WO2015067146A1 - 一种促进植物生根、发芽、生长和延长贮存期的短肽及其应用 - Google Patents

一种促进植物生根、发芽、生长和延长贮存期的短肽及其应用 Download PDF

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WO2015067146A1
WO2015067146A1 PCT/CN2014/090149 CN2014090149W WO2015067146A1 WO 2015067146 A1 WO2015067146 A1 WO 2015067146A1 CN 2014090149 W CN2014090149 W CN 2014090149W WO 2015067146 A1 WO2015067146 A1 WO 2015067146A1
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short peptide
tyr
germination
growth
val
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PCT/CN2014/090149
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French (fr)
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李�昊
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李�昊
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3526—Organic compounds containing nitrogen

Definitions

  • the present invention belongs to the field of biotechnology agriculture, and the present invention more specifically relates to a short peptide which promotes rooting, germination, growth and prolonged storage of plants and its use.
  • auxins on the market are mostly plant hormones, and the most important chemical substance in auxin is 3-indole acetic acid. Plant growth regulators similar in artificial synthesis are also spinesin, cytokinin, gibberellin, sodium naphthaleneacetate, amine fresh ester (DA-6) and the like. This type of auxin has a dual nature, which promotes growth and inhibits growth. That is, low concentration promotes growth and high concentration inhibits growth. Most of these auxins have acute oral toxicity and acute dermal toxicity.
  • bioactive peptides short peptides formed from the amino acid sequences of fragments of proteins, is a new requirement for the application of rooting, germination, growth and storage of plants.
  • preservatives and preservatives are compounds, such as benzoic acid, dibutylhydroxytoluene, etc., these compounds are mostly toxic; there are natural preservatives such as gallate propionate, vitamin E, spice extract, etc. And new preservatives such as nisin, lysozyme, etc.; food preservatives mostly use sodium citrate, sodium metabisulfite and so on.
  • nisin is a polypeptide consisting of 34 amino acids, which has a strong effect on foods with high protein content, and short peptides of less than 10 amino acids are used to prolong the storage of seeds or fruits. In terms of terms, it has not been reported in the literature.
  • bioactive short peptides that can promote plant rooting, germination and growth, and prolong storage period.
  • the object of the present invention is to provide a method for promoting rooting, germination, growth and prolonging storage of plants.
  • Short peptides and their applications are characterized by:
  • amino acid sequences constituting the short peptide are as follows:
  • the nucleotide sequence of the short peptide refers to a gene sequence composed of three codons corresponding to each amino acid sequence of the short peptide.
  • the short peptide fragment of the short peptide for promoting rooting, germination, growth and prolonging storage period of the plant according to the present invention is characterized in that the short peptide fragment is obtained by cutting the sequence of the 7 amino acids from any position. It consists of 3 or 4 or 5 or 6 amino acids and has short peptide fragments that promote plant germination, growth and prolonged storage.
  • a short peptide fragment of a short peptide which promotes rooting, germination, growth and prolonged storage of a plant according to the present invention characterized in that the nucleotide fragment of the short peptide fragment is a correspondence of the seven amino acids
  • the nucleotide sequence is composed of 3 or 4 or 5 or 6 amino acid corresponding base sequences from any position, and has short peptide fragments which promote plant rooting, germination, growth and prolonged storage period.
  • the invention relates to an analog of a short peptide which promotes rooting, germination, growth and prolonged storage period of a plant, wherein the short peptide analog refers to fusion with the short peptide and another compound or Formed by fusing an additional polypeptide or protein with the amino acid sequence of the short peptide, and having a polypeptide sequence or protein that promotes plant germination, growth, and prolonged shelf life.
  • a derivative of a short peptide which promotes rooting, germination, growth and prolonged storage of a plant according to the present invention characterized in that the short peptide derivative refers to an amino acid in the sequence of the amino acid of the short peptide A group of one or several amino acids is substituted with another group and has a short peptide that promotes plant germination, growth, and prolonged storage.
  • a variant of a short peptide which promotes rooting, germination, growth and prolonged storage of plants, according to the present invention Characterized in that the short peptide variant refers to an amino acid sequence having one or several amino acid or nucleotide changes or a nucleotide sequence encoding the same, the alteration being included in an amino acid sequence or a nucleotide sequence An amino acid or nucleotide is deleted, inserted or substituted at any position in the middle of the sequence, or an amino acid or nucleotide is added at both ends of the sequence to have sequence homology or similarity to the short peptide of the present invention.
  • the invention relates to a short peptide which promotes rooting, germination, growth and prolonging storage period of a plant, characterized in that the short peptide, nucleotide, short peptide fragment, short peptide analog, short peptide derivative And short peptide variant preparations have biofunctional products that promote rooting, germination, growth and prolonged storage of seed plants, and promote plant rooting and germination growth in a low concentration range of 0.01 mg/L to 1 mg/L; in a high concentration range of 1 mg/ L ⁇ 1000mg / L inhibition growth is suitable for prolonging the storage period.
  • the invention relates to a short peptide which promotes rooting, germination, growth and prolonging storage period of a plant, wherein the biological function refers to promoting rooting, germination of a seed plant, promoting plant growth and prolonging plant seed or The application function of the storage time of mature fruits and the like.
  • the seed plant comprises angiosperms and gymnosperms, that is, including but not limited to vegetables: leafy vegetables such as Chinese cabbage, rhizomes such as potatoes, radishes, etc., fruits and vegetables such as cucumbers, peppers, beans, beets, etc.; Sugar cane, melons such as watermelon, berries such as strawberries, blueberries, blackberries, grapes, etc., walnuts such as cherries, peaches, jujubes, longan, plums, etc., such as apples, pears, alfalfa, kiwi, etc.; Rice, corn, wheat, soybeans, sorghum, etc.; flowers such as roses, carnations and cosmos.
  • leafy vegetables such as Chinese cabbage, rhizomes such as potatoes, radishes, etc.
  • fruits and vegetables such as cucumbers, peppers, beans, beets, etc.
  • Sugar cane melons such as watermelon, berries such as strawberries, blueberries, blackberries,
  • a significant advantage of the present invention is that the short peptide of the present invention is derived from a bioinformatics method for sequence alignment of proteins of an international protein database, and a novel biologically active peptide having an active function is selected, and the present invention is applied. Short peptides have excellent effects on the rooting, germination, growth and prolonged storage of plant seeds.
  • Fig. 1 is a graph showing the results of experimental results relating to the promotion of cell division and proliferation by short peptides of the present invention
  • FIG. 2 is an experimental diagram of the short peptides of the present invention for promoting rooting and germination of sugarcane; the sugarcane is cultivated by the short peptides of the present invention, and the rooting and germination of sugarcane in the experimental group and the control group are investigated; (1) the sugarcane in the experimental group is contained The short peptide culture solution of the present invention is cultured for 7 days, and the root buds are obviously grown; (2) the control group does not contain the short peptide culture solution of the present invention, and it is obvious that the root buds are short, which is much different from the experimental group;
  • Figure 3 is a diagram showing the effect of short peptide fragments taken by short peptides of the present invention on rooting, germination and growth of sugarcane
  • the experimental chart the sugarcane was cultivated by the short peptide involved in the invention, and the rooting and germination of the sugarcane in the experimental group and the control group were investigated.
  • (1) Experimental group Sugarcane was cultured for 7 days with the short peptide culture solution containing the present invention, and obvious root buds were grown;
  • the control group contained the short peptide culture solution of the present invention, and it was obvious that the root buds were short. , much worse than the experimental group;
  • FIG. 4 is an experimental diagram showing the effect of the short peptide of the present invention on promoting the germination of Chinese cabbage seeds; using the short peptides of the present invention to culture Chinese cabbage seeds, and examining the germination of the Chinese cabbage seeds in the experimental group (1) and the control group (2), Obviously, the germination rate of the experimental group seeds was increased by about 15% compared with the control group.
  • Figure 5 is a graph showing the effect of the short peptide of the present invention on promoting rooting, germination, and growth of radish; using the short peptide solution Tyr-Leu-Tyr-Glu-Val-Val-Arg (experimental group (1)) of the present invention, Tyr-Leu-Tyr-Glu-Val-Ala-Arg (experimental group (2)), and control (3) solution (without the addition of the short peptide solution of the present invention) to cultivate radish; experimental results show that: experimental group (1) The germination rate of the experimental group (2) was significantly higher than that of the control group (3);
  • FIG. 6 is an experimental diagram showing the effect of the short peptide of the present invention on promoting the germination of melon seeds; using the short peptides of the present invention to culture melon seeds, and examining the germination of the melon seeds in the experimental group (1) and the control group (2), it is obvious that The germination rate of the seeds in the experimental group was higher than that in the control group;
  • Figure 7 is a graph showing the effect of the short peptide of the present invention on promoting germination of corn seeds; using the short peptide Gla-Tyr-Ile-Tyr-Glu-Val-Ala-Arg experimental group (1), Tyr-Leu- Tyr-Glu-Val-Ala-Arg (experimental group (2) solution cultured corn seed, compared with control group (3) (without short peptide solution soaking in the present invention), experimental group (1), experimental group (2) The germination rate is much higher than that of the control group; the germination rate of the experimental group (2) can reach over 98%, indicating that the solution containing Tyr-Leu-Tyr-Glu-Val-Ala-Arg short peptide promotes growth and germination. Obvious effect;
  • Fig. 8 is a graph showing the effect of the short peptide of the present invention for prolonging the storage period of sugar cane; soaking the sugar cane section with the short peptide involved in the present invention, and then vacuum-packaging, and examining the results in 7 days.
  • the experimental group sugarcane has less cross-section change, the color is yellow, and the color of the cut surface is the same;
  • the sugarcane that has not been treated by the short peptide involved in the invention has a reddish brown surface, which is polyphenol oxidation of sugarcane.
  • the enzyme causes brown sugar browning reaction;
  • Figure 9 is a graph showing the effect of the short peptide variant of the present invention for prolonging the storage period of sugar cane; soaking the sugar cane section with the short peptide involved in the present invention, and then vacuum-packaging the sample for 7 days; (1) section of the sugarcane of the experimental group No change, the color is yellow, and the color of the cut surface is the same when it is cut; (2) The sugar cane which has not been treated by the short peptide involved in the present invention has a reddish brown surface, which is the sugarcane brown polyphenol oxidase. Change the reaction and affect the storage of sugar cane.
  • the short peptides involved in the present invention are prepared by a chemical synthesis method, that is, a well-established solid phase peptide synthesis method well known in the art, either using the Boc method or the Fmoc method. Specifically, the protected amino acids are coupled to an inert solid phase carrier one by one, and then the peptide chain is cleaved from the carrier by a strong acid while removing side chain protection.
  • Tyr-Ile-Tyr-Glu-Gly-Gly-Lys is a main sequence, and a short peptide fragment having the same biological activity as the short peptide is composed of 3 or 4 or 5 or 6 amino acids from any position.
  • Short peptide analogs, short peptide derivatives, short peptide variants and the like all have the efficacy described in the present invention.
  • nucleotides are prepared synthetically; nucleotides encoding short peptides comprise one of the following groups:
  • the nucleotide consists essentially of a nucleotide encoding a short peptide having the amino acid sequence of ⁇ 1>, and the nucleotide sequence of the present invention is in the form of a DNA comprising cDNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the coding region sequence encoding the short peptide may be the same as or different from the nucleotide in ⁇ 2>, and is referred to as a variant, wherein the variant refers to a coding having the amino acid sequence encoding the claim 1, but may be ⁇ 2
  • the nucleotides in > are different.
  • a variant may be a nucleotide sequence substituted, inserted or deleted by one or several nucleotides, but does not alter the active function of a short peptide encoding the amino acid sequence of ⁇ 1>.
  • a “variant" of a short peptide or nucleotide refers to an amino acid sequence having one or several amino acid or nucleotide changes or a nucleotide sequence encoding the same. Alterations can include deletions, insertions, additions or substitutions of amino acids or nucleotides in an amino acid sequence or nucleotide sequence. Variants may have "conservative" changes in which the substituted amino acid has structural or chemical properties similar to those of the original amino acid, and may also have “non-conservative” changes in which the substituted amino acid does not have a structure similar to the original amino acid or Chemical properties.
  • Missing means A deletion of one or several amino acids or nucleotides in an amino acid sequence or a nucleotide sequence; "insertion” refers to the insertion of one or several amino acids anywhere in the middle of an amino acid sequence or a nucleotide sequence; “addition” means One or several amino acids are added to the N-terminus or C-terminus of the amino acid sequence or nucleotide sequence; “replacement” refers to the replacement of one or several amino acids or nucleotides with a different amino acid or nucleotide.
  • the short peptide, nucleotide, short peptide fragment, short peptide analog, short peptide derivative and short peptide variant are used for preparing functional articles for promoting plant seed rooting, germination, growth and prolonging storage period including but not limited to The use of the effect:
  • This seed plant contains angiosperms and gymnosperms, including but not limited to
  • Vegetables leafy vegetables such as Chinese cabbage, roots such as potatoes, radishes, etc., fruits and vegetables such as cucumber, pepper, beans, beets, etc.;
  • Fruits such as sugar cane, melons such as watermelon, berries such as strawberries, blueberries, blackberries, grapes, etc., walnuts such as cherries, peaches, jujubes, longan, plums, etc., such as apples, pears, clams, etc. Kiwifruit, etc.
  • Food such as rice, corn, wheat, soybeans, sorghum, etc.;
  • percent identity refers to two or more amino acid or nucleic acid sequences. Compare the percentages of the sequences in the same. The sets of sequences are arranged into clusters by examining the distance between all pairs according to the Cluster method (Higging D. G. & Sharp P. M., Gene 1988, 73: 234-237), and then the clusters are distributed in pairs or in groups. The percent identity between two amino acid sequences, such as sequence A and sequence B, is calculated by:
  • the number of residues in the sequence C and the number of residues in the sequence D do not include the number of similar amino acids.
  • LRR-RKs The enzyme activity present on the plasma membrane of the plant cell and the enzyme activity affecting the biological experiment are applied.
  • Kinases (LRR-PKs) are proteases that affect cell proliferation and differentiation in all plant cells.
  • Fig. 1 In the low concentration range of 0.01 mg/L to 1 mg/L, the rooting and germination growth of the plants are promoted; in the high concentration range of 1 mg/L to 1000 mg/L, the growth inhibition is suitable for prolonging the storage period.
  • Example 1 Fruits: such as sugar cane:
  • the MS minimal medium was selected, 3% sucrose was added, and the pH was adjusted to 5.8.
  • a section with a uniform thickness and a length of about 7 cm was cut, and the section was kept flat, and immersed in 5% sodium hypochlorite for 15 mins for disinfection.
  • the sterilized sugarcane stem segments are immersed in a beaker containing the corresponding culture solution, and the liquid surface has not passed through the bottom end section of about 2 cm.
  • the beaker mouth is sealed with plastic wrap to prevent the culture solution from evaporating, and a few empty spaces are poked on the wrap film to maintain aeration state, so as to prevent the sugarcane stem segment from being unable to grow due to lack of oxygen.
  • Incubate room temperature 24 ⁇ 28 ° C daily light, but avoid direct sunlight.
  • MS medium a large amount of element (mother liquor I) mg/L 100 ml. (2) Trace elements (mother liquor II) (3) iron salts (mother liquor III) and the like. Specific steps: mother liquor I is 100ml, mother liquor II, III and other 10ml, the mother liquor is put into the beaker together; weigh 54g carrageenan; 300g sucrose; activated carbon 1g, placed in the plastic bucket; add water to a volume of 1 liter, open the mixer Stir the mixture, dilute to 10 liters, stir well; add white sugar to the mother liquor mixture, continue to stir; then stop the mixer and add carrageenan, stir the mixer; then Stop the mixer and add activated carbon, stir the mixer and stir; adjust the pH with NaOH until the pH of the medium is 5.8; dispense the medium into the selected culture container, cover the bottle mouth; sterilize.
  • the experimental results show that the sugarcane was cultured with the short peptide (Tyr-Leu-Tyr-Glu-Val-Ala-Arg) involved in the present invention, and the short peptide involved in the present invention was observed from the results of the experimental group and the control group after 4 days of culture.
  • the sugarcane has a strong germination promoting effect (shown in Fig. 2), wherein (1) the experimental group of sugar cane is cultured with the short peptide culture solution containing the present invention, and the root bud is obviously grown; (2) the control group is Without the short peptide culture solution of the present invention, it is apparent that the root buds are short and much different from the experimental group.
  • the experimental results show that the sugarcane was cultured with the fragment (Tyr-Glu-Val-Ala-Arg) intercepted by the short peptide of the present invention, and the short peptide pair of the present invention was observed from the results of the experimental group and the control group after 4 days of culture.
  • Sugarcane has a strong role in promoting germination (shown in Figure 3).
  • the experimental group of sugar cane is cultured with the short peptide culture solution containing the present invention, and the root buds are obviously grown, but compared with Fig. 2(1). The short is shown;
  • the control group is a short peptide culture solution which does not contain the present invention, and it is obvious that the root bud is very short, which is much different from the experimental group.
  • the experimental results show that the sugarcane was cultured with the short peptide (Tyr-Leu-Tyr-Glu-Val-Gly-Arg) involved in the present invention, and the short peptide involved in the present invention was observed from the results of the experimental group and the control group after 4 days of culture. It has a strong role in promoting germination of sugar cane.
  • the experimental results are similar to the above results.
  • the short peptide of the present invention intercepts 4 amino acids (Tyr-Glu-Val-Ala-Arg), intercepts 5 amino acids (Tyr-Glu-Val-Ala-Arg) and intercepts 6 amino acids (Tyr- Glu-Val-Ala-Arg) has achieved similar effects in promoting sugarcane germination, and its effect is enhanced with the increase in the number of amino acids in short peptides.
  • Example 2 Vegetables: leafy vegetables such as Chinese cabbage, spinach, celery, spinach, etc.
  • the short peptide of the present invention is chemically synthesized by a solid phase Fmoc method, and the R group of tyrosine Tyr is substituted with a phosphoric acid group, and one and two groups are substituted, and after purification by HPLC, the purity is over 98%, and the solution is dissolved in ultrapure water.
  • the peptides of the invention were prepared as solutions of 10 and 100 ug/ml, respectively.
  • Chinese cabbage experimental group: Chinese cabbage seeds were placed at a temperature of 22 ° C and contained in the short peptide (Tyrp-Leu-Tyr-Glu-Val-Gly-Arg) solution of the present invention, soaked in 6 hours, Placed at a temperature of 15 ° C to germination; control group: take Chinese cabbage seeds at a temperature of 22 ° C, does not contain the present invention The short peptide was soaked in warm water, and was taken out after 6 hours, and placed in a temperature of 15 ° C to germination; the results of the experimental group and the control group observed that the short peptide involved in the present invention has strong promotion of germination to Chinese cabbage seeds.
  • the role shown in Figure 4).
  • the experimental group (1) can germinate in one day, and the germination rate is increased by about 15% compared with the seed of the control group (2).
  • Celery The celery seed is placed in a space at a temperature of 20 ° C to 25 ° C, and is immersed in the short peptide (Tyrp-Leu-Tyrp-Glu-Val-Gly-Arg) solution of the present invention for 8 hours, and then fished out. Wrapped with wet gauze, placed in a cool place at 20 ° C to germination, compared with the soaking seeds without the addition of the short peptide solution of the present invention, the germination time is 4 to 5 days, 2 to 3 days earlier, the germination rate is increased by 20 %about.
  • the short peptide Tyrp-Leu-Tyrp-Glu-Val-Gly-Arg
  • Water spinach Take the spinach seed in 50 ° C ⁇ 55 ° C containing the short peptide of the invention in warm water soaking, after 5 hours, take out wrapped with a damp cloth, germination at 25 ° C temperature, germination rate is not added The seed soaked in the short peptide solution increased by about 24%.
  • Example 3 Fruit vegetables such as cucumber, pepper, tomato, eggplant, beans, beets, etc.;
  • the short peptide Tyr-Ile-Tyr-Glu-Val-Ala-Arg of the present invention was chemically synthesized by a solid phase Fmoc method, and after purification by HPLC, the purity was over 99%, and the peptide of the present invention was dissolved in ultrapure water to prepare 100 ug/ml. The solution.
  • Cucumber Take cucumber seeds and place them in the short peptide solution containing the present invention at a temperature of 28 ° C to 30 ° C, soak seeds for 1 hour, take them out, wrap them in a damp cloth, and germination time is about 8 hours. The germination time is shortened and the germination rate is over 98%.
  • Beans The seeds are placed in the short peptide solution containing the present invention at a temperature of 25 to 30 ° C, soaked for 8 hours, taken out, wrapped with a damp cloth, and the germination time is about 3 days, and new sprouts are emerging. The germination time is shortened and the germination rate is over 95%.
  • Beet The beet seeds were placed in the above short peptide solution containing the present invention at a temperature of 25 ° C, soaked for 12 hours, taken out, wrapped with a damp cloth, and the germination time was about 2 days, and the new shoots emerged and shortened. Germination time, germination rate of more than 99%.
  • the solid phase Fmoc method is used to chemically synthesize the short peptide variant of the present invention, that is, a Gla amino acid is added at the N-terminus, and the short peptide sequence variant sequence Gla-Tyr-Ile-Tyr-Glu-Val-Ala-Arg is synthesized and purified by HPLC.
  • the purity was more than 99.1%, and the peptide of the present invention was dissolved in ultrapure water to prepare a solution of 0.1 mg/ml, which was placed in a spray bottle.
  • Pepper Take the pepper seed in the above-mentioned short peptide variant solution containing the present invention at a temperature of 33 ° C to 35 ° C, soak for 2 hours, take it out, wrap it with a damp cloth, and germination time is about 2 days, both The sprouts are released, shortening the germination time, and the germination rate is over 95%.
  • the short peptide variant of the present invention is chemically synthesized by the solid phase Fmoc method, that is, a Tyr amino acid is inserted in the middle of the short peptide, and the short peptide sequence variant sequence Tyr-Ile-Tyr-Glu-Tyr-Val-Ala-Arg is synthesized and purified by HPLC. Thereafter, the purity was over 98.6%, and the peptide of the present invention was dissolved in ultrapure water to prepare a solution of 0.1 mg/ml, which was placed in a spray bottle.
  • Tomato The tomato seed is placed in a solution containing the above-mentioned short peptide variant of the present invention at a temperature of 28 ° C to 30 ° C, soaked for 2 hours, taken out, wrapped with a damp cloth, and the germination time is about 1 day, and there is a new shoot. Come out, shorten the germination time, and the germination rate is over 95%.
  • the solid phase Fmoc method is used to chemically synthesize the short peptide variant of the present invention, that is, the Glu is replaced with a Tyr amino acid in the middle of the short peptide, and the short peptide sequence variant sequence Tyr-Ile-Tyr-Tyr-Val-Ala-Arg is synthesized. After purification, the purity was over 97.6%, and the peptide of the present invention was dissolved in ultrapure water to prepare a solution of 0.1 mg/ml, which was placed in a spray bottle.
  • Eggplant The eggplant seeds were placed in a solution containing the above-mentioned short peptide variants of the present invention at a temperature of 30 ° C, soaked for 3 hours, taken out, wrapped with a damp cloth, and the germination time was about 2.5 days, and new sprouts appeared. The germination time is shortened and the germination rate is over 95%.
  • the short peptide variants Gla-Tyr-Leu-Tyr-Glu-Val-Ile-Arg, Tyr-Leu-Tyr-Glu-Tyr-Val-Ile-Arg and Tyr-Leu-Tyr-Tyr-Val- Ala-Arg also carried out a corresponding growth-promoting experiment, and the experimental results were not significantly different from the experimental results of the short peptide variant Gla-Tyr-Leu-Tyr-Glu-Val-Ala-Arg in this example, so it is not repeated here. .
  • Rhizomes such as potatoes, sweet potatoes, radishes, etc.
  • the part with the potential bud of the potato is the core, cut into tubers of about 3.5 cm in size, placed in a short peptide solution containing the present invention, and the temperature is 16-18 ° C, soaked for 1 hour, preventing the oxidation of the polyphenols on the cut surface of the potato. Browning of the enzyme, placed in a humid environment, germination after 1 to 2 days, the germination rate reached 95% or more. The germination rate of the control group which was not treated with the short peptide solution of the present invention was increased by more than 15%.
  • Radish The tube with the potential bud of radish as the core, cut into tubers of about 3.5 cm in size, placed in the short peptide solution containing the present invention (experiment group (1), experimental group (2)), and control group (3)
  • the temperature is 18-20 ° C, soaked for 1 hour, placed in a humid environment, and the experimental results are observed after 1 to 2 days, as shown in Fig. 5, the experimental group (1)
  • the germination rate of the experimental group (2) was 95% or more, and the germination rate of the control group which was not treated with the short peptide solution of the present invention was increased by 18.9% and 20.1%, respectively.
  • Example 5 Melons such as watermelon, cantaloupe, cantaloupe
  • the short peptide of the present invention was chemically synthesized by a solid phase Fmoc method, and after purification by HPLC, the purity was over 99.1%, and the peptide of the present invention was dissolved in ultrapure water to prepare a solution of 0.1 mg/ml.
  • Watermelon Take watermelon seeds in a short peptide solution containing the present invention at a temperature of 25 ° C, soak seeds for 1 hour, take them out, wrap them in a damp cloth, and germination time is about 1 day. Both new shoots emerge and the germination is shortened. Time, the germination rate reached more than 99%.
  • Cantaloupe Take the cantaloupe seed in a solution containing the short peptide of the present invention at a temperature of 40 ° C, soak for 12 hours, take it out, wrap it in a damp cloth, place it at a temperature of 30 ° C, and germination time is about 3 days. Come out, shorten the germination time, and the germination rate is over 95%.
  • the melon seeds are placed in a short peptide solution containing the present invention at a temperature of 28 to 30 ° C (experimental group (1)), soaked for 3 hours, taken out, wrapped with a damp cloth, and the germination time is about 1 day;
  • the melon seeds were placed in a short peptide solution not containing the present invention at a temperature of 28 to 30 ° C (control group) (2)), soak seeds for 3 hours, take out, wrap with a damp cloth, germination time is about 1 day; observe the germination of the experimental group and the control group, as shown in Figure 6, the germination rate of the experimental group is significantly higher than the control group Germination rate.
  • Example 6 Berries such as strawberries, blueberries, blackberries, grapes, etc.
  • the short peptide of the present invention was chemically synthesized by a solid phase Fmoc method, and after purification by HPLC, the purity was over 97.1%, and the peptide of the present invention was dissolved in ultrapure water to prepare a solution of 0.1 mg/ml.
  • the strawberry seed is placed in a short peptide solution containing the present invention at a temperature of 60 ° C, and is continuously stirred until the water temperature is lowered to about 25 ° C, taken out, and soaked in the short peptide solution of the present invention.
  • the wet cloth is wrapped and placed at a temperature of 25 ° C to 30 ° C.
  • the germination time is about 3 days. The new shoots are exposed to white, shortening the germination time, and the germination rate is over 95%.
  • Blueberry The blueberry seed is placed in a short peptide solution containing the present invention at a temperature of 23 ° C, soaked for 12 hours, taken out, wrapped with a damp cloth soaked in the short peptide solution of the present invention, and placed at 20 ° C.
  • the germination time is about 15 days, there are sprouts, compared with the control group (not including the short peptide solution soaked in the present invention), the germination time is shortened by half a month, and the germination rate is over 50%.
  • Blackberry The blackberry seed was placed on MS medium at a temperature of 25 ° C (the preparation method of the MS medium was the same as that in the sugar cane of the first embodiment), and the short peptide of the present invention was added to the medium, and the seed was soaked for 12 hours. , taken out, wrapped with a damp cloth soaked in the short peptide solution of the present invention, placed at a temperature of 20 ° C ⁇ 25 ° C, cultured in the dark, germination time of about 15 days, the germination rate of more than 70%.
  • Grape The grape seed was placed in a short peptide solution containing the present invention at a temperature of 40 ° C, soaked for 24 hours, taken out, wrapped with a damp cloth soaked in the short peptide solution of the present invention, and placed at 20 ° C. At 25 ° C, the germination time was about 15 days, and there were sprouts. Compared with the control group (without the short peptide solution soaking in the present invention), the germination time was shortened by half a month, and the germination rate was over 63%.
  • Example 7 Walnuts such as cherries, peaches, jujubes, longan, etc.
  • the short peptide of the present invention was chemically synthesized by a solid phase Fmoc method, and after purification by HPLC, the purity was over 97.1%, and the peptide of the present invention was dissolved in ultrapure water to prepare a solution of 0.1 mg/ml.
  • Cherry The cherry seeds were placed in a short peptide solution containing the present invention at a temperature of 25 ° C, soaked for 12 hours, taken out, wrapped with a damp cloth soaked in the short peptide solution of the present invention, and placed at 20 ° C.
  • the germination time is about 7 days, there are sprouts, compared with the control group (not including the short peptide solution soaked in the present invention), shortening the germination time and increasing the germination rate, which can reach more than 59%.
  • Peach seed is placed in a short peptide solution containing the present invention at a temperature of 25 ° C, soaked for 24 hours, taken out, wrapped with a damp cloth soaked in the short peptide solution of the present invention, placed at 20 ° C ⁇ 25 °C temperature, germination time is about 7 days, there are sprouts, compared with the control group (not including the short peptide solution soaked in the present invention), shortening the germination time and increasing the germination rate, which can reach more than 95%.
  • Jujube The jujube seeds were placed on MS medium at a temperature of 25 ° C (the preparation method of MS medium was the same as that in the sugar cane of Example 1 but the pH was adjusted to 6.5), and the medium was added to the medium.
  • the short peptide involved in the invention is soaked for 12 hours, taken out, wrapped with a damp cloth soaked in the short peptide solution of the present invention, placed at a temperature of 23 ° C to 25 ° C, and the germination time is about 2 weeks, and the rooting germination rate is reached. More than 85%.
  • the longan seeds were placed in a short peptide solution containing the present invention at a temperature of 25 ° C, soaked for 2 days, taken out, wrapped with a damp cloth soaked in the short peptide solution of the present invention, and placed at 20 ° C ⁇ 25 °C temperature, germination time is about 3 days, there are sprouts, compared with the control group (not including the short peptide solution soaked in the present invention), shortening the germination time and increasing the germination rate, which can reach more than 95%.
  • Example 8 Pome fruit such as apple, pear, kiwi, etc.
  • the short peptide of the present invention was chemically synthesized by a solid phase Fmoc method, and after purification by HPLC, the purity was over 97.9%, and the peptide of the present invention was dissolved in ultrapure water to prepare a solution of 0.1 mg/ml.
  • Apple The apple seeds were placed in a short peptide solution containing the present invention at a temperature of 25 ° C, soaked for 12 hours, taken out, peeled off, and wrapped with a damp cloth soaked in the short peptide solution of the present invention.
  • the germination time is about 2 days, there are sprouts, compared with the control group (not including the short peptide solution soaked in the present invention), faster than 2 to 3 days, shortening the germination time and improving the germination The rate can reach more than 95%.
  • Pear seed was placed in a short peptide solution containing the present invention at a temperature of 25 ° C, soaked for 24 hours, taken out, peeled off, peeled, and damp cloth soaked in the short peptide solution of the present invention. wrap up, It is placed at a temperature of 22 ° C ⁇ 25 ° C, the germination time is about 3 days, there is a sprout, compared with the control group (not including the short peptide solution soaked in the present invention), it is 2 to 3 days faster, shortening the germination time, and improving The germination rate can reach more than 80%.
  • Example 9 Grains such as rice, corn, wheat, soybean, sorghum, and the like.
  • the short peptide (Tyr-Leu-Tyr-Glu-Val-Ala-Lys) of the present invention was chemically synthesized by a solid phase Fmoc method, and after purification by HPLC, the purity was over 97.1%, and the peptide of the present invention was dissolved in ultrapure water to prepare 0.1. A solution of mg/ml.
  • the indica rice seeds were placed in a short peptide solution containing the present invention at a temperature of 30 ° C, soaked for 2 hours, taken out, wrapped with a damp cloth soaked in the short peptide solution of the present invention, placed at a temperature of 25 ° C, and germinated time. For about 1 day, there is a new shoot, and compared with the control group (without the short peptide solution soaking in the present invention), the germination rate is increased to 95% or more.
  • the solid phase Fmoc method is used to chemically synthesize the short peptide of the present invention, and a Gla amino acid short peptide variant (Gla-Tyr-Ile-Tyr-Glu-Val-Ala-Arg) is added, and the purity after purification by HPLC exceeds 98.2%.
  • the peptide of the present invention is dissolved in ultrapure water to prepare a solution having a concentration of 0-1 mg/ml, which is named as experimental group (1);
  • the corn seeds were respectively placed in a series of short peptide solutions (1) and (2) with different concentrations at a temperature of 33 ° C, soaked for 6 hours, taken out, and wrapped with a damp cloth soaked in the short peptide solution of the present invention. After being placed at 25 °C, the effects of different concentrations of short peptide solution on the fresh weight and root length of maize seeds were observed after 3 days.
  • the results of the experimental group (1) are shown in Tables 1 and 2; the results of the experimental group (2) are shown in the table.
  • Corn seeds were placed at a temperature of 33 ° C containing the short peptide Gla-Tyr-Ile-Tyr-Glu-Val-Ala-Arg solution of the present invention (experimental group (1)), (Tyr-Leu-Tyr-Glu- Val-Ala-Arg) (experimental group (2)) solution, soaked for 6 hours, taken out, wrapped with a damp cloth soaked in the short peptide solution of the present invention, placed at 25 ° C, germination time is 3 About day, the experimental results are shown in Fig. 7. The experimental group (1) and the experimental group (2) all had sprouts, and compared with the control group (3) (without the short peptide solution soaking in the present invention), the germination time was shortened.
  • the germination rate of the experimental group (2) can reach more than 98%, which is higher than the germination rate of the experimental group (1), indicating that the solution containing Tyr-Leu-Tyr-Glu-Val-Ala-Arg short peptide promotes growth and germination. More remarkable.
  • the short peptide of the present invention is chemically synthesized by a solid phase Fmoc method, and a Tyr amino acid short peptide variant: Tyr-Ile-Tyr-Glu-Tyr-Val-Ala-Arg) is inserted, and after purification by HPLC, the purity exceeds 96.4%.
  • the peptide of the present invention was dissolved in pure water to prepare a solution of 0.1 mg/ml.
  • the wheat seeds were placed in a short peptide solution containing the present invention at a temperature of 30 ° C, soaked for 1 hour, taken out, wrapped with a damp cloth soaked in the short peptide solution of the present invention, placed at a temperature of 25 ° C, and germinated time. For about 2 days, there is a new shoot, compared with the control group (not including the short peptide solution soaked in the present invention), shortening the germination time, one day earlier, and increasing the germination rate, which can reach 98% or more, compared with the control group. Increased by 27%.
  • the short peptide of the present invention was chemically synthesized by the solid phase Fmoc method, and the Glu was replaced with a Tyr short peptide variant Tyr-Ile-Tyr-Tyr-Val-Ala-Arg, which was purified by HPLC and the purity was over 97.1%, and the ultrapure water was used.
  • the peptide of the present invention was dissolved to prepare a solution of 0.1 mg/ml.
  • Soybean seeds were placed in a short peptide solution containing the present invention at a temperature of 20 ° C, soaked for 6 hours, taken out, wrapped with a damp cloth soaked in the short peptide solution of the present invention, placed at a temperature of 15 ° C, germination time For about 3 days, there are sprouts, compared with the control group (not including the short peptide solution soaked in the present invention), shortening the germination time, one day earlier, and increasing the germination rate, which can reach 98% or more, compared with the control group. Increased by 21%.
  • Sorghum The sorghum seed is placed in a short peptide solution containing the present invention at a temperature of 25 ° C, soaked for 6 hours, taken out, wrapped with a damp cloth soaked in the short peptide solution of the present invention, and placed at 25 ° C to 30 ° °C Variable temperature culture, germination time is about 2 days, there are sprouts, compared with the control group (not including the short peptide solution soaked in the present invention), shortening the germination time, one day earlier, and increasing the germination rate, reaching 97% Above, it is 25% higher than the control group.
  • Example 10 Flowers such as roses, carnations, cosmos, and the like.
  • the short peptide of the present invention was chemically synthesized by a solid phase Fmoc method, and after purification by HPLC, the purity was over 96.3%, and the peptide of the present invention was dissolved in physiological saline PBS to prepare a solution of 0.1 mg/ml.
  • Seeds of rose, carnation flower and cosmos flower were respectively placed in the above solution prepared by using the short peptide of the present invention, and after soaking the seed for 2 hours, the gauze soaked with the short peptide solution of the invention was taken out. On the bag, placed at a temperature of 20 ° C ⁇ 25 ° C, there will be white germination after 1 day. The flower germination rate of the above method reaches 90% or more, and the germination rate is greatly improved.
  • the short peptides of the invention (Tyr-Leu-Tyr-Glu-Val-Gly-Arg, Tyr-Leu-Tyr-Glu-Val-Val-Arg and Tyr-Leu-Tyr-Glu-Val-) were chemically synthesized by the solid phase Fmoc method.
  • Ala-Arg and its variants Glu-Val-Ala-Arg, Tyr-Glu-Val-Ala-Arg, Leu-Tyr-Glu-Val-Ala-Arg) after purification by HPLC, purity 99%, 97.2 respectively %, 98.8%, 96.3%, 98%, and 98.5% were prepared as a solution of a short peptide solution having a higher concentration of 10, 50, 100, 500, 1000, 1000 mg/ml.
  • Sugarcane Cut the section of sugarcane stem with uniform thickness and length of about 7cm, keep the section flat, and soak it with 5% sodium hypochlorite for 15mins. The sugar cane is then soaked for 1 hour with a sterile short peptide solution of the present invention and stored under sterile conditions. Compared with the control group which was not soaked by the short peptide solution involved in the present invention, browning occurred only on the 10th day after storage of the sugarcane in the experimental group, which was extended by 6 days compared with the control group, and the elongation rate was 30% to 50%.
  • the short peptide involved in the invention has obvious effect on prolonging the storage period of sugar cane Figure 8 (the liquid prepared by the short peptide (Tyr-Leu-Tyr-Glu-Val-Ala-Arg) of the present invention), wherein (1) the experimental group of sugar cane is soaked for 1 hour with the short peptide solution containing the present invention, On the 9th day, the color remained unchanged; (2) the control group was not soaked with the short peptide solution of the present invention, and it was apparent that it was completely browned.
  • Figure 8 the liquid prepared by the short peptide (Tyr-Leu-Tyr-Glu-Val-Ala-Arg) of the present invention
  • Figure 9 shows a variant of the short peptide of the present invention, which is obtained by cutting a liquid of 4 amino acids (Glu-Val-Ala-Arg), wherein (1) the experimental group of sugar cane is soaked with the short peptide solution containing the present invention. At 1 hour, the color remained unchanged on the 7th day; (2) the control group was not soaked with the short peptide solution of the present invention, and it was apparent that it was completely browned.
  • the short peptide (Tyr-Leu-Tyr-Glu-Val-Ile-Arg) of the present invention was chemically synthesized by the solid phase Fmoc method, and after purification by HPLC, the purity was over 98%, and a short peptide solution having a higher concentration was prepared, which was 10 mg/ Ml solution.
  • the short peptide (Tyr-Leu-Tyr-Glu-Val-Leu-Arg) of the present invention was chemically synthesized by the solid phase Fmoc method, and after purification by HPLC, the purity was over 97%, and a short peptide solution having a higher concentration was prepared, which was 10 mg/ Ml solution.
  • the short peptide (Tyr-Ile-Tyr-Glu-Val-Ala-Lys) of the present invention was chemically synthesized by the solid phase Fmoc method, and after purification by HPLC, the purity was over 97%, and a short peptide solution having a higher concentration was prepared, which was 10 mg/ Ml solution.
  • the short peptide (Tyr-Ile-Tyr-Glu-Val-Ala-Arg) of the present invention was chemically synthesized by the solid phase Fmoc method, and after purification by HPLC, the purity was over 97%, and a short peptide solution having a higher concentration was prepared, which was 10 mg/ Ml solution.
  • spraying the short peptide solution of the invention can extend the shelf life of 2 days, that is, the shelf life of the original 5 days can be extended to 7 days, and the short peptide of the invention also has inhibition The role of bacterial growth.
  • Taking a sufficient amount of grapes and spraying the short peptide solution of the present invention can prolong the storage period of the grapes for more than 3 days.
  • the short peptide (Tyr-Ile-Tyr-Glu-Gly-Gly-Lys) of the present invention was chemically synthesized by the solid phase Fmoc method, and after purification by HPLC, the purity was over 97%, and a short peptide solution having a higher concentration was prepared, which was 10 mg/ Ml solution.
  • the short peptide (Tyr-Leu-Tyr-Glu-Val-Gly-Arg) of the present invention is chemically synthesized by a solid phase Fmoc method, and after purification by HPLC, the purity is over 99%, and a short peptide solution having a higher concentration is prepared, which is 10 mg/ Ml solution.
  • the high-quality apple without internal and external injuries and no pests and diseases is selected, soaked in the solution prepared by the short peptide of the present invention for 5 minutes, and then taken out, placed in a cool place, sweated for 2 days, and then placed in a fresh-keeping bag.
  • the apple treated by the method has a storage period of more than one year, which is longer than the storage period of the apples treated by other methods for one to two months.
  • the short peptide of the present invention was chemically synthesized by a solid phase Fmoc method, and after purification by HPLC, the purity was over 96.3%, and the peptide of the present invention was dissolved in physiological saline PBS to prepare a solution of 10 mg/ml.
  • Rosette, carnation flower and cosmos flower were respectively taken, and the above solution prepared by using the short peptide of the present invention was sprayed onto the flower and the stem and leaf, respectively. This method can extend the flowering period by 2 to 3 days.

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Abstract

本发明公开了一种促进种子植物发芽、生长和延长贮存期的短肽，及其片段、类似物、衍生物和变体。本发明所述短肽能促进植物种子生根、发芽、生长以及延长其贮存期。

Description

一种促进植物生根、发芽、生长和延长贮存期的短肽及其应用 技术领域
本发明属于生物技术农业领域，本发明更具体涉及了一种促进植物生根、发芽、生长和延长贮存期的短肽及其应用。
背景技术
植物的生根、发芽、生长和贮存一直是农业发展最关键的问题。能够促进植物种子快速生根、发芽、生长的物质是当今农业发展的所急需的。而目前市场上的植物生长素大都是植物激素，生长素中最重要的化学物质为3-吲哚乙酸。人工合成作用类似的植物生长调节剂还有芸苔素、细胞分裂素、赤霉素、萘乙酸钠、胺鲜酯(DA-6)等。该类生长素具有双重性，既能促进生长又能抑制生长。即低浓度促进生长，高浓度抑制生长。这些植物生长素大都拥有急性经口毒性和急性经皮毒性，因此，寻求一种无毒无害无副作用的非激素类的植物生长素是农业发展众望所求。而用生物活性肽——来自于蛋白质的片段的氨基酸序列所形成的短肽，做为植物的生根、发芽、生长及贮存的制品应用，是一种新的需求。
对于种子或者果实的长期贮存与短期的储存或者长途运输，在工农业方面也非常重要。主要是防止种子或果实被细菌侵入发生腐败变质。常用的防腐剂和保鲜剂都是化合物，如苯甲酸、二丁基羟基甲苯等，这些化合物大都是有毒性的；还有天然的保鲜剂如没食子丙酸酯、维生素E、香辛料提取物等，以及新型保鲜剂如乳链球菌素、溶菌酶等；粮食保鲜剂多用柠檬酸钠、焦亚硫酸钠等。这些防腐剂或保鲜剂都有其最适合的作用物质，在其特定的范围内，起到较强的延长贮存期或储存期的作用。其中，乳链球菌素(Nisin)是一个由34个氨基酸组成的多肽，对蛋白质含量高的食品有较强作用，而由低于10个氨基酸以下的短肽，用在延长种子或果实的贮存期方面，还未见文献报道。
因此，研制既能够促进植物生根、发芽、生长，又能够延长贮存期的生物活性短肽及其应用具有十分重要的意义与价值。
发明内容
本发明目的是针对上述问题，提供一种促进植物生根、发芽、生长和延长贮 存期的短肽及其应用，其特征在于：
<1>组成短肽的氨基酸序列如下所示:
Tyr-Leu-Tyr-Glu-Val-Gly-Arg或者
Tyr-Leu-Tyr-Glu-Val-Ala-Arg或者
Tyr-Leu-Tyr-Glu-Val-Ile-Arg或者
Tyr-Leu-Tyr-Glu-Val-Val-Arg或者
Tyr-Leu-Tyr-Glu-Val-Leu-Arg或者
Tyr-Ile-Tyr-Glu-Val-Ala-Arg或者
Tyr-Ile-Tyr-Glu-Val-Ala-Lys或者
Tyr-Ile-Tyr-Glu-Gly-Gly-Lys
<2>所述短肽的核苷酸序列是指短肽的每个氨基酸序列所对应的3个密码子构成的基因序列。
本发明所述的一种促进植物生根、发芽、生长和延长贮存期的短肽的短肽片段，其特征在于：所述短肽片段为将所述的7个氨基酸的序列从任意位置截取成3个或4个或5个或6个氨基酸组成的，且具有促进植物发芽、生长和延长贮存期的短肽片段。
本发明所述的一种促进植物生根、发芽、生长和延长贮存期的短肽的短肽片段，其特征在于：所述短肽片段的核苷酸片段为把所述的7个氨基酸的对应的核苷酸序列从任意位置截取成3个或4个或5个或6个氨基酸对应的碱基序列所组成的，且具有促进植物生根、发芽、生长和延长贮存期的短肽片段。
本发明所述的一种促进植物生根、发芽、生长和延长贮存期的短肽的类似物，其特征在于：所述短肽类似物是指在用所述短肽和另一种化合物融合或者用所述短肽的氨基酸序列融合另外的多肽或者蛋白质而形成，且具有促进植物发芽、生长和延长贮存期的多肽序列或蛋白质。
本发明所述的一种促进植物生根、发芽、生长和延长贮存期的短肽的衍生物，其特征在于：所述短肽衍生物是指把所述短肽的氨基酸的序列中的氨基酸的一个或者几个氨基酸的某个基团用另外的基团取代后，且具有促进植物发芽、生长和延长贮存期的短肽。
本发明所述的一种促进植物生根、发芽、生长和延长贮存期的短肽的变体， 其特征在于：所述短肽变体是指一种具有一个或几个氨基酸或核苷酸改变的氨基酸序列或编码它的核苷酸序列，所述改变包括在氨基酸序列或核苷酸序列中，在序列中间的任一位置缺失、插入或替换氨基酸或核苷酸，或在序列两端添加氨基酸或核苷酸，与本发明短肽有序列同源性或相似性。
本发明所述的一种促进植物生根、发芽、生长和延长贮存期的短肽的应用，其特征在于：所述短肽、核苷酸、短肽片段、短肽类似物、短肽衍生物和短肽变体制备具有促进种子植物生根、发芽、生长和延长贮存期的生物功能性制品，在低浓度范围0.01mg/L～1mg/L促进植物生根、发芽生长；在高浓度范围1mg/L～1000mg/L抑制生长适于延长贮存期。
本发明所述的一种促进植物生根、发芽、生长和延长贮存期的短肽的应用，其中所述的生物功能性是指促进种子植物的生根、发芽、促进植物的生长以及延长植物种子或者成熟果实等的贮存时间的应用功能。此种子植物包含被子植物和裸子植物，即包含但不限于蔬菜类：叶菜类如大白菜，根茎类如土豆、萝卜等，果菜类如黄瓜、菜椒、豆角、甜菜等；水果类：如甘蔗，瓜类如西瓜，浆果类如草莓、蓝莓、黑莓、葡萄等，核桃类如樱桃、桃子、大枣、龙眼、梅子等，仁果类如苹果、梨、枇杷、猕猴桃等；粮食类如稻谷、玉米、小麦、大豆、高粱等；花卉类如玫瑰花、康乃馨花和波斯菊等。
本发明的显著优点是：本发明的短肽来自于运用生物信息学的方法把国际蛋白质数据库的蛋白进行序列比对，筛选出来的一种新型的具有活性功能的生物活性肽，运用本发明的短肽对植物种子的生根、发芽、生长及延长贮存期拥有卓越的显著功效。
附图说明
图1是本发明涉及的短肽在不同浓度区间促进细胞的分裂、增殖作用相关实验结果分析图；
图2是本发明涉及的短肽促进甘蔗生根、发芽效果实验图；用本发明涉及的短肽培养甘蔗，考察实验组和对照组甘蔗的生根、发芽情况；(1)实验组甘蔗是用含有本发明的短肽培养液培养7天，明显有根芽长出来；(2)对照组是不含有本发明的短肽培养液，很明显，根芽很短，比实验组相差很多；
图3是本发明涉及的短肽截取的短肽片段促进甘蔗生根、发芽和生长的效 果实验图；用本发明涉及的短肽培养甘蔗，考察实验组和对照组甘蔗的生根、发芽情况。(1)实验组甘蔗是用含有本发明的短肽培养液培养7天，明显有根芽长出来；(2)对照组是不含有本发明的短肽培养液，很明显，根芽很短，比实验组相差很多；
图4是本发明涉及的短肽促进大白菜种子发芽的效果实验图；用本发明涉及的短肽培养大白菜种子，考察实验组(1)和对照组(2)大白菜种子的发芽情况，很明显，实验组种子的发芽率比对照组种子的发芽率提高了15％左右；
图5是本发明涉及的短肽促进萝卜生根、发芽、生长的效果实验图；用本发明涉及的短肽溶液Tyr-Leu-Tyr-Glu-Val-Val-Arg(实验组(1))、Tyr-Leu-Tyr-Glu-Val-Ala-Arg(实验组(2))，以及对照组(3)溶液中(不加本发明的短肽溶液)培养萝卜的情况；实验结果表明：实验组(1)、实验组(2)的发芽率明显高于对照组(3)的发芽率；
图6是本发明涉及的短肽促进甜瓜种子发芽的效果实验图；用本发明涉及的短肽培养甜瓜种子，考察实验组(1)和对照组(2)甜瓜种子的发芽情况，很明显，实验组种子的发芽率比对照组种子的发芽率高；
图7是本发明涉及的短肽促进玉米种子发芽的效果实验图；用本发明涉及的短肽Gla-Tyr-Ile-Tyr-Glu-Val-Ala-Arg实验组(1)、Tyr-Leu-Tyr-Glu-Val-Ala-Arg(实验组(2)溶液培养玉米种子，和对照组(3)(不含本发明的短肽溶液浸泡)比较，实验组(1)、实验组(2)的发芽率远远高于对照组的发芽率；实验组(2)的发芽率可达到98％以上，说明含Tyr-Leu-Tyr-Glu-Val-Ala-Arg短肽的溶液促生长、发芽作用明显；
图8是本发明涉及的短肽延长甘蔗贮存期的效果实验图；用本发明涉及的短肽浸泡甘蔗截面，然后真空无菌包装，7天考察结果。(1)实验组甘蔗的截面变化不到，颜色呈现黄色，和当时切面色泽一致；(2)没有经过本发明涉及的短肽处理的甘蔗，其表面呈现红褐色，这是甘蔗的多酚氧化酶导致甘蔗褐变反应；
图9是本发明涉及的短肽变体延长甘蔗贮存期的效果实验图；用本发明涉及的短肽浸泡甘蔗截面，然后真空无菌包装，7天考察结果；(1)实验组甘蔗的截面没什么变化，颜色呈现黄色，和刚切开时切面色泽一致；(2)没有经过本发明涉及的短肽处理的甘蔗，其表面呈现红褐色，这是甘蔗的多酚氧化酶导致甘蔗褐 变反应，影响甘蔗的贮存。
具体实施方式
本发明涉及的短肽，采用化学合成的方法来制备，即本领域熟知的已经非常成熟的固相肽合成方法，既可以采用Boc方法也可以采用Fmoc方法。具体做法就是将被保护的氨基酸逐个偶联到惰性固相载体上去，然后利用强酸将肽链从载体上裂解下来，同时去除侧链保护。
短肽的氨基酸序列，Tyr-Leu-Tyr-Glu-Val-Gly-Arg或者Tyr-Leu-Tyr-Glu-Val-Ala-Arg或者Tyr-Leu-Tyr-Glu-Val-Ile-Arg或者Tyr-Leu-Tyr-Glu-Val-Val-Arg或者Tyr-Leu-Tyr-Glu-Val-Leu-Arg或者
Tyr-Ile-Tyr-Glu-Val-Ala-Arg或者Tyr-Ile-Tyr-Glu-Val-Ala-Lys或者
Tyr-Ile-Tyr-Glu-Gly-Gly-Lys为主要序列，从任意位置截取成3个或4个或5个或6个氨基酸组成的与所述短肽具有同样生物活性的短肽片段，短肽类似物，短肽衍生物，短肽变体等都具有本发明所述的功效作用。
核苷酸采用人工合成方法制备；编码短肽的核苷酸包含下组中的一种：
(a)编码具有所述氨基酸序列的短肽或其片段、类似物、衍生物或其变体的核苷酸；
(b)与(a)所述核苷酸互补的核苷酸；
(c)与(a)或(b)中所述核苷酸有≥75％相同性的核苷酸；
该核苷酸基本由编码具有<1>所述的氨基酸序列短肽的核苷酸组成，本发明的核苷酸序列，其形式为DNA形式包括cDNA或人工合成的DNA。DNA可以是单链的也可以是双链的。编码短肽的编码区序列可以和<2>中的核苷酸相同，也可以不同，称为变异体，其中变异体是指具有编码权利要求1中的氨基酸序列的编码，但可以和<2>中的核苷酸不同。变异体可以是一个或几个核苷酸取代、插入、缺失的核苷酸序列，但不会改变编码<1>中氨基酸序列的短肽的活性功能。
该短肽或核苷酸的“变体”是指一种具有一个或几个氨基酸或核苷酸改变的氨基酸序列或编码它的核苷酸序列。改变可包括氨基酸序列或核苷酸序列中氨基酸或核苷酸的缺失、插入、添加或替换。变体可具有“保守性”改变，其中替换的氨基酸具有与原氨基酸相类似的结构或化学性质，也可以有“非保守性”改变，其中替换的氨基酸不具有与原氨基酸相类似的结构或化学性质。“缺失”是指在 氨基酸序列或核苷酸序列中一个或几个氨基酸或核苷酸的缺失；“插入”是指在氨基酸序列或核苷酸序列的中间任意位置插入一个或几个氨基酸；“添加”是指在氨基酸序列或核苷酸序列的N端或C端添加一个或几个氨基酸；“替换”是指用不同的氨基酸或核苷酸替换一个或几个氨基酸或核苷酸。
该短肽、核苷酸、短肽片段、短肽类似物、短肽衍生物和短肽变体用于制备促进植物种子生根、发芽、生长以及延长贮存期的功能性制品包括但不限于下述功效的用途：
此种子植物包含被子植物和裸子植物，即包含但不限于
1.蔬菜类：叶菜类如大白菜，根茎类如土豆、萝卜等，果菜类如黄瓜、菜椒、豆角、甜菜等；
2.水果类：如甘蔗，瓜类如西瓜，浆果类如草莓、蓝莓、黑莓、葡萄等，核桃类如樱桃、桃子、大枣、龙眼、梅子等，仁果类如苹果、梨、枇杷、猕猴桃等；
3.粮食类：如稻谷、玉米、小麦、大豆、高粱等；
4.花卉类如玫瑰花、康乃馨花和波斯菊等。
在计算短肽片段、短肽衍生物和短肽变体的氨基酸主序列具有与短肽氨基酸主序列的相同性和相似性时，“相同性百分率”是指两种或多种氨基酸或核酸序列比较中序列相同的百分率。根据Cluster法(Higging D.G.&Sharp P.M.,Gene1988,73:234-237)通过检查所有配对之间的距离将各组序列排列成簇，然后将各簇以成对或成组分配。两个氨基酸序列如序列A和序列B之间的相同性百分率通过下式计算：
Figure PCTCN2014090149-appb-000001
其中，序列C中间隔的残基个数和序列D中间隔的残基个数都不包括相似氨基酸的个数。
下面结合实施例，进一步阐述本发明。这些实施举例仅用于说明本发明而不用于限制本发明的范围。下列所有列举实施例所涉及的所有合成短肽都具有本发明涉及的功能而不限于所列举功能。
(一)本发明涉及的短肽促进细胞的分裂、增殖作用相关实验
应用本发明涉及的短肽与植物细胞的质膜上都存在的激酶(LRR-RKs)进行酶活力影响生物实验。激酶(LRR-PKs)是所有植物细胞中都存在的影响细胞增殖、分化的蛋白酶。实验结果如图1所示，在低浓度范围0.01mg/L～1mg/L促进植物生根、发芽生长；在高浓度范围1mg/L～1000mg/L抑制生长适于延长贮存期。
[0032]说明：本发明涉及的短肽对该植物激酶的作用效果一致，在此不一一列出。
(二)促进种子植物生根、发芽、生长的实验效果
实施例1 水果类：如甘蔗：
促进甘蔗发芽的实验效果(合成本发明涉及的短肽：Tyr-Leu-Tyr-Glu-Val-Gly-Arg和Tyr-Leu-Tyr-Glu-Val-Ala-Arg及其在序列中截取4个氨基酸：Glu-Val-Ala-Arg，截取5个氨基酸：Tyr-Glu-Val-Ala-Arg和截取6个氨基酸：Leu-Tyr-Glu-Val-Ala-Arg)
采用固相Fmoc方法化学合成本发明短肽Tyr-Leu-Tyr-Glu-Val-Gly-Arg、Tyr-Leu-Tyr-Glu-Val-Ala-Arg、Glu-Val-Ala-Arg、Tyr-Glu-Val-Ala-Arg和Leu-Tyr-Glu-Val-Ala-Arg，经过HPLC纯化后，纯度超过98.4％、97.8％、95.9％、99.6％和96.3％，用超纯水分别溶解本发明的肽，分别制备成0.01，0.05，0.1，0.5，1mg/ml的溶液。采用新鲜甘蔗进行发芽实验。
选择MS基本培养基，加入3％蔗糖，调pH至5.8。在甘蔗茎部截取粗细均匀，长度7cm左右的截段，保持截面平整，用5％次氯酸钠浸泡15mins消毒。将消毒后的甘蔗茎段浸泡至盛有相应培养液的烧杯中，液面没过底端截面2cm左右。烧杯口用保鲜膜封住，防止培养液蒸发，并在保鲜膜上戳几个空，保持通气状态，以免甘蔗茎段缺氧无法生长。培养室温度24～28℃，每日光照，但避免阳光直射。
MS培养基的配制：大量元素(母液Ⅰ)mg/L100ml。(2)微量元素(母液Ⅱ)(3)铁盐(母液Ⅲ)等。具体步骤：母液Ⅰ为100ml，母液Ⅱ、Ⅲ等各10ml，母液一起放入烧杯中；称取54g卡拉胶；300g蔗糖；活性炭1g，置于胶桶中；加水定容至1升，开搅拌机，搅匀混合液，定容至10升，搅匀；向母液混合物加入白沙糖，继续搅拌；然后停一下搅拌机再加卡拉胶，开搅拌机混匀；接着再 停搅拌机加活性炭，开搅拌机搅匀；用NaOH调pH一直到培养基的pH为5.8为止；将培养基分装到所选用的培养容器中，盖住瓶口；灭菌。
实验结果表明，用本发明涉及的短肽(Tyr-Leu-Tyr-Glu-Val-Ala-Arg)培养甘蔗，从培养4天后，实验组和对照组的结果观察到，本发明涉及的短肽对甘蔗具有较强的促进发芽的作用(附图2所示)，其中(1)实验组甘蔗是用含有本发明的短肽培养液培养，明显有根芽长出来；(2)对照组是不含有本发明的短肽培养液，很明显，根芽很短，比实验组相差很多。
实验结果表明，用本发明涉及的短肽截取的片段(Tyr-Glu-Val-Ala-Arg)培养甘蔗，从培养4天后，实验组和对照组的结果观察到，本发明涉及的短肽对甘蔗具有较强的促进发芽的作用(附图3所示)，其中(1)实验组甘蔗是用含有本发明的短肽培养液培养，明显有根芽长出来，但较图2(1)所示的短；(2)对照组是不含有本发明的短肽培养液，很明显，根芽很短，比实验组相差很多。
实验结果表明，用本发明涉及的短肽(Tyr-Leu-Tyr-Glu-Val-Gly-Arg)培养甘蔗，从培养4天后，实验组和对照组的结果观察到，本发明涉及的短肽对甘蔗具有较强的促进发芽的作用。实验结果与上述结果类似。
同样地实验方法，本发明涉及的短肽截取4个氨基酸(Tyr-Glu-Val-Ala-Arg)，截取5个氨基酸(Tyr-Glu-Val-Ala-Arg)和截取6个氨基酸(Tyr-Glu-Val-Ala-Arg)，取得了类似的促进甘蔗发芽的作用，其作用效果随着短肽氨基酸数量的增加，效果随之增强。
实施例2 蔬菜类：叶菜类如大白菜、菠菜、芹菜、空心菜等
促进大白菜、菠菜、芹菜、空心菜种子发芽的实验效果(合成本发明涉及的短肽衍生物，在酪氨酸加磷酸类基团修饰：Tyrp-Leu-Tyr-Glu-Val-Gly-Arg和Tyrp-Leu-Tyrp-Glu-Val-Gly-Arg)
采用固相Fmoc方法化学合成本发明短肽，在酪氨酸Tyr的R基团用磷酸基团取代，取代一个和两个基团，经过HPLC纯化后，纯度超过98％，用超纯水溶解本发明的肽，分别制备成10和100ug/ml的溶液。
大白菜：实验组：取大白菜种子置于温度22℃并含有本发明涉及的短肽(Tyrp-Leu-Tyr-Glu-Val-Gly-Arg)溶液中浸种，6个小时后捞出来，置于置于15℃温度下催芽；对照组：取大白菜种子置于温度22℃、不含有本发明涉及的 短肽温水中浸种，6个小时后捞出来，置于置于15℃温度下催芽；实验组和对照组的结果观察到，本发明涉及的短肽对大白菜种子具有较强的促进发芽的作用(附图4所示)。其中，实验组(1)1天即可发芽，发芽率比对照组(2)的种子的发芽率提高了15％左右。
芹菜：取芹菜种子放置在温度20℃～25℃的空间里，用上述本发明涉及的短肽(Tyrp-Leu-Tyrp-Glu-Val-Gly-Arg)溶液浸泡8小时后，捞出来，用湿纱布包好，置于20℃的阴凉处催芽，和不加本发明的短肽溶液浸泡种子想比，其发芽时间为4～5天，提前了2～3天，发芽率提高了20％左右。
菠菜：用还有本发明涉及的短肽溶液浸泡菠菜种子8小时，置于15℃温度下催芽，1～2天即可发芽，其发芽率比不加本发明涉及的短肽溶液提高了32％左右。
空心菜：取空心菜种子置于50℃～55℃含有本发明涉及的短肽的温水中浸种，5个小时后，取出用湿布包好，在25℃温度下催芽，发芽率比不加本发明涉及的短肽溶液浸泡的种子提高了24％左右。
实施例3 果菜类如黄瓜、菜椒、番茄、茄子、豆角、甜菜等；
促进果菜类如黄瓜、菜椒、番茄、茄子、豆角、甜菜种子发芽(合成本发明涉及的短肽：Tyr-Ile-Tyr-Glu-Val-Ala-Arg及其短肽变体)
采用固相Fmoc方法化学合成本发明短肽Tyr-Ile-Tyr-Glu-Val-Ala-Arg，经过HPLC纯化后，纯度超过99％，用超纯水溶解本发明的肽，制备成100ug/ml的溶液。
黄瓜：取黄瓜种子置于温度为28℃～30℃的含有本发明涉及的上述短肽溶液中，浸种1小时，取出，用湿布包好，催芽时间为8小时左右，既有新芽冒出来，缩短了发芽时间，发芽率达到98％以上。
豆角：取豆角种子置于温度为25～30℃的含有本发明涉及的上述短肽溶液中，浸种8小时，取出，用湿布包好，催芽时间为3天左右，既有新芽冒出来，缩短了发芽时间，发芽率达到95％以上。
甜菜：取甜菜种子置于温度为25℃的含有本发明涉及的上述短肽溶液中，浸种12小时，取出，用湿布包好，催芽时间为2天左右，既有新芽冒出来，缩短了发芽时间，发芽率达到99％以上。
采用固相Fmoc方法化学合成本发明短肽变体，即在N端增加一个Gla氨基酸，合成短肽序列变体序列Gla-Tyr-Ile-Tyr-Glu-Val-Ala-Arg，经过HPLC纯化后，纯度超过99.1％，用超纯水溶解本发明的肽，制备成0.1mg/ml的溶液，装入喷剂瓶。
菜椒：取菜椒种子置于温度为33℃～35℃的含有本发明涉及的上述短肽变体溶液中，浸种2小时，取出，用湿布包好，催芽时间为2天左右，既有新芽冒出来，缩短了发芽时间，发芽率达到95％以上。
采用固相Fmoc方法化学合成本发明短肽变体，即在短肽中间插入一个Tyr氨基酸，合成短肽序列变体序列Tyr-Ile-Tyr-Glu-Tyr-Val-Ala-Arg，经过HPLC纯化后，纯度超过98.6％，用超纯水溶解本发明的肽，制备成0.1mg/ml的溶液，装入喷剂瓶。
番茄：取番茄种子置于温度为28℃～30℃的含有本发明涉及的上述短肽变体溶液中，浸种2小时，取出，用湿布包好，催芽时间为1天左右，既有新芽冒出来，缩短了发芽时间，发芽率达到95％以上。
采用固相Fmoc方法化学合成本发明短肽变体，即在短肽中间把Glu替换成一个Tyr氨基酸，合成短肽序列变体序列Tyr-Ile-Tyr-Tyr-Val-Ala-Arg，经过HPLC纯化后，纯度超过97.6％，用超纯水溶解本发明的肽，制备成0.1mg/ml的溶液，装入喷剂瓶。
茄子：取茄子种子置于温度为30℃的含有本发明涉及的上述短肽变体溶液中，浸种3小时，取出，用湿布包好，催芽时间为2.5天左右，既有新芽冒出来，缩短了发芽时间，发芽率达到95％以上。
同样的，对短肽变体Gla-Tyr-Leu-Tyr-Glu-Val-Ile-Arg、Tyr-Leu-Tyr-Glu-Tyr-Val-Ile-Arg和Tyr-Leu-Tyr-Tyr-Val-Ala-Arg也做了相应促长实验，实验结果与本实施例中的短肽变体Gla-Tyr-Leu-Tyr-Glu-Val-Ala-Arg的实验结果无明显差别，故在此不作复述。
实施例4 根茎类如土豆、红薯、萝卜等
促进块茎类如土豆、红薯发芽的效果实验(合成本发明涉及的短肽：Tyr-Leu-Tyr-Glu-Val-Val-Arg实验组(1)、Tyr-Leu-Tyr-Glu-Val-Ala-Arg实验组(2))，分别采用固相Fmoc方法化学合成本发明短肽，经过HPLC纯化后，纯 度超过98.5％，用超纯水溶解本发明的肽，制备成0.1mg/ml的溶液。
土豆：把土豆有潜在芽的部分为核心，切成3.5厘米左右大小的块茎，置于含有本发明涉及的短肽溶液中，温度16～18℃，浸泡1小时，阻止了土豆切面多酚氧化酶的褐变，放置潮湿环境中，1～2天后发芽，发芽率达到95％以上。比没有用本发明短肽溶液处理过的对照组发芽率提高了15％以上。
萝卜：以萝卜有潜在芽的部分为核心，切成3.5厘米左右大小的块茎，分别置于含有本发明涉及的短肽溶液中(实验组(1)、实验组(2))，以及对照组(3)溶液中(不加本发明的短肽溶液)，温度18～20℃，浸泡1小时，放置潮湿环境中，1～2天后观察实验结果，如附图5所示，实验组(1)、实验组(2)的发芽率均达到95％以上，比没有用本发明短肽溶液处理过的对照组发芽率分别提高了18.9％、20.1％以上。
实施例5 瓜类如西瓜、香瓜、哈密瓜
促进瓜类如西瓜、香瓜、哈密瓜种子发芽的效果实验(合成本发明涉及的短肽：
Tyr-Leu-Tyr-Glu-Val-Ile-Arg)
采用固相Fmoc方法化学合成本发明短肽，经过HPLC纯化后，纯度超过99.1％，用超纯水溶解本发明的肽，制备成0.1mg/ml的溶液。
西瓜：取西瓜种子置于温度为25℃的含有本发明涉及的短肽溶液中，浸种1小时，取出，用湿布包好，催芽时间为1天左右，既有新芽冒出来，缩短了发芽时间，发芽率达到99％以上。
香瓜：取香瓜种子置于温度为28～30℃的含有本发明涉及的短肽溶液中，浸种3小时，取出，用湿布包好，催芽时间为1天左右，既有新芽冒出来，缩短了发芽时间，发芽率达到95％以上。
哈密瓜：取哈密瓜种子置于温度为40℃的含有本发明涉及的短肽溶液中，浸种12小时，取出，用湿布包好，置于30℃温度，催芽时间为3天左右，既有新芽冒出来，缩短了发芽时间，发芽率达到95％以上。
甜瓜：取甜瓜种子置于温度为28～30℃的含有本发明涉及的短肽溶液中(实验组(1))，浸种3小时，取出，用湿布包好，催芽时间为1天左右；另取甜瓜种子置于温度为28～30℃的不含有本发明涉及的短肽溶液中(对照组 (2))，浸种3小时，取出，用湿布包好，催芽时间为1天左右；观察实验组和对照组的发芽情况，如附图6所示，实验组的发芽率明显高于对照组的发芽率。
实施例6 浆果类如草莓、蓝莓、黑莓、葡萄等
促进浆果类如草莓、蓝莓、黑莓、葡萄种子发芽的实验效果(合成本发明涉及的短肽：
Tyr-Leu-Tyr-Glu-Val-Leu-Arg)
采用固相Fmoc方法化学合成本发明短肽，经过HPLC纯化后，纯度超过97.1％，用超纯水溶解本发明的肽，制备成0.1mg/ml的溶液。
草莓：取草莓种子置于温度为60℃的含有本发明涉及的短肽溶液中，不停地搅动，直至水温降至25℃左右，取出，用在本发明涉及的短肽溶液中浸泡过的湿布包好，置于25℃～30℃温度，催芽时间为3天左右，既有新芽露白冒出来，缩短了发芽时间，发芽率达到95％以上。
蓝莓：取蓝莓种子置于温度为23℃的含有本发明涉及的短肽溶液中，浸种12小时，取出，用在本发明涉及的短肽溶液中浸泡过的湿布包好，置于20℃～25℃温度，催芽时间为15天左右，有发新芽，和对照组(不含本发明的短肽溶液浸泡)比较，缩短了发芽时间半个月，发芽率达到50％以上。
黑莓：取黑莓种子置于温度为25℃的MS培养基上(MS培养基的配制方法与本实施例1甘蔗中的相同)，在该培养基中加入本发明涉及的短肽，浸种12小时，取出，用在本发明涉及的短肽溶液中浸泡过的湿布包好，置于20℃～25℃温度，暗处培养，催芽时间为15天左右，其发芽率达到70％以上。
葡萄：取葡萄种子置于温度为40℃的含有本发明涉及的短肽溶液中，浸种24小时，取出，用在本发明涉及的短肽溶液中浸泡过的湿布包好，置于20℃～25℃温度，催芽时间为15天左右，有发新芽，和对照组(不含本发明的短肽溶液浸泡)比较，缩短了发芽时间半个月，发芽率达到63％以上。
实施例7 核桃类如樱桃、桃子、大枣、龙眼等
促进核桃类如樱桃、桃子、大枣：龙眼、梅子种子发芽生长的实验效果(合成本发明涉及的短肽：Tyr-Ile-Tyr-Glu-Val-Ala-Lys)
采用固相Fmoc方法化学合成本发明短肽，经过HPLC纯化后，纯度超过97.1％，用超纯水溶解本发明的肽，制备成0.1mg/ml的溶液。
樱桃：取樱桃种子置于温度为25℃的含有本发明涉及的短肽溶液中，浸种12小时，取出，用在本发明涉及的短肽溶液中浸泡过的湿布包好，置于20℃～25℃温度，催芽时间为7天左右，有发新芽，和对照组(不含本发明的短肽溶液浸泡)比较，缩短了发芽时间，提高了发芽率，可达到59％以上。
桃子：取桃子种子置于温度25℃的含有本发明涉及的短肽溶液中，浸种24小时，取出，用在本发明涉及的短肽溶液中浸泡过的湿布包好，置于20℃～25℃温度，催芽时间为7天左右，有发新芽，和对照组(不含本发明的短肽溶液浸泡)比较，缩短了发芽时间，提高了发芽率，可达到95％以上。
大枣：取大枣种子置于温度为25℃的MS培养基上(MS培养基的配制方法与本实施例1甘蔗中的相同，但ph值调到6.5)，在该培养基中加入本发明涉及的短肽，浸种12小时，取出，用在本发明涉及的短肽溶液中浸泡过的湿布包好，置于23℃～25℃温度，催芽时间为2周左右，其生根发芽率达到85％以上。
龙眼：取龙眼种子置于温度25℃的含有本发明涉及的短肽溶液中，浸种2天，取出，用在本发明涉及的短肽溶液中浸泡过的湿布包好，置于20℃～25℃温度，催芽时间为3天左右，有发新芽，和对照组(不含本发明的短肽溶液浸泡)比较，缩短了发芽时间，提高了发芽率，可达到95％以上。
实施例8 仁果类如苹果、梨、猕猴桃等
促进核桃类如仁果类如苹果、梨、枇杷、猕猴桃种子发芽生长的实验效果(合成本发明涉及的短肽：Tyr-Ile-Tyr-Glu-Gly-Gly-Lys)
采用固相Fmoc方法化学合成本发明短肽，经过HPLC纯化后，纯度超过97.9％，用超纯水溶解本发明的肽，制备成0.1mg/ml的溶液。
苹果：取苹果种子置于温度25℃的含有本发明涉及的短肽溶液中，浸种12小时，取出，剥开种皮，用在本发明涉及的短肽溶液中浸泡过的湿布包好，置于22℃～25℃温度，催芽时间为2天左右，有发新芽，和对照组(不含本发明的短肽溶液浸泡)比较，快了2～3天，缩短了发芽时间，提高了发芽率，可达到95％以上。
梨：取砂梨种子置于温度25℃的含有本发明涉及的短肽溶液中，浸种24小时，取出，剥开种皮，去壳，用在本发明涉及的短肽溶液中浸泡过的湿布包好， 置于22℃～25℃温度，催芽时间为3天左右，有发新芽，和对照组(不含本发明的短肽溶液浸泡)比较，快了2～3天，缩短了发芽时间，提高了发芽率，可达到80％以上。
实施例9 粮食类如稻谷、玉米、小麦、大豆、高粱等。
促进粮食类如稻谷、玉米、小麦、大豆、高粱种子发芽生长的实验效果(合成本发明涉及的短肽：Tyr-Leu-Tyr-Glu-Val-Ala-Arg、Tyr-Leu-Tyr-Glu-Val-Ala-Lys，增加一个Gla氨基酸短肽变体(Gla-Tyr-Ile-Tyr-Glu-Val-Ala-Arg，插入一个Tyr氨基酸短肽变体：Tyr-Ile-Tyr-Glu-Tyr-Val-Ala-Arg，Glu替换成一个Tyr短肽变体Tyr-Ile-Tyr-Tyr-Val-Ala-Arg)
稻谷：
采用固相Fmoc方法化学合成本发明短肽(Tyr-Leu-Tyr-Glu-Val-Ala-Lys)，经过HPLC纯化后，纯度超过97.1％，用超纯水溶解本发明的肽，制备成0.1mg/ml的溶液。
取粳稻种子置于温度30℃的含有本发明涉及的短肽溶液中，浸种2小时，取出，用在本发明涉及的短肽溶液中浸泡过的湿布包好，置于25℃温度，催芽时间为1天左右，有发新芽，和对照组(不含本发明的短肽溶液浸泡)比较，提高了发芽率，可达到95％以上。
玉米：
(1)采用固相Fmoc方法化学合成本发明短肽，增加一个Gla氨基酸短肽变体(Gla-Tyr-Ile-Tyr-Glu-Val-Ala-Arg)，经过HPLC纯化后，纯度超过98.2％，用超纯水溶解本发明的肽，制备成浓度为0-1mg/ml的溶液，命名为实验组(1)；
(2)采用固相Fmoc方法化学合成本发明短肽(Tyr-Leu-Tyr-Glu-Val-Ala-Arg)，经过HPLC纯化后，纯度超过97.9％，用超纯水溶解本发明的肽，制备成浓度为0-1mg/ml的溶液，命名为实验组(2)；
取玉米种子分别置于温度33℃的浓度不同的一系列短肽溶液(1)、(2)中，浸种6小时，取出，用在本发明涉及的短肽溶液中浸泡过的湿布包好，置于25℃温度，3天后观察不同浓度的短肽溶液对玉米种子鲜重、根长的影响，实验组(1)的结果如表1、2所示；实验组(2)的结果如表3所示；另外，本实施例选用了其他生长调节剂GA、GGR6、Ca、青霉素培养玉米种子，与本实施例的实验组 (1)、实验组(2)对比，实验结果如表4所示，含本发明涉及的短肽溶液与其他生长调节剂相比，玉米种子的苗长增长率显著高于其他生长调节剂，尤其是含Tyr-Leu-Tyr-Glu-Val-Ala-Arg短肽溶液，说明本发明涉及的Tyr-Leu-Tyr-Glu-Val-Ala-Arg短肽促植物生根发芽生长效果显著。
表1 不同浓度短肽溶液对玉米种子鲜重的影响
Figure PCTCN2014090149-appb-000002
表2 不同浓度短肽溶液对玉米种子根长的影响
Figure PCTCN2014090149-appb-000003
表3 不同浓度短肽溶液对玉米种子鲜重的影响
Figure PCTCN2014090149-appb-000004
表4 不同生长调节剂(浓度相同)对玉米种子苗长的影响
Figure PCTCN2014090149-appb-000005
取玉米种子分别置于温度33℃的含有本发明涉及的短肽Gla-Tyr-Ile-Tyr-Glu-Val-Ala-Arg溶液(实验组(1))、(Tyr-Leu-Tyr-Glu-Val-Ala-Arg)(实验组(2))溶液中，浸种6小时，取出，分别用在本发明涉及的短肽溶液中浸泡过的湿布包好，置于25℃温度，催芽时间为3天左右，实验结果如图7所示，实验组(1)、实验组(2)均有发新芽，和对照组(3)(不含本发明的短肽溶液浸泡)比较，缩短了发芽时间，提前了2天，并提高了发芽率。其中实验组(2)的发芽率可达到98％以上，比实验组(1)的发芽率高，说明含Tyr-Leu-Tyr-Glu-Val-Ala-Arg短肽的溶液促生长、发芽作用更显著。
小麦：
采用固相Fmoc方法化学合成本发明短肽，插入一个Tyr氨基酸短肽变体：Tyr-Ile-Tyr-Glu-Tyr-Val-Ala-Arg)，经过HPLC纯化后，纯度超过96.4％，用超纯水溶解本发明的肽，制备成0.1mg/ml的溶液。
取小麦种子置于温度30℃的含有本发明涉及的短肽溶液中，浸种1小时，取出，用在本发明涉及的短肽溶液中浸泡过的湿布包好，置于25℃温度，催芽时间为2天左右，有发新芽，和对照组(不含本发明的短肽溶液浸泡)比较，缩短了发芽时间，提前了1天，并提高了发芽率，可达到98％以上，比对照组提高了27％。
大豆：
采用固相Fmoc方法化学合成本发明短肽，把Glu替换成一个Tyr短肽变体Tyr-Ile-Tyr-Tyr-Val-Ala-Arg，经过HPLC纯化后，纯度超过97.1％，用超纯水溶解本发明的肽，制备成0.1mg/ml的溶液。
取大豆种子置于温度20℃的含有本发明涉及的短肽溶液中，浸种6小时，取出，用在本发明涉及的短肽溶液中浸泡过的湿布包好，置于15℃温度，催芽时间为3天左右，有发新芽，和对照组(不含本发明的短肽溶液浸泡)比较，缩短了发芽时间，提前了1天，并提高了发芽率，可达到98％以上，比对照组提高了21％。
高粱：取高粱种子置于温度25℃的含有本发明涉及的短肽溶液中，浸种6小时，取出，用在本发明涉及的短肽溶液中浸泡过的湿布包好，置于25℃～30℃ 变温培养，催芽时间为2天左右，有发新芽，和对照组(不含本发明的短肽溶液浸泡)比较，缩短了发芽时间，提前了1天，并提高了发芽率，可达到97％以上，比对照组提高了25％。
实施例10 花卉类如玫瑰、康乃馨和波斯菊等。
促进花卉类如玫瑰、康乃馨和波斯菊种子发芽生长的实验效果(合成本发明涉及的短肽：Tyr-Leu-Tyr-Glu-Val-Ala-Lys)
采用固相Fmoc方法化学合成本发明短肽，经过HPLC纯化后，纯度超过96.3％，用生理盐水PBS溶解本发明的肽，制备成0.1mg/ml的溶液。
分别取玫瑰花、康乃馨花和波斯菊花的种子，分别置于用本发明涉及的短肽制备的上述溶液中，浸泡种子2小时后，捞出来，用该发明短肽溶液浸泡过的纱布包上，置于20℃～25℃的温度下，1天后既会有露白发芽。该方法上述花卉发芽率达到90％以上，大大地提高了发芽率。
(三)延长植物种子或果实的贮存期的实验效果
实施例11
延长水果类如甘蔗贮存期的实验效果(合成本发明涉及的短肽：Tyr-Leu-Tyr-Glu-Val-Gly-Arg、Tyr-Leu-Tyr-Glu-Val-Val-Arg和Tyr-Leu-Tyr-Glu-Val-Ala-Arg及其变体，截取4个氨基酸序列：Glu-Val-Ala-Arg；截取5个氨基酸序列：Tyr-Glu-Val-Ala-Arg；截取6个氨基酸序列：Leu-Tyr-Glu-Val-Ala-Arg)
采用固相Fmoc方法化学合成本发明短肽(Tyr-Leu-Tyr-Glu-Val-Gly-Arg、Tyr-Leu-Tyr-Glu-Val-Val-Arg和Tyr-Leu-Tyr-Glu-Val-Ala-Arg及其变体Glu-Val-Ala-Arg、Tyr-Glu-Val-Ala-Arg、Leu-Tyr-Glu-Val-Ala-Arg)，经过HPLC纯化后，纯度分别为99％、97.2％、98.8％、96.3％、98％和98.5％，制备成浓度较高的短肽溶液，为10,50,100,500，1000，1000mg/ml的溶液。
甘蔗：在甘蔗茎部截取粗细均匀，长度7cm左右的截段，保持截面平整，用5％次氯酸钠浸泡15mins消毒。然后把甘蔗用含有本发明涉及的无菌短肽溶液浸泡1小时，无菌条件保存。和没有经过本发明涉及的短肽溶液浸泡过的对照组相比，实验组甘蔗贮存第10天才有发生褐变现象，比对照组延长了6天，延长率可达30％～50％。本发明涉及的短肽对甘蔗具有明显的延长贮存期的作用附 图8所示(本发明短肽(Tyr-Leu-Tyr-Glu-Val-Ala-Arg)制备的液体)，其中(1)实验组甘蔗是用含有本发明的短肽溶液浸泡过1小时，第9天色泽仍不变；(2)对照组是没有用本发明的短肽溶液浸泡，很明显，其完全褐变。附图9所示(本发明短肽的变体，截取4个氨基酸(Glu-Val-Ala-Arg)制备的液体)，其中(1)实验组甘蔗是用含有本发明的短肽溶液浸泡过1小时，第7天色泽仍不变；(2)对照组是没有用本发明的短肽溶液浸泡，很明显，其完全褐变。
附图8的短肽效果比其变体附图9的效果要好些。
对于短肽Tyr-Leu-Tyr-Glu-Val-Gly-Arg、Tyr-Leu-Tyr-Glu-Val-Val-Arg取得了类似Tyr-Leu-Tyr-Glu-Val-Ala-Arg的实验效果。
对于短肽截取5个氨基酸序列(Tyr-Glu-Val-Ala-Arg)、截取6个氨基酸序列(Leu-Tyr-Glu-Val-Ala-Arg)取得了类似截取4个氨基酸(Glu-Val-Ala-Arg)的作用效果，其延长贮存期的时间随着组成短肽的氨基酸的数量的增加，延长率随之增加。
实施例12
延长蔬菜类——叶菜类如大白菜的贮存期的效果实验(合成本发明涉及的短肽：
Tyr-Leu-Tyr-Glu-Val-Ile-Arg)
采用固相Fmoc方法化学合成本发明短肽(Tyr-Leu-Tyr-Glu-Val-Ile-Arg)，经过HPLC纯化后，纯度超过98％，制备成浓度较高的短肽溶液，为10mg/ml的溶液。
在常温下，把大白菜摆放好，每层喷雾本发明涉及的短肽溶液，不喷的作为对照组，实验结果表明，对照组在第15天开始有发黄、脱帮现象，而实验组到第20天才有叶片发黄现象，本发明的短肽明显延长白菜的贮存期。
实施例13
延长果菜类如黄瓜、菜椒、豆角、甜菜贮存期的效果实验(合成本发明涉及的短肽：Tyr-Leu-Tyr-Glu-Val-Leu-Arg)
采用固相Fmoc方法化学合成本发明短肽(Tyr-Leu-Tyr-Glu-Val-Leu-Arg)，经过HPLC纯化后，纯度超过97％，制备成浓度较高的短肽溶液，为10mg/ml的溶液。
取黄瓜、菜椒、豆角、分别扎成捆，分别喷雾上述本发明制备成的溶液后，放置在塑料袋里，实验组和不喷本发明涉及的短肽溶液的对照组相比，延长5天左右的保存期。
实施例14
延长瓜类如西瓜的贮存期的效果实验(合成本发明涉及的短肽：
Tyr-Ile-Tyr-Glu-Val-Ala-Lys)
采用固相Fmoc方法化学合成本发明短肽(Tyr-Ile-Tyr-Glu-Val-Ala-Lys)，经过HPLC纯化后，纯度超过97％，制备成浓度较高的短肽溶液，为10mg/ml的溶液。
取西瓜数个，在瓜蒂部位喷涂本发明涉及的短肽溶液，在沙藏的条件下，有在瓜蒂部位涂本发明制备的短肽溶液的西瓜比没有喷涂的保鲜期多10天左右，即可达到50天的保鲜期。
实施例15
延长浆果类如草莓、蓝莓、黑莓、葡萄的贮存期的效果实验(合成本发明涉及的短肽：Tyr-Ile-Tyr-Glu-Val-Ala-Arg)
采用固相Fmoc方法化学合成本发明短肽(Tyr-Ile-Tyr-Glu-Val-Ala-Arg)，经过HPLC纯化后，纯度超过97％，制备成浓度较高的短肽溶液，为10mg/ml的溶液。
取草莓、蓝莓和黑莓，喷雾本发明涉及的短肽溶液，可延长2天的货架期，也就是从原本5天的货架期可延长至7天的货架期，本发明的短肽还具有抑制细菌生长的作用。
取葡萄足量，喷雾本发明涉及的短肽溶液，可延长葡萄贮存期3天以上。
实施例16
延长核桃类如樱桃、桃子、大枣、龙眼、梅子的贮存期的实验效果(合成本发明涉及的短肽：Tyr-Ile-Tyr-Glu-Gly-Gly-Lys)
采用固相Fmoc方法化学合成本发明短肽(Tyr-Ile-Tyr-Glu-Gly-Gly-Lys)，经过HPLC纯化后，纯度超过97％，制备成浓度较高的短肽溶液，为10mg/ml的溶液。
取樱桃好果1000个，好果率100％，500个放置在含有本发明涉及的短肽溶 液中，液体贮存；500个置于含有苯甲酸钠食品防腐剂的溶液中。40天后，用本发明涉及的短肽溶液浸泡的樱桃，好果率为98％，而用苯甲酸钠泡的樱桃，好果率在89％。很明显，直观观察既能看到樱桃果之间的差异，该肽明显延长樱桃的贮存期。
同样的方法处理桃子、大枣、梅子获得了类似的延长贮存期的实验效果。
实施例17
延长仁果类如苹果、梨、枇杷的贮存期的实验效果(合成本发明涉及的短肽：Tyr-Leu-Tyr-Glu-Val-Gly-Arg)
采用固相Fmoc方法化学合成本发明短肽(Tyr-Leu-Tyr-Glu-Val-Gly-Arg)，经过HPLC纯化后，纯度超过99％，制备成浓度较高的短肽溶液，为10mg/ml的溶液。
选择无内外伤、无病虫害的优质苹果，置于本发明涉及的短肽配制的溶液中浸泡5分钟，捞出来，放置阴凉处，发汗2天，之后，装入保鲜袋。该方法处理过的苹果贮存期可达1年以上，比其他方式处理的苹果贮存期延长1～2个月之久。
同样的方法处理梨、枇杷也获得了延长贮存期的效果。
实施例18
延长花卉类如玫瑰、康乃馨和波斯菊花期的实验效果(合成本发明涉及的短肽：Tyr-Leu-Tyr-Glu-Val-Ala-Lys)
采用固相Fmoc方法化学合成本发明短肽，经过HPLC纯化后，纯度超过96.3％，用生理盐水PBS溶解本发明的肽，制备成10mg/ml的溶液。
分别取玫瑰花、康乃馨花和波斯菊花，分别用本发明涉及的短肽制备的上述溶液，喷洒到花上及茎叶处。该方法可延长花期2～3天。

Claims (10)

  1. 一种促进植物生根、发芽、生长和延长贮存期的短肽，其特征在于：组成短肽的氨基酸序列如下所示：
    Tyr-Leu-Tyr-Glu-Val-Gly-Arg或者
    Tyr-Leu-Tyr-Glu-Val-Ala-Arg或者
    Tyr-Leu-Tyr-Glu-Val-Ile-Arg或者
    Tyr-Leu-Tyr-Glu-Val-Val-Arg或者
    Tyr-Leu-Tyr-Glu-Val-Leu-Arg或者
    Tyr-Ile-Tyr-Glu-Val-Ala-Arg或者
    Tyr-Ile-Tyr-Glu-Val-Ala-Lys或者
    Tyr-Ile-Tyr-Glu-Gly-Gly-Lys。
  2. 一种编码根据权利要求1所述短肽的核苷酸。
  3. 一种如权利要求1所述的促进植物生根、发芽、生长和延长贮存期的短肽的短肽片段，其特征在于：所述短肽片段为将所述的7个氨基酸的序列从任意位置截取成4个或5个或6个氨基酸组成的，且具有促进植物发芽、生长和延长贮存期的短肽片段。
  4. 一种如权利要求2所述的促进植物生根、发芽、生长和延长贮存期的短肽的短肽片段，其特征在于：所述短肽片段的核苷酸片段为把所述的7个氨基酸的对应的核苷酸序列从任意位置截取成4个或5个或6个氨基酸对应的碱基序列所组成的，且具有促进植物生根、发芽、生长和延长贮存期的短肽片段。
  5. 一种如权利要求1所述的促进植物生根、发芽、生长和延长贮存期的短肽的类似物，其特征在于：所述短肽类似物是指在用所述短肽和另一种化合物融合或者用所述短肽的氨基酸序列融合另外的多肽或者蛋白质而形成，且具有促进植物发芽、生长和延长贮存期的多肽序列或蛋白质。
  6. 一种如权利要求1所述的促进植物生根、发芽、生长和延长贮存期的短肽的衍生物，其特征在于：所述短肽衍生物是指把所述短肽的氨基酸的序列中的氨基酸的一个或者几个氨基酸的某个基团用另外的基团取代后，且具有促进植物发芽、生长和延长贮存期的短肽。
  7. 一种如权利要求1所述的促进植物生根、发芽、生长和延长贮存期的短肽的 变体，其特征在于：所述短肽变体是指一种具有一个或几个氨基酸或核苷酸改变的氨基酸序列或编码它的核苷酸序列，所述改变包括在氨基酸序列或核苷酸序列中，在序列中间的任一位置缺失、插入或替换氨基酸或核苷酸，或在序列两端添加氨基酸或核苷酸，与本发明短肽有序列同源性或相似性。
  8. 一种如权利要求1、2、3、4、5、6或7所述的促进植物生根、发芽、生长和延长贮存期的短肽的应用，其特征在于：所述短肽、核苷酸、短肽片段、短肽类似物、短肽衍生物和短肽变体制备具有促进种子植物生根、发芽、生长和延长贮存期的生物功能性制品，在低浓度范围0.01mg/L～1mg/L促进植物生根、发芽生长；在高浓度范围1mg/L～1000mg/L抑制生长适于延长贮存期。
  9. 根据权利要求8所述的应用，其特征在于：所述的生物功能性是指短肽溶液在低浓度时促进种子植物的生根、发芽、促进植物的生长以及在高浓度时延长植物种子或者成熟果实等的贮存时间的应用功能；所述种子植物包含被子植物和裸子植物。
  10. 根据权利要求8所述的应用，其特征在于：所述种子植物包含但不限于蔬菜类，所述植物种子还包括水果类、粮食类、坚果类和花卉类。
PCT/CN2014/090149 2013-11-05 2014-11-03 一种促进植物生根、发芽、生长和延长贮存期的短肽及其应用 WO2015067146A1 (zh)

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