Porphyra yezoensis thallus cryopreservation technology and porphyra yezoensis thallus resuscitation method

Abstract

The invention relates to the technical field of aquaculture, and particularly discloses a porphyra yezoensis thallus cryopreservation technology and a porphyra yezoensis thallus resuscitation method, and the method comprises the following specific steps: step 1, blow-drying porphyra yezoensis at a low temperature until the water loss rate is 78-82%; 2, performing low-temperature freezing treatment; step 3, resuscitating the thalli of the cryopreserved porphyra yezoensis; through effective cryopreservation and resuscitation technologies, the limitation of seasonal growth can be broken through, annual stable supply is realized, and more reliable guarantee is provided for laver breeding, quality improvement and laboratory research; the cell viability of the porphyra yezoensis thallus treated by the preservation and recovery steps is maintained, the thallus is complete in structure and normal in function, and subsequent culture and growth can be continued.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture, in particular to a freezing preservation technology for Porphyra yezoensis thallus and a resuscitation method thereof. Background Art

[0002] Neopyropia yezoensis belongs to the Rhodophyta and is an important economic seaweed widely distributed along the coast of East Asia. It is rich in protein, vitamins, minerals and other nutrients, and is widely used in the food and pharmaceutical industries. In addition, the cultivation of Neopyropia yezoensis has high economic value and ecological benefits, and helps to improve the marine ecological environment. It plays an indispensable role in the optimization of the fishery economic structure in coastal areas and the sustainable use of marine resources, and has effectively promoted the diversification of industries and the maintenance of ecological balance in related regions.

[0003] Porphyra yezoensis has significant alternation of generations, including two heteromorphic generations: thallus and filaments. Among them, carposporangia can be formed at the edge of the thallus, and carpospora can be released through meiosis. After the carpospora germinates, filaments are formed, and the ends of the filaments swell to produce conchospores, which further develop into thallus. In addition, monospores can be directly formed at the edge of the thallus of Porphyra yezoensis, which can quickly develop into thallus after attachment. The current seedling system mainly relies on the germination of conchospores, but this process is slow, and the conchospores are susceptible to diseases such as yellow spots, causing economic losses. In order to overcome these problems, more and more studies have begun to adopt the monospore seedling method based on the asexual reproduction of Porphyra yezoensis thallus. However, the successful implementation of this method depends on the ability of the thallus to break the limitations of seasonal and environmental conditions and maintain a stable and fresh supply. At present, the production and processing of Porphyra yezoensis mostly adopts high-temperature drying technology, but this method will lead to the loss of nutrients such as vitamins and minerals in the porphyra, thereby reducing its nutritional value, affecting the taste and flavor, and making the porphyra dry, hard and rough. In addition, laboratory research has a continuous demand for a fresh and stable supply of thallus. Therefore, it is of great significance to study the freezing preservation technology and resuscitation methods of Porphyra yezoensis thallus. Through effective freezing preservation and resuscitation technology, it is possible to break through the limitations of seasonal growth and achieve a stable supply throughout the year, which will provide more reliable guarantees for porphyra breeding, quality improvement and laboratory research.

[0004] In summary, the preservation and processing of Porphyra yezoensis thallus in the prior art has the following defects:

[0005] 1. The production and processing of Porphyra yezoensis mostly adopts high-temperature drying technology, but this method will lead to the loss of nutrients such as vitamins and minerals in Porphyra, thereby reducing its nutritional value, affecting the taste and flavor, and making the Porphyra dry, hard and rough. And the revived Porphyra cells are already dead.

[0006] 2. Direct freezing treatment: After the Porphyra yezoensis is collected, the water is simply drained and then directly frozen. This method causes serious cell damage, poor freeze resistance, and a high risk of microbial growth. After recovery, the Porphyra cells become dead cells. Therefore, a Porphyra yezoensis thallus cryopreservation technology and a recovery method are provided. Summary of the invention

[0007] The purpose of the present invention is to provide a cryopreservation technology and a resuscitation method for Porphyra yezoensis thallus in view of the defects of the prior art, so as to solve the problems raised by the above-mentioned background technology.

[0008] To achieve the above object, the present invention provides the following technical solution: a cryopreservation technology for Porphyra yezoensis thallus and a method for its recovery, the specific steps of which are as follows:

[0009] Step 1: Dry the Porphyra yezoensis at low temperature to a water loss rate of 78-82%;

[0010] Step 1.1: After removing excess water from the surface of fresh Porphyra yezoensis, weigh its initial fresh weight m1 g and record it;

[0011] Step 1.2: Start a high-power electric fan and adjust the indoor air conditioner to cooling mode at 16°C. Place several ice packs at the fan outlet to create a low-temperature environment to dry the laver.

[0012] Step 1.3: Every hour, weigh the laver using an electronic balance and record the mass as m2 grams;

[0013] Step 1.4: Calculate the water loss rate of laver according to the following formula: L = (m1-m2) / m1×100%; when the water loss rate reaches 78-82%, the drying process is completed;

[0014] Step 2: low temperature freezing treatment;

[0015] Step 2.1: Pack the dried Porphyra yezoensis into plastic bags and extract an appropriate amount of air from the bags;

[0016] Step 2.1: After completing the subpackaging and properly removing excess air, the plastic bag is stored in a frozen environment at -20°C for long-term storage of Porphyra yezoensis;

[0017] Step 3: revival of cryopreserved Porphyra yezoensis thallus;

[0018] Step 3.1: Sterilize the seawater and pre-cool it to 10°C for later use;

[0019] Step 3.2: Take out the frozen Porphyra yezoensis thallus from the refrigerator and quickly put it into 10°C sterilized seawater for recovery.

[0020] As a preferred technical solution of the present invention, when extracting the air in the bag in step 2.1, the suction force should not be too large to avoid the laver leaves being crushed due to excessive pressure.

[0021] As a preferred technical solution of the present invention, if there is a constant temperature incubator in step 3.2, the recovery process can be placed in the incubator, maintaining an environment of 10°C, and the recovery time is 0.5 hour to 1 hour; if a constant 10°C environment cannot be maintained, an appropriate amount of ice packs can be added to the seawater to maintain low temperature conditions, thereby completing the recovery process of the laver thallus.

[0022] As a preferred technical solution of the present invention, the water loss rate in step 1.4 is preferably 80%.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention can break through the limitation of seasonal growth and realize stable supply throughout the year through effective cryopreservation and resuscitation technology, providing more reliable guarantee for Porphyra breeding, quality improvement and laboratory research; the cell activity of the thallus of Porphyra yezoensis treated by the preservation and resuscitation steps of the present invention is maintained, the thallus structure is intact and functions normally, and subsequent culture and growth can continue. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The present invention shows the changes in the leaves of Porphyra yezoensis thallus after low-temperature freezing and recovery under different degrees of water loss;

[0025] (A) corresponds to the condition of the leaves when the water loss rate is 80%; (B) corresponds to the condition of the leaves when the water loss rate is 60%; (C) corresponds to the condition of the leaves when the water loss rate is 40%; (D) corresponds to the condition of the leaves when the water loss rate is 20%;

[0026] Figure 2 : The leaf cell changes of Porphyra yezoensis thallus after cryopreservation and recovery at different degrees of water loss in the present invention;

[0027] (A) corresponds to the cell state when the water loss rate is 80%; (B) corresponds to the cell state when the water loss rate is 60%; (C) corresponds to the cell state when the water loss rate is 40%; (D) corresponds to the cell state when the water loss rate is 20%;

[0028] Figure 3 : is the thallus growth curve of Porphyra yezoensis with a water loss rate of 80% according to the present invention. DETAILED DESCRIPTION

[0029] The preferred embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0030] Example 1: Porphyra yezoensis is dried at low temperature to a differentiated water loss degree and then frozen at low temperature;

[0031] In the experiment, fresh Porphyra yezoensis was selected and evenly divided into four groups, with three biological replicates in each group. The visible moisture on the surface of the Porphyra was gently wiped off with filter paper, and care should be taken to avoid damaging the Porphyra tissue during the operation. Subsequently, the processed Porphyra was placed on an electronic balance for accurate weighing, and the initial mass was recorded as m1 (unit: g). In the experiment, a high-power electric fan was used, and the indoor air conditioner was adjusted to the lowest temperature of 16°C in the cooling mode. At the same time, several ice bags were placed at the air outlet of the electric fan to create a low-temperature environment for drying the Porphyra. Every half an hour, the weight of the Porphyra was weighed using an electronic balance, and the mass at this time was recorded as m2 (unit: g). The water loss rate (L) of the Porphyra was calculated according to the following formula: (m1-m2) / m1×100%. When the water loss rate of the Porphyra reached 80%, 60%, 40% and 20% respectively, it was immediately transferred to a -20°C refrigerator for freezing treatment, and the freezing time was 3 months.

[0032] Example 2: Changes in the state of Porphyra leaves after cryopreservation and thawing of Porphyra yezoensis;

[0033] In the experiment, the Porphyra yezoensis, which was grouped according to the above water loss rate and subjected to -20℃ low-temperature freezing treatment, was taken out of the refrigerator and placed in natural seawater at a temperature of 10℃ for 30 minutes to recover. After recovery, the Porphyra fronds were carefully spread evenly on a white background for clear photography and careful observation. Figure 1 Then, the leaves were gently spread on a glass slide, and an inverted microscope was used to observe the morphological changes of the leaf cells in depth, and to capture and take photos of the cells in real time, as shown in Figure 1. Figure 2 shown.

[0034] Example 3: Growth and culture study of Porphyra yezoensis thallus under 80% water loss rate;

[0035] In the experiment, the thallus of Porphyra yezoensis, which had been frozen at -20°C and had a water loss rate (L) of 80%, was taken out and placed in 10°C seawater for resuscitation. After the resuscitation was completed, leaves with uniform morphology and a length of 6-8cm were selected, and absorbent paper was used to carefully absorb the residual water on the surface. 0.5g±0.001g of algae was accurately weighed, placed in 1000mL of natural seawater for culture, and 10mL of PES nutrient salt was added. At the same time, in order to ensure the reliability of the experimental results, three biological replicate groups were set up for growth experiments. The sterilized natural seawater was replaced every other day, and PES nutrient salt was supplemented. At the same time, the fresh weight of the algae was accurately weighed and recorded, such as Figure 3 shown.

[0036] Example 4: RNA quality detection of Porphyra yezoensis thallus with a water loss rate (L) of 80%;

[0037] The experiment selected the thallus of Porphyra yezoensis that was frozen at -20°C and had a water loss rate (L) of 80%, and placed it in 10°C seawater for recovery. After 48 hours of cultivation according to the above culture method, fresh algae were selected and quickly added with liquid nitrogen for grinding. Take 50-100 mg of ground powder and place it in a 1.5 mL centrifuge tube, add 1 mL of TRIzol solution, shake it thoroughly and mix it for RNA extraction. The quality and concentration of RNA were first evaluated using a biophotometer (Eppendorf, Hamburg, Germany), and then the integrity of RNA (RIN value) was evaluated by Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), as shown in Table 1.

[0038] Table 1: RNA quality test of Porphyra yezoensis with a water loss rate of 80%

[0039]

[0040]

[0041] Example 5: Quality summary of sequencing data of Porphyra yezoensis samples with a water loss rate (L) of 80%;

[0042] When constructing the library, 1 μg of total RNA extracted as described above was used, and mRNA was isolated from total RNA using Poly-T oligoadenylic acid attached magnetic beads (Invitrogen, Waltham, MA, USA), followed by fragmentation of mRNA using NEB fragmentation buffer (NEB, Ipswich, MA, USA). According to the Illumina library construction requirements, mRNA was converted to complementary DNA for sequencing. Part of the transcriptome sequencing work was completed by Novogene on the Illumina HiSeq 2500 sequencing platform to generate paired-end reads. After removing the adapter sequence, poly-N sequence, and low-quality sequence in the raw data using an internal Perl script, a high-quality read sequence (clean read) was obtained. In addition, fastp (version 0.20.1, https: / / github.com / OpenGene / fastp) was used to calculate the Q20, Q30, and GC content of the clean read, as shown in Table 2.

[0043] Table 2: Sequencing data quality test of Porphyra yezoensis samples with a water loss rate of 80%

[0044]

[0045] It can be seen from Examples 1-5 that:

[0046] (1) Porphyra yezoensis was frozen and revived at water loss rates of 80%, 60%, 40% and 20%. The results showed that the leaves were healthiest and most complete when the water loss rate was 80%; the leaves were in good condition when the water loss rate was 60%; and the leaves were severely damaged and rotten when the water loss rates were 40% and 20%.

[0047] (2) Porphyra yezoensis was frozen and revived at water loss rates of 80%, 60%, 40% and 20%. The results showed that when the water loss rate was 80%, the Porphyra cell structure was intact and the color was bright; when the water loss rate was 60%, some cells turned red and intracellular juice was lost; when the water loss rate was 40%, the cells began to shrink; and when the water loss rate was 20%, the cells were completely dead.

[0048] (3) The thallus of Porphyra yezoensis with a water loss rate of 80% grew well within 0-10 days, and the fresh weight increased from 0.5012 g on day 0 to 0.8157 g on day 10.

[0049] (4) When the water loss rate of Porphyra yezoensis thallus was 80%, the RNA quality was excellent and the minimum RNA integrity could reach 7.4.

[0050] (5) The sequencing data quality of the Porphyra yezoensis thallus sample with a water loss rate of 80% was high, generating an average of about 7.06Gb of raw data, and after filtering, an average of 6.56Gb of clean data was obtained. The Q20 of the clean data was not less than 96%, the Q30 was not less than 92%, and the error rate was only 0.01%.

[0051] The above embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

Claims

1. A cryopreservation technology for Porphyra yezoensis thallus and a method for its recovery, characterized in that: The specific steps are as follows: Step 1: Dry the Porphyra yezoensis at low temperature to a water loss rate of 78-82%; Step 1.1: After removing excess water from the surface of fresh Porphyra yezoensis, weigh its initial fresh weight m1 g and record it; Step 1.2: Start a high-power electric fan and adjust the indoor air conditioner to cooling mode at 16°C. Place several ice packs at the fan outlet to create a low-temperature environment to dry the laver. Step 1.3: Every hour, weigh the laver using an electronic balance and record the mass as m2 grams; Step 1.4: Calculate the water loss rate of laver according to the following formula: L = (m1-m2) / m1×100%; when the water loss rate reaches 78-82%, the drying process is completed; Step 2: low temperature freezing treatment; Step 2.1: Pack the dried Porphyra yezoensis into plastic bags and extract an appropriate amount of air from the bags; Step 2.1: After completing the subpackaging and properly removing excess air, the plastic bag is stored in a frozen environment at -20°C for long-term storage of Porphyra yezoensis; Step 3: revival of cryopreserved Porphyra yezoensis thallus; Step 3.1: Sterilize the seawater and pre-cool it to 10°C for later use; Step 3.2: Take out the frozen Porphyra yezoensis thallus from the refrigerator and quickly put it into 10°C sterilized seawater for recovery.

2. The cryopreservation technology and revival method of Porphyra yezoensis thallus according to claim 1, characterized in that: When extracting the air in the bag in step 2.1, the suction force should not be too large to avoid the laver leaves being crushed due to excessive pressure.

3. The cryopreservation technology and revival method of Porphyra yezoensis thallus according to claim 1, characterized in that: If there is a constant temperature incubator in step 3.2, the recovery process can be placed in the incubator, maintaining an environment of 10°C, and the recovery time is 0.5 hour to 1 hour; if a constant 10°C environment cannot be maintained, an appropriate amount of ice packs can be added to the seawater to maintain low temperature conditions, thereby completing the recovery process of the laver thallus.

4. The cryopreservation technology and revival method of Porphyra yezoensis thallus according to claim 1, characterized in that: The water loss rate in step 1.4 is preferably 80%.

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