High temperature resistant Enteromorpha japonica and its production method

Abstract

To provide a novel high-temperature resistive Ulva prolifera in which maturation is hard to occur and an algae body is increased even in a high water temperature region of 23°C or higher.SOLUTION: A novel high-temperature resistive Ulva prolifera and a method for producing thereof, in which algae body wet weight on day 8 of culture is increased by 6 times or more compared with day 4 of culture in the case of being cultured at the water temperature of 25°C to 30°C for 8 days, and furthermore, a maturation rate is 20% or lower in the case of being cultured at the water temperature of 25°C and light condition 100 μmolphoton / m2 sec for 6 days.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] The present invention relates to high-temperature resistant Enteromorpha platyphylla. The present invention also relates to a method for producing high-temperature tolerant Enteromorpha platyphylla. [Background technology]

[0002] Ulva prolifera is an edible seaweed that belongs to the genus Ulva, family Ulvaceae, order Ulvales, class of green algae. It is the most fragrant and flavorful of all the green seaweed species, and is widely used as a high-quality topping for a variety of foods, from okonomiyaki, takoyaki, yakisoba, and furikake to sweets such as potato chips. It is also known to be rich in nutrients such as vitamins and minerals (potassium, calcium, iron, etc.), as well as dietary fiber.

[0003] Green laver grows well in brackish waters where seawater and freshwater mix, and until a few years ago it was produced in estuaries in Kochi, Tokushima, Okayama, Wakayama, and other prefectures in Japan. The optimum water temperature for green laver to grow without maturing is 15-20°C, and for most green laver, the algae grow largest at this temperature, resulting in the highest yield, and growth slows below 10°C (Non-Patent Document 1).

[0004] In general seaweed, sporophylls or reproductive receptacles are formed after maturation and spores are released from there, but in Enteromorpha, its vegetative cells directly differentiate into reproductive cell capsules and "mature", from which spores are released as swarming cells (reproductive cells with two or four flagella), which separate from the algae and begin to swim (Non-Patent Document 2). Enteromorpha is easily matured at temperatures above 20°C, and releases spores when mature.

[0005] Normally, when Enteromorpha is cultivated at high water temperatures of 25°C and 30°C, maturation occurs in a wide part of the algae body, and the cells that release spores are washed away. As a result, the amount of washed-out mature parts exceeds the amount of growth at 25°C and 30°C, resulting in a decrease in leaf length (Non-Patent Document 1).

[0006] In the wild, spores of Enteromorpha macrocarpa separated from the parent algae attach to a suitable substrate and germinate to form algae. However, as mentioned above, at high water temperatures the amount of maturation exceeds the amount of growth and the algae cannot be maintained, so the spores spend their time in the form of tiny cell masses on the river bottom (Non-Patent Documents 2 and 3).

[0007] In the marine culture method conventionally practiced in major production areas, culture is carried out during the extremely short period from October to December when the water temperature drops from 23°C to around 12°C. Around mid-October, when the water temperature reaches around 23°C, seedlings are harvested by attaching green laver spores released from the parent algae to seed nets, and in November, when the water temperature drops to below 20°C, the seed nets with the spores attached are stretched out in the culture area and main culture begins. Green laver grows to 20-50 cm in about 2-3 weeks after the start of culture (Non-Patent Document 3).

[0008] As mentioned above, the harvest period for conventional green laver and cultivation methods is extremely short, so in Tokushima Prefecture, selective breeding of green laver that can grow even in the high water temperatures of autumn or spring is being promoted with the aim of extending the cultivation period (Non-Patent Document 4).

[0009] In recent years, due to rising seawater temperatures caused by global warming, it has become difficult for the water temperature to drop from 23°C during the traditional main cultivation period of green laver from October to December, which has resulted in a slowdown in the growth of green laver during cultivation, and furthermore, as the algae mature before harvest, the algae fall off, causing a significant drop in production. In 2018, the production volume of natural green laver from Shimanto River, Kochi Prefecture, was only about 10 kg, and the production volume of cultivated green laver from Yoshino River, Tokushima Prefecture, which was about 80 tons per year several years ago, has decreased to 20 tons per year in 2017 and 28 tons per year in 2018, and as a result, the market trading price of green laver has soared to a peak of over 40,000 yen per kg, four times higher than several years ago (Non-Patent Document 5).

[0010] Under these circumstances, in order to stabilize the production volume and price of Green Laver, attention has been focused on land-based cultivation methods that can control to some extent the production environment, such as water temperature, nutrient salt concentration, feeding damage by birds, and attached diatoms. In Kochi Prefecture, a land-based cultivation method of Green Laver has been attempted for several years using a "seaweed cultivation method by spore agglomeration" (Patent Document 1). In the land-based cultivation method by spore agglomeration, Green Laver spores are manipulated in a laboratory, and multiple individuals are allowed to germinate so that they are connected to each other to create a clump, which is then transplanted into an outdoor aquarium for cultivation. Spore clumps grow well when cultivated by floating them in a tank on land (Non-Patent Document 6).

[0011] More specifically, if the spore aggregates of Green Laver prepared as described above are put into an aquarium tank installed on land, and the seawater in the tank is replaced by deep seawater with a temperature of about 12°C taken from the seabed 2 km offshore and about 350 m deep through a pipeline several times a day, and the algae are cultured while moving by aeration, the wet weight of the algae increases about 10 times in one week (Non-Patent Document 7). By using this method and cultivating Green Laver in deep seawater with a temperature of about 12°C while cooling it several times a day, it has become possible to produce Green Laver in the summer, which was impossible with the conventional marine culture method. Currently, it is possible to culture Green Laver all year round using this method. However, since the top of the land-based culture tank is open for the photosynthesis of Green Laver by sunlight, the influence of the outside temperature cannot be completely avoided, and in the summer when the temperature is high, it is necessary to increase the amount of seawater taken in order to bring the water temperature closer to 15°C to 20°C, which is suitable for the maximum growth of Green Laver.

[0012] Land-based cultivation of Green Laver by spore agglomeration requires the electricity required to pump seawater, capital investment in aeration, and energy for growth management, and therefore production costs are high, so production volume per unit volume must be kept high from a profitability perspective.Therefore, there is a demand for high-quality Green Laver that can be produced efficiently with a fast growth rate, does not require frequent cooling, and has high-temperature resistance that allows it to grow even in high water temperatures. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] Patent No. 3828359 [Non-patent literature]

[0014] [Non-Patent Document 1] Masanori Hiraoka et al. "Growth and maturation of clonal algae of Enteromorpha japonica under different temperature conditions," Nippon Suisan Gakkaishi 65(2), 302-303(1999) [Non-Patent Document 2] Akinori Dan, "Verification of aquaculture techniques from the viewpoint of physiology and ecology of green laver (review)," Tokushima Fisheries Research Report No. 10, pp. 15-24 (2015) [Non-Patent Document 3] Tokushima Prefectural Agriculture, Forestry and Fisheries Technical Support Center: Tokushima Prefecture Brand Seafood Knowledge Guide "Suji Aonori" (http: / / www.pref.tokushima.jp / _files / 00875752 / monoshiri_sujiao.pdf) [Non-Patent Document 4] Noboru Murase et al. "Growth, photosynthesis and respiratory characteristics of the green alga Enteromorpha sieboldii in high temperature regions" Journal of National Fisheries University Vol. 53(3): pp. 131-136 (2005) [Non-Patent Document 5] Food Newspaper, February 4, 2019 [Non-Patent Document 6] Masao Ohno, "Development of a Mass Cultivation System for Seaweed Using Deep Ocean Water," Journal of Industrial-Academic-Government Collaboration, November 2006 issue (https: / / sangakukan.jst.go.jp / journal / journal_contents / 2006 / 11 / articles / 0611-05 / 0611-05_article.html) [Non-Patent Document 7] Hiroki Ebata et al., "Potential for land-based cultivation of green algae Enteromorpha japonica using underground seawater," Aquaculture Science 55(1), 103-108 (2007) [Non-Patent Document 8] Ogawa et al. “High heterozygosity and phenotypic variation of zoids in apomictic Ulva prolifera (Ulvophyceae) from brackish environments”, Aquatic Botany 120, 185-192 (2014) [Non-Patent Document 9] Shimada et al. "Phylogeography of the genus Ulva (Ulvophyceae, Chlorophyta), with special reference to the Japanese freshwater and brackish taxa", Journal of Applied Phycology 20, 979-989 (2008) Summary of the Invention [Problem to be solved by the invention]

[0015] Therefore, the present invention aims to provide a novel high-temperature-resistant green laver that has a fast growth rate and is less likely to mature even in high water temperatures of 23°C or higher, as a superior green laver that is suitable for both the conventional marine aquaculture method and the more recent land-based aquaculture method using spore clumping. The object of the present invention is to provide a novel high-temperature-resistant green laver that has a fast growth rate and is less likely to mature even in high water temperatures of 23°C or higher, as well as increases in algal mass. [Means for solving the problem]

[0016] The present inventors have searched for Green Laver having the above-mentioned characteristics and conducted intensive testing and research, and as a result, have succeeded in finding, among natural Green Laver, a Green Laver that has excellent high-temperature resistance and productivity, which differs from previously known strains.

[0017] The present invention includes the following features. [1] A high-temperature-resistant Enteromorpha prolifera or a part thereof, which is characterized in that when cultured for 8 days at a water temperature of 25°C to 30°C, the wet weight of the algae increases by 6 times or more on the 8th day of culture compared to that on the 4th day of culture. [2] Water temperature 25℃, light condition 100μmol photon / m 2 3. The high-temperature-resistant Enteromorpha prolifera or a part thereof according to [1] above, characterized in that the maturity rate of algal bodies is 20% or less when cultured at 20° C. for 6 days. [3] The high-temperature-resistant Enteromorpha spp. or a part thereof according to [1] or [2] above, characterized in that the Enteromorpha spp. contains a base sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 2. [4] The high-temperature-resistant Enteromorpha prolifera or a part thereof according to any one of [1] to [3] above, characterized in that the part is a spore, a cell or a tissue. [5] A high-temperature-resistant Enteromorpha platyphylla Kochi No. 1 strain comprising the base sequence represented by SEQ ID NO: 1 and / or 2, or a descendant thereof. [6] A method for producing high-temperature-resistant Enteromorpha algae bodies, comprising the steps of growing high-temperature-resistant Enteromorpha algae bodies using spores of the high-temperature-resistant Enteromorpha described in any one of [1] to [4] or the high-temperature-resistant Enteromorpha Kochi No. 1 strain described in [5], and recovering the high-temperature-resistant Enteromorpha algae bodies. [7] The method according to [6], further comprising the step of washing and drying the heat-resistant Enteromorpha platyphylla. [8] The method according to [6] or [7], characterized in that the growing step is carried out by marine aquaculture or land aquaculture. Effect of the Invention

[0018] By using the green laver of the present invention, which has excellent high-temperature resistance as described above, it becomes possible to cultivate green laver in the summer, which is a high-water temperature environment, which was previously impossible with marine culture methods, and in land-based culture methods using spore agglomeration, it is possible to reduce the cost of culture in the summer, which is a high-water temperature environment, by reducing the amount of seawater intake for cooling.As a result of this extension of the cultivation period using marine culture methods and the cost reduction using land culture methods, it is expected that an increase in the production volume of green laver and a stabilization of the price will be achieved overall. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 is a graph showing the daily growth rate (%) of the Enteromorpha platyphylla of the present invention when cultured for 6 days in 500 ml of sterilized seawater containing added 100 μmol of nitrogen, 10 μmol of phosphorus, and 10 μmol of iron at temperatures of 10° C., 15° C., 20° C., 25° C., or 30° C., in comparison with the daily growth rates (%) of Enteromorpha platyphylla strains collected in other production areas (control strains 1, 2, 3, and 4). [Diagram 2] FIG. 2 is a graph showing the maturity rate (%) of the Enteromorpha platyphylla of the present invention when cultured for 6 days at 25° C. in 500 ml of sterilized seawater containing added 100 μmol of nitrogen, 10 μmol of phosphorus, and 10 μmol of iron, in comparison with Enteromorpha platyphylla strains collected in other production areas (control strains 1, 5, 6, 7, and 8). [Diagram 3] FIG. 3 is a graph showing the change in algal body length (mm) of the Enteromorpha prolifera of the present invention when cultured for 8 days at 25° C. in 500 ml of sterilized seawater containing added 100 μmol of nitrogen, 10 μmol of phosphorus, and 10 μmol of iron, in comparison with Enteromorpha strains collected in other production areas (control strains 1, 5, and 6). [Figure 4] FIG. 4 is a graph showing the change in wet weight (g) of the algae of the present invention when cultured for 8 days at 25° C. in 500 ml of sterilized seawater containing added 100 μmol of nitrogen, 10 μmol of phosphorus, and 10 μmol of iron, in comparison with the Enteromorpha strains collected in other production areas (control strains 1, 5, and 6). [Diagram 5] FIG. 5 is a graph showing the change in wet weight (g) of algae of the Enteromorpha prolifera of the present invention when cultured for 7 days in 500 ml of sterilized seawater containing added 100 μmol of nitrogen, 10 μmol of phosphorus, and 10 μmol of iron at temperatures of 10° C., 15° C., 20° C., 25° C., 30° C., and 35° C. [Figure 6A-1]Figure 6A-1 is a diagram showing the nucleotide sequence of the heat shock protein (HSP) 90 gene (referred to as "HSP90") of the Lavera spp. Kochi No. 1 strain of the present invention (Kochi No. 1 and Kochi No. 1#3 (described below)) in comparison with the nucleotide sequence of a previously known strain (sequence from DDBJ (DNA Data Bank of Japan) and indicated by the accession number). In the figure, the boxes indicate bases in the above sequence of the Lavera spp. Kochi No. 1 strain of the present invention that differ from the sequence of the previously known strain. [Figure 6A-2] This is a continuation of the sequence shown in Figure 6A-1. [Figure 6A-3] This is a continuation of the sequence shown in Figure 6A-2. [Figure 6B-1] Figure 6B-1 is a diagram showing the nucleotide sequence of the 5S ribosomal DNA intervening region (referred to as "S5S") of the Laver Kochi No. 1 strain of the present invention (Kochi No. 1 and Kochi No. 1#11 (described below)) in comparison with the nucleotide sequence of a previously known strain (sequence from DDBJ (DNA Data Bank of Japan) and indicated by the accession number). In the figure, the boxes indicate bases in the above sequence of the Kochi No. 1 strain of the present invention that differ from the sequences of previously known strains. [Figure 6B-2] This is a continuation of the sequence shown in Figure 6B-1. [Figure 6B-3] This is a continuation of the sequence shown in Figure 6B-2. [Figure 6B-4] This is a continuation of the sequence shown in Figure 6B-3. [Figure 6B-5] This is a continuation of the sequence shown in Figure 6B-4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present invention will now be described in further detail.

[0021] 1. High-temperature resistant seaweed As described above, the high-temperature-resistant Enteromorpha of the present invention has a characteristic that when cultured for 8 days in water at a temperature of 25°C to 30°C, the wet weight of the algae on the 8th day of culture increases by 6 times or more, preferably 7 times or more, 8 times or more, or 9 times or more, and more preferably 10 times or more compared to that on the 4th day of culture ("Characteristic 1"; Figures 4 and 5). This characteristic shows that the Enteromorpha of the present invention has excellent high-temperature resistance.

[0022] In one embodiment of the present invention, the Green Enteromorpha of the present invention further exhibits an average daily growth rate over 6 days at a water temperature of 25°C of more than about 120%, and an average daily growth rate over 6 days at a water temperature of 30°C of about 110%, showing extremely excellent growth rates even at water temperatures of 25°C to 30°C ("Characteristic 2"; Figure 1).

[0023] In another embodiment of the present invention, Enteromorpha platyphylla of the present invention further exhibits a maturation rate of algal bodies of 20% or less when cultured for 6 days in water at a temperature of 25° C. ("Characteristic 3"; FIG. 2).

[0024] Green laver that exhibits the above characteristic 1 in the high temperature range of 25°C to 30°C, or Green laver that exhibits the above characteristic 1 and at least one of characteristic 2 and characteristic 3, preferably Green laver that exhibits the above characteristics 1, 2 and 3, has not been reported until now.

[0025] As described in the Background Art above, in conventional Enteromorpha strains, the algae mature and release spores at water temperatures of 20° C. to 23° C., and the cells in the mature parts are washed out, causing a decrease in the length and weight of the algae, resulting in a significant decrease in the yield of Enteromorpha. For this reason, it is difficult to mass-produce conventional Enteromorpha strains by either marine or land culture methods in environments with high water temperatures of 20° C. or higher, especially 25° C. or higher.

[0026] Non-Patent Document 4 describes the selection and breeding of Green Laver strains that can grow even at high water temperatures with the aim of extending the possible cultivation period, and as a result, the NH strain is described as a high-temperature-resistant Green Laver strain, but as shown in Figure 3 in the document, the growth rate of the NH strain Green Laver on the 6th day of cultivation at temperatures of 20, 25, and 30°C (length of measurement day / length of starting day) is about 5.5 times at 20°C, but about 3.3 times and 2.4 times at high water temperatures of 25°C and 30°C, respectively, decreasing as the temperature increases. Furthermore, this document does not describe the growth rate or wet weight of algae after the 6th day.

[0027] On the other hand, as described above, the Green Enteropore of the present invention has the characteristic that when cultured for 8 days in water at a temperature of 25°C to 30°C, the wet weight of the algae on the 8th day of culture increases by 6 times or more, preferably 7 times or more, 8 times or more, or 9 times or more, and more preferably 10 times or more compared to that on the 4th day of culture (Figures 4 and 5). In view of these characteristics, it differs from the NH strain described in Non-Patent Document 4 and is superior in terms of productivity.

[0028] Furthermore, when the Green Enteropore of the present invention was cultured at a water temperature of 25°C for 6 days, the maturity rate of the algal bodies was 20% or less, whereas in the case of the NH strain of Non-Patent Document 4, mature portions (i.e., 100% maturity rate) were observed in all individuals tested at 25°C, demonstrating a clear difference.

[0029] Thus, the green laver of the present invention can be cultured at high productivity not only in water temperatures of about 15°C to about 20°C, but also in high temperature ranges of 20°C or higher, for example, about 25°C to about 30°C.

[0030] Since high-temperature-resistant Enteromorpha laver of the present invention has not been known in the past, it can be used to increase the production volume of Enteromorpha laver and stabilize the price by cultivating it over a wide temperature range using methods such as marine culture and land culture.

[0031] An example of the high-temperature-resistant Enteropore of the present invention is the high-temperature-resistant Enteropore Kochi No. 1 strain collected in Kochi Prefecture (having the above characteristics 1, 2, and 3) or a descendant thereof. The descendants of the Kochi No. 1 strain are Enteropore strains having at least the above characteristic 1, and may be Enteropore strains having at least one of the above characteristics 2 and 3 in addition to characteristic 1, but preferably have the above characteristics 1, 2, and 3.

[0032] The Laver Kochi No. 1 strain of the present invention has different bases (or nucleotides) from the base sequences of the heat shock protein (HSP) 90 gene (referred to as "HSP90") and the 5S ribosomal DNA intervening region (referred to as "S5S") compared with the conventionally known strains (sequences from the DNA Data Bank of Japan (DDBJ) (SEQ ID NOs: 5 to 33 in Table 1, indicated by accession numbers) (boxes in Figures 6A and 6B). The base sequences in Figure 6A are shown in SEQ ID NO: 1 for HSP90 of Kochi No. 1 (including overlapping bases) and SEQ ID NO: 3 for HSP90 of Kochi No. 1 (no overlapping bases #3). The base sequences in Figure 6B are shown in SEQ ID NO: 2 for S5S of Kochi No. 1 (including overlapping bases) and SEQ ID NO: 4 for S5S of Kochi No. 1 (no overlapping bases #11). Since the Laver Kochi No. 1 strain is a diploid organism, it has more than two types of HSP90 and S5S sequences, and when sequenced, 23 overlapping bases were found in the HSP90 sequence, while 5 overlapping bases were found in the S5S sequence. Furthermore, at least two of the multiple sequences for HSP90 and S5S were cloned, and the sequences #3 and #11, which do not contain overlapping bases, were also determined. In the present specification, in the base sequences of SEQ ID NOs: 1 and 2, the overlapping bases are represented by the symbols R (G or A), Y (T or C), K (G or T), S (G or C), and M (A or C).

[0033] Therefore, the Enteromorpha prolifera of the present invention and its progeny may be characterized in that it contains the nucleotide sequence represented by SEQ ID NO: 1 and / or SEQ ID NO: 2 ("Characteristic 4").

[0034] Alternatively, the Enteromorpha prolifera of the present invention and its progeny may be characterized in that it comprises the nucleotide sequence represented by SEQ ID NO: 3 and / or SEQ ID NO: 4 ("Characteristic 5").

[0035] The descriptions of SEQ ID NOs: 1 to 4 of the Enteromorpha laver Kochi No. 1 strain of the present invention and SEQ ID NOs: 5 to 33 of the conventionally known strains are shown in Table 1 below.

[0036] [Table 1]

[0037] The present invention further includes parts of the high temperature tolerant Enteromorpha prolifera of the present invention.

[0038] The term "portion" is used herein to refer to a portion of Enteromorpha japonica that contains the genetic trait that confers at least characteristic 1. The "portion" may further contain at least one of the genetic traits of characteristic 2 and characteristic 3.

[0039] The part of Enteromorpha commune is selected from the group consisting of, for example, spores (or reproductive cells), cells (other than reproductive cells), and tissues (for example, thallus, adventitious roots, branches, etc.).

[0040] 2. Method for producing high-temperature resistant Enteromorpha platyphylla The present invention further provides a method for producing heat-resistant Enteromorpha algae bodies, the method comprising the steps of growing heat-resistant Enteromorpha algae bodies using spores of the heat-resistant Enteromorpha or the heat-resistant Enteromorpha Kochi No. 1 strain of the present invention described in 1 above, and recovering the heat-resistant Enteromorpha algae bodies.

[0041] In one embodiment of the present invention, the method further comprises washing and drying the thermotolerant Enteromorpha platyphylla to obtain a dried Enteromorpha platyphylla as a product.

[0042] In another embodiment of the present invention, the growing step can be carried out by marine or land-based aquaculture.

[0043] Below, examples of marine aquaculture and land-based aquaculture methods will be described.

[0044] 2.1 Sea culture method An example of the marine culture method for producing the high-temperature tolerant Enteromorpha platyphylla described in 1 above will be described below.

[0045] In this specification, "marine aquaculture" refers to aquaculture carried out in brackish waters near the mouth of a river where seawater and freshwater mix.

[0046] The method includes, for example, the following steps: (a) obtaining spores from Enteromorpha platyphylla as described in 1 above; (b) attaching the spores obtained in step (a) to a support (e.g. a net); (c) growing Enteromorpha platyphylla by marine culture using a support (e.g., a seed net) to which the spores produced in step (b) are attached; and (d) recovering the Enteromorpha platyphylla grown in step (c); Includes.

[0047] Each step of the above method will now be described.

[0048] <Step (a)> In step (a), spores are released from the algal bodies of Enteromorpha prolifera of the present invention as parent individuals for the purpose of reproduction.

[0049] As used herein, the term "algal body" refers to the part of the thallus of Enteromorpha that is composed only of vegetative cells and does not include the parts that have matured and differentiated into reproductive cells.

[0050] As used herein, "spore" refers to a reproductive cell, such as a zoospore, gamete, zygote, tetraspore, carpospore, uniospore, or neutral zoospore, and refers to a type of reproductive cell that can attach to and connect with each other in multiple units.

[0051] The following procedures are carried out to release spores from Enteromorpha platyphylla. If the spores have phototaxis (they move away from or gather towards light), they are aggregated using light to obtain a highly concentrated spore suspension. If they do not have phototaxis, the spores are collected using a centrifuge or other equipment to obtain a highly concentrated spore liquid.

[0052] <Step (b)> In step (b), the spores obtained in step (a) above are attached to a support (eg, a net).

[0053] In this specification, the term "support" includes a woven mesh-like "mesh" of about 20 cm square, made of a material to which spores easily adhere.

[0054] In this specification, the term "seed net" refers to a net-like support having spores of Enteromorpha prolifera of the present invention attached to net threads obtained in step (a).

[0055] <Step (c)> In step (c), the support such as the seed net prepared in step (b) is moved to a marine aquaculture site and set up, and the seedlings of Enteromorpha platyphylla of the present invention are grown until they become algae. The water temperature is, for example, about 10°C to about 35°C, preferably about 15°C to about 30°C.

[0056] In this specification, the term "marine aquaculture site" refers to an area established over a certain area in the brackish coastal waters where seawater and freshwater mix, in which supports such as seed nets with spores attached are fixed and the seedlings are allowed to grow until they become algae bodies.

[0057] <Step (d)> In step (d), the Enteromorpha aquatic plant bodies grown in step (c) are removed and collected from the support such as a net installed in the marine aquaculture site.

[0058] If necessary, the Enteromorpha purpurea algae collected in step (d) are washed with fresh water and dried. Drying may be performed using a dryer or by natural drying.

[0059] 2.2 Land-based aquaculture An example of the land-based culture method for producing the high-temperature tolerant Enteromorpha platyphylla described in 1 above will be described below.

[0060] As used herein, "land-based aquaculture" refers to aquaculture in large containers such as aquariums installed on land using seawater (natural or artificial) containing nutrients for algae cultivation.

[0061] The method includes, for example, the following steps: (a) obtaining spores from Enteromorpha platyphylla as described in 1 above; (b) seeding the spores obtained in step (a) in an appropriate container and culturing them to form a spore mass in which a plurality of spores are connected to each other or a germinant mass in which the germinants formed by germinating the spores are intertwined; (c) culturing the spore clumps or germling clumps obtained in step (b) to produce seedlings; (d) transplanting the seedlings obtained in step (c) into a land-based aquarium; (e) supplying seawater into the land-based water tank of step (d) to suspend the seedlings, and growing Enteromorpha platyphylla from the seedlings by mixing the supplied seawater with a mixing means such as aeration; and (f) recovering the Enteromorpha platyphylla grown in step (e); Includes.

[0062] Each step of the above method will now be described.

[0063] <Step (a)> In step (a), spores are released for reproduction from the Enteromorpha prolifera of the present invention as parent individuals in the same manner as described in step (a) of the marine aquaculture method described above.

[0064] <Step (b)> In step (b), spore clumps or germling clumps are formed from the spores obtained in step (a).

[0065] As used herein, the term "spore clump" refers to a clump of two or more connected spores, and also includes a germinant clump in which a plurality of spores have germinated and become entangled.

[0066] More specifically, the spore concentration of a high-concentration spore suspension of Enteromorpha platyphylla obtained by the same method as step (a) of the above <Marine surface culture method> is measured, the concentration is adjusted, and the suspension is then seeded in a suitable container. After culturing for several days, it is confirmed under a microscope that the spores are attached to each other. Thereafter, it is confirmed that spore clumps have been formed, and the spore clumps are collected. If necessary, the spore clumps are crushed into small clumps, for example, with a diameter of about 5 mm or less.

[0067] <Step (c)> In step (c), seeds for land-based aquaculture are prepared from the spore clumps or germling clumps obtained in step (b). When the small spore clumps are transferred to flasks and cultured, for example with aeration, germlings grow radially from the small clumps. The germlings that grow to about 5 mm are used as seeds.

[0068] <Step (d)> In step (d), the seedlings prepared in step (c) are transferred to an aquatic tank on land for the actual cultivation of Enteromorpha algae.

[0069] The term "land-based aquarium" refers to a large-scale aquarium with an open top that is installed on land near the coast for growing Enteromorpha platyphylla. Preferably, the seeds of the Enteromorpha platyphylla strain of the present invention are placed in a tank with a diameter of 1 m to 30 m and a water depth of 30 cm to 60 cm.

[0070] <Step (e)> In step (e), the seedlings of Enteromorpha japonica of the present invention transplanted in step (d) are grown to a harvestable size in a water tank at a water temperature of, for example, about 10°C to about 35°C, preferably about 15°C to about 30°C.

[0071] The term "seawater" includes both natural seawater (including, for example, deep ocean water and groundwater) and artificial seawater. Deep ocean water is seawater taken from the ocean at a depth of 200m or more, and has characteristics different from surface seawater, such as being clean, rich in nutrients, and having stable water quality at low temperatures. Groundwater can be excavated to a depth of several meters from the beach near the coast, and has similar characteristics to deep ocean water.

[0072] <Step (f)> In step (f), the green laver that has grown to a harvestable size in step (e) is removed from the tank and collected.

[0073] If necessary, the Enteromorpha platyphylla cells collected in step (f) are washed with fresh water and dried. Drying may be performed using a dryer or by natural drying. EXAMPLES

[0074] The present invention will be described in more detail by the following examples, which are merely illustrative and not limiting of the present invention.

[0075] The "Porphyra platyphylla of the present invention" described in the following examples is the high-temperature-resistant Porphyra platyphylla Kochi No. 1 strain.

[0076] Example 1: Comparison of average daily growth rates of Enteromorpha strains at water temperatures of 10°C, 15°C, 20°C, 25°C, and 30°C In order to compare the daily growth rate (%) of the Green Aonori Kochi No. 1 strain of the present invention with the daily growth rate (%) of Green Aonori strains collected in other production areas, the strains were cultured for 6 days at temperatures of 10°C, 15°C, 20°C, 25°C, or 30°C (Figure 1). The daily growth rate (%) is the average of the percentage increase or decrease in the wet weight of the algae body on the test day one day later, when the wet weight of the algae body on the day before the test is taken as 100%, for 6 days. In Figure 1, control strain 1 is a Green Aonori strain collected from Yoshii River in Okayama Prefecture, control strain 2 is a strain collected from Hyogo Prefecture, control strain 3 is a strain collected from Tokushima Prefecture, and control strain 4 is a strain collected from Kagawa Prefecture. Four test plots were set up for each strain.

[0077] Five clumps of agglomerated spores of each Enteromorpha strain (initial wet weight of algae: 0.002 g / 5 clumps) were used as test algae, and were placed in a 500-mL Erlenmeyer flask containing sterilized seawater as a medium to which 100 μmol of nitrogen, 10 μmol of phosphorus, and 10 μmol of iron had been added per 500 mL of seawater. The light / dark period was 12 hours / 12 hours, and the light intensity was 100 μmol photon / m 2 After pre-cultivation for 2 days under the culture conditions of 10 / s and water change every 24 hours, the mixture was cultured with aeration for 6 days at temperatures of 10°C, 15°C, 20°C, 25°C or 30°C.

[0078] 1, the average daily growth rate (%) of the Lavera spp. Kochi No. 1 strain of the present invention exceeded 120% over 6 days at a water temperature of 25° C., and was approximately 110% over 6 days at a water temperature of 30° C., demonstrating extremely excellent high-temperature resistance at water temperatures of 25° C. and 30° C. In contrast, all of the other test plots of Control Strain 1 to Control Strain 4 showed obvious poor condition at a water temperature of 25° C., with the algae turning white or breaking apart rapidly due to rapid maturation, and it was therefore not possible to obtain data on daily growth rates (%) at water temperatures of 25° C. and 30° C.

[0079] The results of Example 1 showed that the Laver Kochi No. 1 strain of the present invention has significant tolerance to high water temperatures of 25°C and 30°C compared to the Laver strains collected in the respective production areas of Okayama Prefecture, Hyogo Prefecture, Tokushima Prefecture, and Kagawa Prefecture.

[0080] Example 2: Comparison of maturity rates of Enteromorpha spp. strains after 6-day cultivation at 25°C The maturity rate (%) of the Green Aonori Kochi No. 1 strain of the present invention after 6 days of cultivation at a water temperature of 25°C was compared with the maturity rate (%) of Green Aonori strains collected in other production areas (Figure 2). In Figure 2, control strain 1 is a Green Aonori strain collected from Yoshii River in Okayama Prefecture, control strain 5 is a Green Aonori strain collected from Yoshino River in Tokushima Prefecture, control strain 6 is a Green Aonori strain collected from Matsuzaki Town in Shizuoka Prefecture, control strain 7 is a Green Aonori strain collected from Kaiyo Town in Tokushima Prefecture, and control strain 8 is a Green Aonori strain collected from Awaji Island. Four test plots were set up for each strain. Each test plot was tested four or more times. Regarding the maturity rate (%), the Green Aonori algae in the flask were checked every day for maturity, and when one or more mature algae were confirmed, the flask test plot was considered to be mature, and the values ​​for each test plot were averaged.

[0081] Five clumps of spores from each Enteromorpha strain were used as test algae, and were placed in a 500-mL Erlenmeyer flask containing sterilized seawater as a medium to which 100 μmol of nitrogen, 10 μmol of phosphorus, and 10 μmol of iron had been added. The light / dark period was 12 hours / 12 hours, and the light intensity was 100 μmol photon / m 2 After pre-cultivation for two days under the culture conditions of 100 / s and water change every 24 hours, the mixture was aerated and cultured at 25°C for six days, and the maturation rate (%) was measured every 24 hours.

[0082] As shown in Figure 2, the average maturity rate (%) of the four test plots of the Lavera purpurea Kochi No. 1 strain of the present invention was only 16.6%, resulting in almost no maturation, even after six days of cultivation at a water temperature of 25°C. In contrast, control strains 1 and 5 to 8 all reached 100% maturity after six days of cultivation at a water temperature of 25°C, with the algal bodies releasing spores from the tips, which then turned white after release.

[0083] The results of Example 2 showed that the Laver Kochi No. 1 strain of the present invention is less likely to mature even when cultured in a high water temperature environment of 25°C for 6 days, compared to Laver strains collected from the respective production areas of Yoshii River in Okayama Prefecture, Yoshino River in Tokushima Prefecture, Matsuzaki Town in Shizuoka Prefecture, Kaiyo Town in Tokushima Prefecture, and Awaji Island.

[0084] Example 3: Comparison of algal body length of Enteromorpha spp. strain after 8 days of cultivation at 25°C The algal body length (mm) of the Kochi No. 1 strain of the Green Aonori of the present invention after 8 days of cultivation at a water temperature of 25°C was compared with the algal body length (mm) of Green Aonori strains collected in other production areas (Figure 3). In Figure 3, control strain 1 is a Green Aonori strain collected from the Yoshii River in Okayama Prefecture, control strain 5 is a Green Aonori strain collected from the Yoshino River in Tokushima Prefecture, and control strain 6 is a Green Aonori strain collected from Matsuzaki Town in Shizuoka Prefecture. Four test plots were set up for each strain. Each test plot was tested three times. Regarding the algal body length (mm), the algal bodies were removed from the flask every 24 hours, and the longest part from the root to the tip was measured as the algal body length, and the values ​​for each test plot were averaged.

[0085] One clump of spores (initial algal body length: 10 mm ± 3 mm) of each Enteromorpha strain was used as the test algal body and placed in a 500-mL Erlenmeyer flask containing sterilized seawater as a medium to which 100 μmol of nitrogen, 10 μmol of phosphorus, and 10 μmol of iron had been added per 500 mL of seawater. The light / dark period was 12 hours / 12 hours, and the light intensity was 300 μmol photon / m 2 After pre-cultivation for 2 days under the conditions of 1000 / s and water change every 24 hours, the algae were aerated and cultured at 25°C for 8 days, and the algal body length (mm) was measured every 24 hours. The algal body length was measured based on the longest clumped algal body length each time.

[0086] 3, the average algal body length of the Lavera purpurea Kochi No. 1 strain of the present invention after 8 days of culture at a water temperature of 25° C. was 68 mm, which was about 7 times the initial algal body length on day 1 of culture. In contrast, the algal body lengths of the other control strains 1, 5, and 6 after 8 days of culture at a water temperature of 25° C. were all less than 50 mm, about 5 times or less the initial algal body length on day 1 of culture.

[0087] As a result, it was shown that the Green Laver Kochi No. 1 strain of the present invention grows to a significantly longer algal body length when cultured for 8 days in a high water temperature environment of 25°C, compared to the Green Laver strains collected from the respective production areas of Yoshii River in Okayama Prefecture, Yoshino River in Tokushima Prefecture, and Matsuzaki Town in Shizuoka Prefecture.

[0088] <Example 4> Comparison of wet weight of algae of Enteromorpha spp. strain after 8 days of cultivation at a water temperature of 25°C The wet weight (g) of the green laver Kochi No. 1 strain of the present invention after 8 days of cultivation at a water temperature of 25°C was compared with the wet weight (g) of green laver strains collected in other production areas (Figure 4). In Figure 4, control strain 1 is a green laver strain collected from the Yoshii River in Okayama Prefecture, control strain 5 is a green laver strain collected from the Yoshino River in Tokushima Prefecture, and control strain 6 is a green laver strain collected from Matsuzaki Town in Shizuoka Prefecture. Four test plots were set up for each strain. Each test plot was tested three times. The wet weight (g) of the green laver was measured by removing the green laver from the flask every 24 hours, wiping off the surface moisture, and then weighing.

[0089] One clump of spores (initial wet weight of algae: average 0.001 g) of each Enteromorpha strain was used as the test algae, and was placed in a 500-mL Erlenmeyer flask containing sterilized seawater as a medium to which 100 μmol of nitrogen, 10 μmol of phosphorus, and 10 μmol of iron had been added per 500 mL of seawater. The light / dark period was 12 hours / 12 hours, and the light intensity was 300 μmol photon / m 2 After pre-cultivation for 2 days under the culture conditions of 100 / s and water change every 24 hours, the mixture was aerated and cultured at 25°C for 8 days, and the wet weight (g) of the algae was measured every 24 hours. The results are shown in FIG.

[0090] [Table 2]

[0091] 4 and Table 2, the average wet weight (g) of the Enteromorpha laver Kochi No. 1 strain of the present invention was approximately 0.276 g after 8 days of culture at a water temperature of 25° C., and the average wet weight (g) of the algal body on day 8 of culture was increased by 12.6 times compared to the average wet weight (g) of the algal body on day 4 of culture. In contrast, the other control strains 1, 5, and 6 all had an average wet weight (g) of approximately 0.152 g or less after 8 days of culture at a water temperature of 25° C., which was 4.74 times or less the average wet weight of the algal body on day 4 of culture.

[0092] The results of Example 4 showed that the Laver Kochi No. 1 strain of the present invention grew into algal bodies with a significantly higher wet weight when cultured for 8 days in a high water temperature environment of 25°C, compared to Laver strains collected from the respective production areas of Yoshii River in Okayama Prefecture, Yoshino River in Tokushima Prefecture, and Matsuzaki Town in Shizuoka Prefecture.

[0093] Example 5: Change in wet weight of Enteromorpha spp. at water temperatures of 10°C, 15°C, 20°C, 25°C, 30°C, and 35°C The changes in wet weight (g) of the algae of the present invention's Enteromorpha spp. Kochi No. 1 strain after 7-day cultivation at water temperatures of 10°C, 15°C, 20°C, 25°C, 30°C, and 35°C were compared (Figure 5). Four test plots were set up for each temperature. Each test plot was performed three times.

[0094] One clump of spores (initial wet weight of algae: average 0.002 g) of each Enteromorpha strain was used as the test algae, and was placed in a 500-mL Erlenmeyer flask containing sterilized seawater as a medium to which 100 μmol of nitrogen, 10 μmol of phosphorus, and 10 μmol of iron had been added per 500 mL of seawater. The light / dark period was 12 hours / 12 hours, and the light intensity was 100 μmol photon / m 2 After pre-cultivation for 2 days under the culture conditions of 10 / s and water change every 24 hours, the mixture was aerated and cultured for 7 days at water temperatures of 10°C, 15°C, 20°C, 25°C, 30°C, and 35°C, and the wet weight (g) of the algae was measured every 24 hours.

[0095] As shown in Fig. 5, after culturing for 6 days at each of the above temperatures, the average wet weight (g) of the algal body of the Kochi No. 1 strain of the present invention was 0.102 g at a water temperature of 25°C and 0.116 g at a water temperature of 30°C, and after culturing for 7 days, the average wet weight (g) of the algal body of the Kochi No. 1 strain of the present invention was 0.2092 g at a water temperature of 25°C and 0.2318 g at a water temperature of 30°C, showing a significant increase to the same level at water temperatures of 25°C and 30°C. This result indicates that the Laver strain of the present invention has significant high temperature resistance at high water temperatures of 25°C to 30°C.

[0096] <Example 6> Determination of the characteristic base sequence of Enteromorpha japonica Kochi No. 1 strain Using the forward primer hsp90-6F (5′-GCAGACCCAGAAAGTGATCTATTAYATCA -3′) (SEQ ID NO: 34) and the reverse primer hsp90-6R (5′-GCAGGYTCATCCAGACTAAATCC -3′) (SEQ ID NO: 35) described in Ogawa et al. ("High heterozygosity and phenotypic variation of zoids in apomictic Ulva prolifera (Ulvophyceae) from brackish environments", Aquatic Botany 120, 185-192 (2014)), PCR was performed using the gene of Enteromorpha spp. Kochi No. 1 strain as a template, and the nucleotide sequence of HSP90 in the PCR product was determined. Duplicate nucleotides were found in HSP90, revealing that it contains multiple nucleotide sequences. The sequence including the duplicated nucleotides was determined and is shown in SEQ ID NO: 1. Furthermore, one of the multiple base sequences was selected and amplified by cloning, and the base sequence of HSP90#3 was determined and shown as SEQ ID NO:3.

[0097] Using the forward primer 5S-F (5'- GGTTGGGCAGGATTAGTA -3') (SEQ ID NO: 36) and the reverse primer 5S-R (5'- AGGCTTAAGTTGCGAGTT -3') (SEQ ID NO: 37) described in Shimada et al. ("Phylogeography of the genus Ulva (Ulvophyceae, Chlorophyta), with special reference to the Japanese freshwater and brackish taxa", Journal of Applied Phycology 20, 979-989 (2008)), PCR amplification was performed using the gene of Enteromorpha spp. Kochi No. 1 strain as a template, and the nucleotide sequence of the PCR product, S5S, was determined. Duplicate nucleotides were found in S5S, and it was found that it contains multiple nucleotide sequences. The sequence including the duplicated nucleotides was determined and shown as SEQ ID NO: 2. Furthermore, cloning was performed to select and amplify one of the multiple nucleotide sequences, and the nucleotide sequence of S5S#11 was determined and shown as SEQ ID NO: 4. [Industrial Applicability]

[0098] The green laver of the present invention having excellent high temperature resistance as described above is useful for increasing the production amount of green laver by marine culture method or land tank culture method and for stabilizing the price of green laver.

Claims

1. A high-temperature resistant Enteromorpha spp. or a part thereof, characterized in that it contains a base sequence represented by sequence numbers 1 and / or 4.

2. The high-temperature resistant Enteromorpha sieboldii or a part thereof according to claim 1, characterized in that the part is a spore, a cell or a tissue.

3. A method for producing high-temperature-resistant Enteromorpha platyphylla, comprising the steps of growing high-temperature-resistant Enteromorpha platyphylla using spores of the high-temperature-resistant Enteromorpha platyphylla described in claim 1 or 2, and recovering the high-temperature-resistant Enteromorpha platyphylla.

4. The method according to claim 3, further comprising the step of washing and drying the thermotolerant Enteromorpha platyphylla.

5. 5. The method according to claim 3 or 4, characterized in that the growing step is carried out by marine or land-based aquaculture.

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