CN113388523B

CN113388523B - Marine nannochloropsis oculata LAMB204 resisting disease ciliates and application thereof

Info

Publication number: CN113388523B
Application number: CN202110639406.4A
Authority: CN
Inventors: 赵璐; 王松; 韩吉昌; 潘克厚
Original assignee: Rizhao Polytechnic
Current assignee: Rizhao Polytechnic
Priority date: 2021-06-08
Filing date: 2021-06-08
Publication date: 2022-03-25
Anticipated expiration: 2041-06-08
Also published as: CN113388523A

Abstract

The invention relates to the technical field of microorganisms, in particular to a marine nannochloropsis LAMB204 resistant to disease ciliates and application thereof. The marine Nannochloropsis oceanica Lamb204 is classified and named as marine Nannochloropsis oceanica, the preservation number is CGMCC NO.20713, the preservation date is 2020, 10 and 23 days, the preservation unit is the common microorganism center of China microorganism culture preservation management committee, the preservation address is No. 3 of West Lu No. 1 of Xinyang district, Beijing, and the postal code is as follows: 100101; the marine Nannochloropsis Lamb204 can be used for inhibiting growth of ciliate population. The marine nannochloropsis has resistance to ciliates, and is suitable for large-scale culture.

Description

Marine nannochloropsis oculata LAMB204 resisting disease ciliates and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a marine nannochloropsis LAMB204 resistant to disease ciliates and application thereof.

Background

Because nannochloropsis has important application value in various fields of food, nutrition, health care and the like, the development of nannochloropsis resources becomes an ideal way for obtaining bioactive substances with high added values and renewable biological energy, and the culture scale of nannochloropsis is gradually enlarged. However, the development of nannochloropsis oculata resources has certain limiting factors, for example, pollution of enemy organisms such as plankton and the like seriously restricts the large-scale culture of nannochloropsis oculata, plankton can feed algae to influence the normal growth of nannochloropsis oculata, and even can completely eat nannochloropsis oculata within a short period of time to cause serious economic loss to culturists.

At present, an effective pollution control method is still lacking in the field of engineering culture of nannochloropsis. Most of the existing researches are focused on the pollution of protozoa in the outdoor culture of nannochloropsis oculata, and the researches mainly focus on the aspects of changing the pH, temperature and nitrogen source of the culture water body, such as reducing the reproduction rate of the protozoa by adding ammonium bicarbonate, temporarily inhibiting the protozoa from eating nannochloropsis oculata, and the like, by mainly researching the physicochemical culture conditions for controlling the harm of the protozoa. The method has a certain control effect on pollution in the culture of freshwater algae strains, but has little effect on controlling protozoan pollution in the engineering culture of nannochloropsis oculata due to the buffer capacity of seawater.

Based on this, breeding a nannochloropsis which can resist ingestion of protozoa becomes a problem to be solved urgently in the current stage of nannochloropsis engineering culture.

Disclosure of Invention

The invention provides a marine nannochloropsis LAMB204 for resisting disease ciliates and application thereof, aiming at the technical problems that the control effect of protozoan pollution in nannochloropsis engineering culture is not ideal, and nannochloropsis is easy to be eaten by protozoans.

In a first aspect, the invention provides a strain of anti-disease ciliate marine Nannochloropsis oceanica LAMB204, which is classified and named as marine Nannochloropsis oceanica, with the collection number of CGMCC NO.20713, the collection date of 10-23 days of 2020 year, the collection unit is the China general microbiological culture Collection center, the collection address is No. 3 of the West Lu No. 1 of the morning district in Beijing, and the postal code is as follows: 100101.

further, the inhibition rate of marine nannochloropsis LAMB204 on the growth of ciliate population was 73.68%.

Further, the method for obtaining marine nannochloropsis LAMB204 comprises the following steps:

culturing mutagenic strain of wild marine nannochloropsis oceanica to 3 × 10⁷～5×10⁷Adding ciliates after cells/mL, then continuing culturing until the density of the ciliates reaches a relatively stable state, and screening the marine nannochloropsis oceanica strains with disease and pest resistance according to the density of the ciliates.

Further, the mutation treatment method of the wild marine nannochloropsis comprises the following steps:

adding ethyl methanesulfonate into the algae solution to obtain a reaction system, placing the reaction system in a shaking table to shake and incubate under the condition of room temperature and darkness, then stopping the reaction by using a sodium thiosulfate solution, centrifugally collecting the algae solution, taking the supernatant, and washing the supernatant by using the sodium thiosulfate solution again.

Further, the method for culturing marine nannochloropsis LAMB204 comprises the following steps:

inoculating marine Nannochloropsis oculata LAMB204 into f/2 culture medium with light intensity of 100 μmol phos/s/m²irradiation。

Further, the light cycle of the marine nannochloropsis LAMB204 is 12: 12.

further, the culture temperature of the marine nannochloropsis LAMB204 is 20-25 ℃.

In a second aspect, the invention provides a use of marine nannochloropsis LAMB204 for inhibiting growth of ciliate population.

The beneficial effect of the invention is that,

the marine nannochloropsis LAMB204 provided by the invention can inhibit growth of ciliate population, and compared with wild strains, the inhibition effect is greatly improved.

Drawings

In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.

FIG. 1 is a graph comparing the inhibitory effect of wild strains and mutant strains on growth of ciliate populations.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1EMS mutagenesis treatment

Before EMS mutagenesis treatment is carried out, separation and purification treatment are carried out on wild marine nannochloropsis, and the method comprises the following steps:

(1) taking 50mL of the mixture with the density of 10⁷centrifuging and collecting cell/mL algae solution at 3000rpm for 10min, washing twice with fresh f/2 seawater culture medium, and suspending in 30mL fresh culture medium;

(2) respectively adding 200nL of the culture medium into a first row of a 96-well plate, and then gradually diluting the culture medium by using a fresh f/2 seawater culture medium according to the dilution ratio of 10 times;

(3) after dilution, observing a 96-well plate by using an inverted microscope, and marking the wells with only single cells in the wells;

(4) placing the 96-well plate in a light incubator for culture, and observing the change of the number of the algae seeds by using a microscope every day;

(5) after 5-7 days, the color of the algae liquid appears in the pore plate, the algae liquid grown from the single cells is diluted by 100 times and coated in a sterilized f/2 solid flat plate, and then the flat plate is placed in an illumination incubator for cultivation;

(6) and after the monoclonal algae colony grows out, inoculating the algae colony into a 50mL conical flask for amplification culture, and preserving the seeds for subsequent mutagenesis experiments, wherein the strain is named nannochloropsis B.

EMS mutagenesis treatment comprises the following steps:

(1) culturing nannochloropsis B in f/2 seawater culture medium, wherein each 1L seawater in the f/2 seawater culture medium contains the following substances:

NaNO₃：75mg、NaH₂PO₄·H₂O：5mg、Na₂SiO₃·9H₂O：20mg、Na₂EDTA：4.36mg、FeCl₃·6H₂O：3.16mg、CuSO₄·5H₂O：0.01mg、ZnSO₄·7H₂O：0.023mg、CoCl₂·6H₂O：0.012mg、MnCl₂·4H₂O：0.18mg、Na₂MoO₄·2H₂o: 0.07mg, vitamin B1: 0.1mg, vitamin B12: 0.5. mu.g, biotin: 0.5 mu g;

the culture conditions were as follows:

the temperature is 23 +/-1 ℃, and the light intensity is 100 mu mol phos/s/m²irradiation, photoperiod 12: 12, salinity of 30 per mill and pH of 7.8;

(2) when the nannochloropsis B grows to the middle exponential phase, carrying out mutagenesis, firstly, centrifugally collecting 10mL of algae solution, then washing twice by using a fresh f/2 seawater culture medium, and suspending in 1mL of f/2 seawater culture medium;

(3) counting with a blood cell counter plate, transferring 10⁶Putting the algae cells into a 1.5mL centrifuge tube to make the final concentration of the algae cells be 10⁶cells/mL；

(4) Adding EMS mother liquor to enable the final concentration of EMS to reach 1mol/L, placing the whole reaction system in a shaking table, and performing shake incubation for 1h under the condition of room temperature darkness at the rotating speed of 300 rpm;

(5) terminating the reaction by using a 10% DMSO (sodium thiosulfate) solution, centrifuging at 5000rpm for 10min to collect algae liquid, taking the supernatant, washing again by using a sodium thiosulfate solution, and repeating for 2 times to remove residual EMS;

(6) resuspending the algae cells in f/2 seawater culture medium, overnight at room temperature in dark condition, and coating the mutagenized algae cells on f/2 seawater solid culture medium the next day;

f/2 seawater solid culture medium prepared from seawater and NaNO₃Mixing the following substances in 1L of seawater:

NaNO₃：75mg、NaH₂PO₄·H₂O：5mg、Na₂SiO₃·9H₂O：20mg、Na₂EDTA：4.36mg、FeCl₃·6H₂O：3.16mg、CuSO₄·5H₂O：0.01mg、ZnSO₄·7H₂O：0.023mg、CoCl₂·6H₂O：0.012mg、MnCl₂·4H₂O：0.18mg、Na₂MoO₄·2H₂o: 0.07mg, vitamin B1: 0.1mg, vitamin B12: 0.5. mu.g, biotin: 0.5 μ g, agar: 10g of a mixture;

(7) growing monoclonal algae colonies on a solid plate 18 days after the algae cells which are not dead in the mutagenesis reaction, randomly selecting the monoclonal algae colonies, putting the monoclonal algae colonies into a 50mL conical flask, and carrying out amplification culture.

Example 2 screening of Marine Nannochloropsis against disease ciliates

(1) After the mutagenic strain and the wild strain obtained in example 1 were cultured in f/2 seawater medium to the initial exponential phase, the cells were separated on a clean bench in a volume ratio of 1: 10 into a 500mL conical flask, placing the algae liquid into a light culture chamber for culture after inoculation, wherein the culture temperature is 23 +/-1 ℃, and the light intensity is 100 mu molphostons/s/m²irradiation, photoperiod 12: 12, culturing for 20 days until the density of algae reaches 3 × 10⁷cells/mL；

(2) Screening is carried out in a 24-hole plate, three repeats are respectively arranged on a mutagenic strain and a wild strain, the fanshaped wandering bug is respectively added into algae liquid of the mutagenic strain and the wild strain, 20 fanshaped wandering bugs are arranged in each hole, then the 24-hole plate is placed under 22 ℃ for illumination culture, and the marine nannochloropsis with good disease and pest resistance performance is determined and named as LAMB 204.

As shown in FIG. 1, in the wild marine Nannochloropsis oceanica-fed ad culture medium, the density of the ad increased from the initial 10ind/mL to 76ind/mL at the end of the index after 60 hours and finally maintained at an average density of 52 ind/mL. Compared with wild marine nannochloropsis, marine nannochloropsis LAMB204 has obvious inhibition effect on the growth of the paragonimus population, the paragonimus exponential growth speed is slower, the time is shorter, the maximum population density is 20ind/mL, and finally the average density is stabilized at 12 ind/mL.

Example 3 culture of Nannochloropsis oceanica LAMB204

Inoculating marine Nannochloropsis oculata LAMB204 in f/2 seawater culture medium at 23 + -1 deg.C and light intensity of 100 μmol phosns/s/m²irradiation, photoperiod 12: 12, salinity of 30 per mill and pH of 7.8;

f/2 seawater culture medium contains the following substances in each 1L of seawater:

NaNO₃：75mg、NaH₂PO₄·H₂O：5mg、Na₂SiO₃·9H₂O：20mg、Na₂EDTA：4.36mg、FeCl₃·6H₂O：3.16mg、CuSO₄·5H₂O：0.01mg、ZnSO₄·7H₂O：0.023mg、CoCl₂·6H₂O：0.012mg、MnCl₂·4H₂O：0.18mg、Na₂MoO₄·2H₂o: 0.07mg, vitamin B1: 0.1mg, vitamin B12: 0.5. mu.g, biotin: 0.5. mu.g.

Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention.

Claims

1. A marine Nannochloropsis oceanica LAMB204 resisting the disease ciliates is characterized in that the marine Nannochloropsis oceanica LAMB204 is classified and named as Nannochloropsis oceanica with the preservation number of CGMCC NO.20713, the preservation date is 10-23 days of 2020, the preservation unit is the common microorganism center of China general microbiological culture Collection center, the preservation address is No. 3 of North West Lu No. 1 of the morning area in Beijing, and the postal code is as follows: 100101.

2. use of the marine Nannochloropsis Lamb204 of claim 1 for inhibiting the growth of a swim worm population.