CN119242552A - Methods of using exogenous osmotic regulating molecules to assist adaptive evolution of microalgae to resist salt stress - Google Patents
Methods of using exogenous osmotic regulating molecules to assist adaptive evolution of microalgae to resist salt stress Download PDFInfo
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Abstract
本发明涉及微藻育种技术领域，并公开了外源渗透调节分子辅助微藻抗盐胁迫适应性进化的方法，包括以下步骤：选用微藻作为初始育种材料；准备含有低浓度外源渗透调节剂分子的培养基；将微藻接种至培养基中，在适宜条件下进行培养，培养过程中，逐步增加盐胁迫压力，促进微藻的适应性进化；筛选耐盐性藻种；通过多次传代和筛选，获得纯度高、耐盐性强的微藻新品种。本发明采用上述的外源渗透调节分子辅助微藻抗盐胁迫适应性进化的方法，通过外源渗透调节分子的辅助，进行微藻抗盐胁迫适应性进化育种，显著提高了微藻的耐盐性，同时缩短了育种周期，提高了育种效率，为耐盐新品种微藻的开发奠定基础。
The present invention relates to the technical field of microalgae breeding, and discloses a method for exogenous osmotic regulating molecules to assist the adaptive evolution of microalgae to resist salt stress, comprising the following steps: selecting microalgae as initial breeding materials; preparing a culture medium containing low-concentration exogenous osmotic regulating molecules; inoculating the microalgae into the culture medium, culturing under suitable conditions, gradually increasing the salt stress pressure during the culturing process, and promoting the adaptive evolution of the microalgae; screening salt-tolerant algae species; obtaining new microalgae varieties with high purity and strong salt tolerance through multiple passages and screening. The present invention adopts the above-mentioned method for assisting the adaptive evolution of microalgae to resist salt stress by exogenous osmotic regulating molecules, and carries out adaptive evolution breeding of microalgae to resist salt stress with the assistance of exogenous osmotic regulating molecules, which significantly improves the salt tolerance of the microalgae, shortens the breeding cycle, improves the breeding efficiency, and lays a foundation for the development of new salt-tolerant microalgae varieties.
Description
Technical Field
The invention relates to the technical field of microalgae breeding, in particular to a method for assisting adaptive evolution of microalgae against salt stress by using exogenous osmotic adjustment molecules.
Background
Microalgae are taken as a microorganism with high-efficiency photosynthesis capability, and have increasingly prominent application value in various industries such as food, cosmetic, medicine and the like. The conventional microalgae cultivation method mainly depends on fresh water resources, which not only increases cultivation cost, but also can be afflicted by water resource shortage and biological pollution. The culture cost can be obviously reduced and the consumption of fresh water resources can be reduced by utilizing the seawater to prepare the culture medium for microalgae culture. However, the high salinity in seawater constitutes a serious stress on microalgae growth and metabolism, limiting its immediate use in seawater environments. Therefore, domesticating new fresh water food resource algae such as spirulina, chlorella, haematococcus pluvialis and the like to endure higher salinity has important significance for promoting sustainable development of algae industry.
The traditional adaptive screening method, such as natural selection and gradual adaptation method, can improve the salt tolerance of microalgae to a certain extent, but has long period and low efficiency, and is difficult to meet the requirement of rapid breeding of new varieties. In addition, mutation breeding is capable of introducing random mutation by a physical or chemical method, but the stability and genetic risk of mutants are difficult to control, and the screening process is cumbersome. These methods involve prolonged acclimatization and adaptation, and the screened algal species often contain adverse variations, requiring further purification. However, the purification selection process is also challenging, requiring accurate screening of good algal species with stable salt tolerance in a large population of algal strains, which is time consuming and labor intensive, and the results are susceptible to fluctuations in environmental conditions.
In order to overcome the limitations of the method, the invention provides an adaptive evolution breeding method for assisting microalgae to resist salt stress by using exogenous osmotic adjustment molecules. When the candidate algae species do not generate enough resistance, the method increases the salt resistance of the candidate algae species in a short period by adding exogenous betaine, nitric oxide and other osmotic adjusting molecules, thereby increasing the probability of generating and accumulating enough mutation. The method has the advantages that the rapid regulation effect of exogenous molecules and the long-term accumulation effect of adaptive evolution are combined, the efficient screening breeding based on the adaptive evolution can be realized, and a new thought and method are provided for the cultivation of new microalgae salt-tolerant varieties.
Disclosure of Invention
The invention aims to provide a method for assisting the adaptive evolution of microalgae against salt stress by using exogenous osmotic adjustment molecules, which is used for carrying out adaptive evolution breeding of microalgae against salt stress by using the assistance of exogenous osmotic adjustment molecules, so that the salt tolerance of the microalgae is remarkably improved, the breeding period is shortened, the breeding efficiency is improved, and a foundation is laid for the development of new species of microalgae with salt tolerance.
In order to achieve the above purpose, the invention provides a method for assisting the adaptive evolution of microalgae against salt stress by using exogenous osmotic adjustment molecules, which comprises the following steps:
a. selecting initial breeding material, namely selecting microalgae as the initial breeding material to ensure clear sources and high purity of algae seeds;
b. Preparing a culture medium, wherein the culture medium contains low-concentration exogenous osmotic regulator molecules, and the exogenous osmotic regulator molecules comprise betaine and/or sodium nitroprusside;
c. inoculating and culturing, namely inoculating the microalgae selected in the step a into the culture medium in the step b, and culturing for 2-4 weeks under proper conditions to ensure that the algae seeds can grow and reproduce normally;
d. Gradually eliminating microalgae cells sensitive to stress along with growth of microalgae and consumption of exogenous osmotic regulator molecules in the culture process, gradually enriching and amplifying the microalgae cells with strong tolerance, taking old culture solution, re-inoculating the old culture solution to a new culture medium every 2-3 weeks, gradually increasing the salinity in the new culture medium, increasing the salt stress pressure, and promoting adaptive evolution of the microalgae;
e. screening salt-tolerant algae, namely observing and recording the growth condition of microalgae under salt stress, taking the OD value, growth rate and chlorophyll content of biomass as screening standards, and selecting algae with good growth and strong salt tolerance as candidate salt-tolerant algae after the biomass OD value, growth rate and chlorophyll content reach the screening standards;
f. And E, purifying the candidate salt-tolerant algae seeds screened in the step E, and carrying out passage and screening for a plurality of times to ensure that the number of the purified algae seeds with the SNP mutation rate of more than 95 percent is close to the number of the original algae seeds, thereby obtaining a new microalgae variety with high purity and strong salt tolerance.
Further, in step b, the medium comprises 50-200. Mu.M betaine or 25-100. Mu.M sodium nitroprusside or 50-200. Mu.M betaine and 25-100. Mu.M sodium nitroprusside.
Further, in the step C, the microalgae comprise freshwater spirulina, chlorella and haematococcus pluvialis, wherein the culture temperature of the freshwater spirulina is 25-30 ℃, the illumination intensity is 3000-6000 lux, the pH value is 8.5-11.5, the culture temperature of the chlorella is 20-25 ℃, the illumination intensity is 5000-10000 lux, the pH value is 6.5-8.0, and the culture temperature of the haematococcus pluvialis is 22-28 ℃, the illumination intensity is 4000-8000 lux, and the pH value is 7.0-8.5.
Further, in step d, the maximum salinity is 30-35 g/L, and the salinity is increased by 5-10 g/L each time.
Further, in step e, the screening criteria further comprise growth rate, chlorophyll content, and detection and measurement by microscopic observation and spectroscopic analysis.
In the step f, the algae seed purification process is realized by the techniques of gradient dilution, plate separation and single cell operation, the candidate salt-tolerant algae seed is subjected to subculture or seed preservation under proper conditions, and then the subcultured algae seed is subjected to whole genome sequencing analysis by the molecular biology technique, so that the purified algae seed is ensured to be cultured and separated.
Further, the number of Single Nucleotide Polymorphism (SNP) sites of the purified algae seeds is close to the Single Nucleotide Polymorphism (SNP) level of the wild-type pure strain before domestication, and meets the requirements.
The invention also provides application of the method in screening new varieties of salt-tolerant microalgae.
The method for assisting the adaptive evolution of microalgae to salt stress by using the exogenous osmotic adjustment molecule has the advantages and positive effects that:
1. According to the invention, by adding exogenous betaine, nitric oxide and other osmotic adjusting molecules, the salt resistance of the osmotic adjusting molecules is improved in a short period, so that the probability of occurrence and accumulation of enough mutation of the osmotic adjusting molecules is increased. The method combines the rapid regulation effect of exogenous molecules and the long-term accumulation effect of adaptive evolution, can realize high-efficiency screening breeding based on the adaptive evolution, and provides a new thought and method for cultivating new microalgae salt-tolerant varieties.
2. The invention realizes adaptive evolution breeding of microalgae against salt stress by the assistance of exogenous osmotic adjusting molecules, obviously improves the salt tolerance of the microalgae, shortens the breeding period and improves the breeding efficiency. Meanwhile, the screened new variety has better growth performance and biomass accumulation capacity in a high-salt environment, and provides a new solution for the application of microalgae in environments such as saline-alkali soil and the like.
The technical scheme of the invention is further described in detail through the drawings and the embodiments.
Drawings
FIG. 1 shows OD 560 of spirulina in the present invention.
Detailed Description
The technical scheme of the invention is further described below through the attached drawings and the embodiments.
Unless defined otherwise, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.
The instruments, devices, reagents used in the present invention are commercially available as usual unless otherwise defined.
Step 1, selecting initial breeding materials, namely selecting freshwater spirulina, chlorella and haematococcus pluvialis as the initial breeding materials, and ensuring clear sources and high purity of algae seeds.
Step 2, preparing a culture medium containing a low concentration of exogenous osmotic regulator molecules, wherein 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 mu M, and the concentration range of sodium nitroprusside is set to 25 to 100 mu M, so that the accuracy of the molecular concentration of the osmotic regulator in the culture medium is ensured. The pH value of the culture medium is adjusted to 8.5-11.5, the illumination intensity is set to 4000-6000 lux, and the temperature is controlled to 25-30 ℃.
And 3, inoculating and culturing, namely inoculating the microalgae selected in the step 1 onto the culture medium in the step 2, and culturing under the conditions of proper temperature, illumination, pH and the like to ensure that the algae can grow and reproduce normally. The specific culture conditions are different according to different algae species, the culture temperature of the freshwater spirulina is 25-30 ℃, the illumination intensity is 3000-6000 lux, the pH is 8.5-11.5, the culture temperature of the freshwater chlorella is 20-25 ℃, the illumination intensity is 5000-10000 lux, the pH is 6.5-8.0, the culture temperature of the freshwater haematococcus pluvialis is 22-28 ℃, and the illumination intensity is 4000-8000 lux, and the pH is 7.0-8.5.
And step 4, increasing the salt stress pressure gradually along with the growth of microalgae and the consumption of exogenous osmotic regulator molecules in the culture process. By gradually increasing the salinity in the culture medium, the salinity change in the natural environment is simulated, and the adaptive evolution of microalgae is promoted. The salinity increase range was set to 0- (30-35) g/L, with 5 g/L sodium chloride increase each time.
And 5, screening salt-tolerant algae, namely observing and recording the growth condition of microalgae under salt stress, and comprehensively evaluating by measuring a biomass OD 560 value as a screening standard and combining with other biological indexes (such as growth rate, chlorophyll content and the like). After the screening standard (similar to the index of a wild algae strain) is reached, selecting algae species with good growth and strong salt tolerance as candidate salt tolerance algae species.
And 6, purifying the screened candidate salt-tolerant algae, namely purifying the screened candidate salt-tolerant algae, carrying out mutation rate detection by combining molecular biology technologies (such as SNP analysis, gene sequencing and the like) through multiple passages and screening, ensuring that the mutation rate of the purified algae meets the requirement (the number of specific SNP reaches more than 70% of that of the original algae), and finally obtaining a new microalgae variety with high purity and strong salt tolerance.
Example 1
Adding exogenous osmotic adjusting molecular betaine to Zarouk culture medium to cultivate freshwater spirulina, gradually increasing salt stress pressure, increasing from 0 g/L,10 g/L,15 g/L,20 g/L,25 g/L to 30 g/L sodium chloride, and simultaneously adding 150 mu M betaine into the culture medium each time to cultivate. The initial concentration of algae cells is controlled at 0.45 g/L, the illumination culture temperature is 28 ℃, the illumination intensity is 5500 lux, and aeration culture is carried out. After the cultivation, the OD 560 of the freshwater spirulina was 2.01.
Example 2
Adding exogenous osmotic regulator sodium nitroprusside into Zarouk culture medium to cultivate freshwater spirulina, gradually increasing salt stress pressure, increasing from 0g/L, 10g/L, 15 g/L,20 g/L,25 g/L to 30 g/L sodium chloride, and simultaneously adding 50 mu M nitroprusside into the culture medium each time to cultivate. The initial concentration of algae cells is controlled at 0.45 g/L, the illumination culture temperature is 28 ℃, the illumination intensity is 5500 lux, and aeration culture is carried out. After the domestication culture, the OD 560 of the freshwater spirulina reaches 1.90.
Example 3
Adding exogenous osmotic regulator sodium nitroprusside into Zarouk culture medium to cultivate freshwater spirulina, gradually increasing salt stress pressure, increasing from 0g/L, 10g/L, 15 g/L,20 g/L,25 g/L to 30 g/L sodium chloride, and simultaneously adding 50 mu M nitroprusside into the culture medium each time to cultivate. The initial concentration of algae cells is controlled at 0.45 g/L, the illumination culture temperature is 28 ℃, the illumination intensity is 5500 lux, and aeration culture is carried out. After the domestication culture, the OD 560 of the freshwater spirulina reaches 1.90.
Comparative example 1
The freshwater spirulina is cultivated by adopting a conventional cultivation mode (namely, no exogenous osmotic adjusting molecule is added to Zarouk culture medium), and the salt stress pressure is gradually increased from 0 g/L,10 g/L,15 g/L,20 g/L and 25 g/L to 30 g/L of sodium chloride. The initial concentration of algae cells is controlled at 0.45 g/L, the illumination culture temperature is 28 ℃, the illumination intensity is 5500 lux, and aeration culture is carried out. After the domestication culture, the OD 560 of the freshwater spirulina reaches 0.79.
Fig. 1 shows the change of OD 560 value of salt-tolerant spirulina obtained in examples 1, 2 and 3 and comparative example 1 under 30 g/L sodium chloride, and it can be seen from fig. 1 that the addition of exogenous osmotic adjustment molecule can regulate adaptive evolution of microalgae, and compared with betaine or sodium nitroprusside alone, the comprehensive use of betaine and sodium nitroprusside further improves salt tolerance of spirulina. The optimal conditions for the use of betaine and sodium nitroprusside together are 150. Mu.M betaine and 50. Mu.M sodium nitroprusside. The number of SNP mutation rates of the optimal salt-tolerant spirulina after purification is more than 95 percent and is consistent with the level of the original spirulina.
Therefore, the method for assisting the adaptive evolution of the microalgae to resist the salt stress by adopting the exogenous osmotic adjustment molecule disclosed by the invention has the advantages that the adaptive evolution breeding of the microalgae to resist the salt stress is carried out by assisting the exogenous osmotic adjustment molecule, the salt tolerance of the microalgae is obviously improved, the breeding period is shortened, the breeding efficiency is improved, and a foundation is laid for the development of new species of microalgae with the salt tolerance.
It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted by the same, and the modified or substituted technical solution may not deviate from the spirit and scope of the technical solution of the present invention.
Claims (8)
1. The method for assisting the adaptive evolution of microalgae against salt stress by using exogenous osmotic adjustment molecules is characterized by comprising the following steps:
Step a, selecting initial breeding materials, namely selecting microalgae as the initial breeding materials, and ensuring clear sources and high purity of algae seeds;
Step b, preparing a culture medium, wherein the culture medium contains low-concentration exogenous osmotic regulator molecules, and the exogenous osmotic regulator molecules comprise betaine and/or sodium nitroprusside;
C, inoculating and culturing, namely inoculating the microalgae selected in the step a into the culture medium in the step b, and culturing for 2-4 weeks under proper conditions to ensure that the algae seeds can grow and reproduce normally;
Step d, increasing the salt stress pressure, namely gradually eliminating microalgae cells sensitive to stress along with growth of the microalgae and consumption of exogenous osmotic regulator molecules in the culture process, gradually enriching and amplifying the microalgae cells with strong tolerance, taking old culture solution to re-inoculate the new culture medium every 2 to 3 weeks, gradually increasing the salinity in the new culture medium, increasing the salt stress pressure and promoting adaptive evolution of the microalgae;
Step e, screening salt-tolerant algae, namely observing and recording the growth condition of microalgae under salt stress, taking the OD value, growth rate and chlorophyll content of biomass as screening standards, and selecting algae with good growth and strong salt tolerance as candidate salt-tolerant algae after the biomass OD value, growth rate and chlorophyll content reach the screening standards;
And f, purifying the algae seeds, namely purifying the candidate salt-tolerant algae seeds screened in the step e, and carrying out passage and screening for multiple times to obtain a new microalgae variety with high purity and strong salt tolerance.
2. The method according to claim 1, wherein in step b, the medium comprises 50-200. Mu.M betaine or 25-100. Mu.M sodium nitroprusside or 50-200. Mu.M betaine and 25-100. Mu.M sodium nitroprusside.
3. The method according to claim 1, wherein in the step C, the microalgae comprise freshwater spirulina, chlorella and haematococcus pluvialis, the culture temperature of the freshwater spirulina is 25-30 ℃, the illumination intensity is 3000-6000 lux, the pH is 8.5-11.5, the culture temperature of the chlorella is 20-25 ℃, the illumination intensity is 5000-10000 lux, the pH is 6.5-8.0, the culture temperature of the haematococcus pluvialis is 22-28 ℃, and the illumination intensity is 4000-8000 lux, and the pH is 7.0-8.5.
4. The method of claim 1, wherein 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 of claim 1, wherein in step e, the screening criteria further comprise growth rate, chlorophyll content, and detection and measurement are performed by microscopic observation and spectroscopic analysis.
6. The method according to claim 1, wherein in the step f, the purification process of the algae seeds is realized by the techniques of gradient dilution, plate separation and single cell operation, the candidate salt-tolerant algae seeds are subjected to subculture or seed preservation under proper conditions, and then the subcultured algae seeds are subjected to whole genome sequencing analysis by the molecular biology technique, so that the purified algae seeds are ensured to be subjected to culture separation.
7. The method of claim 6, wherein the number of single nucleotide polymorphism SNP loci of greater than 95% of the purified algal species corresponds to the single nucleotide polymorphism SNP level of the wild type inbred prior to acclimation.
8. Use of the method according to any one of claims 1-7 for screening new species of salt tolerant microalgae.
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