Method for screening low-temperature-resistant biostimulant composition based on CBFs gene and composition

Abstract

The invention discloses a low-temperature-resistant biostimulant composition screened based on a CBFs gene. The low-temperature-resistant biostimulant composition comprises beta-nicotinamide mononucleotide and at least one of Ectoine and / or a microbial fermentation product containing Ectoine, amino acid and a derivative thereof, sugar and a derivative thereof, and a seaweed extract. The biostimulant composition contains beta-nicotinamide mononucleotide, so that the low-temperature-resistant CBFs gene expression in plants can be promoted, the low-temperature stress resistance of the plants is improved, the plant growth is promoted, and the yield is increased.

Description

Technical Field

[0001] The present application belongs to the field of agricultural production technology, and specifically relates to a method and composition for screening a low-temperature resistant biostimulant composition based on CBFs genes, and more specifically to a biostimulant composition, a fertilizer, a method for screening a target biostimulant substance, and a method for improving the low-temperature resistance of crops. Background Art

[0002] Low temperature, as a major adverse stress, not only affects the growth and development of plants, but also limits the geographical distribution of plants. Plant biostimulants are products that do not rely on plant nutrients. They can stimulate the natural processes of plants, thereby promoting the absorption of plant nutrients, improving the nutrient utilization efficiency of plants or plant rhizosphere, improving the ability of plants to resist abiotic stress, improving crop quality, and promoting the effective absorption of nutrients in the soil or rhizosphere. Biostimulants can solve problems that fertilizers and pesticides cannot solve, help reduce the amount of chemical fertilizers and pesticides and increase their efficiency, and use biostimulants to achieve the functional requirements of crop growth promotion, quality improvement and stress resistance, which has important practical significance. In view of its outstanding role in the growth process of crops, biostimulants are developing rapidly in the global market.

[0003] In the domestic market, the focus is on the development of biostimulant raw materials represented by humic acid, amino acids, and alginic acid, and there is less research on some highly active substances that resist low temperature stress. At the same time, in the evaluation of the low temperature effect of biostimulant products, crop phenotype is often used as a screening indicator. Although mild to moderate low temperature stress has affected the genes and physiological and biochemical indicators in the plant, there is no obvious change in the plant phenotype, which is not easy to observe. At the same time, the crop phenotype is easily disturbed by the external environment, and the accuracy and efficiency are low; gene molecule-related research methods are mostly used in the fields of biological breeding, and are rarely used in the field of crop nutrition and fertilizer.

[0004] Therefore, there is an urgent need to develop a highly active and low-temperature resistant biostimulant composition product. Summary of the invention

[0005] The present application aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present application provides a low-temperature resistant composition based on CBFs gene screening, which can promote the expression of plant low-temperature resistant CBFs genes, improve the ability of crops to resist low-temperature stress, and promote plant growth and increase yield.

[0006] This application is completed based on the following findings of the inventors:

[0007] In order to cope with low temperature stress, plants have evolved complex and sophisticated regulatory networks, among which the most widely studied is the low temperature signaling pathway with the transcription factor CBF / DREB1 (C-repeat binding factor / dehydration-responsive element binding factor 1) as the core. When plants perceive low temperature signals, they can quickly induce the expression of CBFs genes, thereby activating the expression of its downstream COR genes and enhancing the plant's tolerance to low temperatures. The CBFs transcription factors in Arabidopsis contain three members, namely CBF1 / DREB1B, CBF2 / DREB1C and CBF3 / DREB1A, which are tightly arranged in series on chromosome 4 of Arabidopsis. Overexpression of CBFs genes in plants significantly enhances the plant's frost resistance.

[0008] However, there are currently many related genes in the low-temperature signaling pathway, and no research has shown what specific method can be used to screen the anti-cold composition. The inventors of the present application unexpectedly discovered that compared with other genes, the accuracy of using CBFs gene expression to screen the anti-cold composition is high; and compared with traditional phenotypic screening methods, the method of using CBFs gene expression to screen the anti-cold composition has high sensitivity and is less affected by the environment, which can not only improve the screening accuracy, but also improve the screening efficiency.

[0009] β-Nicotinamide mononucleotide (NMN) is a naturally occurring biologically active nucleotide that exists in various organisms and is widely used in the cosmetics field. It is also a key intermediate in the synthesis of coenzyme I-NAD+. However, there has been no relevant research in the agricultural field, especially in the field of crop resistance to cold stress.

[0010] Ecdoin was first discovered in halophilic bacteria. Under extreme conditions of high salt, high temperature, and high ultraviolet radiation, Ecdoin protects halophilic bacteria from harm. After Ecdoin was developed, it was widely used in the pharmaceutical industry, and various drugs were developed and produced, such as eye drops, nasal sprays, oral sprays, etc. Ecdoin has been proven to be an alternative to corticosteroids without any side effects. It can be used to treat eczema and neurodermatitis, and is approved for the treatment and prevention of pollution-induced lung diseases. Studies have shown that Ecdoin also has a good repair and protective effect on the skin, and is one of the bioengineering preparations used in high-end cosmetics. Ecdoin is mostly synthesized by chemical methods and is expensive, so it is mostly used in biomedicine and skin care products. Limited by the application cost, there is no relevant research in the agricultural field, especially in the field of crop resistance to low temperature stress.

[0011] However, when screening for cold-resistant compositions using CBFs gene expression, it was unexpectedly discovered that both NMN and Ecdoin can significantly improve the cold resistance of crops. Therefore, compositions prepared using NMN and / or Ecdoin can promote the expression of plant cold-resistant CBFs genes, improve the ability of crops to resist cold stress, and promote plant growth and increase yield.

[0012] Therefore, in the first aspect of the present application, the present application proposes a use of β-nicotinamide mononucleotide in the preparation of crop products. β-nicotinamide mononucleotide can promote the expression of low-temperature resistant CBFs genes in crops and improve the ability of crops to resist low temperature stress. Thus, crop products (such as fertilizers, etc.) prepared using β-nicotinamide mononucleotide can improve the ability of crops to resist low temperature stress, promote crop growth, and increase yield.

[0013] In the second aspect of the present application, the present application proposes a biostimulant composition. According to an embodiment of the present application, the biostimulant composition includes: β-nicotinamide mononucleotide, and at least one of ectoine and / or microbial fermentation products containing ectoine, amino acids and their derivatives, sugars and their derivatives, and seaweed extracts. The above compositions all contain β-nicotinamide mononucleotide, thereby promoting the expression of cold-resistant CBFs genes in plants (especially crops), improving the ability of plants to resist cold stress, and promoting plant growth and increasing production.

[0014] In the third aspect of the present application, the present application proposes a biostimulant composition. According to an embodiment of the present application, the composition is selected from at least one of the following groups:

[0015] 1) Includes:

[0016] 0.5 to 2.5 parts by weight of β-nicotinamide mononucleotide, 1.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, and 2 of the following:

[0017] 10.0 to 20.0 parts by weight of amino acids and their derivatives,

[0018] 35.0-50.0 parts by weight of sugar and its derivatives,

[0019] 30.0-80.0 parts by weight of seaweed extract;

[0020] 2) Includes:

[0021] 0.5 to 2.5 parts by weight of β-nicotinamide mononucleotide,

[0022] 1.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L,

[0023] 10.0 to 20.0 parts by weight of amino acids and their derivatives,

[0024] 40.0-45.0 parts by weight of sugar and its derivatives,

[0025] 30.0 to 80.0 parts by weight of seaweed extract.

[0026] The above-mentioned biostimulant compositions all contain β-nicotinamide mononucleotide, thereby promoting the expression of low-temperature-resistant CBFs genes in plants (especially crops), improving the ability of plants to resist low-temperature stress, and promoting plant growth and increasing yield.

[0027] In a fourth aspect of the present application, the present application proposes a biostimulant composition. According to an embodiment of the present application, the composition is selected from at least one of the following groups:

[0028] 1) Includes:

[0029] 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide,

[0030] 1.0 to 3.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L,

[0031] 8.0 parts by weight of compound amino acid powder,

[0032] 2.0 parts by weight of an amino acid derivative,

[0033] 40.0 parts by weight of chitosan oligosaccharide,

[0034] 45.0-50.0 parts by weight of seaweed extract;

[0035] 2) Includes:

[0036] 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide,

[0037] 4.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, 40.0 parts by weight of chitosan oligosaccharide,

[0038] 75.0 parts by weight of seaweed 30;

[0039] 3) Includes:

[0040] 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide,

[0041] 4.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, 30.0 parts by weight of polyglutamic acid,

[0042] 75.0 parts by weight of seaweed 30;

[0043] 4) Includes:

[0044] 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide,

[0045] 1.0 to 3.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, 8.0 parts by weight of a composite amino acid powder,

[0046] 2.0 parts by weight of an amino acid derivative,

[0047] 40.0 parts by weight of chitosan oligosaccharide;

[0048] 5) Includes:

[0049] 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide,

[0050] 4.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, 5.0 parts by weight of gamma-aminobutyric acid,

[0051] 5.0 parts by weight of melatonin,

[0052] 40.0 parts by weight of chitosan oligosaccharide,

[0053] 30.0 parts by weight of seaweed 45;

[0054] 6) Includes:

[0055] 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide,

[0056] 0.5.0 to 1.5 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, 5.0 parts by weight of nicotinamide,

[0057] 15.0 parts by weight of melatonin,

[0058] 45.0 parts by weight of oligosaccharides,

[0059] 70.0 parts by weight of seaweed 30;

[0060] 7) Includes:

[0061] 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide,

[0062] 4.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, 5.0 parts by weight of gamma-aminobutyric acid,

[0063] 10.0 parts by weight of ergothioneine,

[0064] 15.0 parts by weight of chitosan oligosaccharide,

[0065] 15.0 parts by weight of chitin,

[0066] 15.0 parts by weight of oligosaccharides,

[0067] 80.0 parts by weight of kelp extract.

[0068] The above compositions all contain β-nicotinamide mononucleotide, thereby promoting the expression of low-temperature resistant CBFs genes in plants (especially crops), improving the ability of plants to resist low-temperature stress, and promoting plant growth and increasing yield.

[0069] In the fifth aspect of the present application, the present application proposes a fertilizer. According to an embodiment of the present application, the fertilizer includes the biostimulant composition described in the second aspect, the third aspect or the fourth aspect. The above fertilizers all contain β-nicotinamide mononucleotide, thereby promoting the expression of cold-resistant CBFs genes in plants (especially crops), improving the ability of plants to resist cold stress, promoting plant growth and increasing production, and the fertilizer has stable properties and a wide range of applications.

[0070] In the sixth aspect of the present application, the present application proposes a method for screening target biostimulant substances. According to an embodiment of the present application, the method includes: contacting the material to be screened with a crop, detecting the CBFs gene expression level before and after the contact treatment of the crop; and determining the target biostimulant substance based on the change in the CBFs gene expression level before and after the contact treatment of the crop. Through the above-mentioned screening method, the present application can screen out a low-temperature resistant biostimulant composition, which can promote the expression of plant low-temperature resistant CBFs genes, improve the ability of crops to resist low-temperature stress, and promote plant growth and increase production. Compared with traditional phenotypic screening methods, the above-mentioned screening method has high sensitivity, is less affected by the environment, and has the advantages of high screening accuracy, time saving, and high stability.

[0071] In the seventh aspect of the present application, the present invention discloses a method for preparing the above-mentioned biostimulant composition. According to an embodiment of the present application, the method comprises: (1) establishing a crop CBFs gene evaluation method, and screening biostimulant substances that resist low temperature stress according to the crop CBFs gene evaluation method; (2) preparing the biostimulant composition using the target biostimulant substance screened in step (1); and (3) detecting the effect of the prepared biostimulant composition on crops resisting low temperature stress. The biostimulant composition prepared by the above method in the present application can promote the expression of plant anti-low temperature CBFs genes, improve the ability of crops to resist low temperature stress, and promote plant growth and increase production. Compared with traditional phenotypic screening methods, the above screening method has high sensitivity, is less affected by the environment, and has the advantages of high screening accuracy, time saving, and high stability.

[0072] In the eighth aspect of the present application, the present application proposes a method for improving the low temperature resistance of crops. According to an embodiment of the present application, the method comprises: cultivating crops with the biostimulant composition described in the second aspect, the third aspect or the fourth aspect, the fertilizer described in the fifth aspect, or the target biostimulant substance screened by the method described in the sixth aspect.

[0073] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0075] Figure 1 The HPLC spectrum of a highly active microbial fermentation product in an example of the present application;

[0076] Figure 2 This is a step for synthesizing ectoine in a microbial fermentation product in an example of the present application;

[0077] Figure 3 This is a graph showing the resistance results of Samples 11 and 15 in Test Example 2 of this application to the sudden cold snap in late spring during the peach tree flowering period. DETAILED DESCRIPTION

[0078] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0079] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0080] In this document, the terms "include" or "comprising" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0081] As used herein, the terms "optionally", "optional", "optionally", "optional" or "optional" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0082] The present application proposes a composition, a fertilizer, a method for screening a target biostimulant substance, and a method for improving the low temperature resistance of crops, which will be described in detail below.

[0083] use

[0084] In the first aspect of the present application, the present application proposes a use of β-nicotinamide mononucleotide in the preparation of crop products. The present application first discovered that β-nicotinamide mononucleotide can promote the expression of cold-resistant CBFs genes in crops and improve the ability of crops to resist cold stress. Therefore, crop products (such as fertilizers, etc.) prepared using β-nicotinamide mononucleotide can improve the ability of crops to resist cold stress, promote crop growth, and increase yield.

[0085] According to an embodiment of the present application, the crop product is used to improve the low temperature resistance of crops.

[0086] According to an embodiment of the present application, the crops include Arabidopsis, tomato, corn, potato, grape, cabbage, and peach.

[0087] According to an embodiment of the present application, the crop product is fertilizer.

[0088] In the second aspect of the present application, the present application proposes a biostimulant composition. According to an embodiment of the present application, the biostimulant composition includes: β-nicotinamide mononucleotide, ectoine and / or a microbial fermentation product containing ectoine; and at least two of amino acids and their derivatives, sugars and their derivatives, and seaweed extracts. The present application first discovered that β-nicotinamide mononucleotide can promote the expression of cold-resistant CBFs genes in plants (especially crops) and improve the ability of plants to resist cold stress. Under the synergistic effect of β-nicotinamide mononucleotide and ectoine and / or a microbial fermentation product containing ectoine, the ability of plants to resist cold stress can be significantly improved. Therefore, the biostimulant composition prepared using β-nicotinamide mononucleotide, ectoine and / or a microbial fermentation product containing ectoine, and at least two of amino acids and their derivatives, sugars and their derivatives, and seaweed extracts can improve the ability of plants to resist cold stress, promote plant growth, and increase production.

[0089] Furthermore, the inventors unexpectedly discovered that ectoine and / or microbial fermentation products containing ectoine and β-nicotinamide mononucleotide work synergistically to stimulate the natural growth process of plants, especially to stimulate gene expression in plants, thereby increasing the physiological activity of the crops themselves, improving the physiological and biochemical characteristics of plants, and increasing the resistance of plants to low temperature stress without causing harm to plants and the environment, thereby achieving the effects of promoting plant growth and increasing production.

[0090] According to an embodiment of the present application, the content of icodine in the microbial fermentation product is 30 g / L to 50 g / L.

[0091] In an optional embodiment of the present application, the biostimulant composition includes β-nicotinamide mononucleotide, microbial fermentation products containing ectoine, amino acids and their derivatives, sugars and their derivatives, and seaweed extracts.

[0092] In an optional embodiment of the present application, the microbial fermentation product containing ectoine (hereinafter referred to as microbial fermentation product) can be obtained by conventional methods in the art.

[0093] In an optional embodiment of the present application, the microbial fermentation product containing ectoine is obtained by: genetically modifying halophiles using synthetic biology methods to obtain modified halophiles; fermenting the modified halophiles to obtain microbial fermentation products. The fermentation content of ectoine in the microbial fermentation product produced by unmodified halophiles under fermentation conditions is low, only reaching 10g / L; however, the fermentation content of ectoine in the microbial fermentation product produced by genetically modified halophiles is 30g / L to 50g / L.

[0094] In an optional embodiment of the present application, the genetic modification is obtained by knocking out the Halomonas salina DSM 5928 of the halophilic bacterium T teaABC genes. Thus, H.salina DSM5928 can be constructed. T The modified halophilic bacteria has a fermentation content of 30 g / L to 50 g / L of ectoine in the high-activity microbial fermentation product. The HPLC spectrum of the high-activity microbial fermentation product is shown in Figure 1 .

[0095] In an optional embodiment of the present application, the genetic modification is performed by Red recombination. For example, by cloning Halomonas salina DSM 5928 T TeaABC gene knockout in Halomonassalina DSM 5928 using Red recombination technology T teaABC genes. For details, see "Xu Rui. Construction of engineering strains for ectoine synthesis [D]. Dalian Maritime University, 2012. DOI: 10.7666 / d.y2089190."

[0096] It should be noted that the process of obtaining ectoine from microbial fermentation products can be found in Figure 2 . Specifically: L-aspartate-β-semialdehyde (ASA) is used as a substrate, catalyzed by 2,4-diaminobutyric acid aminotransferase (EctB) to generate L-2,4-diaminobutyric acid (DABA); under the catalysis of 2,4-diaminobutyric acidacetyltransferase (EctA), DABA is acetylated to generate N-acetyl-L-2,4-diaminobutyric acid (ADABA); finally, ectoine synthase (EctC) cyclizes ADABA to form ectoine. Hydroxyectoine is generated by ectoinehydroxylase (EctD) hydroxylating ectoine.

[0097] According to an embodiment of the present application, the amino acid and its derivatives are selected from at least one of melatonin, nicotinamide, proline, polyglutamic acid, fish protein peptide, gamma-aminobutyric acid, and ergothioneine. Thus, the biostimulant composition contains the above amino acids and their derivatives, which can further improve the plant's resistance to low temperatures and promote plant growth.

[0098] According to an optional embodiment of the present application, the amino acid and its derivatives are selected from at least one of melatonin, nicotinamide, γ-aminobutyric acid, and ergothioneine.

[0099] According to an embodiment of the present application, the sugar and its derivatives are selected from at least one of pantothenic acid polysaccharide, chitosan oligosaccharide, seaweed polysaccharide, chitin, oligosaccharide, chitosan and chitosan derivatives. Therefore, the biostimulant composition contains the above-mentioned sugar and its derivatives, which can further improve the plant's resistance to low temperature and promote plant growth.

[0100] According to an optional embodiment of the present application, the average molecular weight of the chitosan oligosaccharide is 800-1000, and the proportion of chitosan pentose and chitosan hexaose is high (for example, greater than or equal to 80%).

[0101] According to an embodiment of the present application, the seaweed extract is selected from at least one of seaweed essence, seaweed mother liquid, seaweed 30, seaweed 45, kelp extract, giant kelp extract, ascophyllum extract, and sea cystis extract. Therefore, the biostimulant composition contains the above seaweed extract, which can further improve the plant's resistance to low temperature and promote plant growth.

[0102] According to an embodiment of the present application, the biostimulant composition includes β-nicotinamide mononucleotide, ectoine or a microbial fermentation product containing ectoine, amino acids and their derivatives, sugars and their derivatives, and seaweed extract.

[0103] According to an embodiment of the present application, the weight ratio of the β-nicotinamide mononucleotide to ectoine is (0.1-5.0):(0.03-0.25), preferably (0.1-2.0):(0.05-0.2).

[0104] According to an embodiment of the present application, the weight ratio of the β-nicotinamide mononucleotide and the microbial fermentation product is (0.1-5.0): (1.0-5.0), for example (0.1-5.0): (1.0-4.0), (0.1-5.0): (1.0-3.0), (0.1-5.0): (1.0-2.0), (0.1-5.0): (2.0-5.0), (0.1-5.0): (2.0-4.0), (0.1-5.0): (2.0-3.0), (0.1-5.0): (3.0-5.0). )、(0.1~5.0):(3.0~4.0)、(0.1~5.0):(4.0~5.0)、(0.5~5.0):(1.0~4.0)、(0.5~5.0):(1.0~3.0)、(0.5~5.0):(1.0~2.0)、(0.5~5.0):(2.0~5.0)、(0.5~5.0):(2.0~4.0)、(0.5~5.0):(2.0~3.0)、(0.5~5.0):(3.0~5.0)、(0.5~5.0):(3 .0~4.0)、(0.5~5.0):(4.0~5.0)、(0.5~3.0):(1.0~4.0)、(0.5~3.0):(1.0~3.0)、(0.5~3.0):(1.0~2.0)、(0.5~3.0):(2.0~5.0)、(0.5~3.0):(2.0~4.0)、(0.5~3.0):(2.0~3.0)、(0.5~3.0):(3.0~5.0)、(0.5~3.0):(3.0~4.0)、(0.5~3 .0): (4.0~5.0), (0.5~2.5): (1.0~4.0), (0.5~2.5): (1.0~3.0), (0.5~2.5): (1.0~2.0), (0.5~2.5): (2.0~5.0), (0.5~2.5): (2.0~4.0), (0.5~2.5): (2.0~3.0), (0.5~2.5): (3.0~5.0), (0.5~2.5): (3.0~4.0), (0.5~2.5): (4.0~5.0).

[0105] According to an optional embodiment of the present application, the weight ratio of the β-nicotinamide mononucleotide to the microbial fermentation product is (0.5-2.5): (1.0-5.0).

[0106] According to an embodiment of the present application, the weight ratio of the β-nicotinamide mononucleotide and the amino acid and its derivatives is (0.1-5.0): (3.0-30.0), for example (0.1-5.0): (5.0-30.0), (0.1-5.0): (5.0-25.0), (0.1-5.0): (5.0-20.0), (0.1-5.0): (10.0-30.0), (0.1~5.0):(10.0~25.0), (0.1~5.0):(10.0~20.0), (0.5~5.0):(5.0~30.0), (0.5~5.0):(5.0~25.0), (0.5~5.0):(5.0~20.0), (0.5~5.0):(10.0~30.0), (0.5~5.0):(10.0 ~25.0)、(0.5~5.0):(10.0~20.0)、(0.5~3.0):(5.0~30.0)、(0.5~3.0):(5.0~25.0)、(0.5~3.0):(5.0~20.0)、(0.5~3.0):(10.0~30.0)、(0.5~3.0):(10.0~25.0)、(0.5~3. 0): (10.0~20.0), (0.5~2.5): (5.0~30.0), (0.5~2.5): (5.0~25.0), (0.5~2.5): (5.0~20.0), (0.5~2.5): (10.0~30.0), (0.5~2.5): (10.0~25.0), (0.5~2.5): (10.0~20.0).

[0107] According to an optional embodiment of the present application, the weight ratio of the β-nicotinamide mononucleotide to the amino acid and its derivatives is (0.5-2.5): (10.0-20.0).

[0108] According to an embodiment of the present application, the weight ratio of the β-nicotinamide mononucleotide and the sugar and its derivatives is (0.1-5.0): (10.0-50.0), for example, (0.1-5.0): (15.0-50.0), (0.1-5.0): (20.0-50.0), (0.1-5.0): (25.0-50.0), (0.1-5.0): (30.0-50.0), (0.1-5.0): (35.0-50.0), (0.5-5 .0): (10.0~50.0), (0.5~5.0): (15.0~50.0), (0.5~5.0): (20.0~50.0), (0.5~5.0): (25.0~50.0), (0.5~5.0): (30.0~50.0), (0.5~5.0): (35.0~50.0), (0.5~4.0): (10.0~50.0), (0.5~4.0): (15.0~50.0), (0.5 ~4.0):(20.0~50.0)、(0.5~4.0):(25.0~50.0)、(0.5~4.0):(30.0~50.0)、(0.5~4.0):(35.0~50.0)、(0.5~3.0):(10.0~50.0)、(0.5~3.0):(15.0~50.0)、(0.5~3.0):(20.0~50.0)、(0.5~3.0):(25.0~50.0)、(0 .5~3.0): (30.0~50.0), (0.5~3.0): (35.0~50.0), (0.5~2.5): (10.0~50.0), (0.5~2.5): (15.0~50.0), (0.5~2.5): (20.0~50.0), (0.5~2.5): (25.0~50.0), (0.5~2.5): (30.0~50.0), (0.5~2.5): (35.0~50.0).

[0109] According to an optional embodiment of the present application, the weight ratio of the β-nicotinamide mononucleotide to the sugar and its derivatives is (0.5-2.5): (35.0-50.0).

[0110] According to an embodiment of the present application, the weight ratio of the β-nicotinamide mononucleotide and the seaweed extract is (0.1-5.0): (20.0-100.0), for example, (0.1-5.0): (20.0-95.0), (0.1-5.0): (20.0-90.0), (0.1-5.0): (20.0-85.0), (0.1-5.0): (20.0-80.0), (0.1-5.0): (20.0-70.0), (0.1-5.0): (20.0-60.0), (0.1-5.0): (20.0-50.0), (0.1-5.0): (20.0-40.0), (0.1-5.0): (30.0- 100.0)、(0.1~5.0):(30.0~95.0)、(0.1~5.0):(30.0~90.0)、(0.1~5.0):(30.0~85.0)、(0.1~5.0):(30.0~80.0)、(0.1~5.0):(30.0~70.0)、(0.1~5.0):(30.0~60.0)、(0.1~5.0):(30.0~50.0)、(0.5~5.0):(20.0~95.0)、(0.5~5.0):(20.0~90.0)、(0.5~5.0):(20.0~85.0)、(0.5~5.0):(20.0~80.0)、( 0.5~5.0):(30.0~100.0)、(0.5~5.0):(30.0~95.0)、(0.5~5.0):(30.0~90.0)、(0.5~5.0):(30.0~85.0)、(0.5~5.0):(30.0~80.0)、(0.5~4.0):(20.0~95.0)、(0.5~4.0):(20.0~90.0)、(0.5~4.0):(20.0~85.0)、(0.5~4.0):(20.0~80.0)、(0.5~100.0)、(0.5~4.0):(30.0~95.0)、(0.5~4. 0): (30.0~90.0), (0.5~4.0): (30.0~85.0), (0.5~4.0): (30.0~80.0), (0.5~3.0): (20.0~95.0), (0.5~3.0): (20.0~90.0), (0.5~3.0): (20.0~85.0), (0.5~3.0): (20.0~80.0), (0.5~3.0): (30.0~100.0), (0.5~3.0): (30.0~95.0), (0.5~3.0): (30.0~90.0), (0.5~3.0): (30.0~85.0), (0.5~3.0): (30.0~80.0)、(0.5~2.5):(20.0~95.0)、(0.5~2.5):(20.0~90.0)、(0.5~2.5):(20.0~85.0)、(0.5~2.5):(20.0~80.0)、(0.5~2.5):(30.0~100.0)、(0.5~2.5):(30.0~95.0)、(0.5~2.5):(30.0~90.0)、(0.5~2.5):(30.0~85.0)、(0.5~2.5):(30.0~80.0)。 .

[0111] According to an optional embodiment of the present application, the weight ratio of the β-nicotinamide mononucleotide to the seaweed extract is (0.5-2.5): (30.0-80.0).

[0112] According to an embodiment of the present application, the seaweed extract is selected from seaweed essence, and the weight ratio of the β-nicotinamide mononucleotide and the seaweed extract is (0.1-5.0): (30.0-60.0), for example, (0.5-5.0): (30.0-60.0), (0.5-4.0): (30.0-60.0), (0.5-3.0): (30.0-60.0), (0.5-2.5): (30.0-60.0), (0.1-5.0): (40.0-50.0), (0.5-5.0): (40.0-50.0), (0.5-4.0): (40.0-50.0), (0.5-3.0): (40.0-50.0), (0.5-2.5): (40.0-50.0).

[0113] According to an embodiment of the present application, the seaweed extract is selected from seaweed mother liquor, and the weight ratio of the β-nicotinamide mononucleotide and the seaweed extract is (0.1-5.0): (30.0-60.0), for example, (0.5-5.0): (30.0-60.0), (0.5-4.0): (30.0-60.0), (0.5-3.0): (30.0-60.0), (0.5-2.5): (30.0-60.0), (0.1-5.0): (45.0-55.0), (0.5-5.0): (45.0-55.0), (0.5-4.0): (45.0-55.0), (0.5-3.0): (45.0-55.0), (0.5-2.5): (45.0-55.0).

[0114] According to an embodiment of the present application, the seaweed extract is selected from seaweed 30, and the weight ratio of the β-nicotinamide mononucleotide and the seaweed extract is (0.1-5.0): (50.0-100.0), for example, (0.5-5.0): (50.0-100.0), (0.5-4.0): (50.0-100.0), (0.5-3.0): (50.0-100.0). .0), (0.5~2.5): (50.0~100.0), (0.1~5.0): (65.0~80.0), (0.5~5.0): (65.0~80.0), (0.5~4.0): (65.0~80.0), (0.5~3.0): (65.0~80.0), (0.5~2.5): (65.0~80.0).

[0115] According to an embodiment of the present application, the seaweed extract is selected from seaweed 45, and the weight ratio of the β-nicotinamide mononucleotide and the seaweed extract is (0.1-5.0): (20.0-50.0), for example, (0.5-5.0): (20.0-50.0), (0.5-4.0): (20.0-50.0), (0.5-3.0): (20.0-50.0), (0.5-2.5): (20.0-50.0), (0.1-5.0): (25.0-35.0), (0.5-5.0): (25.0-35.0), (0.5-4.0): (25.0-35.0), (0.5-3.0): (25.0-35.0), (0.5-2.5): (25.0-35.0).

[0116] According to an embodiment of the present application, the seaweed extract is selected from kelp extract, and the weight ratio of β-nicotinamide mononucleotide and seaweed extract is (0.1-5.0): (50.0-100.0), for example, (0.5-5.0): (50.0-100.0), (0.5-4.0): (50.0-100.0), (0.5-3.0): (50.0-100.0). .0), (0.5~2.5): (50.0~100.0), (0.1~5.0): (75.0~95.0), (0.5~5.0): (75.0~95.0), (0.5~4.0): (75.0~95.0), (0.5~3.0): (75.0~95.0), (0.5~2.5): (75.0~95.0).

[0117] According to an embodiment of the present application, the biostimulant composition includes β-nicotinamide mononucleotide, a microbial fermentation product containing ectoine, amino acids and their derivatives, sugars and their derivatives, and seaweed extract.

[0118] According to an embodiment of the present application, the biostimulant composition includes β-nicotinamide mononucleotide, microbial fermentation products containing ectoine, amino acids and their derivatives, sugars and their derivatives, and seaweed extract in a weight ratio of (0.1-5): (1-5): (3-30): (10-50): (20-100).

[0119] According to an embodiment of the present application, the biostimulant composition is a fertilizer.

[0120] In a third aspect of the present application, the present application proposes a biostimulant composition. According to an embodiment of the present application, the biostimulant composition is selected from at least one of the following groups:

[0121] 1) Includes:

[0122] 0.5 to 2.5 parts by weight of β-nicotinamide mononucleotide, 1.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, and 2 of the following:

[0123] 10.0 to 20.0 parts by weight of amino acids and their derivatives,

[0124] 35.0-50.0 parts by weight of sugar and its derivatives,

[0125] 30.0-80.0 parts by weight of seaweed extract;

[0126] 2) Includes:

[0127] 0.5 to 2.5 parts by weight of β-nicotinamide mononucleotide,

[0128] 1.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L,

[0129] 10.0 to 20.0 parts by weight of amino acids and their derivatives,

[0130] 40.0-45.0 parts by weight of sugar and its derivatives,

[0131] 30.0 to 80.0 parts by weight of seaweed extract.

[0132] The above-mentioned biostimulant compositions all contain β-nicotinamide mononucleotide, thereby promoting the expression of low-temperature-resistant CBFs genes in plants (especially crops), improving the ability of plants to resist low-temperature stress, and promoting plant growth and increasing yield.

[0133] In a fourth aspect of the present application, the present application proposes a biostimulant composition. According to an embodiment of the present application, the biostimulant composition is selected from at least one of the following groups:

[0134] 1) Includes:

[0135] 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide,

[0136] 1.0 to 3.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L,

[0137] 8.0 parts by weight of compound amino acid powder,

[0138] 2.0 parts by weight of an amino acid derivative,

[0139] 40.0 parts by weight of chitosan oligosaccharide,

[0140] 45.0-50.0 parts by weight of seaweed extract;

[0141] 2) Includes:

[0142] 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide,

[0143] 4.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, 40.0 parts by weight of chitosan oligosaccharide,

[0144] 75.0 parts by weight of seaweed 30;

[0145] 3) Includes:

[0146] 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide,

[0147] 4.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, 30.0 parts by weight of polyglutamic acid,

[0148] 75.0 parts by weight of seaweed 30;

[0149] 4) Includes:

[0150] 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide,

[0151] 1.0 to 3.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L,

[0152] 8.0 parts by weight of compound amino acid powder,

[0153] 2.0 parts by weight of an amino acid derivative,

[0154] 40.0 parts by weight of chitosan oligosaccharide;

[0155] 5) Includes:

[0156] 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide,

[0157] 4.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L,

[0158] 5.0 parts by weight of γ-aminobutyric acid,

[0159] 5.0 parts by weight of melatonin,

[0160] 40.0 parts by weight of chitosan oligosaccharide,

[0161] 30.0 parts by weight of seaweed 45;

[0162] 6) Includes:

[0163] 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide,

[0164] 0.5.0 to 1.5 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L,

[0165] 5.0 parts by weight of niacinamide,

[0166] 15.0 parts by weight of melatonin,

[0167] 45.0 parts by weight of oligosaccharides,

[0168] 70.0 parts by weight of seaweed 30;

[0169] 7) Includes:

[0170] 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide,

[0171] 4.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L,

[0172] 5.0 parts by weight of γ-aminobutyric acid,

[0173] 10.0 parts by weight of ergothioneine,

[0174] 15.0 parts by weight of chitosan oligosaccharide,

[0175] 15.0 parts by weight of chitin,

[0176] 15.0 parts by weight of oligosaccharides,

[0177] 80.0 parts by weight of kelp extract.

[0178] The above-mentioned biostimulant compositions all contain β-nicotinamide mononucleotide, thereby promoting the expression of low-temperature-resistant CBFs genes in plants (especially crops), improving the ability of plants to resist low-temperature stress, and promoting plant growth and increasing yield.

[0179] In the fifth aspect of the present application, the present application proposes a fertilizer. According to an embodiment of the present application, the fertilizer includes the biostimulant composition described in the second aspect, the third aspect or the fourth aspect. The above fertilizers all contain β-nicotinamide mononucleotide, thereby promoting the expression of cold-resistant CBFs genes in plants (especially crops), improving the ability of plants to resist cold stress, and promoting plant growth and increasing production.

[0180] According to an embodiment of the present application, the fertilizer further includes at least one of nitrogen fertilizer, phosphorus fertilizer, and potassium fertilizer.

[0181] According to an embodiment of the present application, the nitrogen fertilizer includes at least one of ammonium nitrogen fertilizer, nitrate nitrogen fertilizer, ammonium nitrate nitrogen fertilizer, amide nitrogen fertilizer and slow-release nitrogen fertilizer.

[0182] According to an embodiment of the present application, the phosphate fertilizer includes at least one of monoammonium phosphate, diammonium phosphate, calcium magnesium phosphate, superphosphate, and ammonium phosphate.

[0183] According to an embodiment of the present application, the potash fertilizer includes at least one of potassium chloride, potassium sulfate, potassium dihydrogen phosphate, sylvite, and potassium-magnesium salt.

[0184] According to an embodiment of the present application, the weight ratio of the total amount of the nitrogen fertilizer, the phosphorus fertilizer and the potash fertilizer to the biostimulant composition is (1-1000): (1-200), for example, (200-1000): (50-200), (300-1000): (50-200), (400-1000): (50-200), (500-1000): (50-200), (600-1000): (50-200), (200-900): (50-200), (300-900): (50-200), (400-900): (50-200), (500-900) : (50-200), (600-900): (50-200), (200-1000): (50-150), (300-1000): (50-150), (400-1000): (50-150), (500-1000): (50-150), (600-1000): (50-150), (200-900): (50-150), (300-900): (50-150), (400-900): (50-150), (500-900): (50-150), (600-900): (50-150).

[0185] According to an embodiment of the present application, the weight percentage of the biostimulant composition in the fertilizer is 0.1% to 20%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or a range value between any two of the endpoints.

[0186] In the sixth aspect of the present application, the present application proposes a method for screening target biostimulant substances. According to an embodiment of the present application, the method includes: contacting the material to be screened with a crop, detecting the CBFs gene expression level before and after the contact treatment of the crop; and determining the target biostimulant substance based on the change in the CBFs gene expression level before and after the contact treatment of the crop. Through the above-mentioned screening method, the present application can screen out a low-temperature resistant biostimulant composition, which can promote the expression of plant low-temperature resistant CBFs genes, improve the ability of crops to resist low-temperature stress, and promote plant growth and increase production. Compared with traditional phenotypic screening methods, the above-mentioned screening method has high sensitivity, is less affected by the environment, and has the advantages of high screening accuracy, time saving, and high stability.

[0187] According to an embodiment of the present application, the target biostimulant substance is used to promote the crop's resistance to low temperatures.

[0188] According to an embodiment of the present application, the contact treatment is spraying the material to be screened on the crops.

[0189] According to an embodiment of the present application, the treatment temperature of the contact treatment is 4° C., and the treatment time is 12 to 48 hours, preferably 24 hours.

[0190] According to an embodiment of the present application, the crop is a crop containing CBFs genes.

[0191] According to an embodiment of the present application, the crops include Arabidopsis, tomato, corn, potato, grape, cabbage, and peach.

[0192] According to an embodiment of the present application, the up-regulation of the CBFs gene expression in the crop after the contact treatment is an indication that the object to be screened is the target biostimulant substance.

[0193] According to an embodiment of the present application, the CBFs gene includes at least one of CBF1, CBF2 and CBF3.

[0194] In some embodiments of the present application, the CBFs gene expression level includes CBF1 qPCR gene expression level, CBF2 qPCR gene expression level, and CBF3 qPCR gene expression level.

[0195] In some embodiments of the present application, the method further comprises detecting the crop phenotype before and after the crop contact treatment, and determining the target biostimulant substance based on the crop phenotype change before and after the crop contact treatment. Exemplarily, the crop phenotype is a leaf frost damage index.

[0196] According to an embodiment of the present application, the target biostimulant substance is β-nicotinamide mononucleotide, ectoine and / or microbial fermentation products containing ectoine, amino acids and their derivatives, sugars and their derivatives, and seaweed extracts.

[0197] According to an embodiment of the present application, the target biostimulant substance is a single component or a combination.

[0198] According to an embodiment of the present application, the target biostimulant substance is the biostimulant composition described in the second aspect, the third aspect or the fourth aspect.

[0199] According to an embodiment of the present application, the target biostimulant substance is the fertilizer described in the fifth aspect.

[0200] In the seventh aspect of the present application, the present invention discloses a method for preparing the above-mentioned biostimulant composition. According to an embodiment of the present application, the method comprises: (1) establishing a crop CBFs gene evaluation method, and screening biostimulant substances that resist low temperature stress according to the crop CBFs gene evaluation method; (2) preparing the biostimulant composition using the target biostimulant substance screened in step (1); and (3) detecting the effect of the prepared biostimulant composition on crops resisting low temperature stress. The biostimulant composition prepared by the above method in the present application can promote the expression of plant anti-low temperature CBFs genes, improve the ability of crops to resist low temperature stress, and promote plant growth and increase production. Compared with traditional phenotypic screening methods, the above screening method has high sensitivity, is less affected by the environment, and has the advantages of high screening accuracy, time saving, and high stability.

[0201] According to an embodiment of the present application, the step (1) is performed using the method described in aspect 6.

[0202] In the eighth aspect of the present application, the present application proposes a method for improving the low temperature resistance of crops. According to an embodiment of the present application, the method comprises: cultivating crops with the biostimulant composition described in the second aspect, the third aspect or the fourth aspect, the fertilizer described in the fifth aspect, or the target biostimulant substance screened by the method described in the sixth aspect.

[0203] In the ninth aspect of the present application, the present application proposes a method for increasing the expression of CBFs genes. According to an embodiment of the present application, the method comprises: contacting crops with β-nicotinamide mononucleotide, the biostimulant composition described in the second aspect, the third aspect or the fourth aspect, the fertilizer described in the fifth aspect, or the target biostimulant substance screened by the method described in the sixth aspect.

[0204] According to an embodiment of the present application, the crop is a crop containing CBFs genes.

[0205] According to an embodiment of the present application, the crops include Arabidopsis, tomato, corn, potato, grape, cabbage, and peach.

[0206] According to an embodiment of the present application, the CBFs gene includes at least one of CBF1, CBF2 and CBF3.

[0207] The scheme of the present application will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If no specific technology or conditions are indicated in the embodiments, the technology or conditions described in the literature in this area or the product specification are carried out. The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0208] Without special instructions, the microbial fermentation products containing different contents of ectoine in this application are purchased from Zhongke Xinyang Biotechnology Co., Ltd.; NMN is purchased from Geneport (Hong Kong, China) Biotechnology Co., Ltd.; polyglutamic acid is purchased from Nanjing Xuankai Biotechnology Co., Ltd.; chitosan oligosaccharides are purchased from Zhongxiang Yipinhong Plant Immunity Technology Co., Ltd., and the molecular weight of chitosan oligosaccharides is 800-1000, and the proportion of chitosan pentaose and chitosan hexaose is >80%; seaweed polysaccharides, seaweed essence, seaweed mother liquid, and seaweed 30 are all purchased from Qingdao Haida Biological Group; compound amino acid powder is purchased from Xuzhou Green Source Agricultural Technology Co., Ltd., and free amino acids are ≥30%; amino acid derivatives are purchased from Guangdong Jianda Biotechnology. Amino acid derivatives are composed of melatonin, nicotinamide, proline, polyglutamic acid, fish protein peptides, γ-aminobutyric acid, and ergothioneine; plant-derived amino acid complexes are purchased from Jiangsu Hanling Biotechnology Co., Ltd., with total amino acids ≥80%.

[0209] Screening Example 1:

[0210] In this embodiment, after Arabidopsis plants were subjected to low temperature stress (4°C low temperature treatment for 24h), the leaves of the plants were taken for the above-mentioned low temperature stress gene PCR quantitative expression test, and the test included RNA extraction, reverse transcription cDNA, RT PCR, and detection of genes related to plant low temperature stress. The results showed that the expression levels of SPT gene, Hd3a gene, OsVP1 gene, OsLEA3 gene, and OsDREB1A / B gene did not have obvious regularity, and had poor repeatability, poor sample parallelism, etc., and low accuracy. The expression level of CBFs gene has obvious regularity, good repeatability, and high accuracy. Therefore, CBFs gene can be used to screen target biostimulant substances.

[0211] Embodiment 1:

[0212] The components and proportions of each sample are shown in Table 1. After weighing the components of each sample, they were physically blended to obtain a biostimulant composition, and samples 1 to 10, 12 and 14 were obtained.

[0213] Among them, the biostimulant composition sample 10 is mixed with the 300-0-20 specification liquid fertilizer, and the addition ratio of sample 10 is 1% of the total amount. The liquid fertilizer with low temperature resistance function is prepared in a reactor, which is sample 11.

[0214] The biostimulant composition sample 12 was mixed with the solid compound fertilizer of specification 18-18-18, with the addition ratio of sample 12 being 2% of the total amount, and a liquid fertilizer with anti-low temperature function was prepared in a reactor, namely sample 13.

[0215] The biostimulant composition sample 14 was mixed with the 300-0-40 specification liquid fertilizer, and the addition ratio of the sample 14 was 10% of the total amount. The liquid fertilizer with low temperature resistance was prepared in a reactor, which was the sample 15.

[0216] Table 1

[0217]

[0218]

[0219] Test Example 1:

[0220] This test example uses samples 1 to 15 prepared in Example 1 to detect the low temperature resistance effect on Arabidopsis plants, specifically:

[0221] Arabidopsis plants were divided into 16 groups, with 10 plants in each group. One group was used as a control, and the other 15 groups were sprayed with samples 1 to 15, respectively. After 24 hours of low temperature treatment at 4°C, the expression of CBFs genes in Arabidopsis plants and the leaf chilling injury index were detected. The specific detection method is as follows:

[0222] Leaf chilling injury index determination method:

[0223] During the experiment, plant morphological changes were observed and recorded every day, the degree of plant damage (leaf color, morphology, wilting, dehydration) was observed and counted, and different chilling injury symptoms were graded.

[0224] Level 0: no chilling injury symptoms (observation of leaf color and morphology);

[0225] Level 1: The first and second leaves (slight water loss) turn yellow, and the third leaf has no (obvious changes) chilling injury symptoms;

[0226] Level 2: The first and second leaves (severe water loss and wilting) have obvious chlorosis, and the third leaf (observe morphological changes) has slight water loss;

[0227] Level 3: Water spots appear on the first and second leaves (not connected into pieces), the edge of the third leaf loses water severely (wilts and curls), and the top loses water slightly;

[0228] Level 4: The water spots in the middle of the first and second leaves (around the veins) are connected into a piece (water-soaked), the leaves wilt, and water spots appear on the third leaf;

[0229] Level 5: All leaves lose water and wilt (the whole plant wilts);

[0230] Chilling injury index = ∑ (chilling injury level × number of plants at that level) / number of stressed plants.

[0231] Gene quantification methods:

[0232] Step 1, RNA extraction process:

[0233] 1) Pretreatment of adsorption column: Add 500 μL of column washing solution TE to the adsorption column, place at room temperature for 2 min, centrifuge at 2-8°C and 12000 rpm for 2 min, and discard the waste liquid.

[0234] (2) Plant tissue: Grind 100 mg of fresh or -70°C frozen tissue in liquid nitrogen. Add the powder to 1 mL of Lysis Buffer RL and mix well.

[0235] (3) The treated samples were placed at room temperature for 5 min to allow the nucleic acid-protein complex to be completely separated.

[0236] (4) Add 200 μL of chloroform to the homogenate sample, cover the tube, shake vigorously for 15 seconds, leave at room temperature for 3-5 minutes, centrifuge at 2-8°C and 12,000 rpm for 10 minutes, and collect the supernatant.

[0237] (5) Add 200 μL of anhydrous ethanol to the collected supernatant and mix well. Add the supernatant to the adsorption column and let it stand for 2 min. Centrifuge at 2-8°C and 12,000 rpm for 2 min. Discard the waste liquid and put the adsorption column back into the collection tube.

[0238] (6) Add 600 μL of rinse solution RW to the adsorption column (please check whether anhydrous ethanol has been added before use), centrifuge at 2-8°C and 12000 rpm for 2 min, discard the waste liquid, and return the adsorption column to the collection tube.

[0239] (7) Add 600 μL of rinse solution RW to the adsorption column, centrifuge at 2-8°C and 12,000 rpm for 2 min, discard the waste liquid, and return the adsorption column to the collection tube.

[0240] (8) Centrifuge at 2-8°C and 12,000 rpm for 2 min, discard the collection tube, and place the adsorption column at room temperature for several minutes to completely dry the residual rinse solution RW in the adsorption material.

[0241] (9) Place the adsorption column in a new tube, add 50-100 μL RNase free ddH2O to the center of the membrane, leave at room temperature for 5 min, and centrifuge at 12000 rpm for 2 min to obtain RNA.

[0242] The second step is the reverse transcription cDNA process:

[0243] The reaction solution should be prepared on ice. For each reaction, the Master Mix should be prepared according to the number of reactions + 2, and then 10 μL should be dispensed into each reaction tube. After gentle mixing, reverse transcription reaction should be carried out immediately.

[0244] Reagents Dosage Reaction solution 10.0μL PrimeScript RT Enzyme Mix I 1μL RT Primer Mix*4 1.0μL 5× PrimeScript Buffer 2 4.0μL <![CDATA[dH2O free of RNase]]> 4.0μL Total 20μL

[0245] Step 3: RT PCR process

[0246] After the reverse transcription reaction, TB Green Premix Ex Taq II (Tli RNaseH Plus) (Code No. RR820A / B) was selected to perform the RT PCR reaction.

[0247] Reagents Usage Final concentration TB Green Premix Ex Taq II(Tli RNaseH Plus) 12.5μL 1× PCR Forward Primer (10 μM) 1.0μL 0.4μM PCR Reverse Primer (10μM) 1.0μL 0.4μM RT reaction solution (cDNA solution) 2μL Sterilized water 8.5μL Total 25μL

[0248] The test results are shown in Table 3.

[0249] Table 3

[0250]

[0251]

[0252] As can be seen from Table 3, the expression multiples of the cold resistance-related genes CBF1, CBF2, and CBF3 in sample 1 were higher than those in sample 2, sample 3, sample 4, sample 5, and sample 6, and the leaf chilling injury index was lower than that in sample 2, sample 3, sample 4, sample 5, and sample 6. Therefore, it can be shown that the combination of β-nicotinamide mononucleotide, microbial fermentation products, amino acids and their derivatives, sugars and their derivatives, and seaweed extracts has the best effect.

[0253] By comparing sample 1 with samples 7-8, it can be seen that the biostimulant composition formed by controlling the weight ratio of β-nicotinamide mononucleotide, microbial fermentation products, amino acids and their derivatives, sugars and their derivatives, and seaweed extract in the range of (0.1-5):(1-5):(3-30):(10-50):(20-100) has a better effect.

[0254] Comparing sample 11 with sample 10, comparing sample 13 with sample 12, and comparing sample 15 with sample 14, combining the high-activity cold-resistant biostimulant composition with nitrogen, phosphorus and potassium fertilizers also has the function of promoting plant CBFs gene overexpression and enhancing crop resistance.

[0255] Test Example 2: Field Trial

[0256] Samples 11 and 15 prepared in Example 1 were used to conduct a low temperature resistance field test on peach trees in Linyi, Shandong. A blank control group, a low temperature resistance fertilizer group 1 (fertilizer sample 11) and a low temperature resistance fertilizer group 2 (fertilizer sample 15) were set up respectively, and the application amount was 500 ml / mu. After the application, the peach trees encountered a sudden cold and low temperature natural disaster during the flowering period of late spring. The peach trees in different groups were tested and observed. The results are shown in Figure 3 The results showed that compared with the blank control, the number of flowers in the treatment of spraying low-temperature resistant fertilizer increased significantly, which shows that low-temperature resistant fertilizer group 1 and low-temperature resistant fertilizer group 2 can effectively improve the ability of peach trees to resist the natural disaster of late spring cold.

[0257] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0258] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. Use of β-nicotinamide mononucleotide in preparing crop products; Optionally, the crop product is used to improve the low temperature resistance of crops; Optionally, the crop product is fertilizer.

2. A biostimulant composition, characterized in that: include: β-nicotinamide mononucleotide, ectoine and / or microbial fermentation products containing ectoine, and At least two of amino acids and their derivatives, sugars and their derivatives, and seaweed extracts.

3. The biostimulant composition according to claim 2, characterized in that The content of icodine in the microbial fermentation product is 30 g / L to 50 g / L; And / or, the microbial fermentation product containing ectoine is obtained by the following method: Using synthetic biology methods to genetically modify halophiles to obtain modified halophiles; The modified halophilic bacteria are subjected to fermentation treatment to obtain a microbial fermentation product; And / or, the genetic modification is obtained by: By cloning Halomonas salina DSM 5928 T teaABC genes, knocked out of the halophilic bacterium Halomonas salina DSM 5928 using Red recombination technology T teaABC genes; And / or, the amino acid and its derivatives are selected from at least one of melatonin, nicotinamide, proline, polyglutamic acid, fish protein peptide, gamma-aminobutyric acid, and ergothioneine; and / or, the sugar and its derivatives are selected from at least one of pantothenate polysaccharide, chitosan oligosaccharide, seaweed polysaccharide, chitin, oligosaccharide, chitosan and chitosan derivatives; And / or, the seaweed extract is selected from at least one of seaweed essence, seaweed mother liquor, seaweed 30, seaweed 45, kelp extract, giant kelp extract, Ascophyllum nodosum extract, and Haematocystis extract.

4. The biostimulant composition according to claim 2, characterized in that The biostimulant composition includes β-nicotinamide mononucleotide, ectoine or a microbial fermentation product containing ectoine, amino acids and their derivatives, sugars and their derivatives, and seaweed extracts; and / or, the weight ratio of the β-nicotinamide mononucleotide to ectoine is (0.1-5.0):(0.03-0.25), preferably (0.1-2.0):(0.05-0.2); and / or, the weight ratio of the β-nicotinamide mononucleotide to the microbial fermentation product is (0.1-5.0):(1.0-5.0), preferably (0.5-2.5):(1.0-5.0); And / or, the weight ratio of the β-nicotinamide mononucleotide to the amino acid and its derivatives is (0.1-5.0):(3.0-30.0), preferably (0.5-2.5):(10.0-20.0); and / or, the weight ratio of the β-nicotinamide mononucleotide to the sugar and its derivatives is (0.1-5.0):(10.0-50.0), preferably (0.5-2.5):(35.0-50.0); and / or, the weight ratio of the β-nicotinamide mononucleotide to the seaweed extract is (0.1-5.0):(20.0-100.0), preferably (0.5-2.5):(30.0-80.0); And / or, the seaweed extract is selected from seaweed essence, and the weight ratio of the β-nicotinamide mononucleotide and the seaweed extract is (0.1-5.0): (30.0-60.0), preferably (0.1-5.0): (40.0-50.0); And / or, the seaweed extract is selected from seaweed mother liquor, and the weight ratio of the β-nicotinamide mononucleotide and the seaweed extract is (0.1-5.0): (30.0-60.0), preferably (0.1-5.0): (45.0-55.0); And / or, the seaweed extract is selected from seaweed 30, and the weight ratio of the β-nicotinamide mononucleotide and the seaweed extract is (0.1-5.0): (50.0-100.0), preferably (0.1-5.0): (65.0-80.0); And / or, the seaweed extract is selected from seaweed 45, and the weight ratio of β-nicotinamide mononucleotide to the seaweed extract is (0.1-5.0): (20.0-50.0), preferably (0.1-5.0): (25.0-35.0); And / or, the seaweed extract is selected from kelp extract, and the weight ratio of β-nicotinamide mononucleotide to the seaweed extract is (0.1-5.0):(50.0-100.0), preferably (0.1-5.0):(75.0-95.0).

5. The biostimulant composition according to claim 2, characterized in that: The biostimulant composition includes β-nicotinamide mononucleotide, microbial fermentation products containing ectoine, amino acids and their derivatives, sugars and their derivatives, and seaweed extracts; And / or, the biostimulant composition comprises β-nicotinamide mononucleotide, microbial fermentation products containing ectoine, amino acids and their derivatives, sugars and their derivatives, and seaweed extract in a weight ratio of (0.1-5): (1-5): (3-30): (10-50): (20-100); And / or, the biostimulant composition is a fertilizer.

6. A biostimulant composition, characterized in that: Select from at least one of the following: 1) Includes: 0.5 to 2.5 parts by weight of β-nicotinamide mononucleotide, 1.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, and two of the following: 10.0 to 30.0 parts by weight of amino acids and their derivatives, 40.0-50.0 parts by weight of sugar and its derivatives, 30.0-80.0 parts by weight of seaweed extract; 2) Includes: 0.5 to 2.5 parts by weight of β-nicotinamide mononucleotide, 1.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, 10.0 to 20.0 parts by weight of amino acids and their derivatives, 40.0-45.0 parts by weight of sugar and its derivatives, 30.0 to 80.0 parts by weight of seaweed extract.

7. A biostimulant composition, characterized in that: Select from at least one of the following: 1) Includes: 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide, 1.0 to 3.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, 8.0 parts by weight of a composite amino acid powder, 2.0 parts by weight of an amino acid derivative, 40.0 parts by weight of chitosan oligosaccharide, 45.0-50.0 parts by weight of seaweed extract; 2) Includes: 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide, 4.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, 40.0 parts by weight of chitosan oligosaccharide, 75.0 parts by weight of seaweed 30; 3) Includes: 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide, 4.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, 30.0 parts by weight of polyglutamic acid, 75.0 parts by weight of seaweed 30; 4) Includes: 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide, 1.0 to 3.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, 8.0 parts by weight of a composite amino acid powder, 2.0 parts by weight of an amino acid derivative, 40.0 parts by weight of chitosan oligosaccharide; 5) Includes: 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide, 4.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, 5.0 parts by weight of gamma-aminobutyric acid, 5.0 parts by weight of melatonin, 40.0 parts by weight of chitosan oligosaccharide, 30.0 parts by weight of seaweed 45; 6) Includes: 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide, 0.5.0 to 1.5 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, 5.0 parts by weight of nicotinamide, 15.0 parts by weight of melatonin, 45.0 parts by weight of oligosaccharides, 70.0 parts by weight of seaweed 30; 7) Includes: 0.5 to 1.5 parts by weight of β-nicotinamide mononucleotide, 4.0 to 5.0 parts by weight of a microbial fermentation product, wherein the concentration of ectoine in the microbial fermentation product is 40 g / L to 50 g / L, 5.0 parts by weight of gamma-aminobutyric acid, 10.0 parts by weight of ergothioneine, 15.0 parts by weight of chitosan oligosaccharide, 15.0 parts by weight of chitin, 15.0 parts by weight of oligosaccharides, 80.0 parts by weight of kelp extract.

8. A fertilizer, characterized in that A biostimulant composition comprising any one of claims 1 to 7; Optionally, further comprising at least one of nitrogen fertilizer, phosphorus fertilizer and potash fertilizer; Optionally, the nitrogen fertilizer includes at least one of ammonium nitrogen fertilizer, nitrate nitrogen fertilizer, ammonium nitrate nitrogen fertilizer, amide nitrogen fertilizer and slow-release nitrogen fertilizer; Optionally, the phosphate fertilizer includes at least one of monoammonium phosphate, diammonium phosphate, calcium magnesium phosphate, superphosphate and ammonium phosphate; Optionally, the potash fertilizer includes at least one of potassium chloride, potassium sulfate, potassium dihydrogen phosphate, sylvite, and potassium-magnesium salt; Optionally, the weight ratio of the total amount of the nitrogen fertilizer, the phosphorus fertilizer and the potash fertilizer to the biostimulant composition is (1-1000): (1-200); optionally, the weight percentage of the biostimulant composition in the fertilizer is 0.1-20%.

9. A method for screening a target biostimulant substance, characterized in that: include: The biostimulant substance to be screened is contacted with the crop, and the expression amount of the CBFs gene of the crop before and after the contact treatment is detected; Based on the changes in the expression levels of the CBFs genes before and after the crop contact treatment, the target biostimulant substance is determined.

10. The method according to claim 9, characterized in that The crops are crops containing CBFs genes; And / or, the crops include Arabidopsis, tomato, corn, potato, grape, cabbage, peach; And / or, the contact treatment is spraying the material to be screened on the crops; And / or, the contact treatment is performed at a temperature of 4° C. and for a time of 12 to 48 hours; And / or, the up-regulation of the CBFs gene expression in the crop after the contact treatment is an indication that the object to be screened is the target object; And / or, the CBFs gene includes at least one of CBF1, CBF2 and CBF3; And / or, the target biostimulant substance is β-nicotinamide mononucleotide, ectoine and / or microbial fermentation products containing ectoine, amino acids and their derivatives, sugars and their derivatives, and seaweed extracts.

11. A method for preparing the biostimulant composition according to any one of claims 1 to 7, characterized in that: include: (1) establishing a crop CBFs gene evaluation method, and screening biostimulant substances that resist low temperature stress according to the crop CBFs gene evaluation method; (2) preparing the biostimulant composition using the target biostimulant substance screened in step (1); as well as (3) detecting the effect of the prepared biostimulant composition on crops against low temperature stress; Optionally, step (1) is carried out using the method described in any one of claims 9 to 10.

12. A method for improving the low temperature resistance of crops, characterized in that: include: Crops are cultured using the biostimulant composition according to any one of claims 1 to 7, the fertilizer according to claim 8, or the target biostimulant substance screened by the method according to any one of claims 9 to 10.

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