶植物。
上述生物制剂在提高植物产品品质中的应用中，所述双子叶植物可为下述a)、b)、c)、d)、e)、f)、g)或h)：
a)葫芦科植物；b)西瓜；c)锦葵科植物；d)棉花；e)葡萄科植物；f)葡萄；g)十字花科植物；h)萝卜。
上文中VdAL或所述生物制剂对不同植物的作用可因VdAL或所述生物制剂浓度的不同而出现不同的结果。在施用VdAL或所述生物制剂时，VdAL或所述生物制剂的浓度可以根据作物或其品种以及施用VdAL或所述生物制剂的目的(如提高所述目的植物产品中营养物质的含量)调节VdAL或所述生物制剂的浓度。
在本发明的实施例中，所述生物制剂稀释2000倍得到的液体可以提高西瓜中营养物质和微量元素的含量。所述生物制剂稀释3000-5000倍得到的液体可以提高棉花的衣分。所述生物制剂稀释3000倍得到的液体可以提高葡萄维生素C的含量。所述生物制剂稀释5000倍得到的液体可以提高罐萝卜的脆度和甜度。
上文中，所述萝卜可为罐萝卜。
本发明所要解决的第三个技术问题是如何提高植物的抗旱性或促进植物果实着色。
为解决上述技术问题，本发明首先提供了蛋白质在调控植物抗旱性或调控植物果实着色中的应用。
本发明所提供的蛋白质在调控植物抗旱性或调控植物果实着色中的应用中，所述蛋白质的名称为VdAL，为如下A1)或A2)或A3)：
所述蛋白质为如下A1)或A2)或A3)：
A1)氨基酸序列为序列1的蛋白质；
A2)在序列1的氨基酸序列中经过取代和/或缺失和/或添加一个或几个氨基酸残基得到的具有相同功能的由A1)衍生的蛋白质；
A3)在A1)或A2)的N端或/和C端连接标签得到的融合蛋白质。
其中，序列1由297个氨基酸组成。
为了使A1)中的蛋白质便于纯化，可在序列表中序列1所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。
上述A2)中的VdAL可人工合成，也可先合成其编码基因，再进行生物表达得到。上述A2)中的VdAL的编码基因可通过将序列表中序列2所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
上述VdAL在调控植物抗旱性或调控植物果实着色中的应用中，所述植物可为双子叶植物或单子叶植物。
为解决上述技术问题，本发明还提供了与VdAL相关的生物材料在调控植物抗旱性或调控植物果实着色中的应用。
本发明所提供的与VdAL相关的生物材料在调控植物抗旱性或调控植物果实着色中的应用；
所述生物材料，为下述B1)至B20)中的任一种：
B1)编码VdAL的核酸分子；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体；
B4)含有B2)所述表达盒的重组载体；
B5)含有B1)所述核酸分子的重组微生物；
B6)含有B2)所述表达盒的重组微生物；
B7)含有B3)所述重组载体的重组微生物；
B8)含有B4)所述重组载体的重组微生物；
B9)含有B1)所述核酸分子的转基因植物细胞系；
B10)含有B2)所述表达盒的转基因植物细胞系；
B11)含有B3)所述重组载体的转基因植物细胞系；
B12)含有B4)所述重组载体的转基因植物细胞系；
B13)含有B1)所述核酸分子的转基因植物组织；
B14)含有B2)所述表达盒的转基因植物组织；
B15)含有B3)所述重组载体的转基因植物组织；
B16)含有B4)所述重组载体的转基因植物组织；
B17)含有B1)所述核酸分子的转基因植物器官；
B18)含有B2)所述表达盒的转基因植物器官；
B19)含有B3)所述重组载体的转基因植物器官；
B20)含有B4)所述重组载体的转基因植物器官。
上述与VdAL相关的生物材料在调控植物抗旱性或调控植物果实着色中的应用中，B1)所述核酸分子为如下b1)或b2)或b3)的基因：
b1)核苷酸序列是序列表中序列2的cDNA分子或DNA分子；
b2)与b1)限定的核苷酸序列具有75％或75％以上同一性，且编码VdAL的cDNA分子或基因组DNA分子；
b3)在严格条件下与b1)限定的核苷酸序列杂交，且编码VdAL的cDNA分子或基因组DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
其中，序列2由894个核苷酸组成，编码序列1所示的蛋白质。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码VdAL的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明分离得到的VdAL的核苷酸序列75％或者更高同一性的核苷酸，只要编码VdAL且具有VdAL功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码VdAL所示的氨基酸序列组成的蛋白质的核苷酸序列具有75％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
上述与VdAL相关的生物材料在调控植物抗旱性或调控植物果实着色中的应用中，所述严格条件是在2×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次5min，又于0.5×SSC，0.1％SDS的溶液中，在68℃下杂交并洗膜2次，每次15min；或，0.1×SSPE(或0.1×SSC)、0.1％SDS的溶液中，65℃条件下杂交并洗膜。
上述75％或75％以上同一性，可为80％、85％、90％或95％以上的同一性。
上述与VdAL相关的生物材料在调控植物抗旱性或调控植物果实着色中的应用中，B2)所述的含有编码VdAL的核酸分子的表达盒(VdAL基因表达盒)，是指能够在宿主细胞中表达VdAL的DNA，该DNA不但可包括启动VdAL基因转录的启动子，还可包括终止VdAL基因转录的终止子。进一步，所述表达盒还可包括增强子序列。可用于本发明的启动子包括但不限于：组成型启动子，组织、器官和发育特异的启动子，和诱导型启动子。启动子的例子包括但不限于：花椰菜花叶病毒的组成型启动子35S：来自西红柿的创伤诱导型启动子，亮氨酸氨基肽酶("LAP",Chao等人(1999)Plant Physiol 120：979-992)；来自烟草的化学诱导型启动子，发病机理相关1(PR1)(由水杨酸和BTH(苯并噻二唑-7-硫代羟酸S-甲酯)诱导)；西红柿蛋白酶抑制剂II启动子(PIN2)或LAP启动子(均可用茉莉酮酸甲酯诱导)；热休克启动子(美国专利5,187,267)；四环素诱导型启动子(美国专利5,057,422)；种子特异性启动子，如谷子种子特异性启动子pF128(CN101063139B(中国专利200710099169.7))， 种子贮存蛋白质特异的启动子(例如，菜豆球蛋白、napin,oleosin和大豆beta conglycin的启动子(Beachy等人(1985)EMBO J.4：3047-3053))。它们可单独使用或与其它的植物启动子结合使用。此处引用的所有参考文献均全文引用。合适的转录终止子包括但不限于：农杆菌胭脂碱合成酶终止子(NOS终止子)、花椰菜花叶病毒CaMV 35S终止子、tml终止子、豌豆rbcS E9终止子和胭脂氨酸和章鱼氨酸合酶终止子(参见，例如：Odell等人(I985)Nature313:810；Rosenberg等人(1987)Gene,56:125；Guerineau等人(1991)Mol.Gen.Genet,262:141；Proudfoot(1991)Cell,64:671；Sanfacon等人Genes Dev.,5:141；Mogen等人(1990)Plant Cell,2:1261；Munroe等人(1990)Gene,91:151；Ballad等人(1989)Nucleic Acids Res.17:7891；Joshi等人(1987)Nucleic Acid Res.,15:9627)。
可用现有的表达载体构建含有所述VdAL基因表达盒的重组载体。所述植物表达载体包括双元农杆菌载体和可用于微弹轰击的植物表达载体等。如pAHC25、pBin438、pCAMBIA1302、pCAMBIA2301、pCAMBIA1301、pCAMBIA1300、pBI121、pCAMBIA1391-Xa或pCAMBIA1391-Xb(CAMBIA公司)等。所述植物表达载体还可包含外源基因的3′端非翻译区域，即包含聚腺苷酸信号和任何其它参与mRNA加工或基因表达的DNA片段。所述聚腺苷酸信号可引导聚腺苷酸加入到mRNA前体的3′端，如农杆菌冠瘿瘤诱导(Ti)质粒基因(如胭脂碱合成酶基因Nos)、植物基因(如大豆贮存蛋白基因)3′端转录的非翻译区均具有类似功能。使用本发明的基因构建植物表达载体时，还可使用增强子，包括翻译增强子或转录增强子，这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的，可以是天然的，也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。为了便于对转基因植物细胞或植物进行鉴定及筛选，可对所用植物表达载体进行加工，如加入可在植物中表达的编码可产生颜色变化的酶或发光化合物的基因(GUS基因、萤光素酶基因等)、抗生素的标记基因(如赋予对卡那霉素和相关抗生素抗性的nptII基因，赋予对除草剂膦丝菌素抗性的bar基因，赋予对抗生素潮霉素抗性的hph基因，和赋予对氨甲喋呤抗性的dhfr基因，赋予对草甘磷抗性的EPSPS基因)或是抗化学试剂标记基因等(如抗除莠剂基因)、提供代谢甘露糖能力的甘露糖-6-磷酸异构酶基因。从转基因植物的安全性考虑，可不加任何选择性标记基因，直接以逆境筛选转化植株。
上述与VdAL相关的生物材料在调控植物抗旱性或调控植物果实着色中的应用中，所述载体可为质粒、黏粒、噬菌体或病毒载体。所述质粒可为载体pET42a(+)。
上述与VdAL相关的生物材料在调控植物抗旱性或调控植物果实着色中的应用中，所述微生物可为酵母、细菌、藻或真菌，如大肠杆菌。所述大肠杆菌可 为大肠杆菌JM109。
上述与VdAL相关的生物材料在调控植物抗旱性或调控植物果实着色中的应用中，所述转基因植物细胞系、转基因植物组织和转基因植物器官既可均不包括繁殖材料，也可均包括繁殖材料。
上述与VdAL相关的生物材料在调控植物抗旱性或调控植物果实着色中的应用中，所述转基因植物器官也可为转基因植物的种子。所述转基因植物可包括种子、愈伤组织、完整植株和细胞。
在本发明的一个实施方式中，VdAL的编码基因通过含有VdAL的编码基因的表达盒的重组载体导入大肠杆菌JM109中得到重组微生物。所述重组载体为将载体pET42a(+)的NdeI和KpnI识别位点间的序列替换为序列表中序列2的第1-894位核苷酸所示的DNA分子(即VdAL基因)，得到的重组载体。所述重组载体表达序列1所示的VdAL。所述重组微生物表达序列1所示的VdAL。
上述与VdAL相关的生物材料在调控植物抗旱性或调控植物果实着色中的应用中，所述植物为单子叶植物或双子叶植物。
为解决上述技术问题，本发明还提供了一种提高植物抗旱性或促进植物果实着色的方法。
本发明所提供的一种提高植物抗旱性或促进植物果实着色的方法，包括对目的植物施用VdAL、所述生物材料或生物制剂，提高所述目的植物产品品质；所述生物制剂的活性成分为VdAL或所述生物材料。
上述提高植物抗旱性或促进植物果实着色的方法中，所述生物制剂可按照方法1)制备，所述方法1)为：培养所述重组微生物，使VdAL的编码基因表达，得到所述生物制剂。
上述提高植物抗旱性或促进植物果实着色的方法中，所述方法1)具体可为如下11)和12)：
11)培养所述重组微生物，使所述编码基因表达，得到表达所述蛋白质的重组微生物培养物；
12)破碎所述微生物培养物中的菌体，得到所述生物制剂。
上述提高植物抗旱性或促进植物果实着色的方法中，所述方法1)还可包括对所述生物制剂进行干燥。
上述提高植物抗旱性或促进植物果实着色的方法中，所述编码基因可为B1)所述核酸分子。
在本发明的一个实施方式中，所述重组微生物为将含有VdAL的编码基因的表达盒的重组载体导入大肠杆菌JM109中得到的重组微生物。所述重组载体为将载体pET42a(+)的NdeI和KpnI识别位点间的序列替换为序列表中序列2的第1-894位核苷酸所示的DNA分子(即VdAL基因)，得到的重组载体。所述重组载体表达序列1所示的VdAL。所述重组微生物表达序列1所示的VdAL。
上述提高植物抗旱性或促进植物果实着色的方法中，所述对目的植物施用 VdAL、所述生物材料或生物制剂具体可为对所述目的植物叶片施用VdAL、所述生物材料或所述生物制剂，如对目的植物喷施VdAL、所述生物材料或所述生物制剂，也可用VdAL、所述生物材料或所述生物制剂处理所述目的植物的种子，如用VdAL、所述生物材料或所述生物制剂的液体制剂对所述目的植物的种子进行浸种。VdAL、所述生物材料或所述生物制剂的液体制剂可为直接用清水溶解或悬浮VdAL、所述生物材料或所述生物制剂得到的液体。对目的植物施用VdAL、所述生物材料或生物制剂的量可以根据所述目的植物的种类和/或生长时期确定。
上述提高植物抗旱性或促进植物果实着色的方法中，所述植物为单子叶植物或双子叶植物。
为解决上述技术问题，本发明还提供了所述生物制剂在提高植物抗旱性或促进果实着色中的应用。
上述生物制剂在提高植物抗旱性或促进果实着色中的应用中，所述植物为单子叶植物或双子叶植物。
上文中VdAL或所述生物制剂对不同植物的作用可因VdAL或所述生物制剂浓度的不同而出现不同的结果。在施用VdAL或所述生物制剂时，VdAL或所述生物制剂的浓度可以根据作物或其品种以及施用VdAL或所述生物制剂的目的调节VdAL或所述生物制剂的浓度。
在本发明的实施例中，所述生物制剂在质量百分比含量为0.2-4％时可以提高小麦的抗旱性。所述生物制剂稀释3000-5000倍得到的液体可以促进樱桃或葡萄的着色。
附图说明
图1为维达利安外观及维达利安中VdAL的western-blot检测结果。其中，A为维达利安外观；B为维达利安中VdAL的western-blot检测结果，风机表示在样品干燥过程中进入出风口部位的较细粉末，主塔表示在样品干燥过程中的正常粉末。
图2为维达利安可以促进西瓜的营养生长。其中，CK表示未处理的西瓜，VdAL表示维达利安处理的西瓜。
图3为维达利安可以促进黄瓜座果与膨大。其中，CK表示未处理的黄瓜，300表示稀释300倍的VdAL处理的黄瓜，600表示稀释600倍的VdAL处理的黄瓜，900表示稀释900倍的VdAL处理的黄瓜。
图4为不同处理的黄瓜的长势。其中，CK表示未处理的黄瓜，300表示稀释300倍的VdAL处理的黄瓜。
图5为不同处理的黄瓜的衰老情况。其中，CK表示未处理的黄瓜，2000×表示稀释2000倍的VdAL处理的黄瓜。
图6为不同处理的圣女果的座果情况。其中，CK表示未处理的圣女果，2000×表示稀释2000倍的VdAL处理的圣女果。
图7为不同处理的草莓。其中，CK表示未处理的草莓，2000×表示稀释2000倍的VdAL处理的草莓。
图8为不同处理的甜瓜。其中，CK表示未处理的甜瓜，2000×表示稀释2000倍的VdAL处理的甜瓜。
图9为不同处理的甜椒。其中，CK表示未处理的甜椒，1000×表示稀释1000倍的VdAL处理的甜椒。
图10为不同处理的大豆种子的发芽情况。其中，CK表示未处理的大豆种子，1000表示稀释1000倍的VdAL处理的大豆种子，3000表示稀释3000倍的VdAL处理的大豆种子，5000表示稀释5000倍的VdAL处理的大豆种子，10000表示稀释10000倍的VdAL处理的大豆种子，20000表示稀释20000倍的VdAL处理的大豆种子。
图11为不同处理的大豆幼苗生长状况。其中，CK表示未处理的大豆幼苗，5000×表示稀释5000倍的VdAL处理的大豆幼苗。
图12为维达利安处理水稻后的亩产量。
图13为控水7天各组小麦的生长状况。
图14为各组小麦叶片中的叶绿素含量。
图15为各组小麦的复水指数。
图16为两种不同处理的葡萄。
图17为两种不同处理的樱桃。
图18为两种处理的罐萝卜的外观。
其中，1/300、1/600、1/900、1/1000、1/200、1/250、1/3000、1/4000、1/5000、1/6000、1/7500、1/10000、1/20000、1/40000倍的VdAL分别表示稀释300倍的VdAL、稀释600倍的VdAL、稀释900倍的VdAL、稀释1000倍的VdAL、稀释2000倍的VdAL、稀释2500倍的VdAL、稀释3000倍的VdAL、稀释4000倍的VdAL、稀释5000倍的VdAL、稀释6000倍的VdAL、稀释7500倍的VdAL、稀释10000倍的VdAL、稀释20000倍的VdAL和稀释40000倍的VdAL。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的大肠杆菌JM109为北京索莱宝科技有限公司产品，产品目录号为C1300。
下述实施例中的载体pET42a(+)为北京碧橙蓝生物科技有限公司产品，产品目录号为S18-16。
下述实施例中的新陆早58号为新疆锦棉种业科技股份有限公司产品，鲁30号、616、鲁25号和鲁研棉24号为山东农兴种业有限责任公司产品。
下述实施例中的西瓜品种金丽都为寿光积善商贸有限公司产品。
下述实施例中的中麦816为北京龙盛源科技发展有限公司产品。
下述实施例中的番茄品种富克斯为寿光市金鹏种业有限公司产品。
下述实施例中的华丽青梗菜F1为上海虹桥天龙种业有限公司产品。
下述实施例中的黄瓜品种津杂1号为山东农兴种业有限公司产品。
下述实施例中的圣女果品种京丹2号为北京北农绿亨科技发展有限公司产品。
下述实施例中的草莓品种丰香为北京北农绿亨科技发展有限公司产品。
下述实施例中的超级翠宝香甜瓜品种为济南睿袤农业科技开发有限公司产品。
下述实施例中的甜椒品种睿优816为济南睿袤农业科技开发有限公司产品。
下述实施例中的大豆品种中华大豆王为济南睿袤农业科技开发有限公司产品。
下述实施例中的水稻品种农大502为北京北农绿亨科技有限公司产品。
实施例1、维达利安的制备
维达利安由中国农业大学研制，由山东惠民湖浩生物科技有限公司开发生产。
1、重组载体及重组菌的构建
人工合成序列表中序列2的第1-894位核苷酸所示的DNA分子，即VdAL基因。将载体pET42a(+)的NdeI和KpnI识别位点间的序列替换为序列表中序列2的第1-894位核苷酸所示的DNA分子(即VdAL基因)，保持pET42a(+)的其他序列不变，得到重组载体，将得到的重组载体命名为pET42a-VdAL。重组载体pET42a-VdAL表达序列表中序列1所示的蛋白质VdAL。
其中，序列2由894个核苷酸组成，编码序列1所示的氨基酸序列。
将pET42a-VdAL导入大肠杆菌JM109中，得到重组菌，将得到的重组菌命名为JM109-pET42a-VdAL，JM109-pET42a-VdAL表达序列1所示的蛋白质。
2、维达利安的制备
将JM109-pET42a-VdAL在37℃条件下进行发酵至OD值为0.6得到预发酵液，向预发酵液中加入IPTG，使IPTG的浓度为1mM，得到诱导液，将诱导液在25℃条件下发酵6小时得到发酵液。将发酵液进行离心，弃上清液，将得到的菌体沉淀破碎，然后在150℃进行喷雾干燥得到干粉制剂(图1中A)，将其命名为维达利安。
利用western-blot的方法对维达利安中的VdAL进行检测，一抗为VdAL抗体(VdAL抗体为序列2所示的蛋白质为免疫原制备得到的多克隆抗体)，结果表明，维达利安中含有VdAL(图1中B)，维达利安中VdAL的含量为7.28mg/g。
用清水将维达利安溶解，分别得到维达利安浓度分别为10g/3kg、10g/6kg、10g/9kg、1g/kg、1g/2kg、1g/2.5kg、1g/3kg、1g/4kg、1g/5kg、1g/6kg、1g/7.5kg、1g/10kg、1g/20kg和1g/40kg的液体，将这些液体分别命名为稀释300倍的VdAL、 稀释600倍的VdAL、稀释900倍的VdAL、稀释1000倍的VdAL、稀释2000倍的VdAL、稀释2500倍的VdAL、稀释3000倍的VdAL、稀释4000倍的VdAL、稀释5000倍的VdAL、稀释6000倍的VdAL、稀释7500倍的VdAL、稀释10000倍的VdAL、稀释20000倍的VdAL和稀释40000倍的VdAL。
实施例2、维达利安可以促进棉花的生长
1、维达利安可以促进棉花种子的发芽
分别用实施例1的稀释5000倍的VdAL、稀释7500倍的VdAL和稀释10000倍的VdAL和清水浸泡新陆早58号种子24小时，在大花盆中进行发芽试验，温度为25℃，在播种第8天，统计棉花种子发芽率。每种处理100粒种子，实验重复三次。
结果显示，稀释5000倍的VdAL、稀释7500倍的VdAL和稀释10000倍的VdAL和清水处理棉花种子后棉花的平均发芽率分别为74.00％、75.50％、71.00％和70.23％，稀释5000倍的VdAL、稀释7500倍的VdAL和稀释10000倍的VdAL处理棉花种子后棉花的平均发芽率分别为清水处理棉花种子后棉花的平均发芽率的1.05倍、1.08倍、1.01倍，表明，维达利安可以提高棉花种子的发芽率，维达利安提高棉花种子发芽率的能力随维达利安浓度的变化而变化。
2、维达利安可以提高棉花的产量
实验一：
田间试验设计如下：实验采用随机区组设计，设置3个重复区，每个重复区随机设置九个处理区，分别为鲁30号VdAL处理1区、鲁30号VdAL处理2区、鲁30号对照处理区、616VdAL处理1区、616VdAL处理2区、616对照处理区、鲁25号VdAL处理1区、鲁25号VdAL处理2区、和鲁25号对照处理区，每个处理区的面积均为0.2亩。
在鲁30号VdAL处理1区的鲁30号的苗期和花铃前期分别按照下述方式进行处理：采用顶喷(机械喷施)的方式对棉花叶片喷施实施例1的稀释3000倍的VdAL，每亩喷施45-50kg，得到稀释3000倍的VdAL处理的鲁30号。苗期和花铃前期各喷施1次；喷施时间在早晨或傍晚，最好避开正午高温时段；喷施时严禁与药物及肥料混合；喷施后若2小时内下雨需要补喷施一次。
按照鲁30号VdAL处理1区的处理方法，将稀释3000倍的VdAL替换为稀释5000倍的VdAL对鲁30号VdAL处理2区进行处理，其他步骤均不变，得到稀释5000倍的VdAL处理的鲁30号。
按照鲁30号VdAL处理1区的处理方法，将稀释3000倍的VdAL替换为清水对鲁30号对照处理区进行处理，其他步骤均不变，得到未处理的鲁30号。
按照鲁30号VdAL处理1区的处理方法，分别对616VdAL处理1区和鲁25号VdAL处理1区进行处理，其他步骤均不变，分别得到稀释3000倍的VdAL处理的616、鲁25号。
按照鲁30号VdAL处理1区的处理方法，将稀释3000倍的VdAL替换为稀释5000倍的VdAL，分别对616VdAL处理2区和鲁25号VdAL处理2区进行处理，其他步骤均不变，分别得到稀释5000倍的VdAL处理的616、鲁25号。
按照鲁30号VdAL处理1区的处理方法，将稀释3000倍的VdAL替换为清水，分别对616对照处理区和鲁25号对照处理区进行处理，其他步骤均不变，分别得到未处理的616、鲁25号。
分别统计稀释3000倍的VdAL处理的鲁30号、稀释5000倍的VdAL处理的鲁30号、未处理的鲁30号、稀释3000倍的VdAL处理的616、稀释5000倍的VdAL处理的616、未处理的616、稀释3000倍的VdAL处理的鲁25号、稀释5000倍的VdAL处理的鲁25号和未处理的鲁25号的霜前花产量以、霜后花产量以及总产量，结果如表2所示。
表2、不同处理的棉花的平均产量
Figure PCTCN2016088074-appb-000001
注：处理1表示稀释3000倍的VdAL进行的处理，处理2表示稀释5000倍的VdAL进行的处理，CK表示未处理(即用清水进行的处理)。
结果显示，适当浓度的维达利安可以提高棉花的产量：用稀释3000倍的VdAL处理鲁30号，霜前花产量提高19.48％，总产量提高17.04％；用稀释5000倍的VdAL进行处理时，鲁30号霜前花产量提高21.25％，总产量提高20.00％，616霜前花产量提高19.06％，总产量提高19.69％，鲁25号霜前花产量提高16.37％，总产量提高17.60％。
实验二：
田间试验设计如下：实验采用随机区组设计，设置3个重复区，每个重复区随机设置四个处理区，分别为VdAL处理1区、VdAL处理2区、VdAL处理3区和对照处理区，每个处理区的面积均为10亩。
在VdAL处理1区的鲁研棉24号的苗期和花铃前期分别按照下述方式进行处 理：采用顶喷(机械喷施)的方式对棉花叶片喷施实施例1的稀释3000倍的VdAL，每亩喷施45-50kg，机车作业速度控制在时速6公里左右，得到稀释3000倍的VdAL处理的鲁研棉24号。苗期和花铃前期各喷施1次；喷施时间在早晨或傍晚，最好避开正午高温时段；喷施时严禁与药物及肥料混合；喷施后若2小时内下雨需要补喷施一次。
按照VdAL处理1区的处理方法，将稀释3000倍的VdAL替换为稀释5000倍的VdAL对VdAL处理2区进行处理，其他步骤均不变，得到稀释5000倍的VdAL处理的鲁研棉24号。
按照VdAL处理1区的处理方法，将稀释3000倍的VdAL替换为稀释10000倍的VdAL对VdAL处理3区进行处理，其他步骤均不变，得到稀释10000倍的VdAL处理的鲁研棉24号。
按照VdAL处理1区的处理方法，将稀释3000倍的VdAL替换为清水对对照处理区进行处理，其他步骤均不变，得到未处理的鲁研棉24号。
统计各处理的鲁研棉24号的每亩植株数、单株成铃数、亩总铃数、单铃重、衣分和亩产量，各项目的平均值以及与对照相比的增量如表3和表4所示。
表3、各处理的鲁研棉24号各项目的平均值和增量
Figure PCTCN2016088074-appb-000002
注：处理1表示稀释3000倍的VdAL进行的处理，处理2表示稀释5000倍的VdAL进行的处理，处理3表示稀释10000倍的VdAL进行的处理，CK表示未处理(即用清水进行的处理)。
表4、各处理的鲁研棉24号各项目的平均值和增量
Figure PCTCN2016088074-appb-000003
Figure PCTCN2016088074-appb-000004
注：处理1表示稀释3000倍的VdAL进行的处理，处理2表示稀释5000倍的VdAL进行的处理，处理3表示稀释10000倍的VdAL进行的处理，CK表示未处理(即用清水进行的处理)。
结果显示，适当浓度的维达利安可以提高棉花的每亩植株数、单株成铃数、亩总铃数、单铃重、衣分和亩产量：用稀释3000倍的VdAL处理时，单株成铃数、亩总铃数、衣分和亩产量均有增加，增加率分别为4.5％、1.98％、0.24％和1.76％；用稀释5000倍的VdAL处理时，每亩植株数、单株成铃数、亩总铃数、单铃重、衣分和亩产量均有增加，增加率分别为1.4％、3.41％、4.41％、1.9％、0.49％和5.22％；用稀释10000倍的VdAL处理时，各项目的增加量没有用稀释3000倍的VdAL处理和稀释5000倍的VdAL处理时各项目的增加量高。表明，施用适当浓度的维达利安可以提高棉花的品质和产量。
实施例3、维达利安可以提高西瓜的品质和产量
田间试验设计如下：实验采用随机区组设计，设置3个重复区，每个重复区随机设置2个处理区，分别为VdLA处理区和对照处理区，每个处理区的面积均为0.2亩。
在VdLA处理区的西瓜品种金丽都按照下述方式进行处理：采用顶喷(机械喷施)的方式对西瓜叶片喷施实施例1的稀释2000倍的VdLA，每亩喷施30kg，得到稀释2000倍的VdAL处理的西瓜。喷施从西瓜的幼苗期，并于第一次喷施15天后再喷施1次，共喷2次；喷施时间在早晨或傍晚，最好避开正午高温时段；喷施时最好不与药物及肥料混合；喷施后若2小时内下雨需要补喷施一次。
按照VdAL处理区的处理方法，将稀释2000倍的VdAL替换为清水对对照处理区进行处理，其他步骤均不变，得到未处理的西瓜。
结果发现，维达利安对西瓜的营养生长和座果具有促进作用(图2)。统计不同处理区的西瓜产量，结果发现，稀释2000倍的VdAL处理西瓜后单株结果数为1.43±0.5个，产量为5200.2±500kg/亩，而未处理的西瓜的单株结果数为1.21±0.5个，产量为3898.2±500kg/亩。与未处理的西瓜相比，维达利安处理的西瓜的单株结果数提高了18.2％，产量提高了33.4％。
在西瓜成熟期分别测定不同处理西瓜的含水量、干物率、Vc含量、可溶性糖含量、可滴定酸含量、可溶性固形物含量、瓜皮厚度、蛋白质含量、粗纤维含量(表5)以及微量元素Ca、Cu、Fe、K、Mg、Mn、Na、P和Zn的含量(表6)。其中，含水量与干物率(西瓜中干物质占西瓜鲜重的百分比)均将西瓜干燥后进行测定，Vc含量按照2,6-二氯靛酚滴定法进行测定，可溶性糖含量按照蒽酮比色法进行测定，可滴定酸含量按照比色法进行测定，可溶性固形物含量、蛋白质含量按照分光光度计量方法进行测定，粗纤维含量按照温德法检测粗纤维方法进 行测定，微量元素Ca、Cu、Fe、K、Mg、Mn、Na、P和Zn的含量均利用微量元素分析仪进行测定。
表5、不同处理西瓜各指标的平均含量
指标 未处理 VdLA处理
含水量(％) 90.22 91.43
干物率(％) 9.78 8.57
Vc(mg/100g) 12.1 18.2
可溶性糖(％) 6.83 7.44
可滴定酸(％) 0.09 0.09
可溶性固形物(％) 9.1 9.1
瓜皮厚(cm) 0.97 1.23
蛋白质(％DW) 5.54 6.33
蛋白质(％FW) 0.54 0.54
粗纤维(％DW) 2.06 2.02
粗纤维(％FW) 0.2 0.17
表6、不同处理西瓜各微量元素的平均含量
编号 指标 未处理 VdAL处理
1 含水率(％) 90.22 91.43
2 Ca(mg/L) 20.13 22.70
3 Ca含量(mg/100gFw) 9.83 9.71
4 Cu(mg/L) 0.13 0.15
5 Cu含量(mg/100gFw) 0.06 0.06
6 Fe(mg/L) 0.50 0.60
7 Fe含量(mg/100gFw) 2.50 2.98
8 K(mg/L) 158.00 184.00
9 K含量(mg/100gFw) 77.19 78.64
10 Mg(mg/L) 22.27 22.50
11 Mg含量(mg/100gFw) 10.88 9.63
12 Mn(mg/L) 0.10 0.12
13 Mn含量(mg/100gFw) 0.05 0.05
14 Na(mg/L) 7.20 7.35
15 Na含量(mg/100gFw) 3.52 3.14
16 P(mg/L) 18.30 20.13
17 P含量(mg/100gFw) 8.93 8.65
18 Zn(mg/L) 0.25 0.30
19 Zn含量(mg/100gFw) 0.12 0.13
注：3、5、7、9、11、13、15、17和19行中各微量元素的含量均指每升新鲜西瓜中各微量元素的质量，2、4、6、8、10、12、14、16和18行中各微量元素的含量均指新鲜西瓜中各微量元素的含量。
结果显示，维达利安处理后，西瓜的含水量略有增加，增加了1.34％；Vc含量显著提高，提高了50.41％；可溶性糖含量提高了8.88％，干物质中蛋白质含量提高了14.2％，新鲜西瓜中蛋白质含量提高了0.62％，可滴定酸和可溶性固形物的含量均没有变化，而干物率下降12.4％，粗纤维的含量有少量下降。表明，维达利安处理后，西瓜中的主要营养成分含量增加；而酸度没有明显增加，干物率和粗纤维的含量均有下降，提高西瓜的适口性。维达利安处理后，每升新鲜西瓜的Ca、Cu、Fe、K、Mg、Mn、Na、P和Zn的质量均有增加，增加量分别为12.75％、16.54％、19.21％、16.46％、1.05％、20.45％、2.13％、10.02％、19.73％；新鲜西瓜中Cu、Fe、K、Mn和Zn的质量含量均有增加，增加量分别为2.09％、19.09％、1.88％、5.72％和5.07％。表明，维达利安可以显著提高西瓜中对人体有益微量元素Cu、Fe、K、Mn和Zn的含量。以上实验证明，维达利安可以提高西瓜的品质。
实施例4、维达利安可以促进小麦的生长、提高小麦的产量
1、维达利安可以促进小麦的发芽与营养生长
实验一：
将中麦816种子分别在实施例1的稀释1000倍的VdAL、稀释2000倍的VdAL、稀释5000倍的VdAL、稀释10000倍的VdAL和清水中浸种12小时，然后在发芽盒(发芽盒中铺有两层用无菌水浸湿的无菌滤纸)中于25℃下催芽96小时，统计小麦种子发芽率(小麦种子发芽标准为：种子芽长为或超过种子长度的一半，并且根长为或超过种子长度)。每种处理100粒种子，实验重复三次。
结果显示，对照与稀释1000倍的VdAL、稀释2000倍的VdAL、稀释5000倍的VdAL、稀释10000倍的VdAL和清水处理的小麦种子的发芽率分别为95.31±1％、83.26±1％、92.51±1％、97.67±1％和99.50±1％，稀释1000倍的VdAL、稀释2000倍的VdAL、稀释5000倍的VdAL、稀释10000倍的VdAL处理小麦种子后，发芽率分别提高了-12.64％、-0.00％、2.48％和4.40％。表明，适宜浓度的维达利安可以促进小麦种子的发芽。
实验二：
将中麦816种子分别在实施例1的稀释5000倍的VdAL、稀释10000倍的VdAL和清水中浸种24小时，然后播种于土壤中，将播种当天记为播种第1天，在播种第3天将小麦幼苗从土壤中取出，称量小麦幼苗鲜重。
结果显示，用稀释5000倍的VdAL、稀释10000倍的VdAL和清水处理的小麦的单株鲜重分别为0.128±0.022g、0.141±0.022g和0.111±0.022g，稀释5000倍的VdAL和稀释10000倍的VdAL浸种后，小麦单株鲜重分别提高了15.32％和27.03％，稀释10000倍的VdAL浸种后的单株鲜重显著高于清水浸种后的小麦单株鲜重。表明，维达利安可以促进小麦的营养生长。
2、维达利安可以提高小麦的产量
田间试验设计如下：实验采用随机区组设计，设置3个重复区，每个重复区随机设置五个处理区，分别为VdAL处理1区、VdAL处理2区、VdAL处理3区、VdAL处理4区和对照处理区，每个处理区的面积均为0.5亩。
在VdAL处理1区的中麦816的扬花期按照下述方式进行对小麦处理1次：采用顶喷(人工喷施)的方式对小麦叶片喷施实施例1的稀释2500倍的VdAL，每亩喷施45-50kg，得到稀释2500倍的VdAL处理的小麦。喷施时间在早晨或傍晚，最好避开正午高温时段；喷施时严禁与药物及肥料混合；喷施后若2小时内下雨需要补喷施一次。
按照VdAL处理1区的处理方法，将稀释2500倍的VdAL替换为稀释5000倍的VdAL对VdAL处理2区进行处理，其他步骤均不变，得到稀释5000倍的VdAL处理的小麦。
按照VdAL处理1区的处理方法，将稀释2500倍的VdAL替换为稀释10000倍的VdAL对VdAL处理3区进行处理，其他步骤均不变，得到稀释10000倍的VdAL处理的小麦。
按照VdAL处理1区的处理方法，将稀释2500倍的VdAL替换为稀释20000倍的VdAL对VdAL处理4区进行处理，其他步骤均不变，得到稀释20000倍的VdAL处理的小麦。
按照VdAL处理1区的处理方法，将稀释2500倍的VdAL替换为清水对对照处理区进行处理，其他步骤均不变，得到未处理的小麦。
统计各处理的小麦产量，结果显示，稀释2500倍的VdAL处理小麦后小麦的产量没有提高，产量为8679kg/ha，稀释5000倍的VdAL、稀释10000倍的VdAL、稀释20000倍的VdAL和清水处理的小麦的平均产量分别为8942kg/ha、9485kg/ha、9231kg/ha和8772kg/ha，稀释5000倍的VdAL、稀释10000倍的VdAL、稀释20000倍的VdAL处理小麦后，产量分别提高了1.94％、8.13％、5.23％。
统计不同处理的小麦的千粒重、有效穗数、穗粒数和穗长，结果如表7所示。结果显示，稀释5000倍的VdAL处理小麦后，有效穗数增加了1.5％，穗粒数增加了0.5％；稀释10000倍的VdAL，有效穗数增加了7％，穗粒数增加了3.5％；稀释20000倍的VdAL，有效穗数增加了2.3％，穗粒数增加了3.7％。
表7、不同处理的小麦的千粒重、有效穗数、穗粒数和穗长的平均值
Figure PCTCN2016088074-appb-000005
Figure PCTCN2016088074-appb-000006
注：处理1表示稀释20000倍的VdAL进行的处理，处理2表示稀释10000倍的VdAL进行的处理，处理3表示稀释5000倍的VdAL进行的处理，处理4表示稀释2500倍的VdAL进行的处理，CK表示未处理(即用清水进行的处理)。
实施例5、维达利安可以增加番茄单株结果数
田间试验设计如下：实验采用随机区组设计，设置3个重复区，每个重复区随机设置三个处理区，分别为VdAL处理1区、VdAL处理2区和对照处理区，每个处理区的面积均为0.1亩。
在VdAL处理1区的番茄品种富克斯的开花初期和第一次处理后第15天分别按照下述方式喷施VdAL：采用顶喷(人工喷施)的方式对番茄叶片喷施实施例1的稀释1000倍的VdAL，每亩喷施45-50kg得到稀释1000倍的VdAL处理的番茄，将第1次喷施当天记为喷施后第0天。喷施时间在早晨或傍晚，最好避开正午高温时段；喷施时严禁与药物及肥料混合；喷施后若2小时内下雨需要补喷施一次。
按照VdAL处理1区的处理方法，将稀释1000倍的VdAL替换为稀释3000倍的VdAL对VdAL处理2区进行处理，其他步骤均不变，得到稀释3000倍的VdAL处理的番茄。
按照VdAL处理1区的处理方法，将稀释1000倍的VdAL替换为清水对对照处理区进行处理，其他步骤均不变，得到未处理的番茄。
在喷施后第20天统计番茄的单株结果数，结果显示，稀释1000倍的VdAL、稀释3000倍的VdAL和清水处理番茄后番茄的平均单株结果数分别为34.9个/株、40.4个/株和33.3个/株，稀释1000倍的VdAL和稀释3000倍的VdAL处理番茄后，番茄的单株结果数分别提高了4.8％和21.3％。表明，维达利安可以促进番茄结果。
实施例6、维达利安可以促进青梗菜的生长
田间试验设计如下：实验采用随机区组设计，设置3个重复区，每个重复区随机设置五个处理区，分别为VdAL处理1区、VdAL处理2区、VdAL处理3区、VdAL处理4区和对照处理区，每个处理区的面积均为0.2亩。
在VdAL处理1区的华丽杂交青梗菜的两叶一心期，按照下述方式VdAL喷施1次：采用顶喷(人工喷施)的方式对番茄叶片喷施实施例1的稀释2000倍的 VdAL，每亩喷施45-50kg得到稀释2000倍的VdAL处理的青梗菜，将维达利安喷施当天记为处理第0天。喷施时间在早晨或傍晚，最好避开正午高温时段；喷施时严禁与药物及肥料混合；喷施后若2小时内下雨需要补喷施一次。
按照VdAL处理1区的处理方法，将稀释2000倍的VdAL替换为稀释2500倍的VdAL对VdAL处理2区进行处理，其他步骤均不变，得到稀释2500倍的VdAL处理的青梗菜。
按照VdAL处理1区的处理方法，将稀释2000倍的VdAL替换为稀释3000倍的VdAL对VdAL处理3区进行处理，其他步骤均不变，得到稀释3000倍的VdAL处理的青梗菜。
按照VdAL处理1区的处理方法，将稀释2000倍的VdAL替换为稀释4000倍的VdAL对VdAL处理4区进行处理，其他步骤均不变，得到稀释4000倍的VdAL处理的青梗菜。
按照VdAL处理1区的处理方法，将稀释2000倍的VdAL替换为清水对对照处理区进行处理，其他步骤均不变，得到未处理的青梗菜。
在处理第20天，分别统计不同处理的50株青梗菜的鲜重，结果显示，稀释2000倍的VdAL、稀释2500倍的VdAL、稀释3000倍的VdAL、稀释4000倍的VdAL和清水处理后平均50株青梗菜鲜重分别为2.960kg、3.270kg、3.835kg、3.895kg和1.912kg，稀释2000倍的VdAL、稀释2500倍的VdAL、稀释3000倍的VdAL和稀释4000倍的VdAL处理青梗菜后，50株青梗菜鲜重分别提高了54.8％、71.0％、100.6％和103.7％。表明，维达利安可以促进青梗菜的生长。
实施例7、维达利安在其他作物中的作用
1、维达利安可以促进黄瓜生长、提高产量、延缓衰老
田间试验设计如下：实验采用随机区组设计，设置3个重复区，每个重复区随机设置五个处理区，分别为VdAL处理1区、VdAL处理2区、VdAL处理3区、VdAL处理4区和对照处理区，每个处理区的面积均为0.1亩。
在VdAL处理1区的黄瓜品种津杂1号的开花初期和第一次处理后第15天分别按照下述方式喷施VdAL：采用顶喷(人工喷施)的方式对黄瓜叶片喷施实施例1的稀释300倍的VdAL，每亩喷施45-50kg，得到稀释300倍的VdAL处理的黄瓜，将第1次喷施维达利安喷施当天记为喷施第0天。喷施时间在早晨或傍晚，最好避开正午高温时段；喷施时严禁与药物及肥料混合；喷施后若2小时内下雨需要补喷施一次。
按照VdAL处理1区的处理方法，将稀释300倍的VdAL替换为稀释600倍的VdAL对VdAL处理2区进行处理，其他步骤均不变，得到稀释600倍的VdAL处理的黄瓜。
按照VdAL处理1区的处理方法，将稀释300倍的VdAL替换为稀释900倍的VdAL对VdAL处理3区进行处理，其他步骤均不变，得到稀释900倍的VdAL处 理的黄瓜。
按照VdAL处理1区的处理方法，将稀释300倍的VdAL替换为稀释2000倍的VdAL对VdAL处理4区进行处理，其他步骤均不变，得到稀释2000倍的VdAL处理的黄瓜。
按照VdAL处理1区的处理方法，将稀释300倍的VdAL替换为清水对对照处理区进行处理，其他步骤均不变，得到未处理的黄瓜。
在喷施第20天，统计黄瓜的结果情况，结果发现，维达利安可以提高黄瓜的座果率、促进黄瓜的膨大，提高黄瓜产量(图3)，表明，维达利安可以促进黄瓜的营养生长和生殖生长。在喷施第20天，观察黄瓜的长势，结果发现，维达利安处理的黄瓜的长势好于未处理的黄瓜的长势(图4)，表明，维达利安可以促进黄瓜的营养生长。在喷施第45天，观察黄瓜的衰老情况，结果发现，未处理的黄瓜的衰老情况比维达利安处理的黄瓜的衰老情况严重(图5)，表明，维达利安可以延缓黄瓜的衰老。
2、维达利安可以提高圣女果的座果率、提高产量
田间试验设计如下：实验采用随机区组设计，设置3个重复区，每个重复区随机设置两个处理区，分别为VdAL处理区和对照处理区，每个处理区的面积均为1亩。
对VdAL处理区的圣女果品种京丹2号的开花期按照下述方式VdAL喷施1次：采用顶喷(人工喷施)的方式对圣女果叶片喷施实施例1的稀释2000倍的VdAL，每亩喷施45-50kg，得到稀释2000倍的VdAL处理的圣女果，将维达利安喷施当天记为处理第0天。喷施时间在早晨或傍晚，最好避开正午高温时段；喷施时严禁与药物及肥料混合；喷施后若2小时内下雨需要补喷施一次。
按照VdAL处理区的处理方法，将稀释2000倍的VdAL替换为清水对对照处理区进行处理，其他步骤均不变，得到未处理的圣女果。
在处理第7-10天，观察圣女果的结果情况，结果发现，维达利安可以提高圣女果的座果率，提高圣女果产量(图6)，表明，维达利安可以促进圣女果的生长。
3、维达利安可以促进草莓的生长
田间试验设计如下：品种为丰香，实验采用随机区组设计，设置3个重复区，每个重复区随机设置两个处理区，分别为VdAL处理区和对照处理区，每个处理区的面积均为1亩。
对VdAL处理区的丰香草莓品种在开花期按照下述方式VdAL喷施1次：采用顶喷(人工喷施)的方式对草莓叶片喷施实施例1的稀释2000倍的VdAL，每亩喷施45-50kg，得到稀释2000倍的VdAL处理的草莓，将维达利安喷施当天记为处理第0天。喷施时间在早晨或傍晚，最好避开正午高温时段；喷施时严禁与药物及肥料混合；喷施后若2小时内下雨需要补喷施一次。
按照VdAL处理区的处理方法，将稀释2000倍的VdAL替换为清水对对照处 理区进行处理，其他步骤均不变，得到未处理的草莓。
在处理第7-10天，观察草莓的生长情况，结果发现，维达利安可以促进草莓的营养生长(图7)。
4、维达利安可以促进甜瓜的生长
田间试验设计如下：甜瓜品种为超级翠宝香，实验采用随机区组设计，设置3个重复区，每个重复区随机设置两个处理区，分别为VdAL处理区和对照处理区，每个处理区的面积均为1亩。
对VdAL处理区的超级翠宝香甜瓜品种，在苗期按照下述方式VdAL喷施1次：采用顶喷(人工喷施)的方式对甜瓜叶片喷施实施例1的稀释2000倍的VdAL，每亩喷施45-50kg，得到稀释2000倍的VdAL处理的甜瓜，将维达利安喷施当天记为处理第0天。喷施时间在早晨或傍晚，最好避开正午高温时段；喷施时严禁与药物及肥料混合；喷施后若2小时内下雨需要补喷施一次。
按照VdAL处理区的处理方法，将稀释2000倍的VdAL替换为清水对对照处理区进行处理，其他步骤均不变，得到未处理的甜瓜。
在处理第30天，观察甜瓜的生长情况，结果发现，维达利安可以促进甜瓜的营养生长(图8)。
5、维达利安可以促进甜椒的生长
田间试验设计如下：实验采用随机区组设计，设置3个重复区，每个重复区随机设置两个处理区，分别为VdAL处理区和对照处理区，每个处理区的面积均为1亩。
在VdAL处理区的甜椒品种睿优816的开花期按照下述方式VdAL喷施1次：采用顶喷(人工喷施)的方式对甜椒叶片喷施实施例1的稀释1000倍的VdAL，每亩喷施45-50kg，得到稀释1000倍的VdAL处理的甜椒，将维达利安喷施当天记为处理第0天。喷施时间在早晨或傍晚，最好避开正午高温时段；喷施时严禁与药物及肥料混合；喷施后若2小时内下雨需要补喷施一次。
按照VdAL处理区的处理方法，将稀释1000倍的VdAL替换为清水对对照处理区进行处理，其他步骤均不变，得到未处理的甜椒。
在处理第7-10天，观察甜椒的生长情况，结果发现，喷施维达利安后，甜椒长势茁壮、叶片深绿(图9)，表明维达利安可以促进甜椒的营养生长。
6、维达利安可以促进大豆的出苗与生长
将大豆品种中华大豆王种子分别在实施例1的稀释1000倍的VdAL、稀释3000倍的VdAL、稀释5000倍的VdAL、稀释10000倍的VdAL、稀释20000倍的VdAL和清水中浸种24小时，然后在托盘(托盘中铺有两层用无菌水浸湿的无菌滤纸)中于25℃下催芽72小时，统计大豆种子发芽率。每种处理100粒种子，实验重复三次。
结果显示(图10)，稀释3000倍的VdAL和稀释5000倍的VdAL大豆种子后，发芽率均有提高。表明，维达利安可以促进大豆种子的发芽。
实验二：
将大豆品种中华大豆王种子分别在实施例1的稀释5000倍的VdAL和清水中浸种24小时，然后播种于土壤中，将播种当天记为播种第1天，在播种第20天将大豆幼苗从土壤中取出，称量大豆幼苗鲜重(图11)。
结果显示，用稀释5000倍的VdAL和清水处理的大豆的单株鲜重分别为1.59±0.3g和1.36±0.3g，稀释5000倍的VdAL浸种后，大豆单株鲜重提高了16.91％；用稀释5000倍的VdAL和清水处理的大豆的地上部分单株鲜重分别为1.36±0.3g和1.06±0.3g，稀释5000倍的VdAL浸种后，大豆地上部分单株鲜重提高了28.30％，显著高于清水处理的大豆的地上部分单株鲜重。表明，维达利安可以促进大豆的营养生长。
实施例8、维达利安可以促进水稻的生长、提高水稻产量
田间试验设计如下：实验采用随机区组设计，设置3个重复区，每个重复区随机设置四个处理区，分别为VdAL处理1区、VdAL处理2区、VdAL处理3区和对照处理区，每个处理区的面积均为0.1亩。
对于VdAL处理1区的农大502，在扬花期后，按照下述方式进行对水稻处理1次：采用顶喷(人工喷施)的方式对水稻叶片喷施实施例1的稀释10000倍的VdAL，每亩喷施45-50kg，得到稀释10000倍的VdAL处理的水稻(1/10000)。喷施时间在早晨或傍晚，最好避开正午高温时段；喷施时严禁与药物及肥料混合；喷施后若2小时内下雨需要补喷施一次。
按照VdAL处理1区的处理方法，将稀释10000倍的VdAL替换为稀释20000倍的VdAL对VdAL处理2区进行处理，其他步骤均不变，得到稀释20000倍的VdAL处理的水稻(1/20000)。
按照VdAL处理1区的处理方法，将稀释10000倍的VdAL替换为稀释40000倍的VdAL对VdAL处理3区进行处理，其他步骤均不变，得到稀释40000倍的VdAL处理的水稻(1/40000)。
按照VdAL处理1区的处理方法，将稀释10000倍的VdAL替换为清水对对照处理区进行处理，其他步骤均不变，得到未处理的水稻(CK)。
统计各处理的水稻产量，结果(图12)显示，稀释10000倍的VdAL、稀释20000倍的VdAL、稀释40000倍的VdAL和清水处理的水稻的平均产量分别为447.29kg/亩、408.35kg/亩、390.14kg/亩和398.37kg/亩，稀释10000倍的VdAL、稀释20000倍的VdAL处理水稻后，产量分别提高了12.28％及2.51％。
实施例9、维达利安可以提高小麦的抗旱性
实验重复三次，每次重复实验的具体步骤如下：
实验一：
将中麦816种子分为4组，即组1、组2、组3和组4，每组50粒种子。
向质量百分比浓度为6％的福戊(北农(海利)涿州种衣剂有限公司)中加入实施例1的维达利安，得到维达利安质量百分比浓度为2％的液体，将该液体命名为2.0％+福戊。利用2.0％+福戊按照如下方式处理组1小麦种子：用2.0％+福戊100-200g，兑水1.5-2kg后搅拌均匀，拌种子100kg，充分拌匀并阴干后播种。然后将组1的小麦种子播种于土壤中，在小麦三叶期时，对小麦停止浇水(即控水处理)。
向质量百分比浓度为6％的福戊中加入实施例1的维达利安，得到维达利安质量百分比浓度为1％的液体，将该液体命名为1.0％+福戊。利用1.0％+福戊按照如下方式处理组2小麦种子：1.0％+福戊100-200g，兑水1.5-2kg后搅拌均匀，拌种100kg，充分拌匀并阴干后播种。然后将组2的小麦种子播种于土壤中，在小麦三叶期时，对小麦停止浇水(即控水处理)。
利用1.0％+福戊按照如下方式处理组3小麦种子：将北农(海利)涿州种衣剂有限公司的质量百分比浓度为6％福戊100-200g，兑水1.5-2kg搅拌均匀，拌种100kg，充分拌匀并阴干后播种。然后将组3的小麦种子播种于土壤中，在小麦三叶期时，对小麦停止浇水(即控水处理)。
利用清水按照如下方式处理组4小麦种子，作为对照(CK)：用水1.5-2kg拌种100kg，充分拌匀后阴干后播种。然后将组4的小麦种子播种于土壤中，在小麦三叶期时，对小麦停止浇水(即控水处理)。
将停止浇水的第1天记为控水1天。结果显示，将维达利安添加在种衣剂中处理小麦种子，可以显著提高小麦的耐旱性：组1小麦在控水20天全部枯死，组2小麦在控水15天全部枯死，组3小麦在控水10天全部枯死，组4小麦在控水12天全部枯死，组1小麦全部枯死的时间均分别显著大于组3和组4小麦全部枯死的时间，组2小麦全部枯死的时间均分别显著大于组3和组4小麦全部枯死的时间。控水7天各组小麦的生长状况如图13所示。
实验二：
将中麦816种子分为6组，即组1、组2、组3、组4、组5和组6，每组50粒种子。
利用清水按照如下方式处理组1小麦种子，作为对照(CK1)：用清水1.5-2kg拌种100kg，充分拌匀并阴干后播种。然后将组1的小麦种子播种于土壤中，在3叶期时，对小麦停止浇水，停止浇水7后，对小麦进行复水处理。
向质量百分比浓度为6％的福戊中加入实施例1的维达利安，得到维达利安质量百分比浓度为0.2％的液体，将该液体命名为0.2％+福戊。利用0.2％+福戊按照如下方式处理组2小麦种子：用0.2％+福戊100-200g，兑水1.5-2kg后搅拌均匀，拌种100kg，充分拌匀阴干后播种。然后将组2的小麦种子播种于土壤中，在3叶期时，对小麦停止浇水，停止浇水7后，对小麦进行复水处理。
向质量百分比浓度为6％的福戊中加入实施例1的维达利安，得到维达利安质量百分比浓度为1％的液体，将该液体命名为1.0％+福戊。利用1.0％+福戊按照如 下方式处理组3小麦种子：1.0％+福戊100-200g，兑水1.5-2kg搅拌均匀，拌种100kg，充分拌匀阴干后播种。然后将组3的小麦种子播种于土壤中，在3叶期时，对小麦停止浇水，停止浇水7后，对小麦进行复水处理。
向质量百分比浓度为6％的福戊中加入实施例1的维达利安，得到维达利安质量百分比浓度为2％的液体，将该液体命名为2.0％+福戊。利用2.0％+福戊按照如下方式处理组4小麦种子：2.0％+福戊100-200g，兑水1.5-2kg搅拌均匀，拌种100kg，充分拌匀阴干后播种。然后将组4的小麦种子播种于土壤中，在3叶期时，对小麦停止浇水，停止浇水7后，对小麦进行复水处理。
向质量百分比浓度为6％的福戊中加入实施例1的维达利安，得到维达利安质量百分比浓度为4％的液体，将该液体命名为4.0％+福戊。利用4.0％+福戊按照如下方式处理组5小麦种子：4.0％+福戊100-200g，兑水1.5-2kg搅拌均匀，拌种100kg，充分拌匀后阴干后播种。然后将组5的小麦种子播种于土壤中，在3叶期时，对小麦停止浇水，停止浇水7后，对小麦进行复水处理。
利用6％福戊按照如下方式处理组6小麦种子，作为对照(CK2)：将北农(海利)涿州种衣剂有限公司的质量百分比浓度为6％福戊100-200g，兑水1.5-2kg搅拌均匀，拌种100kg，充分拌匀阴干后播种。然后将组6的小麦种子播种于土壤中，在3叶期时，对小麦停止浇水，停止浇水7后，对小麦进行复水处理。
在各组停止浇水处理当天，利用SPAD-502Plus(北京博伦经纬科技发展有限公司)测定各组小麦叶片中的叶绿素含量，结果如图14所示，图14中，1表示组1,2表示组2,3表示组3,4表示组4，5表示组5，6表示组6，纵坐标的单位为SPAD。组1、组2、组3、组4、组5和组6小麦的平均叶绿素含量分别为34.1SPAD、34.5SPAD、34.5SPAD、34.4SPAD、33.6SPAD、33.9SPAD，组2、组3和组4小麦的叶绿素含量均显著高于组1和组6，而组5的叶绿素含量低于组1和组6，表明，适当浓度的维达利安可以提高小麦的叶绿素含量。
在各组复水处理第4天计算复水指数，复水指数＝(1×I级株数+2×II级株数+3×III级株数+4×IV级株数)/(4×调查株数)。
植株分级的标准如下：
0级：恢复浇水后，单株叶片卷曲的面积为100％，叶片没有变绿；
I级：恢复浇水后，单株叶片卷曲的面积为100％，但叶片开始变绿；
II级：恢复浇水后，单株叶片卷曲的面积大于等于50％小于100％；
III级：恢复浇水后，单株叶片卷曲的面积大于0％小于50％；
IV级：恢复浇水后，单株叶片卷曲的面积为0％。
结果如图15所示，图15中，1表示组1,2表示组2,3表示组3,4表示组4，5表示组5，6表示组6。组1(CK1)、组2、组3、组4、组5和组6(CK2)小麦的平均复水指数分别为0.348、0.516、0.523、0.43、0.219、0.323，组2、组3和组4小麦的复水指数均显著高于组1和组6，而组5的复水指数低于组1和组6，表明，适当浓度的维达利安可以提高小麦的抗旱性。
实施例10、维达利安可以提高葡萄品质
田间试验设计如下：实验地点为甘肃敦煌，实验采用随机区组设计，设置3个重复区，每个重复区随机设置两个处理区，分别为VdAL处理区和对照处理区，每个处理区的面积均为0.1亩。
对VdAL处理区的葡萄品种红提(甘肃省永登永盛红提葡萄专业合作社)在开花期按照下述方式进行对葡萄处理1次：采用顶喷(人工喷施)的方式对葡萄叶片喷施实施例1的稀释3000倍的VdAL，每亩喷施45-50kg，得到稀释3000倍的VdAL处理的葡萄(1/3000)。喷施时间在早晨或傍晚，最好避开正午高温时段；喷施时严禁与药物及肥料混合；喷施后若2小时内下雨需要补喷施一次。
按照VdAL处理区的处理方法，将稀释3000倍的VdAL替换为清水对对照处理区进行处理，其他步骤均不变，得到未处理的葡萄(对照，CK)。
统计各处理的葡萄成熟时间，结果显示，稀释3000倍的VdAL和清水处理的葡萄的平均成熟时间分别为9月15日(距离开花期85天，或距离VdAL处理75天)和9月20日(距离开花期90天，或距离清水处理80天)，稀释3000倍的VdAL处理葡萄后，葡萄成熟时间提前了5天。
按照2,6-二氯靛酚滴定法测定成熟时葡萄的Vc含量，结果发现，稀释3000倍的VdAL和清水处理的葡萄的平均Vc含量分别为18.20mg/100g鲜重和12.10mg/100g鲜重，稀释3000倍的VdAL处理葡萄后，葡萄Vc含量提高了50.4％。
观察成熟葡萄，发现，喷施VdAL后的葡萄色泽更艳丽、果穗紧凑不易掉粒(图16)。
实施例11、维达利安可以促进樱桃的成熟与着色，提高樱桃产量
田间试验设计如下：实验地点为烟台市福山区，实验采用随机区组设计，设置3个重复区，每个重复区随机设置两个处理区，分别为VdAL处理区和对照处理区，每个处理区的面积均为0.1亩。
在VdAL处理区的樱桃品种龙冠(烟台市栖霞群利果品苗木专业合作社)的开花期按照下述方式进行对樱桃处理1次：采用顶喷(人工喷施)的方式对樱桃叶片喷施实施例1的稀释5000倍的VdAL，每亩喷施45-50kg，得到稀释5000倍的VdAL处理的樱桃(1/5000)。喷施时间在早晨或傍晚，最好避开正午高温时段；喷施时严禁与药物及肥料混合；喷施后若2小时内下雨需要补喷施一次。
按照VdAL处理区的处理方法，将稀释5000倍的VdAL替换为清水对对照处理区进行处理，其他步骤均不变，得到未处理的樱桃(对照，CK)。
统计各处理的樱桃成熟时间，结果显示，稀释5000倍的VdAL和清水处理的樱桃的平均成熟时间分别为5月1日(距离开花期距离VdAL处理52天)和5月6日(距离VdAL处理57天)，稀释5000倍的VdAL处理樱桃后，樱桃成熟时间提前了5天。
统计各处理的樱桃产量，结果显示，稀释5000倍的VdAL和清水处理的樱桃的平均产量分别为2200kg和1715kg，稀释5000倍的VdAL处理樱桃后，樱桃产量提高了28.28％。
在樱桃成熟时(VdAL52天，清水处理57天)，观察樱桃外观，发现VdAL处理的樱桃的颜色明显较未处理的樱桃红(图17)，表明，VdAL可以促进樱桃的着色。
实施例12、维达利安可以促进罐萝卜生长、改善其适口性
田间试验设计如下：实验采用随机区组设计，设置3个重复区，每个重复区随机设置两个处理区，分别为VdAL处理区和对照处理区，每个处理区的面积均为0.1亩。
在VdAL处理区的罐萝卜品种791(郑州市三合农业科技开发有限公司)的根膨大期按照下述方式进行对罐萝卜处理1次：采用顶喷(人工喷施)的方式对罐萝卜叶片喷施实施例1的稀释5000倍的VdAL，每亩喷施45-50kg，得到稀释5000倍的VdAL处理的罐萝卜(1/5000)。喷施时间在早晨或傍晚，最好避开正午高温时段；喷施时严禁与药物及肥料混合；喷施后若2小时内下雨需要补喷施一次。
按照VdAL处理区的处理方法，将稀释5000倍的VdAL替换为清水对对照处理区进行处理，其他步骤均不变，得到未处理的罐萝卜(对照，CK)。
在VdAL/清水处理20天，统计各处理的罐萝卜产量(图18)，结果显示，稀释5000倍的VdAL和清水处理的罐萝卜的平均块根单重分别为0.90kg和0.85kg，稀释5000倍的VdAL处理罐萝卜后，罐萝卜平均块根单重提高了5.88％；稀释5000倍的VdAL和清水处理的罐萝卜的平均单株鲜重分别为1.20kg和1.09kg，稀释5000倍的VdAL处理罐萝卜后，罐萝卜平均块根单重提高了10.09％。
选取30名有经验品评员，对上述两种处理的罐萝卜进行脆度评估(图18)，其中红富士为参比样品，红富士脆度分值为10。每种食品取10份样品，进行10次平行实验，计算评分平均值。结果显示，未处理的罐萝卜的平均脆度分值为5.32，稀释5000倍的VdAL处理的罐萝卜的平均脆度分值为6.55，并且经检验10次平行实验的相对平均偏差均小于20％，符合实验要求。未处理的罐萝卜的平均脆度分值显著小于稀释5000倍的VdAL处理的罐萝卜的平均脆度分值。
另选取30名有经验品评员，对上述两种处理的罐萝卜进行甜度评估，其中红富士为参比样品，红富士甜度分值为10。每种食品取10份样品，进行10次平行实验，计算评分平均值。结果显示，未处理的罐萝卜的平均甜度分值为4.27，稀释5000倍的VdAL处理的罐萝卜的平均甜度分值为5.56，并且经检验10次平行实验的相对平均偏差均小于20％，符合实验要求。未处理的罐萝卜的平均甜度分值显著小于稀释5000倍的VdAL处理的罐萝卜的平均甜度分值。
说明，维达利安可以提高罐萝卜的脆度与甜度，维达利安处理后的罐萝卜具 有较强的适口性。
实施例13、维达利安可以促进菠菜生长、提高其产量
田间试验设计如下：实验采用随机区组设计，设置3个重复区，每个重复区随机设置三个处理区，分别为VdAL处理1区、VdAL处理2区和对照处理区，每个处理区的面积均为0.1亩。
在VdAL处理1区的菠菜品种秋绿菠菜(郑州市三合农业科技开发有限公司)的3叶期按照下述方式进行对菠菜处理1次：采用顶喷(人工喷施)的方式对菠菜叶片喷施实施例1的稀释4000倍的VdAL，每亩喷施15-20kg，得到稀释4000倍的VdAL处理的菠菜(1/4000)。喷施时间在早晨或傍晚，最好避开正午高温时段；喷施时严禁与药物及肥料混合；喷施后若2小时内下雨需要补喷施一次。
按照VdAL处理1区的处理方法，将稀释4000倍的VdAL替换为稀释6000倍的VdAL对VdAL处理2区进行处理，其他步骤均不变，得到稀释6000倍的VdAL处理的菠菜(1/6000)。
按照VdAL处理1区的处理方法，将稀释4000倍的VdAL替换为清水对对照处理区进行处理，其他步骤均不变，得到未处理的菠菜(CK)。
在VdAL/清水处理20天，统计各处理的菠菜产量，结果显示，稀释4000倍的VdAL、稀释6000倍的VdAL和清水处理的菠菜的平均产量分别为0.58kg/0.12m2、0.98kg/0.12m2和0.63kg/0.12m2，稀释4000倍的VdAL、稀释6000倍的VdAL处理菠菜后，产量分别提高了-7.45％、56.38％，表明，适当浓度的VdAL处理菠菜可以提高其产量。
工业应用
实验证明，本发明中以VdAL为活性成分的维达利安生物制剂(VdAL)可以促进多种植物种子发芽、植株生长、果实生长、提高座果数目，并可延缓植株衰老：
1、稀释7500倍的VdAL处理棉花种子后棉花的平均发芽率为清水处理棉花种子后棉花的平均发芽率的1.08倍。用稀释3000倍的VdAL处理鲁30号，霜前花产量提高19.48％，总产量提高17.04％；用稀释5000倍的VdAL进行处理时，鲁30号霜前花产量提高21.25％，总产量提高20.00％，616霜前花产量提高19.06％，总产量提高19.69％，鲁25号霜前花产量提高16.37％，总产量提高17.60％。用稀释3000倍的VdAL处理时，单株成铃数、亩总铃数、衣分和亩产量增加率分别为4.5％、1.98％、0.24％和1.76％；用稀释5000倍的VdAL处理时，单株成铃数、亩总铃数、单铃重、衣分和亩产量增加率分别为3.41％、4.41％、1.9％、0.49％和5.22％。
2、稀释2000倍的VdLA处理西瓜后单株结果数提高18.2％，产量提高33.4％。
3、稀释1000倍的VdLA、稀释2000倍的VdAL、稀释5000倍的VdLA、稀释10000倍的VdAL处理小麦种子后，发芽速率约分别加快了2倍、9倍、12倍和 11倍。稀释5000倍的VdAL、稀释10000倍的VdAL处理的小麦种子的发芽率分别提高了2.48％和4.40％。稀释5000倍的VdAL和稀释10000倍的VdAL浸种后，小麦单株鲜重分别提高了15.32％和27.03％。稀释5000倍的VdAL、稀释10000倍的VdAL、稀释20000倍的VdAL处理小麦后，产量分别提高了1.94％、8.13％、5.23％。稀释5000倍的VdAL处理小麦后，有效穗数增加了1.5％，穗粒数增加了0.5％；稀释10000倍的VdAL，有效穗数增加了7％，穗粒数增加了3.5％；稀释20000倍的VdAL，有效穗数增加了2.3％，穗粒数增加了3.7％。
4、稀释1000倍的VdAL和稀释3000倍的VdAL处理番茄后，番茄的单株结果数分别提高了4.8％和21.3％。
5、稀释2000倍的VdAL、稀释2500倍的VdAL、稀释3000倍的VdAL和稀释4000倍的VdAL处理青梗菜后，50株青梗菜鲜重分别提高了54.8％、71.0％、100.6％和103.7％。
6、VdAL还可以提高黄瓜的座果率、促进黄瓜的膨大，提高黄瓜产量，促进黄瓜植株的生长，延缓黄瓜植株的衰老；VdAL还可以提高圣女果的座果率；促进草莓、甜瓜、甜椒植株的生长；促进大豆的出苗与生长，稀释5000倍的VdAL浸种后，大豆单株鲜重提高了16.91％，大豆地上部分单株鲜重提高了28.30％。
7、稀释10000倍的VdAL、稀释20000倍的VdAL处理水稻后，产量分别提高了12.28％、2.51％、。
8、稀释5000倍的VdAL和清水处理的罐萝卜的平均块根单重分别为0.90kg和0.85kg，稀释5000倍的VdAL处理樱桃后，罐萝卜平均块根单重提高了5.88％；稀释5000倍的VdAL和清水处理的罐萝卜的平均单株鲜重分别为1.20kg和1.09kg，稀释5000倍的VdAL处理樱桃后，罐萝卜平均块根单重提高了10.09％。
9、稀释4000倍的VdAL、稀释6000倍的VdAL和清水处理的菠菜的平均产量分别为0.58kg/0.12m2、0.98kg/0.12m2和0.63kg/0.12m2，稀释4000倍的VdAL、稀释6000倍的VdAL处理菠菜后，产量分别提高了-7.45％、56.38％，表明，适当浓度的VdAL处理菠菜可以提高其产量。
实验证明，本发明中以VdAL为活性成分的维达利安生物制剂(VDLA)可以提高植物产品品质，也可以促进果实成熟：
1、维达利安处理后，西瓜中主要营养成分和微量元素含量增加，适口性提高。维达利安处理后，Vc含量显著提高，提高了50.41％；可溶性糖含量提高了8.88％，干物质中蛋白质含量提高了14.2％，新鲜西瓜中蛋白质含量提高了0.62％，干物率下降12.4％，粗纤维的含量有少量下降。维达利安处理后，每升新鲜西瓜的Ca、Cu、Fe、K、Mg、Mn、Na、P和Zn的质量均有增加，增加量分别为12.75％、16.54％、19.21％、16.46％、1.05％、20.45％、2.13％、10.02％、19.73％；新鲜西瓜中Cu、Fe、K、Mn和Zn的质量含量均有增加，增加量分别为2.09％、19.09％、1.88％、5.72％和5.07％。
2、维达利安处理后，棉花霜前花产量和衣分均增加。用稀释3000倍的VDLA 处理鲁30号，霜前花产量提高19.48％；用稀释5000倍的VDLA进行处理时，鲁30号霜前花产量提高21.25％，616霜前花产量提高19.06％，鲁25号霜前花产量提高16.37％。用稀释3000倍的VDLA处理时，衣分增加率为0.24％；用稀释5000倍的VDLA处理时，衣分增加率为0.49％。
3、稀释3000倍的VdAL处理葡萄后，葡萄Vc含量提高了50.4％，并且色泽更艳丽、果穗紧凑不易掉粒，并且葡萄成熟时间提前了5天。
4、稀释5000倍的VdAL处理樱桃后，樱桃成熟时间提前了5天，产量提高了28.28％。
5、稀释5000倍的VdAL处理的罐萝卜的脆度与甜度增加，表明维达利安处理后的罐萝卜具有较强的适口性。
实验证明，本发明中以VdAL为活性成分的维达利安生物制剂(VDLA)可以提高植物的抗旱性：
维达利安质量百分比浓度为1％-2％的溶液处理的植物在控水15-20天全部枯死，未经维达利安处理的植物在控水10-12天全部枯死，维达利安质量百分比浓度为1％-2％的溶液处理的植物的枯死时间均分别显著大于未经维达利安处理的小麦。维达利安质量百分比浓度为0.2％-2％的溶液处理的植物在停止浇水后再进行复水处理发现，维达利安处理的植物中的叶绿素含量为34.5SPAD-34.4SPAD，显著高于未经维达利安处理的植物；维达利安处理的植物的复水指数为0.43-0.52，显著高于未经维达利安处理的植物。表明，适当浓度的维达利安可以提高小麦的抗旱性。
实验证明，本发明中以VdAL为活性成分的维达利安生物制剂(VDLA)可以促进植物果实着色：
1、稀释5000倍的VdAL处理的樱桃的颜色明显较未处理的樱桃红，表明，VdAL可以促进樱桃的着色。
2、稀释3000倍的VdAL处理葡萄后，葡萄色泽更艳丽。
实验证明，本发明的蛋白质VdAL及其生物制剂可以促进植物生长提高植物产量，可以提高植物产品品质与植物果实的适口性，可以提高植物的抗旱性，还可以促进植物果实的着色，促进果实成熟。

Claims (18)

  1. 蛋白质或其相关生物材料在下述M1、M2、M3或M4中的应用：
    M1、在提高植物产量和/或调控植物生长中的应用；
    M2、在提高植物产品品质中的应用；
    M3、在调控植物抗旱性中的应用；
    M4、在调控植物果实着色中的应用；
    所述蛋白质为如下A1)或A2)或A3)：
    A1)氨基酸序列为序列1的蛋白质；
    A2)在序列1的氨基酸序列中经过取代和/或缺失和/或添加一个或几个氨基酸残基得到的具有相同功能的由A1)衍生的蛋白质；
    A3)在A1)或A2)的N端或/和C端连接标签得到的融合蛋白质；
    所述生物材料，为下述B1)至B20)中的任一种：
    B1)编码所述蛋白质的核酸分子；
    B2)含有B1)所述核酸分子的表达盒；
    B3)含有B1)所述核酸分子的重组载体；
    B4)含有B2)所述表达盒的重组载体；
    B5)含有B1)所述核酸分子的重组微生物；
    B6)含有B2)所述表达盒的重组微生物；
    B7)含有B3)所述重组载体的重组微生物；
    B8)含有B4)所述重组载体的重组微生物；
    B9)含有B1)所述核酸分子的转基因植物细胞系；
    B10)含有B2)所述表达盒的转基因植物细胞系；
    B11)含有B3)所述重组载体的转基因植物细胞系；
    B12)含有B4)所述重组载体的转基因植物细胞系；
    B13)含有B1)所述核酸分子的转基因植物组织；
    B14)含有B2)所述表达盒的转基因植物组织；
    B15)含有B3)所述重组载体的转基因植物组织；
    B16)含有B4)所述重组载体的转基因植物组织；
    B17)含有B1)所述核酸分子的转基因植物器官；
    B18)含有B2)所述表达盒的转基因植物器官；
    B19)含有B3)所述重组载体的转基因植物器官；
    B20)含有B4)所述重组载体的转基因植物器官。
  2. 根据权利要求1所述的应用，其特征在于：B1)所述核酸分子为如下b1)或b2)或b3)的基因：
    b1)核苷酸序列是序列表中序列2的cDNA分子或DNA分子；
    b2)与b1)限定的核苷酸序列具有75％或75％以上同一性，且编码权利要求 1所述蛋白质的cDNA分子或基因组DNA分子；
    b3)在严格条件下与b1)限定的核苷酸序列杂交，且编码权利要求1所述蛋白质的cDNA分子或基因组DNA分子。
  3. 根据权利要求1或2所述的应用，其特征在于：所述调控植物生长为促进植物生长。
  4. 根据权利要求1-3中任一所述的应用，其特征在于：所述生长为营养生长和/或生殖生长。
  5. 根据权利要求1-4中任一所述的应用，其特征在于：所述植物为双子叶植物或单子叶植物。
  6. 根据权利要求1-5中任一所述的应用，其特征在于：所述提高植物产品品质为下述H1和/或H2：
    H1、提高所述植物产品营养成分的含量和/或提高所述植物产品中微量元素的含量；
    H2、通过促进所述植物产品的成熟而提高所述植物产品的品质。
  7. 下述任一方法：
    N1、一种提高植物产量和/或促进植物生长的方法，包括对目的植物施用权利要求1中所述蛋白质、权利要求1或2中所述生物材料或生物制剂，提高所述目的植物产量和/或促进所述目的植物生长；所述生物制剂的活性成分为权利要求1中所述蛋白质或权利要求1或2中所述生物材料；
    N2、一种提高植物产品品质的方法，包括对目的植物施用权利要求1中所述蛋白质、权利要求1或2中所述生物材料或所述生物制剂；
    N3、一种提高植物抗旱性或促进植物果实着色的方法，包括对目的植物施用权利要求1中所述蛋白质、权利要求1或2中所述生物材料或所述生物制剂。
  8. 根据权利要求7所述的方法，其特征在于：所述生物制剂按照方法1)制备，所述方法1)为：培养权利要求1或2中B5)-B8)中任一所述重组微生物，使所述蛋白质的编码基因表达，得到所述生物制剂。
  9. 根据权利要求8所述的方法，其特征在于：所述方法1)为如下11)和12)：
    11)培养权利要求1或2中B5)-B8)中任一所述重组微生物，使所述编码基因表达，得到表达所述蛋白质的重组微生物培养物；
    12)破碎所述微生物培养物中的菌体，得到所述生物制剂。
  10. 根据权利要求7-9中任一所述的方法，其特征在于：所述植物为双子叶植物或单子叶植物。
  11. 根据权利要求7-10中任一所述的方法，其特征在于：所述生长为营养生长和/或生殖生长。
  12. 根据权利要求7-11中任一所述的方法，其特征在于：所述提高植物产品品质为下述H1和/或H2：
    H1、提高所述植物产品营养成分的含量和/或提高所述植物产品中微量元素的含量；
    H2、通过促进所述植物产品的成熟而提高所述植物产品的品质。
  13. 权利要求7-9中任一所述生物制剂的下述O1-O4中的任一应用：
    O1、在提高植物产量和/或调控植物生长中的应用；
    O2、在提高植物产品品质中的应用；
    O3、在提高植物抗旱性中的应用；
    O4、在促进植物果实着色中的应用。
  14. 根据权利要求13所述的应用，其特征在于：所述调控植物生长为促进植物生长。
  15. 根据权利要求13或14所述的应用，其特征在于：所述生长为营养生长和/或生殖生长。
  16. 根据权利要求13-15中任一所述的应用，其特征在于：所述植物为双子叶植物或单子叶植物。
  17. 根据权利要求13-16中任一所述的应用，其特征在于：所述提高植物产品品质为下述H1和/或H2：
    H1、提高所述植物产品营养成分的含量和/或提高所述植物产品中微量元素的含量；
    H2、通过促进所述植物产品的成熟而提高所述植物产品的品质。
  18. 权利要求7-9中任一所述生物制剂。
PCT/CN2016/088074 2015-10-22 2016-07-01 蛋白质VdAL在提高植物产量、产品品质和抗旱性及促进植物果实着色中的应用 WO2017067214A1 (zh)

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