A method for the ultrasonic-assisted ethyl methanesulfonate directed mutagenesis of spirulina

Abstract

The present application relates to the technical field of algal mutagenesis, in particular to a method for ultrasonic-assisted ethyl methanesulfonate (EMS) directed mutagenesis of Spirulina. The Spirulina algal liquid is subjected to ultrasonic treatment to obtain an ultrasonic-treated algal liquid; the ultrasonic-treated algal liquid is mixed with ethyl methanesulfonate and then subjected to directed mutagenesis treatment. According to the results of physiological indicators, PSII function parameters and multivariate analysis, 0.25% and 0.50% EMS treatment can be used as important conditions for screening candidate Spirulina strains under the ultrasonic-assisted EMS system, and are suitable for evaluating photosynthetic performance / antioxidant characteristics and morphological stability, respectively. The present application provides an experimental basis for subsequent identification of excellent algal strains and functional development. In the future, it is still necessary to combine genetic variation detection and long-term stability evaluation to further verify the genetic basis and application potential of the candidate algal strains.

Description

Technical Field

[0001] This invention relates to the field of algal mutagenesis technology, and in particular to a method for directional mutagenesis of Spirulina based on ultrasound-assisted ethyl methanesulfonate. Background Technology

[0002] Microalgae are a class of simple, widely distributed aquatic photosynthetic organisms characterized by high photosynthetic efficiency, short growth cycles, and strong accumulation capacity of bioactive substances. They have significant application potential in CO2 fixation, bioenergy development, functional food production, and the production of high-value-added natural products. Among them, Spirulina (… Spirulinaplatensis Spirulina is a prokaryotic photosynthetic organism with significant economic value. Rich in phycocyanin, polysaccharides, proteins, unsaturated fatty acids, and carotenoids, it holds broad application prospects in functional foods, biomedicine, feed additives, and bioenergy. However, its large-scale cultivation and industrial utilization still face challenges such as limited growth rate, unstable accumulation efficiency of functional substances, insufficient environmental adaptability, and a scarcity of superior algal strains, hindering its further development and utilization. Therefore, screening candidate algal strains with excellent growth performance, strong stress resistance, or outstanding functional traits through mutation breeding is of great significance for improving the utilization efficiency of Spirulina resources.

[0003] Mutagenesis breeding is an important method for microalgal germplasm innovation and the selection of superior algal strains. It mainly involves inducing genetic variation through physical, chemical, or biological methods to obtain candidate materials with stable traits during subsequent screening and passage. Ethyl methanesulfonate (EMS) is a commonly used chemical mutagen that can induce DNA base mismatches through alkylation, thereby inducing mutations and affecting cell growth, morphology, and metabolic characteristics. However, Spirulina has a filamentous structure, and the algal filaments are prone to entanglement and aggregation, which may lead to uneven contact between EMS and algal cells, thus affecting the effectiveness of the mutagenesis treatment. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for ultrasound-assisted ethyl methanesulfonate-induced mutagenesis of Spirulina. This invention establishes EMS concentration gradients of 0%, 0.25%, 0.50%, 0.75%, and 1.00% based on ultrasound pretreatment. After EMS mutagenesis, single algal filament isolation and purification, and continuous subculturing to the M8 generation, the growth characteristics, filament morphology, physiological and biochemical indicators, and PSII function differences of candidate algal strains under different treatments are systematically analyzed. This invention aims to clarify the effects of different EMS concentrations under ultrasound-assisted EMS mutagenesis on the phenotype and photosynthetic function of Spirulina M8 generation candidate strains, and to screen candidate strains with strong antioxidant capacity, stable morphology, or superior PSII function, providing experimental basis for Spirulina mutagenesis breeding and the creation of superior germplasm resources.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for ultrasound-assisted ethyl methanesulfonate-based directed mutagenesis of Spirulina, comprising the following steps: 1) The Spirulina algal solution was subjected to ultrasonic treatment to obtain ultrasonically treated algal solution; 2) The ultrasonically treated algal solution from step 1) is mixed with ethyl methanesulfonate and then subjected to targeted mutagenesis treatment; The final volume percentage of ethyl methanesulfonate after mixing the ultrasonically treated algal solution with ethyl methanesulfonate is 0.25~0.5%.

[0006] Preferably, the conditions for ultrasonic treatment in step 1) include: ice bath conditions, power of 400W, working time of 3 seconds, interval of 3 seconds, and total treatment time of 30 seconds.

[0007] Preferably, the OD of the spirulina solution in step 1) 680 The value is 0.17.

[0008] Preferably, the spirulina in step 1) includes Spirulina platensis.

[0009] Preferably, the conditions for the directed mutagenesis in step 2) include: directed mutagenesis for 40 minutes in the dark.

[0010] The present invention also provides the application of the method described in the above technical solution in increasing the length of Spirulina filaments.

[0011] The present invention also provides the application of the method described in the above technical solution in improving the accumulation of Spirulina biomass.

[0012] The present invention also provides the application of the method described in the above technical solution in increasing the content of soluble protein and soluble sugar in spirulina.

[0013] The present invention also provides the application of the method described in the above technical solution in improving the antioxidant capacity of spirulina.

[0014] The present invention also provides the application of the method described in the above technical solution in promoting the accumulation of chlorophyll a in Spirulina.

[0015] The beneficial effects of this invention are: This invention, based on an ultrasound-assisted EMS treatment, single algal filament isolation, and continuous subculturing system, systematically evaluated the differences in growth, morphology, physiological metabolism, and PSII function of M8 generation Spirulina candidate strains from different EMS concentrations. The results showed that candidate strains from different EMS treatments exhibited significant concentration-dependent phenotypic differentiation. Candidate strains from 0.25% EMS showed superior performance in antioxidant enzyme activity, soluble sugar accumulation, chlorophyll a content, and Fv / Fm, demonstrating better antioxidant and photosynthetic performance. Candidate strains from 0.50% EMS exhibited higher filament length, helix number, and soluble protein content, showing advantages in morphological structure maintenance and metabolic accumulation. In contrast, candidate strains from high-concentration EMS treatments showed higher levels of oxidative damage and weaker photosynthetic performance. Based on comprehensive physiological indicators, PSII functional parameters, and multivariate analysis results, 0.25% and 0.50% EMS treatments can serve as important criteria for screening Spirulina candidate strains under ultrasound-assisted EMS systems, respectively suitable for evaluating photosynthetic performance / antioxidant characteristics and morphological stability. This invention provides an experimental foundation for the subsequent identification and functional development of superior algal strains. Future research, combining genetic variation detection and long-term stability evaluation, is still needed to further verify the genetic basis and application potential of candidate algal strains. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The effect of different EMS concentrations on the filament morphology of candidate algal strains; Figure 2 The effect of different EMS concentrations on the growth of candidate algal strains; Figure 3 The effects of different EMS concentrations on the physiological and biochemical parameters of candidate algal strains; Figure 4 The effects of different EMS concentrations on the OJIP curves and related parameters of candidate algal strains; Figure 5 The effects of different EMS concentrations on the Fv / Fm and chlorophyll a content of candidate algal strains; Figure 6 Correlation analysis of physiological indicators of candidate algal strains under different EMS concentration treatments. Detailed Implementation

[0018] This invention provides a method for ultrasound-assisted ethyl methanesulfonate-based directed mutagenesis of Spirulina, comprising the following steps: 1) The Spirulina algal solution was subjected to ultrasonic treatment to obtain ultrasonically treated algal solution; 2) The ultrasonically treated algal solution from step 1) is mixed with ethyl methanesulfonate and then subjected to targeted mutagenesis treatment; The final volume percentage of ethyl methanesulfonate after mixing the ultrasonically treated algal solution with ethyl methanesulfonate is 0.25~0.5%.

[0019] This invention involves ultrasonically treating a spirulina algal solution to obtain an ultrasonically treated algal solution. The preferred conditions for this ultrasonic treatment include: an ice bath, a power of 400W, a 3-second working time, a 3-second interval, and a total treatment time of 30 seconds. In this invention, the OD value of the spirulina algal solution... 680 The value is preferably 0.17. In this invention, the spirulina preferably includes Spirulina platensis.

[0020] In this invention, the ultrasonically treated algal solution is mixed with ethyl methanesulfonate and then subjected to directed mutagenesis. The final volume percentage of ethyl methanesulfonate after mixing the ultrasonically treated algal solution with ethyl methanesulfonate is 0.25-0.5%. In this invention, the preferred conditions for directed mutagenesis include: directed mutagenesis for 40 minutes in the dark.

[0021] The present invention also provides the application of the method described in the above technical solution in increasing the length of Spirulina filaments.

[0022] The present invention also provides the application of the method described in the above technical solution in improving the accumulation of Spirulina biomass.

[0023] The present invention also provides the application of the method described in the above technical solution in increasing the content of soluble protein and soluble sugar in spirulina.

[0024] The present invention also provides the application of the method described in the above technical solution in improving the antioxidant capacity of spirulina.

[0025] The present invention also provides the application of the method described in the above technical solution in promoting the accumulation of chlorophyll a in Spirulina.

[0026] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1 1. Materials and Methods 1.1 Experimental Materials The tested Spirulina was *Spirulina platensis*, preserved and provided by the Gansu Provincial Microalgae Engineering Technology Research Center. The algal strain was cultured on Zarrouk medium (Wang Lijuan, Zheng Tianxiang, Yang Songqi, et al. Selection of high-yielding temperature-tolerant Spirulina strains by 12C6+ ion beam mutagenesis and optimization of culture conditions [J]. Journal of Radiation Research and Radiation Processing, 2020, 38(2): 27-34.). Before the experiment, the Spirulina was inoculated into fresh Zarrouk medium for activation culture. After the algal solution entered the logarithmic growth phase, it was used for subsequent ultrasonic pretreatment and EMS mutagenesis experiments.

[0028] EMS was selected as the chemical mutagen. It was dissolved in ethanol, then diluted to volume with sterile distilled water, and the mutagenesis reaction was terminated with sodium thiosulfate solution. All reagents used were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0029] 1.2 Experimental Methods 1.2.1 Ultrasonic Pretreatment: Take the Spirulina algal solution in the logarithmic growth phase, dilute it to 120 mL with fresh Zarrouk medium, and adjust the initial OD of the algal solution. 680 The value was set to 0.17. The algal solution was placed in an Erlenmeyer flask and subjected to ultrasonic pretreatment using an ultrasonic cell disruptor (Ningbo Xinyi Ultrasonic Equipment Co., Ltd.) under ice bath conditions. The ultrasonic power was set to 400 W, and intermittent ultrasonic mode was used for treatment. The total treatment time was 30 s, with a working / intermittent time of 3 s / 3 s.

[0030] 1.2.2 EMS Mutagenesis 8 mL of pre-treated Spirulina algal solution was placed in a sterile Petri dish, and EMS was added according to the set final volume fraction. Mutagenesis was performed under dark conditions. Five EMS concentrations were set up for the experiment, with final volume fractions of 0% (T0), 0.25% (T1), 0.50% (T2), 0.75% (T3), and 1.00% (T4), respectively. The mutagenesis treatment time was 40 min. After mutagenesis, 1 mL of 10% sodium thiosulfate solution was added to each treatment to terminate the reaction, and the reaction was allowed to proceed for 10 min. Subsequently, the algal solution was washed three times with fresh Zarrouk medium to thoroughly remove residual EMS. The washed algal solution was then transferred to fresh Zarrouk medium for recovery culture. Three biological replicates were set up for each treatment.

[0031] 1.2.3 Isolation, Purification, and Acquisition of Candidate Algal Strains After EMS mutagenesis treatment and washing, the algal solutions from each treatment were appropriately diluted. A small amount of the algal solution was added to a sterile glass slide or sterile petri dish, and the state of the algal filaments was observed under a microscope under sterile conditions. Single algal filaments with intact morphology, clear structure, and good dispersion were selected and isolated using a sterile micromanipulation needle. The single algal filaments were then transferred to sterile culture plates containing fresh Zarrouk medium for recovery culture. After the single algal filaments recovered growth and formed visible algal bodies, the culture volume was gradually increased to obtain candidate algal strains from different treatments. Each candidate algal strain continued to propagate under the same culture conditions, and preliminary screening was conducted based on growth status, algal filament integrity, and contamination. Candidate algal strains with normal growth, stable morphology, and no obvious contamination were retained for subsequent continuous subculturing.

[0032] 1.2.4 Continuous Subculturing of EMS-Induced Candidate Algal Strains Each candidate algal strain obtained from single algal filament isolation was transferred to fresh Zarrouk medium for recovery culture. After the algal solution recovered growth and reached the logarithmic growth phase, it was transferred to fresh Zarrouk medium at a 10% inoculum for the next generation culture. The culture was continuously subcultured for 8 generations using the above method. The same inoculum ratio and culture conditions were maintained for each subculture. The 8th generation candidate algal strain was used as experimental material for subsequent determination of growth characteristics, filament morphology, physiological and biochemical indicators, chlorophyll content, and chlorophyll fluorescence parameters.

[0033] 1.2.5 Growth Index Determination: Candidate algal strains from each treatment, continuously passaged to the 8th generation, were inoculated into fresh Zarrouk medium and adjusted to the same initial OD value before cultivation. The growth of Spirulina was determined spectrophotometrically. 2.5 mL of algal solution was taken each time, and the absorbance was measured at 680 nm. Measurements were taken every 2 days to continuously monitor changes in OD values ​​during cultivation. Growth curves were plotted, and the differences in growth characteristics among M8 generation candidate algal strains from different EMS mutagenesis treatments were compared.

[0034] 1.2.6 Morphological Index Determination: 10 μL of algal solution from each candidate M8 generation was dropped onto a glass slide, and the morphology of the algal filaments was observed and photographed under an optical microscope. Morphological parameters such as filament length, filament width, number of spirals, and pitch were determined using ImageJ image analysis software. Thirty intact algal filaments from each treatment were selected for statistical analysis.

[0035] 1.2.7 Determination of Physiological and Biochemical Indicators: 0.5 g of fresh algal sludge from each candidate M8 generation was added to 1–2 mL of PBS buffer (pH 7.8) and thoroughly homogenized at 4℃. Subsequently, the mixture was heated at 3700 r·min at 4℃. -1Centrifuge for 20 min, take the supernatant and dilute to 10 mL with PBS buffer, and use it as the test solution for physiological and biochemical index determination. Soluble sugar content was determined using the anthrone-sulfuric acid method (Zou Liuyuan, Wang Jie, Qin Lezheng. Optimization of conditions for determining soluble sugar content in Acorus calamus leaves by anthrone method [J]. Journal of Hainan University (Natural Science Edition, Chinese and English), 2025, 43(4): 405-415.), with glucose as the standard; soluble protein content was determined using the Coomassie Brilliant Blue G-250 method (Jiang Li, Cai Jinyan. Application of biuret method and Coomassie Brilliant Blue staining method in the determination of protein content in dairy beverages [J]. Grain, Oil and Feed Technology, 2023, (2):201-203.), with bovine serum albumin as the standard; malondialdehyde (MDA) content was determined using the thiobarbituric acid method (Zhang Qinghang, Zhang Yongtao. Response of malondialdehyde (MDA) content in plants to drought [J]. Forestry Survey and Design, 2019, (1):110-112.). The activity of superoxide dismutase (SOD) was determined by the photochemical reduction method of nitroblue tetrazolium (Ren Qiuhan. Screening and property study of high-yield superoxide dismutase lactic acid bacteria and yeasts [D]. Jiangsu: Jiangnan University, 2025.); the activity of peroxidase (POD) was determined by the guaiacol method (Lv Chunfang, Li Haixia, Jia Ranran, et al. Improvement of experimental method for determining plant peroxidase activity [J]. Light Industry Science and Technology, 2024, 40(2): 15-18.); the activity of catalase (CAT) was determined by ultraviolet spectrophotometry (Zhao Longfei, Xu Yajun, Yang Jingya, et al. Effects of endophytic bacteria 72 and 146 on catalase activity and proline content in soybean seedlings under salt stress [J]. Acta Microbiologica Sinica, 2025, 65(4): 1726-1741.).

[0036] 1.2.8 Chlorophyll a Content Determination: Algal solutions from each candidate M8 generation were collected by centrifugation, and chlorophyll was extracted with 95% ethanol. The extract was allowed to stand in the dark until the pigments were fully dissolved, and the absorbance was measured by spectrophotometry to calculate the chlorophyll a content.

[0037] 1.2.9 Chlorophyll fluorescence induction kinetics and PSII functional parameter determination: The OJIP and PSII functional parameters of each candidate algal strain in generation M8 were determined using a Plant Efficiency Analyzer (PEA). Before measurement, the algal solutions of each treatment were dark-acclimated for 20 min. Subsequently, the solutions were heated at 3000 μmol·m⁻¹. -2 ·s -1Under excitation intensity and a wavelength of 650 nm, the chlorophyll fluorescence induction kinetics were recorded within 0–2 s. Relevant fluorescence parameters, including Fv / Fm (maximum photochemical quantum yield) and Vj (J-point normalized variable fluorescence), were calculated based on the JIP-test energy flow model. P0 (maximum photochemical efficiency at t=0), PIABS (performance index based on absorbed light energy), ψ0 (electron transport probability at t=0), E0 (electron transfer quantum yield), ABS / RC (light energy absorbed per unit reaction center), TR0 / RC (energy captured per unit reaction center), ET0 / RC (electron transfer energy per unit reaction center), and DIO / RC (energy dissipated per unit reaction center).

[0038] 1.2.10 Data Statistics and Analysis All data were organized using Excel, and statistical analysis was conducted using R software. P A value <0.05 was used as the criterion for statistical significance. Different lowercase letters in the figure indicate significant differences between treatments. Pearson correlation analysis was based on all biological replicates. Before principal component analysis (PCA), all morphological, physiological, biochemical, and chlorophyll fluorescence parameters were standardized using Z-scores. Graphs were generated using Origin, R, and EcoAmp software. Error bars in the graphs represent standard errors (SE).

[0039] 2. Results and Analysis.

[0040] 2.1 Effects of ultrasound-assisted EMS mutagenesis on the growth and morphological characteristics of candidate algal strains Candidate algal strains obtained from different EMS treatments showed significant differences in culture medium color and algal filament morphology. Figure 1 After ultrasound-assisted EMS mutagenesis, single algal filament isolation, and continuous passage to the M8 generation, all candidate algal strains maintained growth, but their filament length and spiral structure showed varying degrees of change. Compared to T0, the filaments of candidate strains T1 and T2 were generally longer, with more complete structures and clearer, relatively regular spiral morphologies. In candidate strain T3, short and broken filaments significantly increased, and the spiral structures of some filaments were relatively loose and irregular. The culture medium of candidate strain T4 was relatively light in color, and the filaments still maintained a certain spiral morphology, but the number and morphological uniformity of filaments decreased. Overall, lower or moderate concentrations of EMS treatment were beneficial for candidate algal strains to maintain a more complete filament structure, while higher concentrations may inhibit algal growth and lead to shortened, broken, and abnormal spiral structures of filaments.

[0041] OD of candidate algal strains treated with different EMS 680 All showed a typical "S"-shaped growth trend with prolonged cultivation time. Figure 2 In the initial stage of cultivation (1–7 days), the growth of each candidate algal strain was relatively slow, and the OD... 680 The differences were small; after entering the rapid growth phase (7–15 days), the OD of each treatment increased. 680 The biomass increased rapidly, and differences between treatments gradually became apparent; after 15 days of cultivation, all candidate algal strains gradually entered a stable phase. Candidate strain T3 showed higher biomass accumulation in the later stages of cultivation, with an OD of [value missing] on day 17. 680 The OD value reached a peak of 0.679, an increase of 2.72% compared to T0. The growth trends of candidate algae T1 and T2 were basically consistent with those of T0, but the OD value increased in the later stages of cultivation. 680 Slightly lower than T0, which was 4.09% and 0.61% higher than T1 and T2, respectively. In contrast, the growth rate and final biomass of candidate algae T4 were relatively low, with an OD of [missing value] on day 19. 680 The value was only 0.610, a decrease of 7.44% compared to T0. The above results indicate that the appropriate concentration of EMS treatment has little effect on the growth of candidate algal strains, and some treatments may promote the accumulation of biomass in the later stage, while higher concentrations of EMS treatment will have a certain inhibitory effect on the growth of algal strains.

[0042] Quantitative morphological analysis further revealed significant differences in the filament morphology parameters of candidate algal strains treated with different EMS (Table 1). The T2 treatment resulted in the longest filament length (634.96 μm), significantly higher than T0 and T3 by 17.33% and 12.14%, respectively; the T1 treatment was the second longest, indicating that 0.25%–0.50% EMS treatment was more conducive to obtaining candidate algal strains with longer filaments. The number of spirals was significantly higher in T1, T2, and T4 treatments than in T0 and T3, with T2 showing the highest mean, indicating relatively good preservation of the spiral structure. The filament diameter was largest in T4 treatment, suggesting that higher concentrations of EMS treatment may have caused some changes in the filament structure. The pitch was largest in T0, significantly lower in T2 and T4 than in T0, indicating that the tightness of the filament spirals changed after EMS treatment. Considering filament length, spiral number, and pitch, the T2 treatment candidate algal strain showed relatively better morphological structure maintenance and overall performance.

[0043] Table 1. Changes in morphological parameters of candidate algal strains under different EMS treatments

[0044] Note: All data in the table are mean ± standard error. Different lowercase letters in the same column after the numerical value indicate significant differences between treatments. P The difference was significant at the <0.05 level.

[0045] 2.2 Changes in physiological and biochemical parameters of M8 generation candidate algal strains under different EMS mutagenesis treatments There were significant differences in the physiological and biochemical indicators of candidate algal strains treated with different EMS. Figure 3 The soluble protein content was highest in treatment T2, significantly higher than in treatments T0, T3, and T4, but not significantly different from treatment T1, indicating that 0.25%–0.50% EMS treatment was beneficial for the accumulation of soluble proteins in candidate algal strains. Soluble sugar content decreased with increasing EMS concentration, reaching a peak of 1.06 mg / g FW in treatment T1, a significant increase of 55.88% compared to T0. Treatment T0 had the lowest content at 0.57 mg / g FW, suggesting that lower concentrations of EMS treatment may enhance the osmotic regulation capacity of candidate algal strains. With increasing EMS concentration, the MDA content of candidate algal strains generally showed a continuous upward trend. Treatment T0 had the lowest MDA content at 0.19 μmol / mg; treatments T1 and T2 showed slight increases compared to T0, but the differences were not significant. Treatment T4 showed a significant increase in MDA content, approximately 131.6% higher than T0, indicating that higher concentrations of EMS treatment may exacerbate cell membrane lipid peroxidation and cause some degree of oxidative damage. Antioxidant enzyme activities showed significant differences among different EMS treatments. SOD activity reached its highest level in treatment T1, significantly increasing by 127.12% compared to T0, and subsequently showed a general decreasing trend with increasing EMS concentration. POD activity was relatively high in treatments T1 and T2, both around 0.41 U / mg FW, with no significant difference compared to T0. CAT activity reached its highest level in treatment T1, at 1.41 U / mg FW, higher than T0, but the difference was not significant. With further increases in EMS concentration, CAT activity decreased significantly, with T3 and T4 treatments showing reductions of 85.96% and 86.84% compared to T1, respectively. This suggests that low-concentration EMS treatment may induce antioxidant defense responses in candidate algal strains, while higher concentrations of EMS may inhibit CAT activity, weakening their ability to scavenge H2O2.

[0046] 2.3 Chlorophyll fluorescence induction kinetics and PSII functional changes in M8 generation candidate algal strains under different EMS mutagenesis treatments Candidate algal strains treated with different EMS all exhibited typical O–J–I–P chlorophyll fluorescence transient curves, but the treatments showed certain differences at different fluorescence stages. Figure 4 (A). The treatments largely overlapped near point O. However, in the O–J phase, the relative variable fluorescence of treatments T3 and T4 was generally higher than that of T0. In the J–I and I–P phases, the fluorescence rise of treatments T3 and T4 was relatively earlier, with T4 reaching point P faster, indicating that higher concentrations of EMS treatment may alter the closing process of the PSⅡ reaction center and the acceptor-side electron transport state. JIP-test parameters further showed that the differences between treatments were mainly in ABS / RC and DI0 / RC, with both parameters being relatively higher in treatment T2, and DI0 / RC in treatments T3 and T4 also higher than that in treatment T0 (A). Figure 4 (B) This indicates that EMS treatment may increase the light energy absorption per unit active reaction center and enhance the dissipation of excess excitation energy. In contrast, VJ, TR0 / RC, ψ0, ETO / RC, P0, The E0 and PIABS parameters showed relatively small overall changes. These results indicate that different concentrations of EMS treatment primarily affect the energy distribution process of the candidate algal strain PSⅡ. Specifically, medium-to-high concentration treatments may enhance light energy dissipation and have some impact on electron transport processes.

[0047] Significant differences were observed in the Fv / Fm and chlorophyll a content of candidate algal strains treated with different EMS. Figure 5 The Fv / Fm ratios reached relatively high levels in treatments T1 and T2, approximately 0.72 and 0.71 respectively, with no significant difference between the two, but both significantly higher than T0. The Fv / Fm ratios in treatments T3 and T4 were approximately 0.67 and 0.65 respectively, significantly lower than T1 and T2, but still significantly higher than T0. These results indicate that EMS treatment generally improved the maximum PSII photochemical efficiency of the candidate algal strains, with the 0.25%–0.50% EMS treatment showing the most significant promoting effect. As the EMS concentration further increased, the Fv / Fm ratio decreased, but not to the T0 level. Chlorophyll a content showed a trend of first increasing and then decreasing with increasing EMS concentration. Treatment T1 had the highest chlorophyll a content, approximately 0.66 mg / L, significantly higher than treatments T0, T3, and T4. With further increases in EMS concentration, the chlorophyll a content in treatments T3 and T4 decreased significantly, with T4 having the lowest content, significantly lower than T0. The results showed that lower concentrations of EMS treatment were beneficial to the accumulation of chlorophyll a, while higher concentrations of EMS treatment may inhibit chlorophyll synthesis or accelerate its degradation.

[0048] 2.4 Correlation analysis of physiological and photosynthetic indicators of candidate algal strains Pearson correlation analysis showed that chlorophyll a content was significantly positively correlated with the activities of soluble protein, soluble sugar, CAT, SOD, and POD, with stronger correlations to soluble sugar and POD activities (r = 0.829 and 0.854, respectively), while it was significantly negatively correlated with MDA content (r = -0.717). Soluble sugar was significantly positively correlated with CAT, SOD, and POD activities, but significantly negatively correlated with MDA content (r = -0.568). MDA content was significantly negatively correlated with CAT and POD activities, respectively (r = -0.858 and -0.599). These results indicate that antioxidant enzyme activity and osmotic regulators have a certain synergistic effect with chlorophyll a accumulation, while MDA accumulation may be related to oxidative stress and decreased photosynthetic pigments caused by high-concentration EMS treatment.

[0049] 2.5 Principal component analysis of physiological and PSII functional indices of candidate algal strains Principal component analysis (PCA) was performed on the physiological indicators of candidate Spirulina strains under different EMS concentrations. The contribution rates of principal component 1 (PC1) and principal component 2 (PC2) were 61.6% and 19%, respectively, with a cumulative contribution rate of 80.6%, indicating that PC1 and PC2 can represent 80.6% of the information for the seven physiological indicators. A comprehensive analysis of membership function values ​​was performed on the physiological indicators of the candidate strains under different EMS concentrations (Table 2). The D values ​​of each treatment group, from largest to smallest, were T1>T2>CK>T3>T4. This comprehensive ranking further indicates that medium-concentration EMS treatment is more conducive to screening dominant strains.

[0050] Table 2. Membership function values ​​and ranking of candidate algal strains under different EMS treatments.

[0051] Note: PC1 and PC2 represent the composite index values ​​of principal component 1 and principal component 2; U1 and U2 represent the membership function values ​​of principal component 1 and principal component 2. 3. Discussion This invention obtained M8 generation candidate algal strains from different EMS concentrations through ultrasound-assisted EMS treatment, single algal filament isolation, and continuous subculturing, and found that they exhibited significant differences in growth, morphology, and physiological function. Since EMS induction may simultaneously cause genetic variation and cellular physiological responses, this study focuses on the phenotypic differences of candidate algal strains from different sources, rather than directly determining their genetic alterations.

[0052] Spirulina has a filamentous structure, and filament aggregation may limit the sufficient contact between mutagens and cells, thus affecting mutagenesis efficiency. Ultrasonic pretreatment may improve the consistency of EMS treatment by improving filament dispersion and increasing cell surface exposure. In this study, candidate algae from low to moderate concentrations of EMS (0.25%–0.50%) exhibited good filament integrity, with the 0.50% EMS-derived candidate algae showing higher filament length and helix number, indicating that moderate EMS treatment is beneficial for screening candidate materials with superior morphological characteristics.

[0053] However, high-concentration EMS treatment led to algal filament breakage and structural irregularities, indicating that strong chemical stress may exceed the cell's tolerance range.

[0054] EMS treatment can induce oxidative stress in cells, further affecting the antioxidant system and metabolic state. This study found that candidate algae treated with 0.25% EMS exhibited higher SOD and CAT activities and soluble sugar content, while having lower MDA accumulation, indicating a stronger oxidative stress response. In contrast, candidate algae treated with high concentrations of EMS showed increased MDA content and significantly decreased CAT activity, suggesting that higher EMS levels may lead to enhanced oxidative damage and weaken the cell's ability to maintain redox balance. It should be noted that the antioxidant indicators in this study primarily reflect the current physiological state of the candidate algae; whether they correspond to stable heritable traits requires further verification using genomic or molecular marker analysis.

[0055] PSII is a crucial functional module for evaluating the light energy utilization efficiency of photosynthetic organisms and is highly sensitive to environmental stress and changes in cell state. This invention found that candidate algae from different EMS sources maintained typical O–J–I–P fluorescence induction curves, but some parameters showed significant differences. Specifically, candidate algae from 0.25% and 0.50% EMS sources exhibited higher Fv / Fm and chlorophyll a content, indicating that algae from moderately EMS-treated sources had better PSII maximum photochemical efficiency and pigment accumulation capacity. JIP-test analysis further showed that EMS treatment mainly affected energy absorption and dissipation parameters such as ABS / RC and DIO / RC, while electron transport-related parameters changed relatively little. This suggests that different EMS treatments may primarily lead to adjustments in PSII energy allocation strategies without causing significant disruption to the electron transport system. Although the decrease in Fv / Fm in candidate algae from high-concentration EMS sources was limited, their chlorophyll a content decreased and growth was inhibited, indicating that a single fluorescence parameter is insufficient to evaluate the overall performance of candidate algae; a comprehensive assessment combining growth, physiological, and PSII indicators is necessary.

[0056] Through principal component analysis and comprehensive evaluation, this invention screened candidate algal strains derived from 0.25% and 0.50% EMS, which exhibited superior overall phenotypes. The 0.25% EMS-derived strains were more suitable for evaluating antioxidant capacity and photosynthetic performance, while the 0.50% EMS-derived strains showed superior morphological stability and protein accumulation. Repeated validation experiments were conducted using 0.25% and 0.50% EMS concentrations combined with sonication. The morphological and index measurement results obtained were 98% similar to the original experimental results, demonstrating the feasibility and reproducibility of the method. However, this invention still has certain limitations. Although the candidate algal strains underwent M8 generations of continuous subculturing, whether their phenotypic differences originate from stable genetic variation requires further verification. Future research could combine whole-genome sequencing, molecular marker analysis, or long-term subculturing stability evaluation to clarify the genetic basis of EMS-induced variations.

[0057] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for ultrasound-assisted ethyl methanesulfonate-directed mutagenesis of Spirulina, characterized in that, Includes the following steps: 1) The Spirulina algal solution was subjected to ultrasonic treatment to obtain ultrasonically treated algal solution; 2) The ultrasonically treated algal solution from step 1) is mixed with ethyl methanesulfonate and then subjected to targeted mutagenesis treatment; The final volume percentage of ethyl methanesulfonate after mixing the ultrasonically treated algal solution with ethyl methanesulfonate is 0.25~0.5%.

2. The method according to claim 1, characterized in that, Step 1) The conditions for ultrasonic treatment include: ice bath conditions, power of 400W, working time of 3 seconds, interval of 3 seconds, and total treatment time of 30 seconds.

3. The method according to claim 1, characterized in that, Step 1) The OD of the Spirulina liquid 680 The value is 0.

17.

4. The method according to claim 1, characterized in that, Step 1) The spirulina mentioned includes Spirulina platensis.

5. The method according to claim 1, characterized in that, Step 2) The conditions for the directed mutagenesis include: directed mutagenesis for 40 minutes in the dark.

6. The application of the method according to any one of claims 1 to 5 in increasing the length of Spirulina filaments.

7. The application of the method according to any one of claims 1 to 5 in improving the accumulation of Spirulina biomass.

8. The application of the method according to any one of claims 1 to 5 in increasing the content of soluble protein and soluble sugar in spirulina.

9. The application of the method according to any one of claims 1 to 5 in improving the antioxidant capacity of spirulina.

10. The application of the method according to any one of claims 1 to 5 in promoting the accumulation of chlorophyll a in Spirulina.

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