CN116751134A - A root-promoting active substance isolated from plant probiotic Trichoderma metabolites and its application - Google Patents
A root-promoting active substance isolated from plant probiotic Trichoderma metabolites and its application Download PDFInfo
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Abstract
The application discloses a root-promoting active substance separated from plant probiotics trichoderma metabolites and application thereof. The molecular formula of the benzene ring root-promoting active substance is C 7 H 7 NO 2 The plant root system growth promoting agent has the function of promoting plant root system growth, obviously increases the total length, total surface area and total root number of the root systems of arabidopsis thaliana, tomatoes and rice, has stable effect and has better application prospect; can be used as a root promoter in green agricultural production.
Description
Technical Field
The application belongs to the field of microorganisms, and particularly relates to a benzene ring root-promoting active substance generated by trichoderma Guizhou NJAU4742 and application thereof.
Technical Field
Providing a healthy food source for an ever-growing global population has become one of the biggest challenges in this century, and therefore the development of new technologies and products with low energy consumption and pollution is critical to the green and sustainable development of agriculture. Trichoderma (Trichoderma spp.) fungi, one of the microbial groups that has a gain in human production and life, have been widely used in agricultural production and exhibit excellent plant growth-promoting and biocontrol effects, and their numbers of related studies and patent applications have been exponentially increased over the last decades. Trichoderma presently identified as beneficial to agricultural production are primarily distributed within the T.atroviride, T.viride, T.aspelelum, T.aspelelide, T.harzianum, T.guizhouense, T.virens populations; some of these species (t.atroviride, t.harzianum, t.guizhouue, t.virens) have plant protection (biological fungicides) and plant growth promoting (biofertilizer) effects, and have been widely used in the biological control of fungal pests in sustainable agricultural production.
Rhizosphere colonization of trichoderma produces more than 120 different types of secondary metabolites, the most common chemical structures of which include terpenes, pyrones, polyketides, and non-ribosomal peptides. Hormones and signal substances that regulate plant root system architecture and promote root nutrient absorption have been identified to date including plant hormones, siderophores, koninginins, 6-pentatyl-alpha-pyrene (6 PP), trichaaranes A-D, harzian operating ridone, cyclonodiol, harzianic acid, swollen, etc.
Disclosure of Invention
The application aims to provide a benzene ring root-promoting active substance produced by fermenting trichoderma guizhou NJAU 4742.
Another object of the present application is to provide a method for extracting and separating the root-promoting active substance.
It is a further object of the present application to provide the use of the root-promoting active substance described above.
The aim of the application can be achieved by the following technical scheme:
a benzene ring type root-promoting active substance is a compound with the structure shown as follows:
the extraction and separation method of the benzene ring root-promoting active substance comprises the steps of fermenting and culturing trichoderma guizhou with the preservation number of CGMCC No.12166, collecting supernatant of fermentation liquor, loading the supernatant on a macroporous resin column, eluting with industrial alcohol gradient, collecting eluent of 50% concentration industrial alcohol, evaporating the collected eluent to remove a solvent, dissolving the eluent with acetonitrile, and further separating and purifying by using an HPLC method to obtain the benzene ring root-promoting active substance.
As a preferable technical scheme, the separation and purification are carried out to obtain the benzene ring type root-promoting active substance under the conditions of acetonitrile-water 60:40, flow rate of 2ml/min, detection wavelength of 280nm and Rt=26.6.
As a preferable technical scheme, the culture medium adopted in the fermentation culture is an oligosaccharide inorganic salt culture medium, and 1L of the oligosaccharide inorganic salt culture medium comprises the following formula: 5g of ammonium sulfate, 0.6g of magnesium sulfate heptahydrate, 15g of potassium dihydrogen phosphate, 1g of calcium chloride, 5mg of ferric sulfate heptahydrate, 1.6mg of manganese sulfate monohydrate, 1.4mg of zinc sulfate heptahydrate, 2mg of cobalt chloride hexahydrate and 10g of glucose.
As a preferable technical scheme, the macroporous resin column is eluted with 30%,50% and 80% concentration industrial alcohol gradient respectively, and 50% concentration eluent is collected.
As a preferable technical scheme, the process of fermenting and culturing trichoderma guizhou NJAU4742 with the preservation number of CGMCC NO.12166 is as follows: transferring NJAU4742 seed liquid into an oligosaccharide inorganic salt culture medium according to an inoculum size of 1%, and culturing at 28 ℃ and 170rpm for 2d; inoculating the cultured bacterial liquid into a fermentation tank filled with an oligosaccharide inorganic salt culture medium for fermentation culture, wherein the temperature is 28 ℃ at the beginning, the rotating speed is 200rpm, the dissolved oxygen value is 75, when the dissolved oxygen value begins to fall below 30, the rotating speed is gradually increased, the oxygen introducing amount is increased, the dissolved oxygen value is kept above 10, the temperature is adjusted to 20 ℃ after 2d of culture, the rotating speed is adjusted to 200rpm, the oxygen introducing amount is kept at the minimum level, the bacterial liquid enters a stationary phase, a large amount of secondary metabolites are produced, and the fermentation is continued for 3d.
The application of the benzene ring root-promoting active substance in promoting plant root system development or preparing a preparation for promoting plant root system development.
The plant is dicotyledon or monocotyledon, and the dicotyledon is Arabidopsis thaliana or Lycopersicon esculentum; the monocotyledonous plant is rice.
The application of Trichoderma Guizhou NJAU4742 strain with the preservation number of CGMCC NO.12166 in the production of the benzene ring root-promoting active substance is provided.
The root-promoting active substances are screened and verified by the following method: knocking out the related gene and 6PP synthetic gene of the trichoderma Guizhou NJAU4742 auxin with the preservation number of CGMCC NO.12166, and constructing the trichoderma Guizhou NJAU 4742-delta iaaH2& ald1& ald2& nit 1-delta ctf1 mutant. Fermenting mutant strain, filtering the fermentation liquor with 4 layers of sterile gauze after fermentation is finished, collecting supernatant, loading the supernatant to a macroporous resin column of 100-200 meshes of about 500g, respectively eluting with 30% industrial alcohol, 50% industrial alcohol and 80% industrial alcohol in gradient, collecting eluent of 50% industrial alcohol, removing solvent by rotary evaporation, dissolving with acetonitrile, further separating and purifying the biomass by HPLC method, and obtaining the benzene ring biomass under conditions of acetonitrile-water 60:40, flow rate of 2ml/min, detection wavelength of 280nm and Rt=26.6. About 500g of the active components separated by HPLC with 100-200 meshes are subjected to biological activity function tracking, and finally the pure product of the benzene ring root-promoting active substance is obtained.
The application has the beneficial effects that:
the application verifies the life-promoting property of the fermentation liquor of trichoderma Guizhou NJAU4742 through a gene knockout experiment and a growth-promoting experiment, searches the optimal culture medium and culture conditions for material fermentation, and separates and purifies a benzene ring type root-promoting active material C 7 H 7 NO 2 The molecular structure of the substance is identified. The root-promoting active substance can promote the total length, total surface area and total root number of root systems of arabidopsis, tomatoes and rice.
Biological preservation information:
NJAU4742, classified and named as Trichoderma Guizhou Trichoderma guizhouense, is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation address of Beijing, chaoyang, no.1, west Lu, 3, and a collection number of CGMCC No.12166, and a collection date of 2016, 4, 11.
Drawings
FIG. 1 shows the growth promoting effect of wild type NJAU4742 and mutant ΔiaaH2& ald1& ald2& nit1- Δ ctf1 on Arabidopsis root development.
FIG. 2 is a benzene ring class C 7 H 7 NO 2 Promoting growth of tomato root system.
FIG. 3 is a benzene ring class C 7 H 7 NO 2 Promoting the growth of rice root system.
Detailed Description
The application discloses a benzene ring root-promoting active substance, and a person skilled in the art can refer to the content of the benzene ring root-promoting active substance and properly improve parameters. In order to enable those skilled in the art to better understand the technical solution of the present application, the present application will be further described in detail with reference to specific embodiments.
Example 1: the benzene ring substances of the application are verified to promote the life of the root system of the arabidopsis:
trichoderma guizhou NJAU4742 extracellular auxin (IAA) and analogues thereof and 6-pendyl-alpha-pyrene have the activity of promoting plant root development, and in order to explore whether other active substances in the extracellular secretion of NJAU4742 have the function of promoting growth, three-fragment fusion method replaced by hygromycin B resistance is sequentially utilized to knock out trichoderma guizhou NJAU 4742-delta iaaH2 synthesis related genes and 6PP synthesis genes&ald1&ald2&nit 1-delta ctf1 mutant 【1】 . Firstly, fusion PCR method is adopted to fuse the gene of hygromycin B resistance screening mark with the upstream and downstream homology arms of the gene to be knocked out, the fusion fragment is transferred into competent NJAU4742, 200 mu l of the mixed solution is sucked into a PDA plate containing 1M sucrose, the PDA plate is evenly coated, and the mixture is cultivated in the dark at 28 ℃ for 12-16h. The next day, PDA medium (without sucrose) containing hygromycin B was slowly poured into the PDA medium to a thickness of about 2mm, and the dark culture was continued at 28℃for 24-48 hours until small NJAU4742 transformants were grown on PDA plates containing hygromycin B. Picking up the transformant for verification to obtain the correct mutant delta iaaH2&ald1&ald2&nit1-Δctf1。
NJAU4742 and ΔiaaH2& ald1& ald2& nit1- Δ ctf1 were inoculated into an inorganic salt medium of oligosaccharides (formulation of 1L medium: 5g ammonium sulfate, 0.6g magnesium sulfate heptahydrate, 15g potassium dihydrogen phosphate, 1g calcium chloride, 5mg ferric sulfate heptahydrate, 1.6mg manganese sulfate monohydrate, 1.4mg zinc sulfate heptahydrate, 2mg cobalt chloride hexahydrate, 10g glucose), cultured at 28℃at 170rpm for 2d, then at 20℃at 200rpm for 3d, and finally the fermentation broth was filtered with 4 layers of sterile gauze for mycelium. Loading the supernatant onto macroporous resin column, gradient eluting with industrial alcohol (30%, 50% and 80% in sequence), and subjecting 50% industrial alcohol effluent to rotary evaporation and leaching with dimethyl sulfoxide (DMSO) to obtain crude extract. Filtering the crude extract with 0.22 μm aseptic organic phase filter membrane, removing precipitate, impurities and mycelium, and storing at-20deg.C. DMSO is a control group, and NJAU4742 and ΔiaaH2& ald1& ald2& nit1- Δ ctf1 crude extracts are experimental groups.
The model plant Arabidopsis thaliana in root development research was selected in 1/2MS medium (1L medium formula: 2.15)4g MS salt,0.5gMES,0.1g inositol, 10g sucrose, 8-10g agar, pH 5.7). Firstly adding 1/2MS culture medium with an addition amount of 4%v/v, pouring into a plate (13 x 13 cm), cooling, transplanting 5 d-aged Arabidopsis seedlings into a culture dish (4 pieces/dish), placing the culture dish in a artificial climate chamber (16 hr:8hr, daytime: night), and heating to 22deg.C and light intensity of 100 μm -2 s -1 After culturing for 7d in a vertical position, the experimental results were observed.
The experimental results of the growth promotion effect of the wild type NJAU4742 and the mutant delta iaaH2& ald1& ald2& nit 1-delta ctf1 on the root system development of the Arabidopsis are shown in figure 1, and the wild type NJAU4742 and the mutant delta iaaH2& ald1& ald2& nit 1-delta ctf1 have promotion effects on the root system development of the Arabidopsis, which indicates that the fermentation liquor of the mutant delta iaaH2& ald1& ald2& nit 1-delta ctf1 contains other unknown promotion substances.
Example 2: preparing, separating and purifying the benzene ring root-promoting active substance:
the mutant ΔiaaH2& ald1& ald2& nit1- Δ ctf1 fermentation liquor still has the capacity of promoting the root system development of arabidopsis, so that living-promoting substances contained in the ΔiaaH2& ald1& ald2& nit1- Δ ctf1 fermentation liquor can be separated and purified in a biological activity tracking mode, namely, the activity of promoting the root system development of arabidopsis is verified in each separation step, and active components are further separated and purified.
Transferring NJAU4742 seed liquid into a fresh oligosaccharide inorganic salt culture medium conical flask according to the inoculation amount of 1% (v/v), and culturing for 2d at 28 ℃ and 170 rpm; inoculating the cultured bacterial liquid into a 50L small-sized fermentation tank, filling 30L of oligosaccharide inorganic salt culture medium into the fermentation tank, wherein the temperature is 28 ℃ at the beginning, the rotating speed is 200rpm, the dissolved oxygen value is 75, when the dissolved oxygen value begins to fall below 30, gradually increasing the rotating speed, increasing the oxygen introducing amount, keeping the dissolved oxygen value above 10, regulating the temperature to 20 ℃ after 2d of culture, regulating the rotating speed to 200rpm, keeping the oxygen introducing amount at the lowest level, starting mass production of secondary metabolites, and continuing fermentation for 3d.
After fermentation, the broth was sterilized by filtration through 4 layers of sterile gauze and the supernatant was collected. Loading the supernatant into a macroporous resin column with 100-200 meshes of about 500g, carrying out gradient elution by using industrial alcohol (the elution sequence is 30%,50% and 80%) and taking an effluent liquid of 50% industrial alcohol for rotary evaporation to remove the solvent, thereby obtaining 100 g of extract. Adding proper amount of acetonitrile to fully dissolve and filtering by a 0.22 mu m aseptic organic phase filter membrane to remove sediment, impurities and mycelium. The crude extract obtained is further separated and purified by an HPLC chromatographic column, the flow rate is 2ml/min, the detection wavelength is 280nm, the crude material is divided into 8 sections, the components of each section are sequentially collected to verify the growth promotion activity on the root system development of the arabidopsis thaliana (the method is referred to in example 1), the 5 th section has stronger activity, and the 5 th section (1.5 g) is further separated and purified by the HPLC chromatographic column after the acetonitrile is fully dissolved after the acetonitrile is added into the 5 th section (1.5 g). Compound 5- (3) with more remarkable activity is obtained by acetonitrile-water concentration gradient elution (60:40), flow rate of 2ml/min and detection wavelength of 280nm and rt=26.6. 5- (3) has good growth promoting activity on the root system development of the arabidopsis thaliana (the method is referred to in example 1), and finally, the pure product of the active component is obtained. The active components are subjected to mass spectrum and NMR (including two-dimensional NMR structural analysis) tests, and the final active compound is a benzene ring compound, and the molecular structure is shown as follows:
by adopting a 600MHz nuclear magnetic resonance (MHz) spectrometer to carry out hydrogen spectrum, carbon spectrum, 1 H- 1 The H COSY spectrum, HSQC spectrum and HMBC spectrum were tested, and the analytical results are shown in Table 1:
TABLE 1 identification of molecular Structure of Compound 5- (3)
By passing through 1 H-NMR coupling information 1 H- 1 H COSY spectrum, the H-3 (. Delta.) in 5- (3) can be determined H 7.68)、H-4(δ H 6.49)、H-5(δ H 7.20)、H-6(δ H 6.72 Are coupled to each other. And by combining the HSQC spectra, the hydrocarbon attribution of the HSQC spectra can be determined, namely the chemical shifts of C-3, C-4, C-5 and C-6 are 131.7,115.0,134.1,116.8 respectively. In HMBC spectroscopy: hydrocarbon remote correlation of H-3 to C-1, C-5, C-7, hydrocarbon remote correlation of H-5 to C-3, C-7, hydrocarbon remote correlation of H-6 to C-2, C-4 corroborates the above speculation and determines chemical shifts of quaternary carbons C-1, C-2, and C-7 in the structure as 170.2,110.3 and 152.0, respectively. Finally, the active substance for promoting root is determined to be benzene ring compound by separation, purification and identification, and the molecular formula is C 7 H 7 NO 2 。
Example 3: the activity of the benzene ring substances in promoting plant root system development is verified:
1 dicotyledonous crop and 1 monocotyledonous crop are selected to verify the root promoting activity of the benzene ring matters. The 2 crops selected were:
tomato (Solanum lycopersicum), 903 variety;
rice (Oryza sativa), japan variety;
preparing a benzene ring substance separated and purified into a 40mM mother solution, carrying out gradient dilution until the final concentration is 0 mu M,5 mu M,15 mu M,30 mu M and 45 mu M, adding a 1/2MS culture medium, pouring a flat plate (13 x 13 cm), respectively dibbling the sterilized tomatoes and rice seeds into a culture dish (10 particles/dish) after the flat plate is cooled, placing the culture dish into an artificial climate chamber (16 hr:8hr, daytime: night), wherein the temperature is 22 ℃ and the light intensity is 100 mu mol M -2 s -1 After 10d of vertical placement and culture, the plates were collected and the experimental results were observed after the seeds germinated and the top of the root system contacted the bottom of the culture dish.
Benzene rings C 7 H 7 NO 2 The experimental result of the growth promotion effect on the root system development of the tomatoes is shown in figure 2, and the separated benzene ring substances have better growth promotion activity on the total root length, the total root surface area and the total root number of tomato plants.
Benzene rings C 7 H 7 NO 2 The experimental result of promoting the growth of the rice root system is shown in figure 3, and the separated benzene ring substances have better promotion on the total root length, the total root surface area and the total root number of the rice plantsActivity.
1.Zhu,H.et al.Intracellular kynurenine promotes acetaldehyde accumulation,further
inducing the apoptosis in soil beneficial fungi Trichoderma guizhouense NJAU4742
under acid stress.Environ.Microbiol.(2022)doi:10.1111/1462-2920.16286.
Claims (9)
1. A benzene ring type root-promoting active substance is a compound with the structure shown as follows:
2. the method for extracting and separating benzene ring root-promoting active substances as claimed in claim 1, characterized in that the method comprises the steps of fermenting trichoderma guizhou with the preservation number of CGMCC No.12166, collecting supernatant of fermentation liquor, loading the supernatant on a macroporous resin column, eluting with industrial alcohol gradient, collecting eluent of 50% concentration industrial alcohol, evaporating the collected eluent to remove solvent, dissolving with acetonitrile, and separating and purifying by an HPLC method to obtain the benzene ring root-promoting active substances.
3. The method according to claim 2, wherein the separation and purification are performed at a flow rate of 2ml/min in acetonitrile-water of 60:40, a detection wavelength of 280nm, and rt=26.6 to obtain the benzene ring type root-promoting active substance.
4. The method according to claim 2, wherein the medium used for the fermentation culture is an oligosaccharide inorganic salt medium.
5. The method of claim 2, wherein the macroporous resin column is eluted with a 30%,50% and 80% industrial alcohol gradient, respectively, and the 50% concentration eluate is collected.
6. The method according to claim 2, wherein the process of fermenting trichoderma guizhou NJAU4742 with the preservation number of CGMCC No.12166 is: transferring NJAU4742 seed liquid into an oligosaccharide inorganic salt culture medium according to an inoculum size of 1%, and culturing at 28 ℃ and 170rpm for 2d; inoculating the cultured bacterial liquid into a fermentation tank filled with an oligosaccharide inorganic salt culture medium for fermentation culture, wherein the temperature is 28 ℃ at the beginning, the rotating speed is 200rpm, the dissolved oxygen value is 75, when the dissolved oxygen value begins to fall below 30, the rotating speed is gradually increased, the oxygen introducing amount is increased, the dissolved oxygen value is kept above 10, the temperature is adjusted to 20 ℃ after 2d of culture, the rotating speed is adjusted to 200rpm, the oxygen introducing amount is kept at the minimum level, the bacterial liquid enters a stationary phase, a large amount of secondary metabolites are produced, and the fermentation is continued for 3d.
7. Use of the benzene ring root-promoting active substance as claimed in claim 1 for promoting plant root system development or for preparing a preparation for promoting plant root system development.
8. The use according to claim 7, wherein the plant is a dicotyledonous plant or a monocotyledonous plant, the dicotyledonous plant being Arabidopsis or Lycopersicon esculentum; the monocotyledonous plant is rice.
9. The application of Trichoderma Guizhou NJAU4742 strain with the preservation number of CGMCC No.12166 in the production of the benzene ring root-promoting active substance as defined in claim 1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016035685A1 (en) * | 2014-09-01 | 2016-03-10 | 雪印種苗株式会社 | Adventitious root formation inducer and root system growth promoter |
| CN107686406A (en) * | 2017-09-20 | 2018-02-13 | 南京农业大学 | A kind of trichoderma all-element bio-organic fertilizer special for garlic and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016035685A1 (en) * | 2014-09-01 | 2016-03-10 | 雪印種苗株式会社 | Adventitious root formation inducer and root system growth promoter |
| CN107686406A (en) * | 2017-09-20 | 2018-02-13 | 南京农业大学 | A kind of trichoderma all-element bio-organic fertilizer special for garlic and its preparation method and application |
Non-Patent Citations (1)
| Title |
|---|
| 张豪等: "高产ＩＡＡ 绿色木霉工程菌株的构建及其应用", 科学技术与工程, vol. 22, no. 20, 31 December 2022 (2022-12-31), pages 8623 - 8631 * |
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