Coral source calcified algae Z1 and culture method and application thereof
By culturing and isolating and purifying calcified algae Z1, the problem of unknown functional characteristics of calcified algae in coral reef ecosystems is solved, efficient culture and calcification rate are achieved, new materials for research on coral reef repair and symbiotic relationships are provided, coral skeleton strength is enhanced, and coral bleaching is prevented and controlled.
Patent Information
- Application Number
- CN202510451944.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-11
- Publication Date
- 2025-07-11
AI Technical Summary
In the prior art, there is a lack of systematic understanding of the functional characteristics and ecological effects of calcified algae in coral reef ecosystems, which leads to fragility of coral structures and aggravates ecological degradation. Especially under the influence of marine acidification and human activities, the rate of coral bleaching and calcification has dropped seriously.
A method for culturing algae Z1 is provided, and culturing algae Z1 with improved f/2 liquid culture medium and specific light conditions is used to obtain efficient purified algae cells through isolation and purification technology to achieve efficient culture and enlarged culture under laboratory conditions.
The efficient culture of calcified algae Z1 was achieved, the cell density reached 8×104 cells/mL, and the calcification rate was improved, providing the basis for the study of new coral reef repair materials and symbiotic relationships, enhancing the strength of coral skeletons, and having the potential to prevent and control coral bleaching and reef reconstruction.
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Figure CN120290325A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the fields of marine biotechnology and ecological restoration technology, and particularly relates to a coral-derived calcifying alga Z1, a cultivation method thereof, and an application thereof. Background Art
[0002] As a core component of the global marine ecosystem, the three-dimensional reef-building structure of coral reefs forms a physical barrier through calcification, and supports human social and economic activities such as fishery resources, coastal protection, and marine drug development through ecological service functions such as carbon and nitrogen cycling and energy flow. It is estimated that global coral reefs only account for 0.25% of the ocean area, yet maintain more than 25% of marine species diversity. However, in 2023, the global area of coral bleaching reached a historical peak, and the coral coverage rate in the South China Sea of our country has decreased by more than 60% compared with 30 years ago; ocean acidification has led to a 40% decrease in the calcification rate of reef-building corals, and human activities (such as overfishing and coastal development) have accelerated the process of ecological degradation.
[0003] In the coral reef ecosystem, the symbiotic relationship between corals and symbiotic algae is the core basis for the stability of the coral reef ecosystem: coral polyps provide a living space and inorganic nutrients for the algae, and the algae transfer up to 70% of the organic carbon source to the corals through photosynthesis. Calcifying algae belong to the order Bryopsidales of the class Ulvophyceae in the phylum Chlorophyta, and have a unique extracellular calcification pattern, where calcium deposits in the vesicle spaces formed by cell wall folds. In the coral reef ecosystem, calcifying algae not only participate in the mineralization process of coral skeletons, and the calcium carbonate crystals secreted by them can enhance the mechanical strength of the coral matrix, but also calcifying algae can use their own secretions to bond biological remains and organic matter fragments together, and the dead algal bodies fill the reef gaps, playing an important role in reef-building and reef-fixing processes. Research shows that when symbiotic algae are lost, the calcification rate of corals can decrease by 40%-70%, leading to the weakening of the coral structure and ultimately death. Therefore, analyzing the composition of symbiotic algal communities and their functional characteristics has important theoretical and practical value for coral reef ecological restoration. In recent years, molecular biology techniques have promoted the progress of research on the taxonomy of coral symbiotic algae. For example, multi-gene joint analysis techniques have revealed the existence of a large number of cryptic species in epiphytic Acropora nana that have not been identified by traditional morphological methods. However, for symbiotic green algal groups with calcification ability, their functional characteristics and ecological roles are still lacking systematic understanding. Summary of the Invention
[0004] The purpose of the present invention is to provide a coral-derived calcifying alga Z1, a cultivation method thereof, and an application thereof, so as to solve the problems existing in the above-mentioned prior art.
[0005] To achieve the above purpose, the present invention provides the following solutions:
[0006] The present invention provides a calcified alga Z1 (Acrochaete zhaohaiensis sp. nov Z1), and the preservation number of the calcified alga Z1 is CCTCC NO: M 2025578.
[0007] The present invention provides a cultivation method of the above-mentioned calcified alga Z1, which includes the step of inoculating the calcified alga Z1 into a culture medium for cultivation.
[0008] Preferably, the culture medium is a modified f / 2 liquid medium;
[0009] The modified f / 2 liquid medium comprises components with the following concentrations:
[0010] 75 mg / L NaNO3, 1.5 g / L NaHCO3, 8 mg / L KH2PO4, 0.01 mg / L FeCl3·6H2O, 1 mL / L complex trace element solution, 0.1 μg / L vitamin B 12 , 0.02 μg / L biotin, 0.5 μg / L folic acid, 50 μg / mL kanamycin, 100 μg / mL ampicillin, 50 μg / mL streptomycin, and 2.5 μg / mL amphotericin b.
[0011] Preferably, the cultivation temperature is 25 °C, the light intensity is 100 μmol / (m 2 ·s), and the light cycle is 12 h light and 12 h darkness.
[0012] The present invention provides the application of the above-mentioned calcified alga Z1 in the preparation of a microbial preparation.
[0013] The present invention provides a microbial preparation, which comprises the above-mentioned calcified alga Z1.
[0014] The present invention provides the application of the above-mentioned calcified alga Z1 in the research on the symbiotic relationship and action mechanism between corals and calcified algae.
[0015] The present invention provides the application of the above-mentioned calcified alga Z1 or the above-mentioned microbial preparation in the protection of coral ecosystems.
[0016] The present invention provides the application of the above-mentioned calcified alga Z1 or the above-mentioned microbial preparation in preventing and / or repairing coral bleaching caused by ocean acidification.
[0017] The present invention provides the application of the above-mentioned calcified alga Z1 or the above-mentioned microbial preparation in coral artificial cultivation and / or coral reef reconstruction.
[0018] The present invention discloses the following technical effects:
[0019] The present invention discovers and identifies a new species of coral-derived calcifying alga - a calcifying alga Z1 (Acrochaete zhaohaiensis sp. nov Z1), with a deposit number of CCTCC NO: M 2025578. The cell surface of the calcifying alga Z1 has unique nanoscale calcium carbonate needle-like protrusions and a hexagonal lattice arrangement structure. Its 18S rRNA sequence shows 100% homology with Acrochaete endozoica. However, through detailed alignment and analysis of the sequences, it is found that there are base differences at multiple internal positions in the gene sequences between the two, that is, the calcifying alga Z1 forms an independent branch on the phylogenetic tree with known coral symbiotic algae, and the calcification rate of the calcifying alga Z1 is significantly increased compared with typical zooxanthellae. Specifically, obvious calcification phenomena start to appear on the 7th day during small-scale culture, enter the rapid mineralization stage on the 14th day, and complete calcified tissues are formed on the 20th day. The calcification rate is relatively high. The reproductive characteristics of its aplanosporangia provide potential technical reserves for subsequent symbiotic relationship research and artificial reef construction. At the same time, this unique calcification metabolism characteristic provides a new functional material for coral reef restoration.
[0020] The present invention also provides a cultivation method for the calcifying alga Z1. The present invention realizes the goal of highly purifying and culturing the calcifying alga Z1 under laboratory conditions for the first time. After cultivation, the algal cell density can reach 8×10 4 cells / mL, achieving the purpose of large-scale culturing of this strain. Brief Description of the Drawings
[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.
[0022] Figure 1 It is a sample coral tissue diagram;
[0023] Figure 2 It is an inverted fluorescence microscope diagram of the calcifying alga Z1; among them, the scale bar is 100.00 μm, and the objective lens is 40×;
[0024] Figure 3 It is a scanning electron microscope diagram of the calcifying alga Z1; among them, A and B are scanning electron microscope diagrams of the calcifying alga Z1 under different scale conditions; the scale bar of A is 100 μm, and the scale bar of B is 50.0 μm;
[0025] Figure 4Transmission electron micrograph of calcified alga Z1; among them, A is the transmission electron micrograph of the whole calcified alga Z1; B is the transmission electron micrograph of the top of calcified alga Z1; C and D are the transmission electron micrographs of parts of calcified alga Z1; the scale bar of A is 2μm, the scale bar of B is 1μm, the scale bar of C is 2μm, and the scale bar of D is 1μm;
[0026] Figure 5 Phylogenetic tree (18S rRNA gene) diagram of calcified alga Z1; among them, Z1 is calcified alga Z1. Detailed implementation manners
[0027] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.
[0028] It should be understood that the terms described in the present invention are only used to describe specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.
[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.
[0030] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.
[0031] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.
[0032] Unless otherwise required, the reagents or components used in the present invention are all routinely purchased by those skilled in the art, and sigma or Solarbio can be selected.
[0033] Isolation and Identification of Calcifying Algae Z1 (Acrochaete zhaohaiensis sp. nov Z1) in Example 1
[0034] The isolation and identification steps are as follows:
[0035] S1. First, rinse the Acropora cervicornis collected from Meiji Reef in the South China Sea ( Figure 1 ) three times with sterilized seawater (pH 7.8 - 8.0) to thoroughly remove the impurities and microbial contamination attached to the surface. Subsequently, use coral scissors to cut the coral tissue into small pieces (about 1×1×1 mm 3 ), and place them in a mortar for thorough grinding. After grinding, add 4 mL of sterilized seawater, filter preliminarily to remove the residue, filter using a 40 μm nylon mesh. To further remove fine residues, pass the filtrate through a 23 μm nylon mesh again, and collect the filtrate for later use;
[0036] S2. Take 200 μL of the filtrate prepared in step S1 and evenly spread it on a solid plate containing modified f / 2 solid medium (the modified f / 2 solid medium contains 75 mg / L NaNO3, 1.5 g / L NaHCO3, 8 mg / L KH2PO4, 0.01 mg / L FeCl3·6H2O, 1 mL / L complex trace element solution (mainly containing EDTA-Fe, ZnSO4, MnCl2, etc.), 0.1 μg / L vitamin B 12 , 0.02 μg / L biotin, 0.5 μg / L folic acid, 50 μg / mL kanamycin, 100 μg / mL ampicillin, 50 μg / mL streptomycin, and 2.5 μg / mL amphotericin b). Place the plate in an environment with a temperature of 25 °C, a light intensity of 150 μmol / (m 2 ·s), and a light cycle of 12 hours of light / 12 hours of darkness for cultivation, and the cultivation time is 15 - 20 days.
[0037] S3. Pick the algal colonies grown on the solid plate in step S2 and inoculate them into sterilized modified f / 2 liquid medium (the modified f / 2 liquid medium contains 75 mg / L NaNO3, 1.5 g / L NaHCO3, 8 mg / L KH2PO4, 0.01 mg / L FeCl3·6H2O, 1 mL / L complex trace element solution, 0.1 μg / L vitamin B 12 , 0.02 μg / L biotin, 0.5 μg / L folic acid, 50 μg / mL kanamycin, 100 μg / mL ampicillin, 50 μg / mL streptomycin, and 2.5 μg / mL amphotericin b), and culture at 25 °C and a light intensity of 200 μmol / (m 2·s), and cultured under the condition of 12-hour light / 12-hour darkness for the logarithmic growth phase (about 7 - 10 days) to obtain a microalgae seed solution, and then coated it onto the modified f / 2 solid medium (the medium components are the same as in step S2).
[0038] S4. Inoculate the microalgae seed solution pre-cultured in step S3 into the sterilized modified f / 2 liquid medium (the components of the modified f / 2 liquid medium are the same as in step S2) and continue to culture for 10 days. When the cell density reaches 8×10 4 cells / mL, gradient centrifugation (centrifuging at 4000×g for 10 min to break the cell wall and at 12000×g for 15 min to precipitate) combined with flow cytometry sorting technology (screening with the threshold of SSC≥300 / FSC≥500) is used to obtain a single-cell level purified algal strain with a purity of ≥98%. Subsequently, coat the purified algal strain onto a solid plate containing the modified f / 2 solid medium (the components of the modified f / 2 solid medium are the same as in step S2), and culture it at 25°C, light intensity 200 μmol / (m 2 ·s), and under the condition of 12-hour light / 12-hour darkness for 15 - 20 days for further separation and purification.
[0039] S5. After the algal colonies grow on the solid plates in steps S2, S3, and S4, perform streak isolation culture under the conditions of 25°C, light intensity 200 μmol / (m 2 ·s), and 12-hour light / 12-hour darkness for 10 - 15 days until monoclonal algal colonies grow. Then pick the monoclonal colonies into fresh modified f / 2 liquid medium for liquid culture.
[0040] S6. Take samples from step S5 for observations such as morphological observations by transmission electron microscopy and scanning electron microscopy. The morphological identification results are as Figures 2 - 4 shown. The results show that when cultured in the modified f / 2 liquid medium, the calcified alga Z1 presents an irregular oval to long rod shape, with a maximum diameter of 25±3 μm and a minimum diameter of 5±2 μm. The surface is covered with dense nano-scale concave and convex textures (spacing 50 - 200 nm), and has typical characteristic structures of calcified algae. The cytoplasm of the calcified alga Z1 contains multiple round to oval calcified particles (diameter 0.5 - 2 μm, number 8 - 12 per cell), and the particles are connected by matrix proteins and distributed in a chain or network shape; the central nuclear region shows a high electron density signal; the chloroplast presents a sheet structure (size 2 - 5 μm), and starch grains can be seen aggregated in the internal matrix. The outer layer of the double membrane is covered with nano-scale calcium carbonate needle-like protrusions (average length 80±15 nm), forming an irregular hexagonal lattice arrangement (lattice spacing 0.5 - 1 μm), and sheet structures formed by crystal fusion can be seen in local areas.
[0041] S7. Molecular identification of calcified alga Z1: The algal solution of monoclonal cells was collected by centrifugation at 5000 rpm for 10 min, and the genomic DNA of calcified alga Z1 was extracted according to the standardized operation procedure in the instruction manual of the Plant DNA Extraction Kit (OMEGA Plant DNA Kit). The extracted DNA was detected for quality and purity by a QuBit 4 fluorometer and a Nanodrop spectrophotometer to ensure that it met the requirements of subsequent experiments. The extracted DNA was used as a template for polymerase chain reaction (PCR) to amplify the 18S rDNA sequence. The amplified product was purified and used for evolutionary analysis and molecular identification, providing reliable data for the construction of subsequent phylogenetic trees.
[0042] S8. Phylogenetic analysis of calcified alga Z1: Based on the 18S rRNA gene sequence of calcified alga Z1, a phylogenetic tree was constructed ( Figure 5 ). The results of phylogenetic analysis showed that the similarity of the 18S rRNA gene sequence between calcified alga Z1 and Acrochaete endozoica reached 100%. However, through detailed alignment and analysis of the sequences, it was found that there were base differences at multiple internal positions of the gene sequences. For example, in the V4 conserved domain (positions 120 - 125), calcified alga Z1 showed a continuous 6 - base repeat sequence (TTTTTT), while Acrochaete endozoica was AGGTCT. This region is highly specific in the Chlorophyta, and the largest continuous repeat unit of known congeneric species is 3 Ts, indicating that there is a unique base expansion event at this position in calcified alga Z1; variation of the 3' - end termination codon, calcified alga Z1 used UGA as the termination codon (positions 1,420 - 1,422), while Acrochaete endozoica was UAA. This difference in codon preference is extremely rare in the Chlorophyta (only found in 12.3% of species), suggesting that calcified alga Z1 may have a unique post - translational modification mechanism. Combining the above molecular markers, calcified alga Z1 formed an independent branch on the phylogenetic tree. These differences indicate that calcified alga Z1 is a new species in the same genus, and its taxonomic name is Acrochaete zhaohaiensis sp. nov. The inventor named this calcified alga as calcified alga Z1 (Acrochaete zhaohaiensis sp. nov Z1), which was deposited in the China Center for Type Culture Collection (CCTCC) on March 24, 2025. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2025578.
[0043] S9. Take 1 mL of the filtrate prepared in step S1 and inoculate it into 25 cm 3In a sterile cell culture flask, add 20 mL of modified f / 2 liquid medium (the composition of the modified f / 2 liquid medium is the same as in step S3) for culturing. The culture conditions are: 25 °C, light intensity 100 μmol / (m 2 ·s), photoperiod 12 h light / 12 h dark, and the culture time is 7 - 10 days. At this stage, by gradually reducing the nutrient salt ratio in the modified f / 2 liquid medium (NO3 - / PO4 3- from 20:1 to 16:1), the calcifying alga Z1 isolated from the filtrate is induced to initiate the biofilm mineralization process. The calcium carbonate crystals secreted by it form a natural physical barrier to inhibit the attachment of miscellaneous bacteria, while maintaining its symbiotic relationship with corals. In small-scale laboratory culture, obvious calcification phenomena of the calcifying alga Z1 start to appear on the 7th day of culture, enter the rapid mineralization stage on the 14th day (the calcification rate is 3 - 5 times higher than that in the initial stage), and complete calcified tissues are formed on the 20th day. The calcium carbonate mineralization structure and symbiotic adaptability characteristics of the calcifying alga Z1 provide a new technical path for coral skeleton repair and artificial reef construction, especially having important application prospects under the background of ocean acidification.
[0044] Through continuous separation and screening by optimizing conditions, the present invention realizes the efficient purification culture of the calcifying alga Z1 under laboratory conditions for the first time, and the algal cell density can reach 8×10 4 cells / mL. Using the separation and purification method provided by the present invention can completely retain the biomineralization ability and symbiotic signal transduction function of the calcifying alga Z1, providing a standardized algal species resource library for the research on coral bleaching prevention and reef restoration projects.
[0045] Example 2 Symbiotic function and potential application of the calcifying alga Z1
[0046] The successful isolation and culture of the calcifying alga Z1 provide important materials for studying its symbiotic relationship with corals and potential applications. Through techniques such as coral tissue pretreatment, algal species preculture, solid plate primary screening, single-cell purification culture, flow cytometry sorting and purification, and monoclonal algal colony identification, the laboratory purification culture of the calcifying alga Z1 is achieved. At the molecular level, the full-length sequence of the ribosomal small subunit gene (18S rRNA gene) of the calcifying alga Z1 is cloned, and its morphological characteristics are obtained through scanning electron microscopy and transmission electron microscopy observations. By comparing the gene sequence of the calcifying alga Z1 with the published algal gene sequences and combining the comprehensive analysis of microscopic morphological photos, it is confirmed that the calcifying alga Z1 is a new species belonging to the genus Acrochaete. The successful isolation and culture of the calcifying alga Z1 not only provide ideal materials for the functional research of marine algae, but also lay a foundation for further exploring its symbiotic relationship with corals. In addition, the calcifying alga Z1 has potential application values in fields such as coral artificial cultivation, enhancing reef-building ability, coral reef reconstruction and repair such as repairing coral bleaching caused by ocean acidification, and the development of new algal materials.
[0047] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.
Claims
1. A calcified alga Z1 (Acrochaete zhaohaiensis sp.nov Z1), characterized in that, The preservation number of the calcified alga Z1 is CCTCC NO: M 2025578.
2. The cultivation method of the calcified alga Z1 according to claim 1, characterized in that, It includes the step of inoculating the calcified alga Z1 into a culture medium for cultivation.
3. The cultivation method according to claim 2, wherein The culture medium is a modified f / 2 liquid medium; The modified f / 2 liquid medium comprises components with the following concentrations: 75 mg / L NaNO3, 1.5 g / L NaHCO3, 8 mg / L KH2PO4, 0.01 mg / L FeCl3·6H2O, 1 mL / L complex trace element solution, 0.1 μg / L vitamin B 12 , 0.02 μg / L biotin, 0.5 μg / L folic acid, 50 μg / mL kanamycin, 100 μg / mL ampicillin, 50 μg / mL streptomycin, and 2.5 μg / mL amphotericin b.
4. The culturing method according to claim 2, characterized in that, The temperature of the cultivation is 25 °C, the light intensity is 100 μmol / (m 2 ·s), and the photoperiod is 12 h of light and 12 h of darkness.
5. Use of the calcified alga Z1 according to claim 1 in the preparation of a microbial preparation.
6. A microbial preparation, characterized in that, The microbial preparation comprises the calcified alga Z1 according to claim 1.
7. Use of the calcified alga Z1 according to claim 1 in the study of the symbiotic relationship and mechanism of action between corals and calcified algae.
8. Use of the calcified alga Z1 according to claim 1 or the microbial preparation according to claim 6 in the protection of coral ecosystems.
9. Use of the calcified alga Z1 according to claim 1 or the microbial preparation according to claim 6 in the prevention and / or repair of coral bleaching caused by ocean acidification.
10. Use of the calcified alga Z1 according to claim 1 or the microbial preparation according to claim 6 in the artificial cultivation of corals and / or the reconstruction of coral reefs.