CN113430120A - Use of gibberellin metabolism modulators - Google Patents

Use of gibberellin metabolism modulators Download PDF

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CN113430120A
CN113430120A CN202110592922.6A CN202110592922A CN113430120A CN 113430120 A CN113430120 A CN 113430120A CN 202110592922 A CN202110592922 A CN 202110592922A CN 113430120 A CN113430120 A CN 113430120A
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路延笃
周文序
甘琴华
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Hainan University
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Abstract

The invention relates to the technical field of biology, in particular to application of gibberellin metabolism regulators. According to the invention, researches show that the physiological processes of the algae can be influenced by exogenously administering the gibberellin or by administering gibberellin metabolism regulators, and the physiological processes comprise the regulation of the growth of the algae, the regulation of the accumulation of secondary metabolites of the algae, the regulation of the stress resistance of the algae and the like. Experiments show that the exogenous gibberellin synthesis inhibitor can inhibit the growth of algae, and the gibberellin synthesis inhibitor can promote the growth of algae.

Description

Use of gibberellin metabolism modulators
Technical Field
The invention relates to the technical field of biology, in particular to application of gibberellin metabolism regulators.
Background
The microalgae absorbs solar energy, captures carbon dioxide, synthesizes various high-value chemicals, and has wide application prospect in the fields of environment, agriculture and aquaculture. The marine microalgae can be cultured in the coastal zone beach and saline-alkali land, does not use fresh water, does not compete with grains for land, and has the possibility of industrial popularization. However, the economic feasibility of the method still faces huge challenges, and the main technical bottlenecks include low microalgae growth density, poor stress resistance, low oil content and the like. The existing method for regulating the growth and stress resistance of microalgae is complex in process and high in cost. Therefore, there is a need to develop a novel method for regulating microalgae growth.
Gibberellin (GA) is a plant hormone used in agricultural production that stimulates the growth of leaves and buds and increases yield. At the end of the 19 th century, gibberellin is used externally to improve the growth rate and the stress resistance of crops, so that the yield of the crops is improved, and the grain crisis is finally solved.
However, the correlation between gibberellins and the life activities of algae has not been elucidated, and it is not clear whether the gibberellin synthesis pathway of algae is different from that of higher plants. Therefore, whether gibberellin modulators can be applied to algae, especially microalgae industrial production, is still to be further researched.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide the use of gibberellin metabolism-regulating substances, including substances that increase or decrease gibberellin levels.
The invention provides application of gibberellin or a metabolic regulator thereof in physiological regulation of algae.
In the present invention, the gibberellin metabolism-regulating substances include at least one of the following i) to vi):
i) key enzymes in gibberellin metabolism or signaling pathways;
ii) a gene encoding the key enzyme of i);
iii) an expression vector comprising the gene of ii);
iv) a promoter or enhancer to enhance expression of the gene of ii);
v) an inducer which promotes the expression of ii) said gene;
vi) an agent that increases the activity of i) the key enzyme.
i) Vi) the physiological regulation of algae by said gibberellin metabolism modulators includes: promoting the growth of algae, increasing the accumulation of secondary metabolites of algae or improving the stress resistance of algae.
In the present invention, the gibberellin metabolism-regulating substances include at least one of the following I) to VI):
I) an expression vector for knocking out or knocking down a key enzyme gene in gibberellin metabolism or a signal transmission pathway;
II), a host cell or viral vector comprising I);
III), nucleic acid molecules that interfere with transcription of key enzyme genes in gibberellin metabolism or signaling pathways;
IV), gibberellin metabolism or key enzyme gene expression in the signaling pathway;
v), agents that inhibit the expression of key enzyme genes in gibberellin metabolism or signaling pathways;
VI), gibberellin metabolism or key enzyme activity inhibitors in the signaling pathway.
In some embodiments, the modulator of gibberellin metabolism is an inhibitor of gibberellin synthesis, including: chlormequat chloride or paclobutrazol.
In the present invention, the physiological regulation of algae by the gibberellin metabolism-regulating substances in I) to VI) includes: inhibiting the growth of algae, reducing the accumulation of secondary metabolites of algae or reducing the stress resistance of algae.
In the present invention, the key enzymes in the gibberellin metabolism or signaling pathway include:
gibberellin synthases, including: ent-phosphate diphosphite synthsase, ent-kaurene synthsase, ent-kaurenic oxidase, GAI-RGA like giberellin response module, ent-kaurenic acid oxidase, GA 20-oxidase or GA 3-oxidase;
gibberellin-degrading enzymes, including: GA 2-oxidases, gibberellin 16 alpha or 17-epoxydase;
gibberellin methylases, including: GA methyltransferase.
Gibberellin signaling and regulatory proteins, including: gibberellin receptor or GA intensive Dwarf 1;
other signaling and regulatory related proteins, including: n-acetyl glucosamine transferase, F-box protein, GA intense, Reresolver of GA, RGA-LIKE1, RGA-LIKE2, RGA-LIKE3, SLENDER RICE 1, Slender protein 1, MADS-box protein, CHD3-type chromamat-modifying factor PICKLE or Katan p60 ATPase-linking subenit.
In the present invention, the algae include: green algae, red algae, diatom, brown algae, or euglena.
The invention also provides a method for promoting the growth of algae, improving the accumulation of secondary metabolites of algae or improving the stress resistance of algae, which comprises the following steps: exogenously administering gibberellin or increasing endogenous levels of gibberellin in algae using at least one of the following modulators of gibberellin metabolism i) to vi):
i) key enzymes in gibberellin metabolism or signaling pathways;
ii) a gene encoding the key enzyme of i);
iii) an expression vector comprising the gene of ii);
iv) a promoter or enhancer to enhance expression of the gene of ii);
v) an inducer which promotes the expression of ii) said gene;
vi) an agent that increases the activity of i) the key enzyme.
The invention also provides a method for inhibiting the growth of algae, reducing the accumulation of secondary metabolites of algae or reducing the stress resistance of algae, which comprises the following steps: reducing endogenous gibberellin levels in the algae using at least one gibberellin metabolism modulator of the following I) -VI):
I) an expression vector for knocking out or knocking down a key enzyme gene in gibberellin metabolism or a signal transmission pathway;
II), a host cell or viral vector comprising I);
III), nucleic acid molecules that interfere with transcription of key enzyme genes in gibberellin metabolism or signaling pathways;
IV), gibberellin metabolism or key enzyme gene expression in the signaling pathway;
v), agents that inhibit the expression of key enzyme genes in gibberellin metabolism or signaling pathways;
VI), gibberellin metabolism or key enzyme activity inhibitors in the signaling pathway.
According to the invention, researches show that the physiological processes of the algae can be influenced by exogenously administering the gibberellin or by administering gibberellin metabolism regulators, and the physiological processes comprise the regulation of the growth of the algae, the regulation of the accumulation of secondary metabolites of the algae, the regulation of the stress resistance of the algae and the like. Experiments show that the exogenous gibberellin synthesis inhibitor can inhibit the growth of algae, and the gibberellin synthesis inhibitor can promote the growth of algae.
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FIG. 1 gibberellin metabolism and signaling pathways in photosynthetic eukaryotes. Black indicates that e-value is more than or equal to 1 e-10; red indicates e-value ≦ 1 e-50. Abbreviations: s, synthesizing; d, degradation; c, conjugation; t, transporter; r, receptors; SC, signaling components.cm, red algae c.merolae; pt, diatom p. Tp, diatom t. Fc, diatom f. No, true eyedrop n.oceanica; es, brown algae e.silicalosus; mi, green alga Micromonas sp. rcc 299; ot, green alga o.tauri; cv, green alga c.variabilis NC 64A; cp, green alga c. Cs, green alga C. subellipsoidea C-169; cr, green algae c.reinhardtii; vc, green algae v. Pp, moss p.patents; sm, marchantia s. Zm, monocot z.mays and At, dicot a.thaliana;
FIG. 2 is a differential expression profile of gibberellin metabolism and signal transmission and regulation related genes after nitrogen deficiency induction of nannochloropsis, red and green indicating up-regulation or down-regulation of transcription level, respectively;
FIG. 3 is a graph showing the effect of gibberellins on the growth of nannochloropsis in accordance with an embodiment of the present invention;
FIG. 4 is a graph showing the effect of paclobutrazol (Pacloutrazole), an inhibitor of gibberellin synthesis, on the growth of nannochloropsis;
FIG. 5 is a graph showing the effect of Chlormequat chloride (gibberellin synthesis inhibitor) on the growth of nannochloropsis.
Detailed Description
The invention provides the application of gibberellin metabolism regulators, and the technical personnel in the field can use the content to reference the content and appropriately improve the process parameters to realize the gibberellin metabolism regulators. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides a method for improving stress resistance of microalgae and increasing growth rate and accumulation capacity of biomass, grease and high value-added compounds. The novel gibberellin metabolism and signal transmission path in microalgae is discovered by a functional genomics means; identifying key gibberellin metabolism and signal transmission related genes in the microalgae stress resistance and oil synthesis process by a transcriptomics means; improving the economic characters of microalgae by regulating the gibberellin level of cells.
The gibberellins comprise GA1, GA3, GA4, GA5, GA6, GA7, GA8, GA9, GA12, GA13, GA15, GA19, GA20, GA24, GA29, GA34, GA44, GA51 and GA 53.
The microalgae comprise all autotrophic, heterotrophic or facultative nutritional unicellular or multicellular algae such as green algae, red algae, diatoms, brown algae, euglena, dinoflagellates, yellow algae, golden algae, true eyespot algae and the like.
The physiological processes of the microalgae comprise growth, stress resistance, grease synthesis and high-value compound synthesis. The method for identifying genes related to gibberellin metabolism and signal transmission is described. The identification method utilizes equivalent means of functional genomics means, including comparative functional genomics means, molecular biology means, proteomics means, yeast hybridization technology, genetic engineering technology, chemical engineering technology, cytology technology and other known or yet-to-be-developed technologies which can be used for identifying the metabolic pathway, the signal transmission pathway and the regulation and control network of gibberellin. The means for identifying key enzymes and proteins for the metabolic pathway, signaling pathway and network regulation of gibberellin described in the methods include transcriptome techniques or their equivalents. The equivalent technologies of the transcriptome technology comprise a high-throughput sequencing technology, a chip technology, a fluorescence quantification technology, a molecular biology technology, a cell biology technology, a plant physiology technology, a proteomics means, a yeast hybridization technology, a genetic engineering technology, a chemical engineering technology and other known or yet-to-be-developed technologies which can be used for identifying key enzymes and proteins of a gibberellin metabolic pathway, a signal transmission pathway and network regulation.
Means of increasing gibberellin levels according to the present invention include exogenous addition or stimulation of endogenous gibberellin production. For example: genetically engineering its metabolism and signal transmission path, chemically physically mutagenizing its metabolism and signal transmission path, applying its metabolism or signal transmission chemical inhibitor, applying microorganism capable of producing gibberellin, etc.
The characters improved by disturbing gibberellin metabolism or signal transmission in the method are stress resistance, growth rate or other characters which can improve the economic value and reduce the industrial cost of the microalgae. The characters that can improve the economic value of the microalgae and reduce the industrial cost comprise biomass synthesis, high-energy density substance synthesis, high value-added compound synthesis and the like.
The method identifies key enzymes or proteins participating in stress response and oil accumulation by gibberellin synthesis and signal paths in microalgae. And the growth of the microalgae is successfully regulated and controlled by using gibberellin. Compared with the prior art, the invention realizes key breakthrough of the algology technology and has the following beneficial effects:
1) the invention provides methods for identifying microalgal gibberellins and their metabolic and signaling pathways. The method can be used for explaining enzymes, signal proteins and related regulation and control mechanisms of gibberellin metabolism closely related to algae physiology, and is used for improving the agronomic traits (improving light energy absorption and growth rate and the like) of organisms (such as plants and microorganisms (such as blue algae, unicellular eukaryotic microalgae and the like)).
2) The invention provides the species and content of gibberellins which can be used for regulating and controlling the physiology of microalgae. Can be used for pertinently regulating and controlling the metabolism and signal path of corresponding gibberellin and improving the physiological characters of microalgae.
3) The present invention provides methods that can be used to identify key enzymes and proteins that regulate gibberellin metabolism and signaling in microalgae physiological processes. Can screen and obtain the enzyme or protein for regulating and controlling the specific physiological property, and improve the specific physiological property of the microalgae in a targeted way.
4) The invention utilizes the method of applying gibberellin from an external source to regulate and control the physiological properties of microalgae and can effectively improve the growth rate of the microalgae.
5) The invention can improve the economic value of engineering algae by regulating gibberellin metabolism or signal pathways, and is used for mass production of various target proteins, biological agents, high value-added compounds and high energy density compounds.
6) By the technology, the microalgae can have more excellent large-scale culture properties, such as insect resistance, disease resistance, salt resistance and the like. The design and construction of the low-cost high-light-efficiency reaction facility for the specific microalgae are combined, and the large-scale culture cost is reduced.
7) The microalgae has the characteristics of high photosynthetic efficiency, quick propagation and strong environmental adaptability, and by means of the method, the photosynthetic efficiency of the microalgae can be further improved by regulating gibberellin metabolism and a signal path, so that carbon dioxide can be effectively fixed, and the concentration of the carbon dioxide in the atmosphere is reduced.
8) Various microalgae genome sequences are determined, and the functional genomics research can be carried out in microalgae by the method, so that enzymes and proteins related to gibberellin metabolism and signal pathways and a network regulation mechanism are further developed.
9) The novel gibberellin metabolism and signaling pathway related genes found in algae can be used to improve various economic traits in microalgae, crops, or other plants.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:
example 1: identification of key enzymes or proteins for metabolism and regulation of algae gibberellins
Step 1, selecting algae:
the algae species related to the invention comprise:
(1) green algae such as Micromonas sp. RCC299, Ostreococcus tauri, Chlorella variabilis NC64A, Haematococcus pluvialis, Chlorella pyrenoidosa, Chlorella subllipsoides C-169, Chlamydomonas reinhardtii, Volvox cateri, etc.;
(2) red algae such as Cyanidioscheyzon merone, Porphyridium purpureum, Gelidium amansii, Gloiopeltis furcata, Caloglossa lepipeurii, Digenea simples, and the like;
(3) diatoms, such as Phaeodactylum tricornutum, Thialassia pseudonana, Fragilariopsis cylindrus, and the like;
(4) brown algae such as Pelvetia canalicula, Ecklonia hornem, Undaria pinnatida, echocarpus silicaulosus, etc.;
(5) euglena, such as Nannochloropsis oceanica, but not limited to the above species.
And 2, data collection:
the data sources related to The invention include The Conserved Domain Database (http:// www.ncbi.nlm.nih.gov/cdd /), SMART (http:// SMART. embedded electronic berg. de /), DOE journal Genome Institute (http:// Genome. jgi-psf. org/phase 2/phase 2.home. html), A functional genetics Database for energy micro-organism (http:// www.bioenergychina.org:8989/), and The like.
Step 3, exploring key enzymes or proteins for metabolism and regulation of algae gibberellins:
by means of comparing functional genomics, key enzymes or proteins for gibberellin metabolism and regulation are discovered in the genome of algae. The software used therein includes localized BLAST software package, Pfam, CLUSTALW, gBlock, ProtTest, PhyML 3.0, etc.
And 4, gibberellin-related enzyme and signal transmission and regulation:
the metabolic key enzymes identified in the present invention include gibberellin synthase, gibberellin degrading enzymes, and gibberellin derivatizing enzymes (glycosylation, acylation, etc.) (FIG. 1), which include:
gibberellin synthetases, for example: ent-carboxyphosphine synthase (CPS), ent-Kaurene Synthase (KS), ent-Kaurene Oxidase (KO), GAI-RGA like giberellin response modulator (GA4), ent-kaurene acid oxidase (KAO1), ent-kaurene acid oxidase (KAO2), GA 20-oxidase (GA20ox), GA 3-oxidase (GA3ox), etc.;
gibberellin-degrading enzymes, for example: GA 2-oxidase (GA2ox), gibberellin 16 α, 17-epoxydase (CYP714D1), and the like.
Gibberellin methylases, for example: GA Methyltransferase (GAMT).
Gibberellin signaling and regulatory proteins include gibberellin receptors, such as: GA intensive Dwarf 1(GID 1);
other signaling and regulatory related proteins, such as: n-acetyl glucosamine transferase (SPY), F-box protein (SLY1), F-box protein (SLY2), GA sensory (GAI), Rerestor of GA (RGA), RGA-LIKE1(RGL1), RGA-LIKE2(RGL2), RGA-LIKE3(RGL3), SLENDER RICE 1(SLR1), Slender protein 1(SLN1), MADS-box protein (SOC1), Katan p60 ATPase-relating reagent (LUE1), PKL (CHD3-type chromoprotein-relating reagent KLE mode). But are not limited to, the above enzymes or proteins.
Genes associated with gibberellin metabolism that may be present in the microalgae listed in FIG. 1; the genes can be used for regulating and controlling the level of gibberellin so as to influence the characters of microalgae such as growth and the like
Example 2: identification of key enzyme or protein participating in synthesis, signal transmission and regulation of gibberellin in stress resistance and oil accumulation process of microalgae
Step 1. algal species culture and sample collection an example is Nannochloropsis oceanica of the genus Euglena. An optimized F/2 seawater culture medium is adopted, and the formula is as follows: 35g/L sea salt, 1g/L NaNO3，67mg/L NaH2PO4·H2O，3.65mg/L FeCl3·6H2O，4.37mg/L Na2EDTA·2H2O，trace metal mix(0.0196mg/L CuSO4·5H2O，0.0126mg/L NaMoO4·2H2O，0.044mg/L ZnSO4·7H2O，0.01mg/L CoCl2，0.36mg/L MnCl2·4H2O), and vitamin mix (2.5. mu.g/L VB12, 2.5. mu.g/L biotin, 0.5. mu.g/L thiamine HCl). Cells were incubated at 50. mu. mol photons m-2Culturing at 25 ℃ under continuous illumination per second. Will be cultured to logarithmic phase (OD)7503.0), washing with sterilized seawater for 3 times, inoculating into fresh nitrogen-free and nitrogen-containing culture solution, culturing in a photobioreactor with an inner diameter of 3.5cm, and timing. Collecting 200ml of algae cells at 3, 4, 6, 12, 24 and 48 hours, freezing with liquid nitrogen, and preservingIn-80 ℃, 3 biological replicates were taken at each time point.
And 2, extracting total RNA, namely fully grinding the frozen and preserved algae liquid in liquid nitrogen, and extracting RNA by using a Trizol (Invitrogen) kit. After quality testing, the library is used for constructing a transcriptome library.
And 3, constructing and sequencing a transcription library, enriching mRNA by using Sera-mag Magnetic Oligo (dT) beads (thermo scientific), randomly breaking the mRNA into short segments of 250bp by using an RNA Fragmentation reagent, carrying out reverse transcription by using a random primer to obtain cDNA, and further synthesizing double-stranded cDNA. The Library was constructed using the instruction manual of NEBNext mRNA Library Prep Reagent Set (NEB). Following quality testing, Illumina HiSeq 2000(2X90bp) paired-end sequencing was performed.
And 4, performing low-quality reads filtration and read length correction by adopting an Illumina quality control system and Fast _ trimmers in data quality control. Raw reads are uploaded to NCBI GEO. And (3) adopting TopHat 2.0.4 to transfer the reads align after QC to the nannochloropsis minitans genome.
Step 5. analyzing the gene expression abundance by using Cufflinks 2.0.2, and obtaining the FPKM (fragments Per Kilobase of exon model Per Million mapped fragments) value through normalization processing. Spearman correlation coefficients were calculated based on FPKM values and correlations between biological replicates were calculated. One of the following conditions is satisfied: (1) significant up-regulation: 2 times and above, the FDR (pulse discovery rate) is less than or equal to 5 percent, and the FPKM is more than or equal to 10 percent; (2) significant downregulation: 2 times or more, FDR is less than or equal to 5 percent, and FPKM is more than or equal to 10 percent; (3) significant upregulation can be considered: at least 2 time points, the rising of 1.5 times or more occurs, the FDR is less than or equal to 5 percent, and the FPKM is more than or equal to 10; (4) significant downregulation can be considered: at least 2 time points, a 1.5-fold or more reduction occurred, with FDR < 5% and FPKM > 10 (FIG. 2). The results show that the genes are related to the gibberellin metabolism of microalgae and are involved in nitrogen deficiency stress or oil synthesis of the microalgae.
And 6, identifying key genes of gibberellin synthesis, signal transmission and regulation in the microalgae stress resistance and oil accumulation process: besides the genes listed in step 4 of example 1, the key enzymes or proteins involved in gibberellin synthesis, signal transmission and regulation in the microalgae stress tolerance and lipid accumulation process also include various gibberellin-related genes associated with microalgae stress tolerance and lipid accumulation obtained by means of transcriptomic, proteomic, metabolomics, and the like, or methods of genetic engineering, metabolic engineering, biochemistry, molecular biology, and the like.
Example 3: application of gibberellin in promoting growth of algae
Step 1. algal species culture and sample collection take Nannochloropsis oculata (N.oceanica) as an example. An optimized F/2 seawater culture medium is adopted, and the formula is as follows: 35g/L sea salt, 1g/L sodium nitrate (NaNO)3) 67mg/L sodium dihydrogen phosphate monohydrate (NaH)2PO4·H2O), 3.65mg/L ferric chloride hexahydrate (FeCl)3·6H2O), 4.37mg/L sodium citrate dihydrate (Na)2EDTA·2H2O), trace elements [0.0196mg/L copper sulfate pentahydrate (CuSO)4·5H2O), 0.0126mg/L sodium molybdate dihydrate (NaMoO)4·2H2O), 0.044mg/L Zinc sulfate heptahydrate (ZnSO)4·7H2O), 0.01mg/L cobalt chloride (CoCl)2) And 0.36mg/L manganese chloride tetrahydrate (MnCl)2·4H2O)]And vitamin mixture (2.5. mu.g/L vitamin B12, 2.5. mu.g/L biotin and 0.5. mu.g/L thiamine hydrochloride). Cells were incubated at 50. mu. mol photons m-2s-1At 25 ℃ to logarithmic phase (OD)750＝3.0)。
Step 2. gibberellin application to dilute log phase nannochloropsis to OD750To the culture broth, gibberellin (0.02, 0.2, 2, 20, and 40mg L) was added-1)。
Step 3. Biometrics determination of OD Per 24 hours750. The results showed 20mg L-1GA promotes nannochloropsis growth (FIG. 3).
Example 4: application of gibberellin synthesis inhibitor paclobutrazol in regulation and control of algae growth
Step 1. algal species culture and sample collection take Nannochloropsis oculata (N.oceanica) as an example. An optimized F/2 seawater culture medium is adopted, and the formula is as follows: 35g/L sea salt, 1g/L sodium nitrate (NaNO)3) 67mg/L monosodium phosphateWater (NaH)2PO4·H2O), 3.65mg/L ferric chloride hexahydrate (FeCl)3·6H2O), 4.37mg/L sodium citrate dihydrate (Na)2EDTA·2H2O), trace elements [0.0196mg/L copper sulfate pentahydrate (CuSO)4·5H2O), 0.0126mg/L sodium molybdate dihydrate (NaMoO)4·2H2O), 0.044mg/L Zinc sulfate heptahydrate (ZnSO)4·7H2O), 0.01mg/L cobalt chloride (CoCl)2) And 0.36mg/L manganese chloride tetrahydrate (MnCl)2·4H2O)]And vitamin mixture (2.5. mu.g/L vitamin B12, 2.5. mu.g/L biotin and 0.5. mu.g/L thiamine hydrochloride). Cells were incubated at 50. mu. mol photons m-2s-1At 25 ℃ to logarithmic phase (OD)750＝3.0)。
Step 2 gibberellin inhibitor application logarithmic phase nannochloropsis dilution to OD750The gibberellin synthesis inhibitor Paclobutrazole (0.5, 5, 20, and 40mg/L) was added to the broth at 0.2.
Step 3. Biometrics determination of OD Per 24 hours750. The results show that paclobutrazol at 0.5mg/L, 5mg/L and 20mg/L reduced the growth rate of nannochloropsis (FIG. 4).
Example 5: application of gibberellin synthesis inhibitor Chlormequat chloride in regulation of algae growth
Step 1. algal species culture and sample collection take Nannochloropsis oculata (N.oceanica) as an example. An optimized F/2 seawater culture medium is adopted, and the formula is as follows: 35g/L sea salt, 1g/L sodium nitrate (NaNO)3) 67mg/L sodium dihydrogen phosphate monohydrate (NaH)2PO4·H2O), 3.65mg/L ferric chloride hexahydrate (FeCl)3·6H2O), 4.37mg/L sodium citrate dihydrate (Na)2EDTA·2H2O), trace elements [0.0196mg/L copper sulfate pentahydrate (CuSO)4·5H2O), 0.0126mg/L sodium molybdate dihydrate (NaMoO)4·2H2O), 0.044mg/L Zinc sulfate heptahydrate (ZnSO)4·7H2O), 0.01mg/L cobalt chloride (CoCl)2) And 0.36mg/L manganese chloride tetrahydrate (MnCl)2·4H2O)]And vitamin mixture (2.5. mu.g/L vitamin B12, 2.5. mu.g/L biotin and 0.5. mu.g/L thiamine hydrochloride)Plain). Cells were incubated at 50. mu. mol photons m-2s-1At 25 ℃ to logarithmic phase (OD)750＝3.0)。
Step 2 gibberellin inhibitor application logarithmic phase nannochloropsis dilution to OD750To the culture broth was added the gibberellin synthesis inhibitor chlorequat chloride (0.5, 5, 20 and 40 mg/L).
Step 3. Biometrics determination of OD Per 24 hours750. The results showed that 0.5mg/L and 5mg/L Chlormequat chloride decreased the growth rate of nannochloropsis (FIG. 5)
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims (10)

1. Use of gibberellin or its metabolic regulators in the physiological regulation of algae.
2. Use according to claim 1, wherein the gibberellin metabolism-modulators comprise at least one of the following i) -vi):
i) key enzymes in gibberellin metabolism or signaling pathways;
ii) a gene encoding the key enzyme of i);
iii) an expression vector comprising the gene of ii);
iv) a promoter or enhancer to enhance expression of the gene of ii);
v) an inducer which promotes the expression of ii) said gene;
vi) an agent that increases the activity of i) the key enzyme.
3. The use of claim 2, wherein the physiological regulation of algae by said gibberellin metabolism-regulating compounds of i) -vi) comprises: promoting the growth of algae, increasing the accumulation of secondary metabolites of algae or improving the stress resistance of algae.
4. Use according to claim 1, wherein the gibberellin metabolism-modulators include at least one of the following I) -VI):
I) an expression vector for knocking out or knocking down a key enzyme gene in gibberellin metabolism or a signal transmission pathway;
II), a host cell or viral vector comprising I);
III), nucleic acid molecules that interfere with transcription of key enzyme genes in gibberellin metabolism or signaling pathways;
IV), gibberellin metabolism or key enzyme gene expression in the signaling pathway;
v), agents that inhibit the expression of key enzyme genes in gibberellin metabolism or signaling pathways;
VI), gibberellin metabolism or key enzyme activity inhibitors in the signaling pathway.
5. The use of claim 1 wherein the modulator of gibberellin metabolism is an inhibitor of gibberellin synthesis, comprising: chlormequat chloride or paclobutrazol.
6. The use of claim 4 or 5, wherein the physiological regulation of algae by the gibberellin metabolism-regulating agents of I) -VI) comprises: inhibiting the growth of algae, reducing the accumulation of secondary metabolites of algae or reducing the stress resistance of algae.
7. The use according to claim 2 or 4, wherein the key enzymes in the gibberellin metabolism or signaling pathway include:
gibberellin synthases, including: ent-phosphate diphosphite synthsase, ent-kaurene synthsase, ent-kaurenic oxidase, GAI-RGA like giberellin response module, ent-kaurenic acid oxidase, GA 20-oxidase or GA 3-oxidase;
gibberellin-degrading enzymes, including: GA 2-oxidases, gibberellin 16 alpha or 17-epoxydase;
gibberellin methylases, including: GA methytranferase;
gibberellin signaling and regulatory proteins, including: gibberellin receptor or GA intensive Dwarf 1;
other signaling and regulatory related proteins, including: n-acetyl glucosamine transferase, F-box protein, GA intense, Reresolver of GA, RGA-LIKE1, RGA-LIKE2, RGA-LIKE3, SLENDER RICE 1, Slender protein 1, MADS-box protein, CHD3-type chromamat-modifying factor PICKLE or Katan p60 ATPase-linking subenit.
8. The use according to any one of claims 1 to 7, wherein the algae comprises: green algae, red algae, diatom, brown algae, or euglena.
9. A method of promoting algal growth, increasing algal secondary metabolite accumulation, or increasing algal stress tolerance, comprising: exogenously administering gibberellin or increasing endogenous levels of gibberellin in algae using at least one of the following modulators of gibberellin metabolism i) to vi):
i) key enzymes in gibberellin metabolism or signaling pathways;
ii) a gene encoding the key enzyme of i);
iii) an expression vector comprising the gene of ii);
iv) a promoter or enhancer to enhance expression of the gene of ii);
v) an inducer which promotes the expression of ii) said gene;
vi) an agent that increases the activity of i) the key enzyme.
10. An algal growth inhibitor, algal secondary metabolite accumulation reduction, or algal stress tolerance reduction, comprising: reducing endogenous gibberellin levels in the algae using at least one gibberellin metabolism modulator of the following I) -VI):
I) an expression vector for knocking out or knocking down a key enzyme gene in gibberellin metabolism or a signal transmission pathway;
II), a host cell or viral vector comprising I);
III), nucleic acid molecules that interfere with transcription of key enzyme genes in gibberellin metabolism or signaling pathways;
IV), gibberellin metabolism or key enzyme gene expression in the signaling pathway;
v), agents that inhibit the expression of key enzyme genes in gibberellin metabolism or signaling pathways;
VI), gibberellin metabolism or key enzyme activity inhibitors in the signaling pathway.
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