CN114480132B - Reef-building coral co-living yellow algae species and separation and purification method and culture method thereof - Google Patents
Reef-building coral co-living yellow algae species and separation and purification method and culture method thereof Download PDFInfo
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Abstract
The invention relates to the field of research of coral symbiotic algae in marine biology, in particular to a reef-building coral co-living yellow algae species, a separation and purification method and a culture method thereof. According to the characteristics of symbiotic and corresponding physiological mechanisms of coral and chlorella, the invention specifically separates, purifies and cultures chlorella in vitro in coral, according to the compact symbiotic relationship, no impurity is removed at the initial stage of culture, so as to ensure that the chlorella can gradually adapt to the environment of in vitro culture, and after entering an index growth period, the chlorella is separated and purified to obtain pure reef-building chlorella species. The research result of the invention is to promote the physiological function of the yellow worm algae, and the research of the symbiotic relationship of the coral and the yellow worm algae, thereby providing a feasible thought for protecting the coral reef ecosystem.
Description
Technical Field
The invention relates to the field of research of coral symbiotic algae in marine biology, in particular to a reef-building coral co-living yellow algae species, a separation and purification method and a culture method thereof.
Background
The yellow worm algae is unicellular dinoflagellate widely symbiotic with marine invertebrates such as coral, giant clams, sponges and the like. In the coral reef ecological system, the reciprocal symbiotic relationship is the basis of the coral reef ecological system and is an important component of the physiological life activities of the corals. The photosynthesis of the yellow worm algae can provide oxygen for corals, and the fixed carbon is transferred to various substances required by the synthesis of life activities in the corals. In exchange, coral can provide protection for yellow algae and provide corresponding metabolites of yellow algae as raw materials for photosynthesis. The cooperation relationship enables coral to be better suitable for survival in oligotrophic water bodies. However, this symbiotic relationship is very susceptible to disruption, and minor environmental changes may lead to loss of yellow algae or destruction of pigments, leading to coral whitening and even death. Therefore, the study on the yellow worm algae becomes an important part of the study and protection of the coral, but the related study is difficult to develop and is greatly interfered by various factors because the yellow worm algae and the coral are in close symbiosis in a natural state.
The yellow worm algae contains a plurality of lines, and at present, the E line (Effrenium) mainly symbiotic in giant clams has been studied intensively, and separation and in vitro large-scale culture have been realized (Zhang Yuehuan, et al, 2018). However, for the A, B, C, D line group which is widely co-generated in hermatypic coral, only one strain of coral yellow algae is cultured in vitro in 2014 worldwide, and is limited to the laboratory culture stage (Lin et al, 2014). Therefore, the difficulty of related research of the yellow algae can be reduced by separating and culturing the yellow algae in the coral and culturing the yellow algae in vitro, the physiological function of the yellow algae is practically promoted, and the research of the symbiotic relationship of the coral and the yellow algae provides a feasible thinking for protecting the coral reef ecological system and has important practical significance.
Disclosure of Invention
In view of the above, the invention provides a reef-building coral coculture yellow algae species, a separation and purification method and a culture method thereof, which provide a practical thought for promoting physiological functions of yellow algae and researching symbiotic relationship between coral and yellow algae and protecting coral reef ecosystem.
In order to achieve the above object, the present invention provides the following technical solutions:
in a first aspect, the invention provides a reef-building coral coincidental chrysophyta species with a preservation number of: CGMCC 23075.
In a second aspect, the invention also provides application of the hermatype coral co-produced xanthomonas algae species in research of symbiotic relationship between coral and xanthomonas, coral physiology research or ecological protection.
In a third aspect, the invention also provides a separation and purification method of the reef-building coral yellow algae species, which comprises the following steps:
step 1: obtaining coral tissue, mixing and grinding with buffer solution to obtain original suspension;
step 2: taking the original suspension for microscopic examination, and determining that the original suspension contains free unicellular algae;
step 3: inoculating the original suspension into an F/2 culture medium, and carrying out low-speed shaking culture at a temperature of 26 ℃ and a speed of 30 revolutions per minute for 10-15 days under 6000lx illumination to obtain an exponential growth phase algae liquid;
step 4: taking the index growth period algae liquid for microscopic examination, and after the swimming individuals with flagella are subjected to dilution separation, namely sucking single algae by a capillary tube, respectively putting the single algae into a 1.5mL centrifuge tube filled with 200 mu L of F/2 culture medium, sucking for more than 30 times, and obtaining at least 30 single algae species subjected to dilution separation;
step 5:6000lx light, 26 ℃, can be kept still for 3-5 days. The cells that are mitotic are examined microscopically and free of protozoa, etc.
Step 6: all of the unicellular algae culture tubes were transferred to 3mL glass test tubes, and the nozzles were plugged with sterile small tampons, during which 20 microliters of fresh medium was added daily. 6000lx light, 26 ℃,30 r/min low-speed shaking culture and continuous culture for 5-7 days. The microscopic concentration can reach hundreds/ml, and no protozoa exists.
Step 7: pure worm Huang Zaozao liquid is obtained, and the obtained liquid is amplified step by step and used for the next experiment or production. The inoculation proportion is that: f/2 medium = 1:1 to 1:2.
in a fourth aspect, the invention also provides an in vitro culture method of the reef-building coral yellow algae species, which comprises the following steps:
step 1: obtaining coral tissue, mixing and grinding with buffer solution to obtain original suspension;
step 2: taking the original suspension for microscopic examination, and determining that the original suspension contains free unicellular algae;
step 3: inoculating the original suspension into an F/2 culture medium, and carrying out low-speed shaking culture at a temperature of 26 ℃ and a speed of 30 revolutions per minute for 10-15 days under 6000lx illumination to obtain an exponential growth phase algae liquid;
step 4: taking the index growth period algae liquid for microscopic examination, and after the swimming individuals with flagella are subjected to dilution separation, namely sucking single algae by a capillary tube, respectively putting the single algae into a 1.5mL centrifuge tube filled with 200 mu L of F/2 culture medium, sucking for more than 30 times, and obtaining at least 30 single algae species subjected to dilution separation;
step 5:6000lx light, 26 ℃, can be kept still for 3-5 days. The cells that are mitotic are examined microscopically and free of protozoa, etc.
Step 6: the unicellular algae culture tube was transferred entirely into a 3mL glass test tube, and the tube mouth was plugged with a sterile small cotton plug, during which 20 microliters of fresh medium was added daily. 6000lx light, 26 ℃,30 r/min low-speed shaking culture and continuous culture for 5-7 days. The microscopic concentration can reach hundreds/ml, and no protozoa exists.
Step 7: pure worm Huang Zaozao liquid is obtained, and the obtained liquid is amplified step by step and used for the next experiment or production. The inoculation proportion is that: f/2 medium = 1:1 to 1:2.
in some embodiments of the invention, the buffer in step 1 comprises EDTA buffer or sterile seawater, and the ratio of coral tissue to buffer is about 1:10 by volume.
In some embodiments of the present invention, in step 2, the cells of the original suspension containing the unicellular algae dissociated from the mucus of the insect body tissue are round, the nail plate is not obvious, no transverse groove exists, the whole body is yellow, the brown oil color is bright, the size is 10-30 μm, no flagella are visible, and no activity exists (as shown in fig. 1).
In some embodiments of the invention, the index-enhanced algae liquid in step 3 and step 4 is marked by the presence of single cells with motility, about 8-20 μm, green overall under an optical microscope, yellowish, free movement stage of individual individuals, less obvious nail, but more obvious transverse furrows (as shown in fig. 2), and the swimming state visible flagella at the transverse furrows (as shown in fig. 3), and spherical at rest.
In some embodiments of the invention, the cells in the division stage described in step 5 are in the form of a binary-divided calabash (as shown in FIGS. 4 and 5).
In some embodiments of the invention the culture broth of step 6 at this point exhibits a intermixing of a plurality of cell morphologies (calabash, motile, resting, etc.). The cultured 6000lx light is irradiated, the temperature is 26 ℃, and the low-speed shaking culture is carried out at 30 revolutions per minute for 5 to 7 days. The microscopic concentration can reach hundreds/ml, and no protozoa exists.
In some embodiments of the invention the concentration amplified in step 7 may eventually reach >10 tens of thousands/mL.
According to the characteristics of symbiotic and corresponding physiological mechanisms of coral and chlorella, the invention specifically separates, purifies and cultures chlorella in vitro in coral, according to the compact symbiotic relationship, no impurity is removed at the initial stage of culture, so as to ensure that the chlorella can gradually adapt to the environment of in vitro culture, and after entering an index growth period, the chlorella is separated and purified to obtain pure reef-building chlorella species. The invention provides a practical thought for the study of the symbiotic relationship of the coral and the yellow-worm algae for promoting the physiological function of the yellow-worm algae and protecting the coral reef ecosystem.
Description of biological preservation
Biological material: symbiotic algae (yellow worm algae) in the body of the Chinese flat brain coral (Platygyra sinensis); classification naming: cladocapium sp.HNPS-C1-A; the microorganism strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection) at the 12 th month of 2021, and the preservation center address is: the institute of microorganisms of national academy of sciences of China, national institute of sciences, no.1, no. 3, north Chen West Lu, the Korean region of Beijing; the preservation number is CGMCC NO.23075.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows free Monomonas in the original suspension in step 2;
FIG. 2 shows the exponential growth phase of algae (with transverse grooves) after 10-15 days of cultivation in steps 3 and 4;
FIG. 3 shows an exponentially increasing microalgae (flagella) after 10 to 15 days of cultivation in step 3 and step 4, step 4;
FIG. 4 shows the algae cells dividing in step 5; wherein a shows dividing cells (30 micron diameter single cells); b shows dividing cells (10 micron diameter single cells);
figure 5 shows the effect of different temperatures on symbiotic algae density.
Detailed Description
The invention discloses a reef-building coral coculture yellow algae species, a separation and purification method and a culture method thereof, and a person skilled in the art can refer to the content of the invention and properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The invention provides a separation and purification and in-vitro culture method of hermatypic coral yellow, which comprises the following steps:
a. firing tweezers, grinding pestles and mortar for standby;
b. clamping coral tissue with forceps, adding buffer solution into a mortar, and grinding uniformly to obtain original suspension A1;
c. observing A1 under a microscope to determine that free monad exists in the suspension;
d. inoculating 1ml of suspension into 9ml of F/2 culture medium, and culturing at 28deg.C under 3000lx illumination for 10-15 days to obtain index-increasing algae liquid B1;
e. after culturing to an index increasing period, placing the algae liquid B1 under a microscope for observation, and diluting and separating after the swimming individual with flagella is observed;
f. the single algae are sucked by a capillary tube and respectively put into a 1.5mL centrifuge tube filled with 200 mu L of F/2 culture medium, and the single algae are repeatedly taken for more than 30 times to respectively obtain at least 30 pipe algae species which are diluted and separated, so as to obtain the unicellular algae;
g.6000lx illumination, 26 ℃, and can be subjected to static culture for 3-5 days. The cells that are mitotic are examined microscopically and free of protozoa, etc.
h. The unicellular algae culture tube was transferred entirely into a 3mL glass test tube, and the tube mouth was plugged with a sterile small cotton plug, during which 20 microliters of fresh medium was added daily. 6000lx light, 26 ℃,30 r/min low-speed shaking culture and continuous culture for 5-7 days. The microscopic concentration can reach hundreds/ml, and no protozoa exists.
i. Pure worm Huang Zaozao liquid is obtained, and the obtained liquid is amplified step by step and used for the next experiment or production. Amplifying step by step, and inoculating according to the ratio of the algae liquid: f/2 medium = 1:1 to 1:2.
in some embodiments of the present invention, the firing in step a is performed with 95% alcohol to fire the surface and wait for the article to cool before using, as much as possible, on an ultra clean bench.
In some embodiments of the invention, the buffer in step b is EDTA buffer or sterile seawater.
In some embodiments of the present invention, the unicellular algae in the step c is characterized in that the isolated mature cells are round under an optical microscope, the nail plate is not obvious, the whole is yellowish green, the size is 10-30 μm, and no flagella are visible and no activity exists.
In some embodiments of the invention, the medium in step d is F/2. The sign of the algae cell entering the index growth period is that single cells with movement capability appear, the whole is green and yellowish under an optical microscope, a single individual has a free movement stage, the nail is not obvious, the transverse groove is obvious, flagella are visible in a swimming state, and the algae cell is spherical when the algae cell is static.
In some embodiments of the present invention, the step e may be performed by dilution to obtain a pure algae solution containing single-digit algae cells.
In some embodiments of the invention, the centrifuge tube in step f controls the medium content to be about 200 microliters, and is continuously replenished with 20 microliters per day.
In some embodiments of the present invention, the algae solution C in the step g cannot contain impurities such as miscellaneous algae, protist, coral tissue fragments, etc.
In some embodiments of the invention, the step-wise amplification time in step h should be 10-15 days apart.
According to the characteristics of symbiotic growth of coral and yellow-worm algae and corresponding physiological mechanisms, the invention performs targeted separation, purification and in-vitro culture on yellow-worm algae in the middle Hua Bian brain coral, and according to the compact symbiotic relationship, no impurity removal is performed at the initial stage of culture so as to ensure that the algae can gradually adapt to the in-vitro culture environment, and after entering an index growth period, separation and purification are performed to obtain pure reef-building coral-coculture yellow-worm algae species. In order to promote the physiological functions of the yellow worm algae, the study of the symbiotic relationship of the coral and the yellow worm algae provides a feasible thought for protecting the coral reef ecosystem.
The reef-building coral coculture yellow algae species, the separation and purification method and the culture method thereof can be obtained from the market.
The invention is further illustrated by the following examples:
example 1
Taking the live coral on a tray, and spraying seawater during the experiment to avoid affecting the health of the coral. Igniting the alcohol lamp to sterilize the tools such as the grinding bowl, the tweezers and the like. After disinfection, selecting full and undamaged coral, taking out the tissue, grinding the tissue in a mortar, adding buffer solution in the grinding process, wherein the buffer solution is not more than 5ml, and grinding to be uniform to obtain tissue suspension. The tissue suspension was poured into a test tube and vigorously shaken. 1ml of the suspension is inoculated into 9ml of F/2 culture medium, and cultured for 10 to 15 days under 3000lx illumination and 28 ℃ to obtain the exponential growth phase algae liquid. After culturing to an index increasing period, taking the algae liquid, observing under a microscope, and after observing the swimming individual with flagella, obtaining the unicellular algae by dilution and separation. The unicellular algae were placed in 1.5ml centrifuge tubes for cultivation, the growth condition was observed every day, and the culture medium was added. After 10-15 days, obviously increased algae cells are observed, and when no other impurities exist, the algae cells are expanded to a 5ml test tube, the culture is continued, and then the algae cells are expanded to a 25ml conical flask, so that the algae seeds which can be subjected to the next experiment are obtained.
The result of 16SrDNA sequencing is shown as SEQ ID No. 1.
EXAMPLE 2 separation and purification and in vitro culture of reef-building coral yellow algae
a. Firing tweezers, grinding pestles and mortar for standby;
the surface is burnt by adopting 95% alcohol and can be used after the articles are cooled, and the process is carried out on an ultra-clean workbench as much as possible;
b. clamping coral tissue with forceps, adding buffer solution (EDTA buffer solution or sterile seawater) into a mortar, and grinding uniformly to obtain original suspension A1;
c. observing A1 under a microscope to determine that free monad exists in the suspension;
the unicellular algae is characterized in that under an optical microscope, the separated mature cells are round, the formazan piece is not obvious, the whole body is yellowish green, the size is 10-30 mu m, flagella cannot be seen, and the activity capacity is not available;
d. inoculating 1ml of suspension into 9ml of F/2 culture medium, and culturing at 26deg.C under 6000lx illumination for 10-15 days to obtain index-increasing algae liquid B1;
the markers of the algae cell entry index growth phase are: single cells with movement capability appear, the whole is green, slightly yellowish under an optical microscope, a single individual has a free movement stage, a formazan piece is not obvious, a transverse ditch is obvious, flagella are visible in a swimming state, and the single cells are spherical when the single cells are static;
e. after culturing to an index increasing period, placing the algae liquid B1 under a microscope for observation, and diluting and separating after the swimming individual with flagella is observed;
f. the single algae are sucked by a capillary tube and respectively put into a 1.5mL centrifuge tube filled with 200 mu L of F/2 culture medium, and the single algae are repeatedly taken for more than 30 times to respectively obtain at least 30 pipe algae species which are diluted and separated, so as to obtain the unicellular algae;
g.6000lx illumination, 26 ℃, and can be subjected to static culture for 3-5 days. The cells that are mitotic are examined microscopically and free of protozoa, etc.
h. The unicellular algae culture tube was transferred entirely into a 3mL glass test tube, and the tube mouth was plugged with a sterile small cotton plug, during which 20 microliters of fresh medium was added daily. 6000lx light, 26 ℃,30 r/min low-speed shaking culture and continuous culture for 5-7 days. The microscopic concentration can reach hundreds/ml, and no protozoa exists.
i. Pure worm Huang Zaozao liquid is obtained, and the obtained liquid is amplified step by step and used for the next experiment or production. Amplifying step by step, and inoculating according to the ratio of the algae liquid: f/2 medium = 1:1 to 1:2.
example 3 broad-spectrum temperature-adaptive experiment of reef-building coral yellow algae
Under the conditions of 28 deg.C, 3000lx illumination and 12 illumination cycles, four temperature gradient groups are set up, respectively at 18+ -0.2 deg.C and 22+ -0.2The culture volume is 100ml by adopting F/2 culture medium at the temperature of 26+/-0.2 ℃ and 30+/-0.2 ℃. The initial concentration of algae cells is 2×10 3 Each of the cells was cultured for 10 days. Starting on day 1 of the experiment, 0.1mL of algae solution was taken every 2 days in a 0.1mL plankton counting frame, and the cell density (in/mL) was counted under a microscope, and the average was taken in three replicates. The results were as follows:
TABLE 1 Effect of different temperatures on symbiotic algae Density
The analysis result shows that the symbiotic algae (yellow worm algae) separated from the Chinese flat brain coral in Wenchang sea area has a wide temperature application range and can survive at 18-30 ℃. Wherein the growth speed is the fastest at 26-28 ℃. The winter water temperature of Wenchang sea area can reach 18-19 deg.c and 30 deg.c in summer, so that coral has wide temperature adaptability to grow and survive. The temperature adaptability of the yellow algae in the bodies also shows that the square adaptability of the coral in Wenchang sea area to the temperature comes from the symbiotic insect Huang Zaoan temperature adaptability.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> university of Hainan
<120> reef-building coral coculture yellow algae species, and separation and purification method and culture method thereof
<130> MP21026620
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 636
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ggtgaacctg cggaaggatc attcgcactg attcaatttt gactacgtga attttggctg 60
tgagggatgc ttgcggtgag atatctgctt gcagaactcg agggcgacag atgctcactc 120
accaaccctt gttggggctg gtgcgagtgt cgctctgtgg ccatgggtcc gtgttcagcg 180
ggccggggaa tgaaggctaa gctgctttag tgacaactgt ctgggaaggg cattgttcca 240
acttccacaa ctttcagcga tggatatctt ggctcgggca cctgtgaagg gcgcagcgaa 300
gcgcgatagt ctttgtgaat tgcagaactc cgtgaaccaa tggcctcctg aacgtgcgtt 360
gcactcttgg gatttcctga gagtatgtct gcttcagtgc ttaacttgcc ccaactttgc 420
aagcaggatg tgtttctgcc ttgcgttctt atgagctatt gccctctgag ccaatggctt 480
gttaattgct tggttcttgc aaaatgcttt gcgcgctgtt attcaggttt ctaccttcgt 540
ggttttactt gagtgacgct gctcatgctt gcaaccgctg ggatgcaggt gcatgcctct 600
agcatgaagt cagacaagtg aacccgctga atttaa 636
Claims (2)
1. The reef-building coral co-growing yellow algae species is characterized in that the preservation number is: CGMCC 23075.
2. The use of a reef-building coral-co-growing xanthomonas sp in the study of the symbiotic relationship of coral and xanthomonas sp, the study of coral physiology or the ecological protection of the species of xanthomonas sp.
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| CN110373329A (en) * | 2019-06-04 | 2019-10-25 | 浙江山诺生物科技有限公司 | A kind of breeding method of heat resistanceheat resistant zooxanthellae |
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