Enzymatic hydrolysate for preparing eucheuma or kappaphycus algae protoplast and preparation method
By using enzyme extract from the digestive system of the horse-foot snail to prepare enzymatic hydrolysate, the problem of low efficiency in preparing protoplasts from algae of the genera *Euphorbia* and *Kappaella* was solved, achieving efficient preparation of high-quality protoplasts that meet the sample requirements for third-generation genome sequencing.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-24
- Publication Date
- 2026-03-24
AI Technical Summary
Existing technologies cannot effectively prepare protoplasts of algae in the genera *Euphorbia* and *Kappaella* with high carrageenan content, making third-generation genome sequencing difficult. Existing enzymatic digestion methods are inefficient and have poor reproducibility.
Enzymatic extracts from the digestive system of Trochus pyramis Born (family Trochusidae), PBS, and mannitol were used to prepare an enzymatic hydrolysate through specific steps. This hydrolysate was then used to treat algae, and combined with antibiotics and filtration methods, to achieve efficient collection of protoplasts.
High-quality protoplasts were obtained, meeting the requirements of third-generation genome sequencing. The cells were in good condition with few dead cells, and the cell collection volume reached ≥10⁶ cells/100 mL, providing a solid foundation for genome research.
Smart Images
Figure CN121718484A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of enzymatic hydrolysate and protoplast preparation, and particularly relates to a protoplast preparation enzymatic hydrolysate suitable for tropical macroalgae of the genus Eucheuma or the genus Kappaphycus, a preparation method thereof, and protoplast preparation and application. BACKGROUND
[0002] Due to the extremely high carrageenan content, the sequencing of the third-generation high-quality genome of the tropical macroalgae of the genus Kappaphycus / Eucheuma has been difficult to carry out so far. The only high-quality genome reported so far is that of E. dimorphus, and the lack of genome seriously hinders the development of basic research work such as genetic breeding and functional gene mining of the genus Kappaphycus / Eucheuma.
[0003] Obtaining fresh and live protoplast cells with low carrageenan residues and low cell wall polysaccharide content can provide raw materials for the third-generation genome sequencing, greatly improve the success probability of HIC sequencing, and provide a solid foundation for the basic research work of the genus Kappaphycus / Eucheuma. Obtaining the protoplast of Kappaphycus / Eucheuma is the only way and method to realize the sequencing of the third-generation high-quality genome.
[0004] In the existing protoplast preparation technology of algae, the commonly used enzyme solution is mainly plant enzymes such as cellulase, hemicellulase, pectinase, and two animal enzymes, namely, abalone enzyme and snail enzyme. Such research has been carried out for several years, and there has been no update in recent years. There are few literatures on the preparation of algal protoplasts, and most of them are in Chinese and are too brief to be repeated. There are many operations that cannot be repeated in the process of reproducing the experimental steps, and the reproducibility is poor. The use of existing enzymes and enzymatic methods in the literature for enzymatic hydrolysis of tropical macroalgae Kappaphycus / Eucheuma shows that the cell clusters or protoplasts of Kappaphycus / Eucheuma cannot be separated, and the enzymatic efficiency is almost 0. Therefore, it is an urgent need to develop a protoplast enzymatic hydrolysate for high-carrageenan-content tropical macroalgae to break through the bottleneck of protoplast preparation. SUMMARY
[0005] The present application provides a special enzymatic hydrolysate and a preparation method thereof, and a protoplast preparation process, which realizes the efficient and high-quality preparation of the protoplast of the alga of the genus Eucheuma / Kappaphycus.
[0006] In view of the technical pain points in the prior art, such as low protoplast preparation efficiency of the alga of the genus Eucheuma / Kappaphycus and the inability to meet the requirements of the third-generation genome sequencing, the present application provides a special enzymatic hydrolysate and a preparation method thereof, and a protoplast preparation process, which realizes the efficient and high-quality preparation of the protoplast of the alga of the genus Eucheuma / Kappaphycus. TECHNICAL SCHEME
[0007] The present application provides an enzymatic solution for preparing Eucheuma or Kappaphycus algae protoplasts, which makes it possible to prepare and collect Eucheuma / Kappaphycus protoplasts for the first time, and the obtained cell sample contains no common salt and protein residues, the amount of collected intact cells is ≥10 6 cells / 100 mL, the cell state is good, the number of dead cells is small, and the sample meets the requirements of three-generation genome HIC sequencing.
[0008] The Eucheuma or Kappaphycus algae in the present application grow in the shallow sea area of the tropics and subtropics, and prefer warm, clear, stable salinity, and a certain water flow.
[0009] Preparation of a Trochus maculosus digestive system enzyme extract
[0010] The present application provides an enzymatic solution for preparing Eucheuma or Kappaphycus algae protoplasts, which makes it possible to prepare and collect Eucheuma / Kappaphycus protoplasts for the first time, and the obtained cell sample contains no common salt and protein residues, the amount of collected intact cells is ≥10 Trochus pyramis cells / 100 mL, the cell state is good, the number of dead cells is small, and the sample meets the requirements of three-generation genome HIC sequencing. Trochus pyramis The present application provides an enzymatic solution for preparing Eucheuma or Kappaphycus algae protoplasts, which makes it possible to prepare and collect Eucheuma / Kappaphycus protoplasts for the first time, and the obtained cell sample contains no common salt and protein residues, the amount of collected intact cells is ≥10 cells / 100 mL, the cell state is good, the number of dead cells is small, and the sample meets the requirements of three-generation genome HIC sequencing.
[0011] Further, the preparation method of the Trochus maculosus digestive system enzyme extract is as follows: Trochus pyramis The present application provides an enzymatic solution for preparing Eucheuma or Kappaphycus algae protoplasts, which makes it possible to prepare and collect Eucheuma / Kappaphycus protoplasts for the first time, and the obtained cell sample contains no common salt and protein residues, the amount of collected intact cells is ≥10 cells / 100 mL, the cell state is good, the number of dead cells is small, and the sample meets the requirements of three-generation genome HIC sequencing. Trochus pyramis The present application provides an enzymatic solution for preparing Eucheuma or Kappaphycus algae protoplasts, which makes it possible to prepare and collect Eucheuma / Kappaphycus protoplasts for the first time, and the obtained cell sample contains no common salt and protein residues, the amount of collected intact cells is ≥10 cells / 100 mL, the cell state is good, the number of dead cells is small, and the sample meets the requirements of three-generation genome HIC sequencing. Trochus pyramis The present application provides an enzymatic solution for preparing Eucheuma or Kappaphycus algae protoplasts, which makes it possible to prepare and collect Eucheuma / Kappaphycus protoplasts for the first time, and the obtained cell sample contains no common salt and protein residues, the amount of collected intact cells is ≥10 cells / 100 mL, the cell state is good, the number of dead cells is small, and the sample meets the requirements of three-generation genome HIC sequencing. The present application provides an enzymatic solution for preparing Eucheuma or Kappaphycus algae protoplasts, which makes it possible to prepare and collect Eucheuma / Kappaphycus protoplasts for the first time, and the obtained cell sample contains no common salt and protein residues, the amount of collected intact cells is ≥10 cells / 100 mL, the cell state is good, the number of dead cells is small, and the sample meets the requirements of three-generation genome HIC sequencing.
[0012] Further, the centrifugation in (3) and (4) is performed at 2000-3000 rpm.
[0013] Further, the centrifugation in (3) and (4) is performed at 2500 rpm.
[0014] Further, the leaching time in (1) is 12 h. Method for preparing protoplast of eucheuma / capra algae
[0015] The application also provides a method for preparing protoplast of eucheuma or capra algae by using the enzymatic hydrolysate, 2 g of the eucheuma or capra algae can be enzymatically hydrolyzed per 40 mL of the enzymatic hydrolysate; the specific method is as follows: (1) Algae pretreatment: fresh and full eucheuma or capra algae is selected, and after brushing to remove surface impurities, overnight sterilization treatment is performed; the algae is cut into 0.1 cm*0.1 cm fragments for standby.
[0016] (2) Enzymatic hydrolysis reaction: the enzymatic hydrolysate is prepared according to the proportion, 2 g of the pretreated algae fragments is added into 40 mL of the enzymatic hydrolysate, and the centrifugal tube is placed in a 26℃ shaking bed for enzymatic hydrolysis at a speed of 45 r / min for 10-16 h.
[0017] (3) Protoplast collection and purification: after the enzymatic hydrolysis is completed, the cell state is confirmed by microscopic observation, and then the protoplast is collected by centrifugation, and bacteria and other impurities are removed by applying antibiotics and filtering to obtain high-purity protoplast.
[0018] Further, the enzymatic hydrolysis time of the enzymatic hydrolysate and the eucheuma or capra algae is 10-16 h.
[0019] The application also provides the enzymatic hydrolysate or the protoplast of eucheuma or capra algae prepared by the above method.
[0020] Further, the protoplast can be subjected to genome sequencing, and the quality and purity meet the sample requirements of third-generation genome HIC sequencing.
[0021] Further, the protoplast has a cell collection amount of ≥10 6 per 100 mL.
[0022] Here, the cell collection amount refers to the intact cell collection amount.
[0023] This invention screens and prepares enzyme solutions for various algae-eating snails and shellfish in Hainan Province. Enzymatic hydrolysis of thalli from the genera *Euphorbia* and *Kappaella* was performed, with the most effective *Heliotropium* species (family Heliotropidae) selected as the hydrolysis raw material. The enzyme preparation method was updated and simplified, resulting in higher hydrolysis efficiency, a large number of protoplast cells were dissociated, and these cells exhibited good activity, reducing cell shrinkage, water absorption and swelling, cell rupture, and leakage of contents. A large number of protoplast cells (up to 10 cells collected) were obtained while removing carrageenan. 6 (each cell per 100 mL) provides clean experimental material for subsequent experiments (such as high-quality genome sequencing, single-cell sequencing, etc.), free from common salt residues, and provides a solid foundation for research on genetic breeding of algae such as Euphorbia / Kappaella. Attached Figure Description
[0024] Figure 1 The images show the dissociation fields of enzyme extracts from different species of *Kappaella asiatica* cells under a 40x microscope. The red bar in the image represents 50µm. AB. Dissociation fields of abalone enzyme extract; CD. Dissociation fields of horseshoe snail enzyme extract; EJ. Dissociation fields of enzyme extracts from other species such as whelk. Figure 2 Fields of view of dissociated Kappaella cells from enzyme extracts of different organs of *Heliotropium indicum* under a 40x microscope; A. Fields of digestive gland enzyme solution; B. Fields of gastric enzyme solution; C. Fields of intestinal enzyme solution; D. Fields of esophageal enzyme solution; E. Fields of gastric + intestinal + esophageal enzyme solution; F. Fields of digestive gland + gastric + intestinal enzyme solution; Figure 3 This is a 40x magnification view of Kappa algae cells after 6 hours of enzymatic hydrolysis. Figure 4 The image shows a 40x microscope view of Kappa algae cells after 10-12 h of enzymatic hydrolysis. Figure 5 The image shows a 40x magnification view of Kappa algae cells after 24 hours of enzymatic hydrolysis. Figure 6 The field of view of the Kappa algae cell clusters collected under a 4x microscope; Figure 7 The field of view of the Kappa algae cell clusters collected under a 40x microscope; Figure 8 For data quality assessment; Figure 9 Evaluation of the second and third generation genome assembly results of *Kappa longiflora*; A. Genomic base statistics; B. Sequencing depth distribution; C. Busco evaluation results; Figure 10 Genome GC content and depth distribution map; main plot, GC content distribution map; horizontal axis, GC content; vertical axis, sequencing depth; top bar chart, GC content distribution map; right bar chart, sequencing depth distribution map; Figure 11 The field of view shows the effect of plant enzymes on the dissociation of Euphorbia milii / Kappaella cells; the red scale bar in the figure is 50 µm. Detailed Implementation
[0025] Example 1: *Heliotropium* genus, family Heliotropidae Trochus pyramis Determination of Born's enzymatic hydrolysis raw materials (1) Preparation of experimental materials: Abalone, horseshoe snail (commonly known as white snail), whelk, wavy barnacle (commonly known as mango snail) and Ceylon snail with uniform size, good vitality and strong adsorption capacity were obtained at Tanmen Wharf, Tanmen Town, Qionghai City, Hainan Province (110.60029°N, 19.24896°E). Two of each species were placed in a flowing water pool at 28°C for starvation treatment and synchronous culture for 2 days. The samples were treated according to the grouping shown in Table 1 below. The whole process was carried out on ice.
[0026] Table 1. Species, dosage, and selected parts for each group
[0027] (2) Preparation of enzyme extract: The “digestive glands + stomach + intestines” taken from each group were soaked in sterile fresh water for 10 min, then soaked in antibiotic solution for 10 min, then ground on ice, and 1×PBS solution with the same volume as the grinding solution was added for extraction. (3) Take two times the volume of 1×PBS solution from step (2) into a new centrifuge tube, add ammonium sulfate powder on ice, dissolve it and mix it with the "digestive gland + stomach + intestine + PBS" extract from the previous step. At this time, the ratio of ammonium sulfate added is 13.4 g / 100 mL.
[0028] (4) After immersion at 4℃ for 12 h, centrifuge at 2500 rpm and 4℃ for 20 min to remove the precipitate and retain the supernatant; (5) Add ammonium sulfate (47.8 g / 100 mL) again on ice, let stand at 4°C for 5.5 h, centrifuge at 2500 rpm at 4°C for 20 min, and collect the precipitate; resuspend the precipitate with 10 mL of 1×PBS, and dialyze overnight at 4°C using a 3.5 kDa dialysis bag. The dialysate was changed 4 times during the dialysis process, which was carried out on ice throughout.
[0029] (6) Use a 0.45 µm diameter filter membrane to filter the dialyzed enzyme solution, and store the filtered enzyme solution at 4℃ for later use.
[0030] Experimental results: Enzymatic hydrolysates of different species were used to hydrolyze the algae of the genera *Euphorbia* and *Kappaella*. The number of individual algal cells that were dissociated, from most to least, was: horseshoe snail > abalone > whelk / wasp clam / Ceylon snail. Among these, the abalone enzyme extract showed a small number of cells that were dissociated, and many cells were broken and had contents leaking out, resulting in poor morphology and condition. Figure 1 AB); The enzyme extract of horseshoe snails showed that a large number of cells were dissociated, with intact cell morphology and good color and vitality. Figure 1 CD); almost no cell dissociation was observed in the enzyme extracts of whelk, wavy barbarian clam, and Ceylon whelk. Figure 1 EJ). The cellular fields of view for the dissociation of various enzymes are as follows. Figure 1 As shown.
[0031] Example 2: *Heliotropium* (family Heliotropidae) Trochus pyramis Determination of Born's extract (1) Experimental grouping: Seven horseshoe snails of uniform size, good vitality and strong adsorption capacity were obtained and placed in a flowing water pool at 28℃ for starvation treatment and synchronous culture for 2 days; the shells were soaked in sterile fresh water for 10 min and then soaked in antibiotic solution for 10 min; the samples of each group were dissected according to the grouping shown in Table 2 below, and the enzymatic hydrolysis effect of different combinations of digestive glands, stomach, intestines, esophagus and gonads on algae was tried. The whole process was carried out on ice.
[0032] Table 2. Selection of body parts for each group
[0033] (2) Enzyme extraction: The enzyme solution was extracted, precipitated, dialyzed and purified according to steps (2)-(6) in Example 1.
[0034] Experimental results: The enzymatic hydrolysates from each group were used to enzymatically hydrolyze the algae of the genera *Euphorbia* and *Kappaella*. The number of individual algal cells that were dissociated, from most to least, was: digestive gland + stomach + intestine. Figure 2 F) ≈ Digestive glands ( Figure 2 A) > Stomach + Intestine + Esophagus ( Figure 2 E) ≈ Stomach ( Figure 2 B) ≈ Intestine ( Figure 2 C) ≈ Esophagus ( Figure 2 D); where "stomach + intestines + esophagus ( Figure 2 In the enzyme extract of "E"), a very small number of cells dissociated, including those from the stomach, intestines, and esophagus. Figure 2 Almost no cell dissociation was observed in the single enzyme extract of BD. The field of view of enzyme-dissociated cells for each group is shown below. Figure 2 As shown.
[0035] Example 3: *Heliotropium* (family Heliotropidae) Trochus pyramisDetermining the volume ratio of Born's digestive glands, stomach, and intestines to PBS solution. Ammonium sulfate powder was dissolved in 1×PBS solution at 4℃ and mixed with the horseshoe snail grinding slurry at ratios of 1:1.5, 1:2, and 1:3. At a ratio of 1:3, ammonium sulfate was completely dissolved, and the horseshoe snail grinding slurry and the PBS mixture containing ammonium sulfate were fully blended without coagulation or adhesion to the walls. When PBS was added at ratios of 1:1.5 and 1:2, the mixture became too viscous, leading to coagulation and adhesion to the walls, and the ammonium sulfate powder was not fully dissolved. The reagent amounts added at each ratio are shown in Table 3 below.
[0036] Table 3. Reagent dosage for different proportions Example 4: Determination of Rotation Speed
[0037] In determining the family Hordeidae and the genus Hordeum. Trochus pyramis After determining the volume ratio of Born's digestive glands, stomach, and intestines to PBS solution, and using the optimal volume ratio of 1:3, four different centrifugation speeds were tested: 1000, 2000, 3000, and 5000 rpm. At 1000 and 2000 rpm, the precipitate could not be completely centrifuged to the bottom of the tube, with a large amount of precipitate fragments suspended in the supernatant. At 3000 and 5000 rpm, the precipitate was completely centrifuged to the bottom of the tube, and the supernatant was clear and transparent. Therefore, 2000-3000 rpm is the optimal centrifugation speed. Centrifugation was then conducted at 2200, 2500, and 2800 rpm, and finally, 2500 rpm was determined to be the optimal speed that ensured the precipitate was centrifuged while minimizing the impact of centrifugation on the protein structure. Example 5: Determination of the optimal enzymatic hydrolysis system
[0038] This study determined the optimal enzymatic digestion volume within a total centrifuge tube volume of 50 mL.
[0039] Enzyme solution volume optimization: After multiple dissections and extractions, it was found that the digestive glands and other organs of the two snails had a volume of ≈5 mL after grinding. After adding 3 times the volume of PBS and 30% mannitol, the volume was ≈25 mL. After extraction and centrifugation, the supernatant volume was ≈20 mL. At this point, 90% ammonium sulfate powder was added. It was necessary to dissolve the powder in PBS beforehand and mix it with the supernatant. Adding the powder directly to the supernatant would cause the protein in the supernatant to precipitate out instantly, affecting the enzymatic hydrolysis effect of the enzyme extract. When ammonium sulfate powder was dissolved in 10 mL of 1×PBS (14.34 g of ammonium sulfate powder was added, resulting in a final volume of 30 mL, with an ammonium sulfate concentration of 95% at 4℃), the powder did not completely dissolve. However, when ammonium sulfate powder was dissolved in 20 mL of 1×PBS (19.12 g of ammonium sulfate powder was added, resulting in a final volume of 40 mL, with an ammonium sulfate concentration of 95% at 4℃), the powder dissolved completely. The centrifuge tube had a capacity of 50 mL, and algae would be added later. To avoid contamination, the total volume was not increased further, so two snails were selected.
[0040] Algal sample dosage optimization: When the algal sample is 2 g, it can be spread evenly on the bottom layer of a horizontally placed centrifuge tube. At this time, the contact area between the algal sample and the enzyme solution is maximized and there is no redundancy. Therefore, 2 g of algal sample was selected for protoplast cell separation.
[0041] Mannitol concentration optimization: Mannitol concentration was tested on a small scale (as shown in Table 4 below): Table 4 Comparison of Mannitol Concentration and Mass (per 40 mL solution)
[0042] Literature (An efficient protocol for perennial ryegrass mesophyllprotoplast isolation and transformation, and its application on interactionstudy between LpNOL and LpNYC1, A highly efficient protocol for isolation of protoplast from China, Assam and Cambod types of tea plants, Establishment of an Efficient Protoplast Isolation and Transfection Method for Eucommia ulmoidesThe mannitol concentration used in Oliver's enzymatic hydrolysis was 0.6 M. However, in this manual's enzymatic hydrolysis system, the number of cells ruptured was minimized when the mannitol concentration was 0.45 M. Therefore, 0.45 M mannitol was selected for the enzymatic hydrolysis system. The CaCl2 concentration of 5 mM was maintained as in the literature. The final enzymatic hydrolysis system is shown in Table 5 below.
[0043] Table 5 Enzymatic hydrolysis system Example 6: Determination of Enzymatic Hydrolysis Time
[0044] Using the optimal enzymatic hydrolysis system determined in Example 5 above, enzymatic hydrolysis times of 6 h, 10 h, 12 h, 16 h, and 24 h were tried. It was found that even after 6 h, the enzymatic hydrolysis was not fully completed, and the number of cells was relatively low (e.g., ...). Figure 3 As shown); the cell lysis ability is similar at 10-16 h, the number of lysed cells increases significantly, the cells are viable, and the cell structure is clear, intact, and brightly colored. At this time, the background remains clean and free of impurities (e.g. Figure 4 As shown); after 24 hours of enzymatic hydrolysis, the cell contents flowed out, and most cells were deformed or dead (as shown). Figure 5 (As shown). Example 7: Preparation and Verification of Protoplasts from Tropical Macroalgae of the Genus *Euphorbia* or *Cappa* Protoplast preparation:
[0045] (1) The genus *Heliotropium* of the family Heliotropidae Trochus pyramis Clean Born with fresh water, soak in sterile fresh water for 10 minutes, remove internal organs in an ice bath, place the whole internal organs in an antibiotic solution and soak for another 10 minutes, remove glands, stomach and intestines, cut into 1-2 cm pieces and grind into a paste, the grinding process should not exceed 5 minutes. (2) Add 1×PBS solution at 4℃ sterile and enzyme-free ratio in a 1:1 ratio and mix well; (3) Take twice the volume of 1×PBS solution from (2) (at this time, the volume ratio of PBS solution to snail viscera is 3:1), dissolve ammonium sulfate powder (add 13.4 g / 100 mL on ice), and mix it evenly with snail grinding extract after the ammonium sulfate powder is completely dissolved.
[0046] (4) Place the mixture obtained in (3) in a refrigerator at 4°C for 12 h, and then centrifuge at 2500 rpm, 20 min, and 4°C until the supernatant is clear. Remove the precipitate and retain the supernatant for the next experiment.
[0047] (5) Add 47.8 g / 100 mL of ammonium sulfate to the supernatant on ice, stir until fully dissolved, and let stand at 4℃ for 6 h; centrifuge 1-2 times at 2500 rpm, 20 min, and 4℃ to collect the precipitate.
[0048] (6) Resuspend the precipitate in 1 mL of 4℃ pre-cooled 1×PBS, dialyze for 12 h in a 3.5 kDa dialysis bag at 4℃, and change the dialysate 3 times during the period, changing it every 2 h for the first two times; after dialysis for 12 h, change the dialysate again and dialyze for more than two hours, and store it at 4℃ for later use.
[0049] (7) Select fresh and plump algae of the genus Euphorbia / Kappa, scrub them clean and leave them overnight to sterilize; (8) Cut off the upper epidermis of the algae and chop it into 0.1 cm × 0.1 cm fragments, add it to the enzyme solution prepared in (6), place it horizontally in a shaker at 26℃, and incubate overnight at 45 r / min.
[0050] (9) Observe the state of the dissociated cells under a microscope and collect the cells by centrifugation. In this step, pay attention to the separation of cells from bacteria to avoid the contamination of bacteria during collection, which may affect the genome sequencing results. Sequencing verification:
[0051] (1) The collected cells were examined under a microscope, and the cell density reached ≥102. 6 Cells were collected at a concentration of ≥10 cells / µL, with a target density of 10 cells / 100 mL. After collection, the cells were resuspended in 10 mL of 1×PBS with 0.45 M mannitol solution. The cell field of view is shown below. Figure 6 and 7 As shown.
[0052] (2) Add 280 µL of formaldehyde solution (37%), gently invert and mix 10 times, crosslink at room temperature for 10 min (at 2.5 min, 5 min and 7.5 min respectively, gently invert and mix 10 times). (3) Add 560 µL of 2.5 M glycine, mix well, and terminate cross-linking at room temperature for 5 min; (4) Centrifuge at 2000 rpm at room temperature for 5 min, discard the supernatant, and recover the cells; (5) Quick-freeze in liquid nitrogen and store at -80°C for genome sequencing.
[0053] (6) Using PacBio technology, select qualified high-quality DNA samples (main band > 30 kb) and sequence them using the PacBioRevio / Sequel II / IIe platform. Data quality assessment shows that ( Figure 8The number of Hifi reads was 3,325,925, and the average read length was approximately 9,124 bp. The data showed good overall uniformity, with prominent peaks and a stable decreasing trend afterward. There were no multiple peaks or abnormal fluctuations, indicating that the length distribution of the sequencing sequences was relatively regular.
[0054] The quality-controlled data were sequenced, and the total sequencing volume of the third-generation sequencing results of *Kappa longiflora* genome was 30.35 G. Based on the estimated genome size of 407.25 M from Survey, the coverage depth was 74.52 × 10⁻⁶. An Illumina small fragment library was constructed, and sequencing was performed using the Illumina platform. The statistical results are shown in Table 6.
[0055] Table 6. Statistics on the amount of genome sequencing data
[0056] Sequences longer than 100 bp were assembled, and the total genome length of *Kappa longiflora* was 343.05 Mbp, with a Contig N50 of 5.55 Mbp (Table 7).
[0057] Table 7 Genome assembly results
[0058] Statistical results of the base content analysis of the genome show that ( Figure 9 The proportion of GC was 44.24%, and the proportions of A, T, G, and C bases were within the normal range. The proportion of N was 0.00, which is within the acceptable range for sequencing (<10%). Sequencing depth distribution map ( Figure 9 B) shows that the average sequencing depth is 37.12.
[0059] The coverage of gene regions of the assembled genome was evaluated according to the BUSCO (Benchmarking Universal Single-Copy Orthologs) assessment method. Figure 9 C), to examine the integrity of the assembled genome. A C value above 90% in the BUSCO assessment indicates good genome assembly integrity, while a D value above 50% suggests the species may be polyploid. Among existing algal genomes, high-quality genomes are relatively few; therefore, the alignment reached 72.2%. Considering published or uploaded genomes, this percentage is considered reliable, and the genome results are relatively accurate.
[0060] To further evaluate the accuracy of the assembly, small-fragment library reads were aligned to the assembled genome using BWA software. The alignment rate, genome coverage, and depth distribution of the reads were statistically analyzed to assess the integrity of the assembly and the uniformity of sequencing. Table 8 shows that the alignment rate of the small-fragment reads to the genome was approximately 92.53%, and the genome coverage was approximately 99.91%, indicating good consistency between the reads and the assembled genome.
[0061] Table 8. Statistics on genomic read coverage
[0062] Note: Mapping rate, the proportion of reads aligned to the genome; Average sequence depth, the average depth of each base on the genome covered by reads; Coverage, the proportion of the genome covered by reads; Coverage at least NX (%), the proportion of the genome covered by NX reads.
[0063] The BWA alignment results were processed using tools such as samtools, including chromosome coordinate sorting and read deduplication, followed by SNP calling. The original results were then filtered and statistically analyzed. The results showed that the heterozygous SNP ratio of the *Kappa longiflora* genome was 0.002824%, and the homozygous SNP ratio was 0.000000%. A lower homozygous SNP ratio indicates higher genome assembly accuracy, suggesting a high single-base accuracy in the assembly.
[0064] The GC content and average depth of the assembled genome sequences were calculated and plotted using 10k windows. Figure 10 It can be seen that the GC content is concentrated around 44.24%, and there is no obvious separation in the scatter plot, that is, there is no obvious separation of GC, indicating that there is no exogenous pollution in the genome.
[0065] The accuracy of the sequence was assessed using the formula Qv = -10 * log E (where E represents the error rate). When the accuracy was 99%, the qv (quality value) was 20; at 99.9%, the qv was 30; and at 99.99%, the qv was 40. The genome accuracy was 43.6653, indicating an accuracy of over 99.999%, making the results accurate and reliable.
[0066] Conclusion: Denovo assembly was performed using 48.86 G of Kappaella longicornis genome sequencing data. The total contig length was 343.05 Mbp, and the contig N50 length reached 5.55 Mbp. Multiple methods were used to evaluate the assembled version, and the results showed good genome consistency, integrity, and accuracy. The protoplast biomass and purity of the provided data were sufficient for genome sequencing and met the sample requirements for third-generation genome sequencing (HIC). However, directly providing *Euphorbia milii* / Kappaella thallus resulted in high gel content, making genome sequencing impossible. Example 8: Following the reported enzymatic hydrolysis method and all the enzyme types mentioned above, we performed protoplast cell dissociation on the thallus of the tropical macroalgae *Euphorbia* / *Cappa* containing carrageenan.
[0067] (1) Add cellulase, pectinase, hemicellulase, pectinase and snail enzyme to 1×PBS buffer to prepare enzyme hydrolysate according to the enzyme type and required concentration in Table 9 below (add to each 12 mL phosphate buffer). Then add mannitol and 5 mM calcium chloride (CaCl2) of the required concentration to the enzyme hydrolysate. Filter through a 0.22 μm microporous membrane for later use. Table 9. Composition of the enzymatic hydrolysate added to each group
[0068] Note: 5 M CaCl2 stock solution: Add 55.49 g CaCl2 to 500 mL phosphate buffer; add 0.6 g of enzyme at a concentration of 5%.
[0069] (2) Add fragments of the same strain and weight of *Euphorbia tirucalli* / *Kappaella* thallus to each group of enzyme solutions, perform cell dissociation, and observe under a microscope. The results are as follows. Figure 11 As shown. All plant enzyme combinations had no dissociative effect on Euphorbia lactea / Kappaella cells.
Claims
1. An enzymatic hydrolysate for preparing protoplasts of algae in the genera *Euphorbia* or *Kappaella*, characterized in that: The enzymatic hydrolysate comprises an enzyme extract of the digestive system of *Trochus pyramis Born* (family Trochiridae), 1×PBS (0.01 mol / L), 0.45 M mannitol, and 5 mM CaCl2. Each 40 mL of the enzymatic hydrolysate contains 20 mL of the enzyme extract of the digestive system of *Trochus pyramis Born* (family Trochiridae), 20 mL of 1×PBS, 3.3 g of 0.45 M mannitol, and 0.028 g of 5 mM CaCl2.
2. The enzymatic hydrolysate according to claim 1, characterized in that: The method for preparing the extract of Trochus pyramis Born (family Trochilliidae, genus Trochus) is as follows: (1) Select the digestive glands, stomach and intestines of Trochus pyramis Born from the family Trochusidae, grind them into a paste, and add an equal volume of 1×PBS solution to extract the paste; (2) Under ice bath conditions, dissolve ammonium sulfate powder with twice the volume of 1×PBS solution in (1), and then mix it with the extract obtained in (1) to further extract and precipitate the proteins that can be separated from algae cells. (3) Centrifuge to remove the precipitate and retain the supernatant; (4) Add ammonium sulfate again on ice and collect the precipitate; resuspend the precipitate with 1×PBS and dialyze overnight on ice or at 4°C to obtain the enzyme extract.
3. The enzymatic hydrolysate as described in claim 2, characterized in that: The specific steps of centrifugation in (3) and (4) are as follows: centrifugation at 2000-3000 rpm.
4. The enzymatic hydrolysate according to claim 3, characterized in that: The specific steps of centrifugation in (3) and (4) are as follows: centrifugation at 2500 rpm.
5. The enzymatic hydrolysate as described in claim 2, characterized in that: The specific extraction time in (1) is 12 h.
6. A method for preparing protoplasts of *Euphorbia* or *Kappaella* algae using the enzymatic hydrolysate as described in any one of claims 1-5, characterized in that: Each 40 mL of enzymatic hydrolysate can enzymatically hydrolyze 2 g of the aforementioned *Euphorbia* or *Kappa* genus algae.
7. The method as described in claim 6, characterized in that: The enzymatic hydrolysis time for *Euphorbia milii* or *Kappaella* algae is 10-16 h.
8. Protoplasts of *Euphorbia* or *Kappa* algae prepared using the enzymatic hydrolysate as described in any one of claims 1-5 or the method as described in any one of claims 6-7.
9. The protoplast of *Euphorbia* or *Kappa* algae as described in claim 8, characterized in that: The protoplasts can be directly used for genome sequencing and meet the sample requirements for third-generation genome HIC sequencing.
10. The protoplast of *Euphorbia* or *Kappa* algae as described in claim 9, characterized in that: The number of intact cells collected from the protoplasts is ≥10 6 Each 100 mL contains one sample, and there is no obvious salt or protein residue.