Method for assisting salt stress resistant adaptive evolution of microalgae by exogenous osmotic regulation molecules

Abstract

The invention relates to the technical field of microalgae breeding, and discloses a method for assisting salt stress resistant adaptive evolution of microalgae by exogenous osmoregulation molecules, which comprises the following steps: selecting microalgae as an initial breeding material; preparing a culture medium containing low-concentration exogenous osmotic regulator molecules; the method comprises the following steps: inoculating microalgae into a culture medium, culturing under proper conditions, gradually increasing salt stress pressure in the culture process, and promoting adaptive evolution of the microalgae; screening salt-tolerant algae species; through multiple passage and screening, a new microalgae variety with high purity and strong salt tolerance is obtained. According to the method for assisting the salt-stress-resistant adaptive evolution of the microalgae by the exogenous osmoregulation molecules, the salt-stress-resistant adaptive evolution breeding of the microalgae is carried out under the assistance of the exogenous osmoregulation molecules, so that the salt tolerance of the microalgae is remarkably improved, meanwhile, the breeding period is shortened, the breeding efficiency is improved, and the breeding cost is reduced. And a foundation is laid for development of new salt-resistant microalgae.

Description

Technical Field

[0001] The invention relates to the technical field of microalgae breeding, in particular to a method for exogenous osmotic regulating molecules to assist the adaptive evolution of microalgae to resist salt stress. Background Art

[0002] As a type of microorganism with efficient photosynthesis ability, the application value of microalgae in multiple industries such as food, beauty and medicine is becoming increasingly prominent. Traditional microalgae cultivation methods mainly rely on freshwater resources, which not only increases the cultivation cost, but may also be plagued by water shortages and biological pollution. Using seawater to prepare culture medium for microalgae cultivation can significantly reduce the cultivation cost and reduce the consumption of freshwater resources. However, the high salinity in seawater poses a serious threat to the growth and metabolism of microalgae, limiting its direct application in seawater environments. Therefore, domesticating new freshwater food resource algae species such as Spirulina, Chlorella, and Haematococcus pluvialis to tolerate higher salinity is of great significance for promoting the sustainable development of the algae industry.

[0003] Traditional adaptive screening methods, such as natural selection and stepwise adaptation, can improve the salt tolerance of microalgae to a certain extent, but they are often long-term and inefficient, and it is difficult to meet the needs of rapid breeding of new varieties. In addition, although mutagenesis breeding can introduce random mutations through physical or chemical methods, the stability and genetic risks of mutants are difficult to control, and the screening process is cumbersome. These methods involve long-term domestication and adaptation, and the screened algae species often contain adverse mutations and need to be further purified. However, the purification selection process is also full of challenges. It requires accurate screening of excellent algae species with stable salt tolerance from a large population of algae strains. This process is not only time-consuming and labor-intensive, but the results are also easily affected by fluctuations in environmental conditions.

[0004] In order to overcome the limitations of the above methods, the present invention proposes an adaptive evolution breeding method for salt stress tolerance of microalgae assisted by exogenous osmotic regulatory molecules. This method improves the salt resistance of candidate algae in a short period of time by adding exogenous osmotic regulatory molecules such as betaine and nitric oxide when the candidate algae species have not yet developed sufficient resistance, thereby increasing the probability of occurrence and accumulation of sufficient mutations. The advantage of this method is that it combines the rapid regulatory effect of exogenous molecules with the long-term cumulative effect of adaptive evolution, and can achieve efficient screening and breeding based on adaptive evolution, providing new ideas and methods for the cultivation of new salt-tolerant microalgae varieties. Summary of the invention

[0005] The purpose of the present invention is to provide a method for the adaptive evolution of microalgae to resist salt stress assisted by exogenous osmotic regulating molecules. With the assistance of exogenous osmotic regulating molecules, the adaptive evolution breeding of microalgae to resist salt stress is carried out, which significantly improves the salt tolerance of microalgae, shortens the breeding cycle, improves the breeding efficiency, and lays a foundation for the development of new salt-tolerant microalgae varieties.

[0006] To achieve the above object, the present invention provides a method for using exogenous osmotic regulating molecules to assist the adaptive evolution of microalgae to resist salt stress, comprising the following steps: a. Select initial breeding materials: Select microalgae as the initial breeding materials to ensure that the source of the algae is clear and the purity is high; b. preparing a culture medium: the culture medium contains a low concentration of exogenous osmotic regulator molecules, wherein the exogenous osmotic regulator molecules include betaine and / or sodium nitroprusside; c. Inoculation and cultivation: The microalgae selected in step a are inoculated into the culture medium in step b, and cultured under suitable conditions for 2-4 weeks to ensure that the algae can grow and reproduce normally; d. Increasing salt stress pressure: During the culture process, as the microalgae grow and consume exogenous osmotic regulator molecules, the microalgae cells that are sensitive to stress are gradually eliminated, and the microalgae cells with strong tolerance are gradually enriched and expanded. Every 2-3 weeks, the old culture medium is taken and re-inoculated into the new culture medium, and the salinity in the new culture medium is gradually increased to increase the salt stress pressure and promote the adaptive evolution of the microalgae; e. Screening of salt-tolerant algae species: Observe and record the growth of microalgae under salt stress, use biomass OD value, growth rate and chlorophyll content as screening criteria, and select algae species with good growth and strong salt tolerance as candidate salt-tolerant algae species after meeting the screening criteria; f. Algae purification: The candidate salt-tolerant algae selected in step e are purified, and through multiple passages and screening, the number of SNP mutation rates of the purified algae is ensured to be greater than 95% and close to that of the original algae, so as to obtain new microalgae varieties with high purity and strong salt tolerance.

[0007] Furthermore, in step b, the culture medium includes 50-200 μM betaine or 25-100 μM sodium nitroprusside or 50-200 μM betaine and 25-100 μM sodium nitroprusside.

[0008] Further, in step c, the microalgae include freshwater spirulina, chlorella, and haematococcus pluvialis; the culture temperature of freshwater spirulina is 25-30°C, the light intensity is 3000-6000 lux, and the pH is 8.5-11.5; the culture temperature of chlorella is 20-25°C, the light intensity is 5000-10000 lux, and the pH is 6.5-8.0; the culture temperature of haematococcus pluvialis is 22-28°C, the light intensity is 4000-8000 lux, and the pH is 7.0-8.5.

[0009] Further, in step d, the maximum salinity is 30-35 g / L, and the salinity is increased by 5-10 g / L each time.

[0010] Furthermore, in step e, the screening criteria also include growth rate and chlorophyll content, which are detected and measured through microscopic observation and spectral analysis.

[0011] Furthermore, in step f, the process of purifying algae species is achieved through gradient dilution, plate separation, and single-cell operation techniques; the candidate salt-tolerant algae species are subcultured or preserved under suitable conditions, and then the whole genome sequencing analysis of the subcultured algae species is performed through molecular biology techniques to ensure that the purified algae species have completed culture and separation.

[0012] Furthermore, the number of single nucleotide polymorphism (SNP) sites in the purified algae species was close to the single nucleotide polymorphism (SNP) level of the wild-type pure strain before domestication, which met the requirements.

[0013] The present invention also provides application of the above method in screening new varieties of salt-tolerant microalgae.

[0014] The advantages and positive effects of the method of the present invention for assisting the adaptive evolution of microalgae to resist salt stress with exogenous osmotic regulating molecules are: 1. The present invention improves salt resistance in a short period of time by adding exogenous osmotic regulatory molecules such as betaine and nitric oxide, thereby increasing the probability of occurrence and accumulation of sufficient mutations. This method combines the rapid regulatory effect of exogenous molecules with the long-term cumulative effect of adaptive evolution, and can achieve efficient screening and breeding based on adaptive evolution, providing new ideas and methods for the cultivation of new salt-tolerant varieties of microalgae.

[0015] 2. The present invention achieves adaptive evolution breeding of microalgae to resist salt stress with the assistance of exogenous osmotic regulating molecules, significantly improves the salt tolerance of microalgae, shortens the breeding cycle, and improves the breeding efficiency. At the same time, the new varieties selected have better growth performance and biomass accumulation capacity in high-salt environments, providing a new solution for the application of microalgae in saline-alkali land and other environments.

[0016] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the OD of freshwater Spirulina in the embodiment of the present invention 560 value. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0019] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0020] Unless otherwise defined, the instruments, equipment and reagents used in the present invention are conventionally commercially available.

[0021] Step 1, selection of initial breeding materials: freshwater Spirulina, Chlorella, and Haematococcus pluvialis are selected as initial breeding materials to ensure that the source of the algae species is clear and the purity is high.

[0022] Step 2, medium preparation: Prepare medium containing low concentration of exogenous osmotic regulator molecules, where the exogenous osmotic regulator molecules are betaine and / or sodium nitroprusside (nitric oxide donor). The concentration range of betaine is set to 50 to 200 μM, and the concentration range of sodium nitroprusside is set to 25 to 100 μM to ensure the accuracy of the concentration of osmotic regulator molecules in the medium. Adjust the pH value of the medium to 8.5-11.5, set the light intensity to 4000-6000 lux, and control the temperature at 25-30°C.

[0023] Step 3, inoculation and cultivation: inoculate the microalgae selected in step 1 onto the culture medium in step 2, and cultivate under suitable temperature, light, pH and other conditions to ensure that the algae can grow and reproduce normally. The specific culture conditions vary according to the algae species. The culture temperature of freshwater Spirulina is 25-30℃, the light intensity is 3000-6000 lux, and the pH is 8.5-11.5; the culture temperature of freshwater Chlorella is 20-25℃, the light intensity is 5000-10000 lux, and the pH is 6.5-8.0; the culture temperature of freshwater Haematococcus pluvialis is 22-28℃, the light intensity is 4000-8000 lux, and the pH is 7.0-8.5.

[0024] Step 4, increasing salt stress pressure: During the culture process, as the microalgae grow and consume exogenous osmotic regulator molecules, the salt stress pressure is gradually increased. By gradually increasing the salinity in the culture medium, the salinity changes in the natural environment are simulated to promote the adaptive evolution of the microalgae. The salinity increase range is set to 0-(30-35) g / L, and 5 g / L of sodium chloride is added each time.

[0025] Step 5: Screening of salt-tolerant algae: Observe and record the growth of microalgae under salt stress, and measure the biomass OD 560 The value is used as the screening criterion, and a comprehensive evaluation is performed in combination with other biological indicators (such as growth rate, chlorophyll content, etc.). After reaching the screening criteria (similar to the indicators of wild-type algae strains), algae species with good growth and strong salt tolerance are selected as candidate salt-tolerant algae species.

[0026] Step 6, algae purification: purify the selected candidate salt-tolerant algae species, perform mutation rate detection through multiple passages and screening, and combine molecular biology techniques (such as SNP analysis, gene sequencing, etc.) to ensure that the mutation rate of the purified algae species meets the requirements (the number of unique SNPs reaches more than 70% of the original algae species), and finally obtain new microalgae varieties with high purity and strong salt tolerance.

[0027] Example 1 Add exogenous osmotic regulating molecule betaine to Zarrouk medium to cultivate freshwater Spirulina, and gradually increase the salt stress pressure from 0 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L to 30 g / L sodium chloride, and add 150 μM betaine to the culture medium each time for cultivation. The initial concentration of algal cells was controlled at 0.45 g / L, the light culture temperature was 28 ℃, the light intensity was 5500 lux, and aeration culture was performed. After the culture was completed, the OD of freshwater Spirulina 560 is 2.01.

[0028] Example 2 Add exogenous osmotic regulating molecule sodium nitroprusside to Zarrouk medium to cultivate freshwater Spirulina, gradually increase salt stress pressure from 0 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L to 30 g / L sodium chloride, and add 50 μM sodium nitroprusside to the culture medium each time for cultivation. The initial concentration of algal cells was controlled at 0.45 g / L, the light culture temperature was 28℃, the light intensity was 5500 lux, and aeration culture was performed. After the acclimation culture, the OD of freshwater Spirulina was 560 Reached 1.90.

[0029] Example 3 Add exogenous osmotic regulating molecule sodium nitroprusside to Zarrouk medium to cultivate freshwater Spirulina, gradually increase salt stress pressure from 0 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L to 30 g / L sodium chloride, and add 50 μM sodium nitroprusside to the culture medium each time for cultivation. The initial concentration of algal cells was controlled at 0.45 g / L, the light culture temperature was 28℃, the light intensity was 5500 lux, and aeration culture was performed. After the acclimation culture, the OD of freshwater Spirulina was 560 Reached 1.90.

[0030] Comparative Example 1 Freshwater Spirulina was cultivated using conventional culture methods (i.e., without adding any exogenous osmotic regulating molecules to Zarrouk medium), and the salt stress pressure was gradually increased from 0 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L to 30 g / L sodium chloride. The initial concentration of algal cells was controlled at 0.45 g / L, the light culture temperature was 28 ℃, the light intensity was 5500 lux, and aeration culture was performed. After the acclimation culture, the OD of freshwater Spirulina was 560 Reached 0.79.

[0031] Figure 1 OD of the salt-tolerant freshwater Spirulina obtained after acclimation and culture in Examples 1, 2, 3 and Comparative Example 1 under 30 g / L sodium chloride 560 The value changes due to Figure 1 It can be seen that the addition of exogenous osmotic regulating molecules can regulate the adaptive evolution of microalgae. Compared with the use of betaine or sodium nitroprusside alone, the combined use of betaine and sodium nitroprusside further improved the salt tolerance of freshwater Spirulina. At the same time, the optimal conditions for the use of betaine and sodium nitroprusside together are: 150 μM betaine and 50 μM sodium nitroprusside. The number of SNP mutation rates greater than 95% in the purified best salt-tolerant Spirulina is consistent with the level of the original algae species.

[0032] Therefore, the present invention adopts the above-mentioned method of using exogenous osmotic regulating molecules to assist the adaptive evolution of microalgae to resist salt stress. With the assistance of exogenous osmotic regulating molecules, the adaptive evolution breeding of microalgae to resist salt stress is carried out, which significantly improves the salt tolerance of microalgae, shortens the breeding cycle, improves the breeding efficiency, and lays the foundation for the development of new salt-tolerant microalgae varieties.

[0033] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for the adaptive evolution of microalgae to resist salt stress assisted by exogenous osmotic regulating molecules, characterized in that: The following steps are involved: Step a, selecting initial breeding materials: selecting microalgae as initial breeding materials to ensure that the source of the algae is clear and the purity is high; Step b, preparing a culture medium: the culture medium contains a low concentration of exogenous osmotic regulator molecules, wherein the exogenous osmotic regulator molecules include betaine and / or sodium nitroprusside; Step c, inoculation and cultivation: inoculating the microalgae selected in step a into the culture medium in step b, and culturing for 2-4 weeks under suitable conditions to ensure that the algae can grow and reproduce normally; Step d, increasing salt stress pressure: during the culture process, as the microalgae grow and consume exogenous osmotic regulator molecules, microalgae cells that are sensitive to stress are gradually eliminated, and microalgae cells with strong tolerance are gradually enriched and expanded. Every 2 to 3 weeks, the old culture solution is taken and re-inoculated into a new culture medium, and the salinity in the new culture medium is gradually increased to increase the salt stress pressure and promote the adaptive evolution of the microalgae; Step e, screening salt-tolerant algae species: observe and record the growth of microalgae under salt stress, use biomass OD value, growth rate and chlorophyll content as screening criteria, and after meeting the screening criteria, select algae species with good growth and strong salt tolerance as candidate salt-tolerant algae species; Step f, algae purification: purify the candidate salt-tolerant algae selected in step e, and obtain new microalgae varieties with high purity and strong salt tolerance through multiple passages and screening.

2. The method according to claim 1, characterized in that: In step b, the culture medium includes 50-200 μM betaine or 25-100 μM sodium nitroprusside or 50-200 μM betaine and 25-100 μM sodium nitroprusside.

3. The method according to claim 1, characterized in that: In step c, the microalgae include freshwater spirulina, chlorella, and haematococcus pluvialis; the culture temperature of freshwater spirulina is 25-30°C, the light intensity is 3000-6000 lux, and the pH is 8.5-11.5; the culture temperature of chlorella is 20-25°C, the light intensity is 5000-10000 lux, and the pH is 6.5-8.0; the culture temperature of haematococcus pluvialis is 22-28°C, the light intensity is 4000-8000 lux, and the pH is 7.0-8.

5.

4. The method according to claim 1, characterized in that: In step d, the maximum salinity is 30-35 g / L, and the salinity is increased by 5-10 g / L each time.

5. The method according to claim 1, characterized in that: In step e, the screening criteria also include growth rate and chlorophyll content, which are detected and measured through microscopic observation and spectral analysis.

6. The method according to claim 1, characterized in that In step f, the process of purifying algae species is achieved through gradient dilution, plate separation, and single-cell operation techniques; the candidate salt-tolerant algae species are subcultured or preserved under suitable conditions, and then the whole genome sequencing analysis of the subcultured algae species is performed through molecular biology techniques to ensure that the purified algae species have completed culture and separation.

7. The method according to claim 6, characterized in that More than 95% of the single nucleotide polymorphism (SNP) sites in the purified algae were consistent with the SNP levels of the wild-type pure strain before domestication.

8. Use of the method according to any one of claims 1 to 7 in screening new varieties of salt-tolerant microalgae.

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