Method for rapidly extracting high-quality genomic DNA from thick-walled microalgae and application thereof

Abstract

The application belongs to the field of genetic engineering and biotechnology, and particularly relates to a method for rapidly extracting high-quality genomic DNA from a thick-walled microalgae and application. In the present study, a set of rapid genomic DNA extraction method is optimized by combining snail enzyme with vacuum treatment to lyse Chlorella cells. Compared with the method of combining cellulase with vacuum treatment and two commercial genomic DNA extraction kits, the genomic DNA obtained by the method has higher integrity. In addition, the genomic DNA extracted by the optimized method is completely suitable for downstream applications such as gene cloning and genotyping, which significantly improves the extraction integrity of Chlorella genomic DNA, and provides a new idea for the extraction of high-quality genomic DNA of other types of microalgae.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and biotechnology, specifically relating to a method and application for rapidly extracting high-quality genomic DNA from thick-walled microalgae. Background Technology

[0002] Microalgae, as photosynthetic organisms, can produce a variety of high-value natural bioactive compounds, including vitamins, essential amino acids, polyunsaturated fatty acids, minerals, carotenoids, enzymes, and cellulose, making them a promising source of innovation for novel foods and functional products. Environmental disturbances and abiotic stresses, such as temperature, pH, salinity, ultraviolet radiation, light, and nutrient availability, affect the growth rate, metabolic level, and biochemical composition of microalgae. Microalgae range in size from a maximum of 1000 micrometers to a minimum of only 0.95 micrometers. They are widely distributed in various habitats, including freshwater, marine, and terrestrial environments, such as snow cover, sea ice (psychrophiles), hot springs (thermophiles), and salt lakes (halophiles), providing ideal models for studying the evolution of ecological tolerance and the biosynthesis of functional products. Sufficient genetic information is needed to deeply understand the characteristics of microalgae.

[0003] However, the complex cell wall structure of microalgae often hinders the effective extraction of their genomic material, which has become a limiting factor in the in-depth exploration of microalgal resources. Traditional DNA extraction methods mostly employ physical disruption of cells, including liquid homogenization, sonication, glass bead grinding, and liquid nitrogen grinding. Genomic DNA is then purified using chloroform / isoamyl alcohol and / or cesium chloride (CsCl), a time-consuming process that makes it difficult to process multiple samples simultaneously. Furthermore, the obtained genomic DNA often suffers from severe contamination and shearing breaks. These methods have also been applied to microalgal DNA extraction. To obtain high-quality microalgal DNA, studies have used a complex enzyme containing lysin, Onozuka R-10 cellulase, pectinase, and pectin lyase for cell lysis, but this enzyme treatment scheme failed to improve the extraction quality of microalgal genomic DNA. This study establishes a rapid extraction method to obtain high-quality microalgal genomic DNA: replacing the cell homogenization step with snail enzyme combined with vacuum treatment for lysing JB6 and other microalgal species. The obtained DNA has good integrity and is suitable for downstream applications such as PCR amplification and genotyping of new genes. Summary of the Invention

[0004] To address the problems existing in the prior art, the purpose of this invention is to design and provide a method and technical solution for rapidly extracting high-quality genomic DNA from thick-walled microalgae.

[0005] The present invention is implemented using the following technical solutions: The first aspect of this invention provides a method for rapidly extracting high-quality genomic DNA from thick-walled microalgae, comprising the following steps: S.1 The microalgae to be treated are cultured in liquid culture medium with shaking for 4-5 days until A 600 The optical density value reaches 14-16; S.2 Centrifuge the culture medium described in S.1 to collect microalgae cells, add an enzyme solution and then vacuum process to obtain an enzymatic hydrolysate; the enzyme solution is snail enzyme or cellulase Onozuka R-10. S.3 Centrifuge the enzymatic hydrolysate described in S.2, add DNA extraction solution to the precipitate, centrifuge after sufficient reaction, add isopropanol and mix well, centrifuge to collect DNA precipitate, wash with ethanol and dry thoroughly at room temperature. S.4 The DNA precipitate was resuspended in nuclease-free water to obtain microalgal genomic DNA.

[0006] Furthermore, the oscillation culture conditions described in step S.1 are 20-25℃ and 100-150 rpm.

[0007] Furthermore, the enzyme solution concentration in step S.2 is 0.05-0.5%, and the amount added is 100-300 μL.

[0008] Furthermore, the vacuum treatment conditions described in step S.2 are -80~-100 kPa, and the treatment time is 5-50 min.

[0009] Furthermore, the DNA extraction solution described in step S.3 includes 100-150 mM Tris-HCl, pH 7.5, 200-300 mM NaCl, 20-30 mM EDTA, and 0.4-0.6% SDS.

[0010] Furthermore, the sufficient reaction conditions described in step S.3 are: standing at -15~-25℃ for 10 min, and heating at 90-100℃ for 2-4 min.

[0011] Furthermore, the centrifugation conditions described in step S.2 are 4000-6000 g for 2-5 min; the centrifugation conditions described in step S.3 are 4000-6000 g for 2-5 min and 10000-15000 g for 5-15 min.

[0012] Furthermore, the thick-walled microalgae include Chlorella ( Chlorella sp. ), slender Euglena ( Euglena gracilis ), Anabaena ( Anabaena sp.), Brown finger algae (sp.), Phaeodactylum tricornutum ) and Chlamydomonas reinhardtii ( Chlamydomonas reinhardtii Any one of the following.

[0013] A second aspect of the present invention provides the application of the above method in the rapid extraction of high-quality genomic DNA from thick-walled microalgae.

[0014] Furthermore, the thick-walled microalgae mentioned include Chlorella ( Chlorella sp. ), slender Euglena ( Euglena gracilis ), Anabaena ( Anabaena sp.), Brown finger algae (sp.), Phaeodactylum tricornutum ) and Chlamydomonas reinhardtii ( Chlamydomonas reinhardtii Any one of the following.

[0015] The present invention has the following beneficial effects: This invention utilizes a combination of snail enzyme and vacuum treatment to lyse Chlorella cells. Compared to cellulase combined with vacuum treatment and two commercial genomic DNA extraction kits, the genomic DNA obtained using this method exhibits higher integrity. Furthermore, the genomic DNA extracted using this optimized method is fully suitable for downstream applications such as gene cloning and genotyping, providing new insights for high-quality genomic DNA extraction research from other microalgae species. Attached Figure Description

[0016] Figure 1 JB6 morphological characteristics: Laser scanning confocal microscopy image of JB6 cells expressing blue fluorescent protein (BFP); Red: autofluorescence of chloroplasts; Blue: BFP; Figure 2 Growth of JB6 after treatment with snail enzyme and cellulase (under normal pressure and vacuum conditions); 0: untreated control; Figure 3 Electrophoretic analysis of genomic DNA extracted by different methods; M: 15 kbp DNA label; H2O+V: JB6 vacuum-treated without snail enzyme, with water replacing the DNA extraction buffer; V: JB6 vacuum-treated without snail enzyme, then DNA extraction buffer added; S+V: Snail enzyme combined with vacuum treatment, JB6 treated with both snail enzyme and vacuum before being added to the DNA extraction buffer; C+V: Cellulase combined with vacuum treatment, JB6 treated with both cellulase and vacuum before being added to the DNA extraction buffer; KitA: Kit A; KitB: Kit B; Figure 4 Optimization of snail enzyme concentration; Figure 4 In this context, A represents the lytic effect of snail enzyme on JB6 cells; Figure 4 In this context, B represents the lytic effect of cellulase on JB6 cells; Figure 4 In the image, C represents the electrophoretic pattern of genomic DNA treated with snail enzyme in combination with vacuum; M represents a 15000 bp DNA marker; 0 represents the control without enzyme; lanes 3-6 represent DNA extracted from genomic DNA treated with different concentrations of snail enzyme. Figure 4 In the image, D represents the electrophoretic pattern of genomic DNA treated with cellulase in combination with vacuum; M represents a 15000 bp DNA marker; 0 represents the control without enzyme; lanes 3-6 represent DNA extracted from DNA treated with different concentrations of cellulase. Figure 5 Vacuum processing time optimization; Figure 5 In the figure, A represents the lysis effect of 0.5% snail enzyme combined with different vacuum treatment times (10, 20, 30, 40 minutes) on JB6 cells; Figure 5 In the figure, B represents the lysis effect of 0.5% cellulase combined with different vacuum treatment times (10, 20, 30, 40 minutes) on JB6 cells; Figure 5 In the table, C represents the electrophoretic analysis of genomic DNA extracted after vacuum treatment for different times (10, 20, 30, 40 minutes); M represents 15000 bp DNA labeling; lanes 2 and 7: unenzyme control (0); lanes 3-6: DNA extracted after treatment with 0.5% snailase for different vacuum treatment times; lanes 8-11: DNA extracted after treatment with 0.5% cellulase for different vacuum treatment times. Figure 6 Electrophoretic analysis of PCR products from the amplification of genes of different lengths using snail enzymatic method to extract genomic DNA; M: 10000 bp DNA marker; Gene A: 4472 bp; Gene B: 3197 bp; Gene C: 3236 bp; Gene D: 2563 bp; Gene E: 1381 bp; Gene F: 1433 bp; Figure 7 In the image, A represents an agarose gel electrophoresis image of DNA extracted from other plants using the snail enzymatic method; M represents a 15000bp DNA marker; 1: Populus tomentosa; 2: Actinopterygium indicum; 3: Arabidopsis thaliana; 4: Rice; 5: Peach; 6: Tobacco. Figure 7 B in the image represents an agarose gel electrophoresis image of DNA extracted from four other algal strains using the snail enzyme method; M: 15000 bp DNA marker; 1: Euglena scabra; 2: Anabaena natans; 3: Phaeodactylum tricornutum; 4: Chlamydomonas reinhardtii; Figure 8 Schematic diagram of DNA extraction process. Detailed Implementation

[0017] To better understand the present invention, the following specific embodiments further illustrate the invention, but the content of the present invention is not limited to the following embodiments.

[0018] Example 1: Snail enzyme combined with vacuum treatment severely affects the growth of JB6. 1. Microalgae materials This study used Chlorella ( Chlorellasp.) JB6, this algal strain was originally isolated from soil in the Songnen Plain of Northeast China (43°30'-48°40' N, 121°30'-127°00' E). JB6 was cultured in BG11 liquid medium (Table 1) at 22°C and 110 rpm for 4-5 days until the optical density at A600 reached approximately 15. Other algal strains include Euglena scabra ( Euglena gracilis ), Anabaena ( Anabaena sp.), Brown finger algae (sp.), Phaeodactylum tricornutum ) and Chlamydomonas reinhardtii ( Chlamydomonas reinhardtii All of them were purchased from the Freshwater Algae Culture Bank (FACHB) of the Institute of Hydrobiology, Chinese Academy of Sciences.

[0019] Table 1 BG11 Culture Medium Formulation .

[0020] 2. Enzyme solution preparation Most algal cell walls are composed of cellulose, hemicellulose, and polysaccharides. Snail enzyme (Sangon, China) and cellulase Onozuka R-10 (Yakult, Japan) were used to digest the cell walls in this experiment. 2% stock solutions of snail enzyme and Onozuka R-10 cellulase were prepared separately and then diluted to final concentration gradients of 0.05%, 0.1%, 0.25%, and 0.5%.

[0021] 3. Growth detection of JB6 after enzyme treatment Collect JB6 cells by centrifuging at 5,000 g for 3 min in 1.5 mL centrifuge tubes. Add 200 μL of enzyme solutions of different concentrations to each tube and incubate for different times under -90 kPa vacuum or normal pressure. After centrifugation, discard the supernatant and wash the cell pellet twice with sterile water. Then, perform serial dilutions (5... -1 5 -2 5 -3 Take 4 μL of the stock solution and cell sap of various dilutions and seed them on BG11 solid medium containing 1% D-glucose and 300 mM NaHCO3. Incubate at 22℃ for 7-10 days and observe growth.

[0022] The results showed that JB6 cells were 5-10 μm in diameter and possessed a single chloroplast and a rigid cell wall. Figure 1 Traditional freeze-thaw methods or mortar grinding are insufficient for effectively extracting high-quality genomic DNA. This invention utilizes snail enzymes and cellulase to lyse cells. JB6 cells were treated for 30, 60, 90, 120, and 150 minutes, followed by gradient dilutions at different concentrations (5... -1 5 -2 5 -3For the untreated control group, 4 μL of stock culture and dilution were inoculated onto BG11 solid medium containing 1% D-glucose or 300 mM NaHCO3. Experiments showed that compared to cellulase (…),… Figure 2 In the B group, snail enzyme significantly inhibited the growth of JB6 on high-concentration NaHCO3 (300 mM) medium. Figure 2 (A in the text). Further research showed that 0.5% snail enzyme combined with vacuum treatment for 30 min could completely eliminate the viability of JB6 on 300 mM NaHCO3 medium. Figure 2 (C in the text), while cells treated with cellulase under the same conditions can still survive ( Figure 2 (D in the text). This indicates that 0.5% snail enzyme combined with vacuum treatment for 30 min can completely destroy the JB6 cell wall, while cellulase failed to achieve the same effect. Therefore, snail enzyme was chosen for cell lysis before DNA extraction.

[0023] Example 2: Optimization of Genomic DNA Extraction Protocol Transfer 200 μL of liquid-cultured JB6 cells to a 1.5 mL centrifuge tube, add 200 μL of enzyme solution, and vacuum treat. Centrifuge at 5,000 g for 3 min, discard the supernatant completely, and add 200 μL of DNA extraction buffer (200 mM Tris-HCl pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS). Incubate the cell pellet and extraction buffer at -20°C for 10 min, then heat at 95°C for 3 min. Centrifuge at 5,000 g for 3 min, transfer the supernatant to a new tube, and add 200 μL of isopropanol to mix. Centrifuge at 12,000 g for 10 min to collect the DNA pellet, wash with 500 μL of 70% ethanol, and dry thoroughly at room temperature. Resuspend the DNA pellet in 50 μL of nuclease-free water.

[0024] To investigate whether snail enzyme and vacuum treatment can improve DNA quality, JB6 cells were lysed with 0.5% snail enzyme (with vacuum and non-vacuum controls) instead of the grinding step. The DNA extraction flowchart is shown below. Figure 8 As shown. Figure 3 The largest DNA marker band in the sample was 15 kbp (lane 1), and the expected genomic DNA band should be higher than this position. The sample treated with 0.5% snailase combined with vacuum for 30 min showed the highest DNA quality, with a single, clear band and no tailing (lane 4). Compared to the control group without snailase (lanes 2 and 3), snailase combined with vacuum treatment significantly improved DNA extraction; while 0.5% cellulase combined with vacuum treatment failed to extract DNA (lane 5).

[0025] To determine the cost-effective enzyme concentration, cells were treated with different concentrations of snail enzyme (0.05%, 0.1%, 0.25%, and 0.5%) under vacuum for 10 minutes. DNA extraction buffer was then added, and the cells were heated at 95°C for 5 minutes. After centrifugation, images were taken. DNA was extracted, separated by 1% agarose gel electrophoresis, and observed using SYBR safe DNA gel red staining. The results showed that low-concentration enzyme solution combined with vacuum treatment failed to effectively extract DNA, indicating that 0.5% was the more suitable lysis concentration. Figure 4 ).

[0026] To optimize the enzyme treatment time for extraction, different time gradients were set: JB6 cells were treated with 0.5% snailase and 0.5% cellulase for 10, 20, 30, and 40 min, respectively, followed by the addition of DNA extraction buffer and heating at 95℃ for 5 min. After centrifugation, images were taken and recorded. DNA was extracted, separated by 1% agarose gel electrophoresis, and observed by SYBR safe DNA gel red staining to assess genomic DNA integrity. The results showed that high-quality genomic DNA could be obtained by using 0.5% snailase combined with 20 min of vacuum treatment. Figure 5 Under the same conditions, cellulase combined with vacuum treatment did not improve DNA extraction efficiency. Figure 4 , Figure 5 ).

[0027] Example 3: Extraction of genomic DNA using a commercially available kit Two commercially available genomic DNA extraction kits were used to extract JB6 genomic DNA: Plant DNA Isolation Reagent (Cas NO. 100-44-7, TaKaRa, Japan) and Ykang minEasy Universal Genomic DNA Extraction Kit (No. YK00A3, Ykang, China). All steps were strictly performed according to the manufacturer's instructions, and the integrity of the extracted DNA was verified by 1% agarose gel electrophoresis.

[0028] The results showed that the DNA fragments extracted by both commercial kits (lanes 6 and 7) were smaller than those extracted by the snail enzymatic method (lane 4), indicating that the DNA extracted by the snail enzymatic method had higher integrity. Figure 3 ).

[0029] kitA: Plant DNA Isolation Reagent (Cas NO.100-44-7, TaKaRa, Japan) Steps: Weigh 10-100 mg of cells into a 1.5 mL microcentrifuge tube and freeze at -20 °C.

[0030] Place the frozen plant tissue at room temperature for 5 minutes until thawed.

[0031] Brief centrifugation causes the plant tissue to aggregate at the bottom of the tube.

[0032] Use a pipette tip to physically grind the plant tissue at the bottom of the tube, repeatedly pressing and crushing the sample 10 times.

[0033] Add 400 μL of Extract 1 and vortex at maximum speed for 5 seconds. If the plant tissue clumps together at the bottom of the tube, gently tap the bottom of the tube with your finger to resuspend it.

[0034] After brief centrifugation, add 80 μL of Extract 2 and vortex at maximum speed for 5 seconds. A white precipitate should appear in the solution after adding Extract 2, and the solution will become cloudy during vortexing. If the plant tissue clumps together at the bottom of the tube, gently tap the bottom with your finger to resuspend it.

[0035] After a brief centrifugation, add 150 μL of extraction buffer 3 and vortex at maximum speed for 5 seconds. If the plant tissue clumps together at the bottom of the tube, gently tap the bottom of the tube with your finger to resuspend it.

[0036] Centrifuge gently (for no more than 2 seconds), then incubate at 50°C for 15 minutes.

[0037] Centrifuge at 12,000 rpm for 15 minutes at 4°C to separate the aqueous phase (upper layer) from the organic phase (lower layer).

[0038] Carefully collect as much of the clear supernatant as possible, being careful not to aspirate the lower liquid phase or tissue debris. Approximately 400 microliters of liquid can be recovered.

[0039] Add an equal volume of isopropanol to the collected aqueous phase and mix gently.

[0040] Centrifuge at 12,000 rpm for 10 minutes at 4°C.

[0041] Remove the supernatant, being careful not to disturb the precipitate.

[0042] Add 1 ml of 70% ethanol to wash the precipitate.

[0043] Centrifuge at 12,000 rpm for 3 minutes at 4°C.

[0044] Remove the supernatant, being careful not to disturb the precipitate.

[0045] Dry the precipitate and resuspend it with an appropriate amount of TE buffer (about 20 μL).

[0046] kitB: Ykang minQuick Plant Genomic DNA Extraction Kit (Yk00A5) Grind with liquid nitrogen, 50-150 mg fresh sample.

[0047] Add 700 μL of Buffer PD1 to the ground sample, vortex to mix, incubate at 60°C for 30 min, add 700 μL of chloroform, vortex to mix, and centrifuge.

[0048] Add 500 μL of Buffer PD2 and 10 μL of RNase A to the supernatant, mix by inversion, and then add an equal volume of anhydrous ethanol and mix by inversion.

[0049] Transfer <700 μL of the mixture, centrifuge, add 500 μL of buffer PW1, centrifuge, discard the filtrate, add 500 μL of buffer PW2, centrifuge, discard the filtrate.

[0050] Add 60 μL of preheated Buffer DE and centrifuge at 12,000 rpm for 1 min to collect genomic DNA.

[0051] The purity and yield of JB6 cell DNA obtained by the above DNA extraction method are shown in Table 2.

[0052] Table 2. Purity and yield of JB6 cell DNA obtained by various DNA extraction methods .

[0053] Example 4: Gene cloning based on DNA extraction using snail enzymatic method To verify DNA quality, six genes of varying lengths (1381-4472 bp) from JB6 were selected, with nucleotide sequences shown in SEQ ID NO.7-12. DNA extracted using the snail enzyme method was diluted 10-fold as a template for amplification. Primer sequences are shown in Table 3, corresponding to SEQ ID NO.1-6 in sequence. PCR amplification was performed using KOD FX neo polymerase (KFX-201, TOYOBO, Japan), and the products were analyzed by 1% agarose gel electrophoresis.

[0054] Table 3 Primer Sequences .

[0055] The results showed that all target genes were successfully amplified. Figure 6 This demonstrates that the DNA extracted by this method is suitable for downstream work such as gene cloning and typing.

[0056] Example 5: Application of snail enzymatic method in other organisms This method was not successfully applied to various higher plants, including Populus tomentosa, Actinidia chinensis, Arabidopsis thaliana, rice, peach, and tobacco. Figure 7 (A in the text), but it can effectively extract genomic DNA from microalgae such as Euglena scabra, Anabaena, Brown Finger Algae, and Chlamydomonas reinhardtii. Figure 7 (B in the middle).

Claims

1. A method for rapidly extracting high-quality genomic DNA from thick-walled microalgae, characterized in that, Includes the following steps: S.1 The microalgae to be treated are cultured in liquid culture medium with shaking for 4-5 days until A 600 The optical density value reaches 14-16; S.2 The culture medium described in S.1 is centrifuged to collect microalgal cells. An enzyme solution is added and then vacuum-treated to obtain an enzymatic hydrolysate. The enzyme solution is a 0.5% snail enzyme solution. The vacuum treatment lasts for 20-40 minutes. S.3 Centrifuge the enzymatic hydrolysate described in S.2, add DNA extraction solution to the precipitate, centrifuge after sufficient reaction, collect the supernatant, add isopropanol and mix well, centrifuge to collect the DNA precipitate, wash with ethanol and dry thoroughly at room temperature; the DNA extraction solution includes 200 mM Tris-HCl, pH 7.5, 250 mM NaCl, 25 mM EDTA, and 0.5% SDS; the sufficient reaction conditions are -20℃ for 10 min and 95℃ for 3 min. S.4 The DNA precipitate was resuspended in nuclease-free water to obtain microalgal genomic DNA.

2. The method as described in claim 1, characterized in that, The shaking culture conditions described in step S.1 are 20-25℃ and 100-150 rpm.

3. The method as described in claim 1, characterized in that, The centrifugation conditions described in step S.2 are 4000-6000 g for 2-5 min; the centrifugation conditions described in step S.3 are 4000-6000 g for 2-5 min and 10000-15000 g for 5-15 min.

4. The method as described in claim 1, characterized in that, The thick-walled microalgae include any one of Euglena scabra, Anabaena, Phaeodactylum tricornutum, and Chlamydomonas reinhardtii.

5. The application of the method according to any one of claims 1-4 in the rapid extraction of high-quality genomic DNA from thick-walled microalgae.

6. The application as described in claim 5, characterized in that, The thick-walled microalgae mentioned include any one of Euglena scabra, Anabaena, Phaeodactylum tricornutum, and Chlamydomonas reinhardtii.

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