A culture and result determination method for promoting photosynthetic efficiency of lichen symbiotic green algae

Abstract

The application relates to a culture and result determination method for promoting the photosynthetic efficiency of lichen symbiotic green algae, which uses subzero temperature to treat lichen symbiotic algae Diplosphaera chodatii A07020, the microbial preservation registration number is CGMCC No. 3892. The culture cycle comprises normal temperature 23 DEG C, light intensity 2000 lx, 16h light and 8h dark alternation treatment, culture for 3 weeks, transfer to low temperature-4 DEG C: the temperature condition is-4 DEG C, 16h light and 8h dark alternation treatment, the light intensity is 2000 lx, continuous treatment for 1 week, and then return to normal temperature 23 DEG C, light intensity 2000 lx, 16h light and 8h dark alternation treatment, and culture for 2 days. The lichen symbiotic green algae is treated by subzero temperature in the application, the implementation cost is low, the operation is easy, the environment is safe, the photosynthetic efficiency can be remarkably promoted, and therefore the lichen symbiotic green algae can be more efficiently utilized.

Description

Technical Field

[0001] This invention belongs to the field of algal photosynthesis technology, and relates to a cultivation method that utilizes sub-freezing temperatures to promote the photosynthetic efficiency of lichen-symbiotic green algae. Background Technology

[0002] Lichens are symbiotic organisms composed of lichen-type fungi and corresponding algae and / or cyanobacteria. They can withstand harsh environments such as extreme cold, low nutrition, drought, and strong ultraviolet radiation, and are widely distributed on Earth, serving as pioneer organisms in rock weathering and soil formation. Among the photosynthetic organisms (green algae and cyanobacteria) that live in symbiosis with lichens, the vast majority are microalgae of the green algae class. Of the nearly 20,000 known lichen species, 98% live in symbiosis with green algae, with the most prevalent green algae belonging to the class Chlorophyta.

[0003] Coccella kosei Diplosphaera chodatii It is a species of algae in the class Chlorophyta, and is used as a lithospermum. Endocarpon pusillum This symbiotic algae, *Lithocarpus spp.*, is also a common symbiotic algae in the order Bottlenose Lithocarpus, belonging to the genus *Lithocarpus*. *Lithocarpus spp.* is a common lichen in deserts. Previous studies have reported that both the symbiotic fungus and algae of *Lithocarpus spp.* exhibit strong drought tolerance. Furthermore, *Diplococcus kurstii* also demonstrates good salt and alkali tolerance. However, current research lacks on the algae's tolerance to low temperatures and its biological effects at low temperatures.

[0004] A comprehensive survey of the temperature response of green algae indicates that the optimal growth temperature for green algae is typically 20-25°C, and low temperatures generally inhibit their photosynthetic performance. Even psychrophilic algae adapted to polar environments, such as... Chlamydomonas raudensis UWO241 and Chlorella sp. strain BI has an optimal growth temperature of 8-15℃, and its photosynthetic activity decreases at sub-zero temperatures.

[0005] Research revealed no reports on how low temperatures below freezing point can promote photosynthetic efficiency in green algae. This invention is the first to study the photosynthetic effect of treating lichen symbiotic algae at temperatures below freezing point. Summary of the Invention

[0006] This invention aims to provide a method for cultivating and measuring the photosynthetic efficiency of lichen symbiotic green algae. The method uses low-temperature treatment below freezing point to treat lichen symbiotic green algae, which is low-cost, easy to operate, environmentally friendly and safe, and can significantly promote its photosynthetic efficiency, thereby making more efficient use of lichen symbiotic green algae.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a cultivation method for promoting the photosynthetic efficiency of lichen symbiotic green algae, which utilizes a temperature below freezing point to treat the lichen symbiotic green algae: the temperature condition is -4℃, with alternating treatment of 16h light and 8h darkness, and a light intensity of 2000 lx, for a continuous treatment of 1 week; or the temperature condition is 4℃, with alternating treatment of 16h light and 8h darkness, and a light intensity of 2000 lx, for a treatment of 4 days, and then transferred to -4℃, with alternating treatment of 16h light and 8h darkness, and a light intensity of 2000 lx, for a treatment of 3 days.

[0008] Furthermore, the lichen symbiotic green algae is a Lithocarpus symbiotic algae. Diplosphaera chodatii A07020 has the preservation registration number CGMCCNo.3892 at the China General Microbiological Culture Collection Center.

[0009] Furthermore, the symbiotic algae of the stone fruit skin Diplosphaera chodatii The culture period for A07020 CGMCC No.3892 is 30 days.

[0010] Furthermore, the method specifically includes the following steps: (1) Activation of symbiotic algae in Lithocarpus: The symbiotic algae of Lithocarpus spp. were transferred to PDA medium and cultured at 23°C with alternating light and dark cycles of 16 h and 8 h, with a light intensity of 2000 lx for 19 days. (2) Large-scale culture of Lithocarpus symbiotic algae: The activated Lithocarpus symbiotic algae were scraped into sterile water. 20 μL of Lithocarpus symbiotic algae with OD600=0.6 was inoculated into a new PDA medium and placed in an artificial climate chamber for cultivation. Under the conditions of 23℃, the algae were treated with alternating 16h light and 8h dark, and the light intensity was 2000lx for 3 weeks. (3) Grouped control culture of Lithocarpus symbiotic algae: The *Lithocarpus spp.* symbiotic algae were divided into four groups and cultured under different temperature conditions: -4℃, 4℃, 4℃ / -4℃, and 23℃. In the -4℃ group, the expanded-cultured *Lithocarpus spp.* symbiotic algae were transferred to a -4℃ refrigerator with an external light source, using a 16-hour light-8-hour dark cycle with a light intensity of 2000 lx, and cultured for one week. In the 4℃ group, the expanded-cultured *Lithocarpus spp.* symbiotic algae were transferred to a 4℃ refrigerator with an external light source, using a 16-hour light-8-hour dark cycle with a light intensity of 2000 lx, and cultured for one week. The 4℃ / -4℃ group... The expanded cultured *Lithocarpus spp.* symbiotic algae were transferred to a 4°C freezer with an external light source, and treated with alternating 16 hours of light and 8 hours of darkness at a light intensity of 2000 lx for 4 days. Then, they were transferred to a -4°C freezer with an external light source, and treated with alternating 16 hours of light and 8 hours of darkness at a light intensity of 2000 lx for 3 days. The 23°C control group consisted of *Lithocarpus spp.* symbiotic algae cultured for 3 weeks kept in an artificial climate chamber at 23°C, treated with alternating 16 hours of light and 8 hours of darkness at a light intensity of 2000 lx for another week. (4) Room temperature culture of Lithocarpus symbiotic algae: The *Lithocarpus spp.* symbiotic algae treated in the -4℃, 4℃, and 4℃ / -4℃ culture groups were transferred to an incubator at 23℃ and subjected to alternating light and dark conditions of 16 hours and 8 hours at a light intensity of 2000 lx for 2 days. The *Lithocarpus spp.* symbiotic algae in the 23℃ control group were kept in an artificial climate chamber and subjected to alternating light and dark conditions of 16 hours and 8 hours at a light intensity of 2000 lx for 2 days.

[0011] Furthermore, the PDA culture medium is prepared by mixing 39.0g of PDA culture medium powder with 1L of distilled water.

[0012] Secondly, this invention also provides a method for determining the results of a cultivation method that promotes the photosynthetic efficiency of lichen symbiotic green algae. Chlorophyll fluorescence was measured on four groups of lichen symbiotic algae colony samples under different temperature cultivation conditions. The specific steps include: (1) Place the culture dish containing the algal colony sample with the symbiotic algae in a completely dark environment for 20-30 minutes for dark adaptation, and then place the culture dish on the measuring stage of the chlorophyll fluorescence-imaging-gas exchange synchronous measurement system. After the measuring spot evenly covers the algal colony sample to be tested, the measurement will begin. (2) Calculation of photosynthetic efficiency value Fv / Fm: After the measurement starts, first turn on the measuring light of the pulse-modulated fluorometer, obtain and record the initial basal fluorescence value Fo of the algal colony sample when all photosystem II reaction centers are in the open state. Subsequently, apply a saturating pulse to the algal colony sample to instantaneously and completely close the photosystem II reaction centers, obtain and record the maximum fluorescence value Fm, and automatically calculate the maximum quantum yield Fv / Fm of photosystem II, which reflects the potential maximum photosynthetic efficiency of the algae. The calculation formula is Fv / Fm = (Fm - Fo) / Fm; (3)Perform statistical analysis on the measurement results of the -4°C culture group, 4°C culture group, 4°C / -4°C culture group, and 23°C culture group: Analyze 6 biological replicates of the algal colony samples under each temperature culture condition, use GraphPad prism 10.0 software for one-way ANOVA, and use Duncan's multiple range test for post hoc comparison. **** indicates P < 0.0001, *** indicates 0.0001 < P < 0.001, ** indicates 0.001 < P < 0.01, and * indicates 0.01 < P < 0.05.

[0013] Furthermore, perform real-time fluorescence quantitative PCR analysis on the algal colony samples of Diploschistes muscorum symbiotic algae under four different temperature culture conditions, and analyze the photosynthesis gene expression of Diploschistes muscorum symbiotic algae under four different temperature culture conditions of -4°C, 4°C, 4°C / -4°C, and 23°C, including the following operating steps: Use Trizol TM reagent to extract total RNA; use ABScript Neo RT Master Mix Kit to synthesize complementary DNA; to detect the expression of two genes rbcL and Lhc related to photosynthesis, design primers for real-time fluorescence quantitative PCR analysis; after confirming the expression stability of the actin gene, use it as an internal reference; perform real-time fluorescence quantitative PCR analysis on a Bio-Rad CFX96 TM system using SYBR Green I Master Mix; the results of real-time fluorescence quantitative PCR analysis show that the photosynthetic efficiency of photosystem II of Diploschistes muscorum symbiotic algae in the -4°C culture group and the 4°C / -4°C culture group is significantly improved compared with the 23°C control group.

[0014] Furthermore, quantitatively characterize the expression levels of two genes rbcL and Lhc related to the photosynthesis of Diploschistes muscorum symbiotic algae. Gene rbcL shows an increase in transcript abundance in all low-temperature treatment groups, rbcL and the relative expression level of Lhc is negatively correlated with the photosynthetic efficiency; while the transcription of gene Lhc The relative expression level is positively correlated with photosynthetic efficiency.

[0015] Furthermore, during the real-time fluorescence quantitative PCR analysis process, the thermal cycling conditions of the real-time fluorescence quantitative PCR analyzer were set as follows: the temperature was set to 37°C for 2 minutes, then the temperature was set to 95°C for 3 minutes, and then 40 cycles were carried out. One cycle included setting the temperature to 95°C for 5 seconds and setting the temperature to 60°C for another 30 seconds; the amplification specificity was confirmed by melting curve analysis, and the relative expression level was calculated using the 2 - ΔΔCt method, and 3 biological replicates were set for the samples under each temperature culture condition; the statistical analysis of the relative expression level was performed using GraphPad prism 10.0 software for one-way ANOVA and Tukey's post hoc multiple comparison test, with **** indicating P<0.0001, *** indicating 0.0001<P<0.001, ** indicating 0.001<P<0.01, and * indicating 0.01<P<0.05.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: Compared with the normal temperature culture at 23°C, all low-temperature treatments in the method of the present invention result in rbcL an increase in transcript abundance, which is consistent with the general cold stress response at the transcriptional level. However, rbcL the up-regulation amplitude is negatively correlated with photosynthetic performance: at 4°C rbcL the expression level is the highest, while the photosynthetic efficiency Fv / Fm is inhibited; in the -4°C treatment rbcL the expression level is the lowest, and Fv / Fm reaches the maximum. This pattern is contrary to the typical cold stress response in most plants. Low temperature usually inhibits photosynthesis by inhibiting Rubisco activation and Calvin cycle enzymes, and the up-regulation of cold-induced photosynthetic genes is usually associated with acclimation adaptation rather than immediate functional enhancement.

[0017] The symbiotic alga of the present invention, Pleurococcus kochii Diplosphaera chodatii ), can achieve the maximum photosynthetic efficiency with the minimum transcriptional investment at -4°C, which represents a unique adaptation strategy that is not common in free-living algae and plants. In contrast, compared with the normal culture at 23°C, the expression of Lhc is significantly down-regulated under all low-temperature conditions. This down-regulation is consistent with the recognized photoprotection mechanism, that is, reducing the light capture ability under cold stress to prevent the accumulation of excess excitation energy. The down-regulation amplitude is significantly positively correlated with the Fv / Fm value: the down-regulation is the largest at 4°C, corresponding to the lowest Fv / Fm; while the down-regulation is the smallest at -4°C, corresponding to the highest Fv / Fm. The alternating 4°C / -4°C treatment shows a medium level of Lhc expression and Fv / Fm.

[0018] The symbiotic algae of the stone fruit skin (Diplococcus koraiensis) of this invention Diplosphaera chodatii The photosynthetic promotion effect was maximized precisely at -4°C, below freezing, while the photosynthetic effect was inhibited at 4°C, above freezing. This reveals an extremely narrow and finely tuned low-temperature adaptation mechanism, with an adaptation range far exceeding that of previously reported free-living green algae. Alternating temperature treatments of 4°C / -4°C also produced a photosynthetic promotion effect, but its effect was lower than that of -4°C, indicating that the algae can regulate the balance between photosynthetic inhibition (4°C) and promotion (-4°C) as the temperature exposure progresses.

[0019] The cultivation method for promoting the photosynthetic efficiency of lichen-symbiotic green algae of the present invention is low in implementation cost, easy to operate, environmentally friendly and safe, and can significantly promote the photosynthetic efficiency of Lithocarpus symbiotic algae. Diplosphaera chodatii The photosynthetic efficiency of A07020 CGMCC No.3892 provides a new method for the efficient utilization of lichen-symbiotic green algae. Attached Figure Description

[0020] Figure 1 Comparison of the colors of the algal colonies of the symbiotic algae *Lithocarpus spp.* after cultivation at different temperatures.

[0021] Figure 2 A statistical graph showing the photosynthetic efficiency of the symbiotic algae *Lithocarpus spp.* after cultivation at different temperatures.

[0022] Figure 3 The bar chart shows the expression levels of two genes related to photosynthesis after cultivation at different temperatures. The expression levels are normalized with respect to the internal reference gene Actin. The vertical axis represents the value of log2 (fold change). Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0024] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available. The main equipment models and parameters used in the embodiments of this invention are as follows: Artificial Climate Chamber: Ningbo Jiangnan Instrument Co., Ltd., GXZ Intelligent Model; Refrigerator: Taizhou LG Electronics Refrigeration Co., Ltd., BCD-240H (GR-S25EHPD); Clean bench: Beijing Yatai Kelong Instrument Technology Co., Ltd., YT-CJ-1N model; Portable light source: AUX Group Co., Ltd., AS-528; Chlorophyll fluorescence-imaging-gas exchange synchronous measurement system: WALZ, GFS-DUAL; Description of the main reagent kits used in the quantitative real-time fluorescence PCR analyzer in this embodiment of the invention: ABScript Neo RT Master Mix Kit (ABclonal, item number RK20433); SYBR Green I Master Mix (ABclonal, item number RK221219); The culture medium formulation used in the embodiments of this invention is as follows: PDA medium: 39.0g PDA medium powder (Difco) Potato Dextrose Agar (BD Company), 1L of distilled water.

[0025] This embodiment discloses a cultivation method for promoting the photosynthetic efficiency of lichen-symbiotic green algae. The specific cultivation method is as follows: In a clean bench, an inoculation needle was used to inoculate the culture medium with the preservation number […]. Diplosphaera chodatii A suitable amount of algal seed from the symbiotic algae strain A07020 CGMCCNo.3892 was scraped and transferred to fresh solid PDA medium. The medium was then placed in an artificial climate chamber for activation under the following conditions: 23°C, 16h light (intensity 2000 lx) / 8h dark alternation, and cultured for 19 days.

[0026] The activated Lithocarpus symbiotic algae were placed in an ultra-clean workbench. Diplosphaera chodatii A07020CGMCC No.3892 was scraped into sterile water, and 20 μL of the Lithocarpus symbiotic algae was taken. Diplosphaera chodatii The algal culture A07020CGMCC No.3892 (OD600=0.6) was inoculated onto new solid PDA medium and placed in an artificial climate chamber for expansion culture. The culture conditions were 23℃, 16h light (intensity 2000lx) / 8h dark alternation, and cultured for 3 weeks.

[0027] The -4℃ culture group contained Lithocarpus symbiotic algae cultured at 23℃ for 3 weeks. Diplosphaera chodatii Transfer A07020CGMCC No.3892 to a -4℃ freezer with an external light source inside, light intensity 2000 lx, alternating between 16h light and 8h dark, and incubate for 1 week.

[0028] To verify the effect of different temperature cultivation conditions on the photosynthetic efficiency of *Lithocarpus spp.*, three experimental groups and control groups were set up at different temperatures: ① The 23℃ control group (CK) maintained a constant cultivation temperature of 23℃, a light intensity of 2000 lx, and a 16h light / 8h dark alternation for 4 weeks; ② The 4℃ group consisted of *Lithocarpus spp.* cultured at 23℃ for 3 weeks. Diplosphaera chodatii A07020 CGMCC No.3892 was transferred to a 4℃ refrigerator with an external light source inside, with a light intensity of 2000 lx, and alternated between 16h light and 8h dark for 1 week; ③ The 4℃ / -4℃ culture group consisted of Lithocarpus symbiotic algae cultured at 23℃ for 3 weeks. Diplosphaera chodatii A07020 CGMCC No.3892 was transferred to a 4°C freezer with an external light source inside, a light intensity of 2000 lx, and a 16-hour light / 8-hour dark cycle for 4 days. Then it was transferred to a -4°C freezer with an external light source inside, a light intensity of 2000 lx, and a 16-hour light / 8-hour dark cycle for 3 days.

[0029] The symbiotic algae of Lithocarpus spp. after the -4℃ culture group, 4℃ culture group, and 4℃ / -4℃ culture group were cultured. Diplosphaera chodatii A07020 CGMCC No.3892 was transferred to a 23℃ incubator and cultured alongside a 23℃ control group of symbiotic algae, *Lithocarpus spp.* Diplosphaera chodatii Continue culturing A07020 CGMCC No.3892 together, with a light intensity of 2000 lx, alternating between 16 hours of light and 8 hours of darkness, for 2 days.

[0030] Symbiotic algae of *Lithocarpus spp.* cultured in the 23℃ control group, -4℃ culture group, 4℃ culture group, and 4℃ / -4℃ culture group. Diplosphaera chodatii A07020 CGMCC No.3892 underwent chlorophyll fluorescence measurement and real-time quantitative PCR analysis (qRT-PCR) to calculate the photosynthetic efficiency Fv / Fm value for each group and to detect two genes related to low-temperature photosynthesis. rbcL and Lhc The amount of expression of ).

[0031] See Figure 1 and Figure 2 The results of this embodiment are as follows: After 3 weeks of expanded culture, 1 week of low-temperature stimulation and 2 days of recovery, the symbiotic algae *Lithocarpus spp.* in the four groups cultured at different temperatures (23℃ control group, -4℃ culture group, 4℃ culture group and 4℃ / -4℃ culture group) showed significant differences. Diplosphaera chodatiiStatistical analysis revealed significant differences in the chlorophyll photosynthetic efficiency (Fv / Fm) of A07020 CGMCC No.3892. Compared with the 23℃ control group, the Fv / Fm values ​​of the -4℃ and 4℃ / -4℃ culture groups were significantly increased, with the -4℃ group showing a greater increase. This indicates that the -4℃ low temperature below freezing point can effectively improve the symbiotic algae growth of *Lithocarpus spp.* Diplosphaera chodatii The photosynthetic efficiency of A07020 CGMCC No.3892 was significantly lower than that of the 23℃ control group in the 4℃ culture group, indicating that the low temperature above freezing (4℃) reduces the photosynthetic efficiency of the Lithocarpus symbiotic algae. Diplosphaera chodatii The photosynthetic efficiency of A07020 CGMCC No.3892. Additionally, qRT-PCR results revealed two genes related to cryoregulation. rbcL Increased transcript abundance was observed in all low-temperature treatment groups. rbcL The relative expression level of was negatively correlated with photosynthetic efficiency; while Lhc Transcription was significantly downregulated under all low-temperature conditions. This downregulation reduces light-harvesting capacity under cold stress to prevent the accumulation of excess excitation energy. Lhc The relative expression level of [a substance] is positively correlated with photosynthetic efficiency. See [reference needed]. Figure 3 .

[0032] This invention is based on the effect of changes in culture temperature on symbiotic algae of Lithocarpus spp. Diplosphaera chodatii The growth regulation of A07020CGMCC No.3892, using the optimal growth temperature of green algae (20℃ to 25℃) as a control (23℃ in this experiment), and the fine regulation at -4℃ below freezing and 4℃ above freezing, revealed the symbiotic algae of *Lithocarpus spp.* Diplosphaera walking The phenomenon of fine-tuned low-temperature adaptation, and at the gene transcription level. rbcL and Lhc The expression patterns of the two genes explain the symbiotic algae *Lithocarpus spp.* Diplosphaera chodatii Regarding the mechanism of low-temperature adaptation, it is proposed that low-temperature treatment below the freezing point of -4℃ is a significant promoter of symbiotic algae growth in Lithocarpus spp. Diplosphaera chodatii A07020 CGMCC No.3892 Methods for photosynthetic efficiency.

[0033] Instructions for microbial preservation: Strain name: *Diplococcus kurstii* (a symbiotic algae of *Lithocarpus spp.*) Latin name: Diplosphaera chodatii Strain number: A07020 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection institution abbreviation: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Date of preservation: May 31, 2010 Collection Center Registration Number: CGMCC No. 3892 Those skilled in the art can make various adjustments and improvements to the types of raw materials, mineral levels, process parameters, and dosage forms without departing from the core ideas of this invention, and all such adjustments and improvements should fall within the protection scope of this invention.

Claims

1. A cultivation method for promoting the photosynthetic efficiency of lichen-symbiotic green algae, characterized in that, Treatment of lichen-symbiotic green algae using sub-freezing temperatures: Temperature conditions: -4℃, with alternating 16h light and 8h darkness, light intensity: 2000 lx, for 1 week; or Temperature conditions: 4℃, with alternating 16h light and 8h darkness, light intensity: 2000 lx, for 4 days, then transferred to -4℃, with alternating 16h light and 8h darkness, light intensity: 2000 lx, for 3 days.

2. The cultivation method for promoting the photosynthetic efficiency of lichen-symbiotic green algae according to claim 1, characterized in that, The lichen symbiotic green algae is *Lithocarpus symbiotic algae*. Diplosphaera chodatii A07020 has the preservation registration number CGMCC No.3892 at the China General Microbiological Culture Collection Center.

3. The cultivation method for promoting the photosynthetic efficiency of lichen-symbiotic green algae according to claim 2, characterized in that, The symbiotic algae of Lithocarpus spp. Diplosphaera chodatii The culture period for A07020 CGMCC No.3892 is 30 days.

4. The cultivation method for promoting the photosynthetic efficiency of lichen-symbiotic green algae according to claim 3, characterized in that, The method specifically includes the following steps: (1) Activation of symbiotic algae in Lithocarpus: The symbiotic algae of Lithocarpus spp. were transferred to PDA medium and cultured at 23°C with alternating light and dark cycles of 16 h and 8 h, with a light intensity of 2000 lx for 19 days. (2) Large-scale culture of Lithocarpus symbiotic algae: The activated Lithocarpus symbiotic algae were scraped into sterile water. 20 μL of Lithocarpus symbiotic algae with OD600=0.6 was inoculated into a new PDA medium and placed in an artificial climate chamber for cultivation. Under the conditions of 23℃, the algae were treated with alternating 16h light and 8h dark, and the light intensity was 2000lx for 3 weeks. (3) Grouped control culture of Lithocarpus symbiotic algae: The *Lithocarpus spp.* symbiotic algae were divided into four groups and cultured under different temperature conditions: -4℃, 4℃, 4℃ / -4℃, and 23℃. In the -4℃ group, the expanded-cultured *Lithocarpus spp.* symbiotic algae were transferred to a -4℃ refrigerator with an external light source, using a 16-hour light-8-hour dark cycle with a light intensity of 2000 lx, and cultured for one week. In the 4℃ group, the expanded-cultured *Lithocarpus spp.* symbiotic algae were transferred to a 4℃ refrigerator with an external light source, using a 16-hour light-8-hour dark cycle with a light intensity of 2000 lx, and cultured for one week. The 4℃ / -4℃ group... The expanded cultured *Lithocarpus spp.* symbiotic algae were transferred to a 4°C freezer with an external light source, and treated with alternating 16 hours of light and 8 hours of darkness at a light intensity of 2000 lx for 4 days. Then, they were transferred to a -4°C freezer with an external light source, and treated with alternating 16 hours of light and 8 hours of darkness at a light intensity of 2000 lx for 3 days. The 23°C control group consisted of *Lithocarpus spp.* symbiotic algae cultured for 3 weeks kept in an artificial climate chamber at 23°C, treated with alternating 16 hours of light and 8 hours of darkness at a light intensity of 2000 lx for another week. (4) Room temperature culture of Lithocarpus symbiotic algae: The *Lithocarpus spp.* symbiotic algae treated in the -4℃, 4℃, and 4℃ / -4℃ culture groups were transferred to an incubator at 23℃ and subjected to alternating light and dark conditions of 16 hours and 8 hours at a light intensity of 2000 lx for 2 days. The *Lithocarpus spp.* symbiotic algae in the 23℃ control group were kept in an artificial climate chamber and subjected to alternating light and dark conditions of 16 hours and 8 hours at a light intensity of 2000 lx for 2 days.

5. The cultivation method for promoting the photosynthetic efficiency of lichen-symbiotic green algae according to claim 4, characterized in that, The PDA medium was prepared by mixing 39.0 g of PDA medium powder with 1 L of distilled water.

6. A method for determining the results of a cultivation method for promoting the photosynthetic efficiency of lichen-symbiotic green algae according to any one of claims 1-5, characterized in that, Chlorophyll fluorescence measurements were performed on four groups of lichenicolous algal colony samples under different temperature cultivation conditions, which specifically included the following operating steps: (1) After placing the petri dish with the lichenicolous algal colony sample in a completely dark environment for 20 - 30 minutes for dark adaptation, then place the petri dish on the measuring stage of the chlorophyll fluorescence - imaging - gas exchange synchronous measurement system. After the measuring spot evenly covers the algal colony sample to be measured, start the measurement; (2) Calculation of the photosynthetic efficiency value Fv / Fm: After the measurement starts, first turn on the measuring light of the pulse - modulated fluorometer to obtain and record the initial basal fluorescence value Fo of the algal colony sample when all photosystem II reaction centers are in the open state. Subsequently, apply a saturating pulse to the algal colony sample to instantaneously and completely close the photosystem II reaction centers, and obtain and record the maximum fluorescence value Fm. Automatically calculate the maximum photochemical quantum yield Fv / Fm of photosystem II, which reflects the potential maximum photosynthetic efficiency of the algae, and its calculation formula is Fv / Fm = (Fm - Fo) / Fm; (3) Statistical analysis was performed on the measurement results of the - 4°C cultivation group, 4°C cultivation group, 4°C / -4°C cultivation group, and 23°C cultivation group: 6 biological replicates of the algal colony samples under each temperature cultivation condition were analyzed. One - way analysis of variance was performed using GraphPad prism 10.0 software, and Duncan's multiple - range test was used for post - hoc comparison. **** indicates P < 0.0001, *** indicates 0.0001 < P < 0.001, ** indicates 0.001 < P < 0.01, and * indicates 0.01 < P < 0.

05.

7. The result determination method according to claim 6, characterized in that, Real-time quantitative PCR analysis was performed on algal colony samples of *Lithocarpus spp.* symbiotic algae under four different temperature cultivation conditions. The analysis included the expression of photosynthetic genes in *Lithocarpus spp.* symbiotic algae at -4℃, 4℃, 4℃ / -4℃, and 23℃, and the following steps were performed: Trizol... TM Reagents were used to extract total RNA; complementary DNA was synthesized using the ABScript Neo RT Master Mix Kit; this was done to detect two genes related to photosynthesis. rbcL and Lhc To determine the expression of actin, primers were designed for real-time quantitative PCR analysis; after confirming the stability of actin gene expression, it was used as an internal control. Real-time quantitative PCR analyzer on Bio-Rad CFX96 TM The system was operated on SYBR Green I Master Mix; real-time quantitative PCR analysis showed that the photosynthetic efficiency of the Lithocarpus symbiotic algae photosystem II in the -4℃ and 4℃ / -4℃ culture groups was significantly higher than that in the 23℃ control group.

8. The result determination method according to claim 7, characterized in that, Two genes related to photosynthesis in the symbiotic algae Lithocarpus. rbcL and Lhc Quantitative characterization of gene expression levels rbcL Increased transcript abundance was observed in all low-temperature treatment groups. rbcL The relative expression level of the gene was negatively correlated with photosynthetic efficiency; while the gene Lhc Transcription was significantly downregulated under all low-temperature conditions. This downregulation reduced light-harvesting capacity under cold stress to prevent the accumulation of excess excitation energy. Lhc The relative expression level of [a substance] is positively correlated with photosynthetic efficiency.

9. The result determination method according to claim 8, characterized in that, During the process of real-time fluorescence quantitative PCR analyzer, the thermal cycling conditions of the real-time fluorescence quantitative PCR analyzer are set as follows: the temperature is set to 37 °C and run for 2 minutes, then the temperature is set to 95 °C and run for 3 minutes, followed by 40 cycles. One cycle process includes setting the temperature to 95 °C and running for 5 seconds, and setting the temperature to 60 °C and running for another 30 seconds; the amplification specificity is confirmed by melting curve analysis, and the relative expression level is calculated using the 2 - ΔΔCt method, and 3 biological replicates are set for the samples under each temperature culture condition; the statistical analysis of the relative expression level is performed using one-way ANOVA and Tukey's post hoc multiple comparison test with GraphPad prism 10.0 software. **** indicates P < 0.0001, *** indicates 0.0001 < P < 0.001, ** indicates P < 0.01, and * indicates 0.01 < P < 0.05.

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