JP2021013338A - Transgenic microalgae and method for culturing the transgenic microalgae - Google Patents
Transgenic microalgae and method for culturing the transgenic microalgae Download PDFInfo
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Abstract
Description
本発明は、例えばオイル等の物質生産に利用されうる微細藻類を宿主とした形質転換微細藻類及び当該形質転換微細藻類の培養方法に関する。 The present invention relates to transformed microalgae using microalgae as a host, which can be used for producing substances such as oil, and a method for culturing the transformed microalgae.
近年、CO2排出削減及び環境への負荷軽減の観点から、再生可能な生物資源（バイオマス）を用いた物質生産が注目されている。光合成を行う単細胞生物である微細藻類は、光エネルギーを利用して、水とCO2から様々な代謝産物を産生することが知られている。微細藻類を用いて生産される物質としては、バイオ燃料の原料としての利用が可能なトリアシルグリセロール（TAG）やデンプン、健康サプリメント等に利用可能なタンパク質やアミノ酸、抗酸化作用等多様な生理活性を有するカロテノイド類等を挙げることができる。 In recent years, from the viewpoint of reducing CO 2 emissions and reducing the burden on the environment, material production using renewable biological resources (biomass) has attracted attention. It is known that microalgae, which are unicellular organisms that perform photosynthesis, use light energy to produce various metabolites from water and CO 2 . Substances produced using microalgae include triacylglycerol (TAG) and starch that can be used as raw materials for biofuels, proteins and amino acids that can be used for health supplements, and various physiological activities such as antioxidant activity. Carotenoids having the above can be mentioned.
例えば、オイル高含有海洋珪藻であるFistulifera solaris JPCC DA0580株 (以下F. solaris)は、屋外での大量培養試験が試みられている。また、F. solarisについては全ゲノム解析やトランスクリプトーム解析の対象として網羅的な解析がなされており、様々な物資生産の宿主として期待されている。 For example, Fistulifera solaris JPCC DA0580 strain (F. solaris), which is an oil-rich marine diatom, has been attempted to be mass-cultured outdoors. In addition, F. solaris has been comprehensively analyzed as a target of whole genome analysis and transcriptome analysis, and is expected as a host for various material production.
但し、F. solaris等の微細藻類を使用する系では、屋外培養におけるバイオマス生産性は屋内と比較して顕著に低下する傾向にあり、コスト収支の低さの一因となっている。したがって、微細藻類を利用した有用物質生産の実現に向け、屋外大量培養におけるバイオマス生産性の向上は必要不可欠である。より具体的には、微細藻類の培養において最も問題視されているのが、培養系に対する他の生物種の混入（コンタミネーション）である。特に、屋外培養におけるバイオマス生産性の低下の要因には、栄養源の供給の不足、pHやCO2濃度など培養条件の変化、気候変動といった非生物的要因、及び生物的要因として他生物種のコンタミネーションが挙げられる。この中でもコンタミネーションは、“Pond crash”と呼ばれる、特に甚大な藻類バイオマス量の減少を引き起こす要因とされており、微細藻類の屋外開放系大量培養における大きな課題の一つである。 However, in a system using microalgae such as F. solaris, the biomass productivity in outdoor culture tends to be significantly lower than that in indoor culture, which contributes to the low cost balance. Therefore, it is indispensable to improve the biomass productivity in outdoor mass culture in order to realize the production of useful substances using microalgae. More specifically, the most problematic aspect of culturing microalgae is contamination of other species into the culture system. In particular, factors that reduce biomass productivity in outdoor culture include insufficient supply of nutrient sources, changes in culture conditions such as pH and CO 2 concentration, abiotic factors such as climate change, and other biological factors as biological factors. Contamination can be mentioned. Among them, contamination is considered to be a factor called "Pond crash", which causes a particularly large decrease in the amount of algae biomass, and is one of the major problems in the large-scale outdoor open culture of microalgae.
したがって、微細藻類を利用した物質生産において、コンタミネーションの抑制が微細藻類のバイオマス生産性の向上、並び実用化に大きく寄与できると考えられる。微細藻類の培養系にコンタミネーションする具体的な生物種には、ワムシや繊毛虫、鞭毛虫、アメーバ等の動物プランクトン、真菌類や真正細菌等の従属栄養生物、他の微細藻類種、またはウイルスが挙げられる。なお、これら生物種の微細藻類に対する影響も様々であり、直接的な捕食、栄養源の競合、宿主としての感染がある。これらの中でも動物プランクトンによる微細藻類の直接的な捕食は特に甚大な被害をもたらす。藍藻Oscillatoria属に対する繊毛虫Nassula属、緑藻Scenedesmus属に対するアメーバVampyrellida属によって、いずれの場合も数日間でほとんどの藻類バイオマスが失われたことが報告されている。 Therefore, in the production of substances using microalgae, it is considered that the suppression of contamination can greatly contribute to the improvement of biomass productivity of microalgae and the practical application. Specific species that contaminate the microalgae culture system include zooplankton such as worms, ciliates, flagellates, and amoeba, heterotrophic species such as fungi and eubacteria, other microalgae species, or viruses. Can be mentioned. The effects of these species on microalgae also vary, including direct predation, competition for nutrient sources, and infection as a host. Of these, direct predation of microalgae by zooplankton causes particularly great damage. It has been reported that most of the algae biomass was lost within a few days by the ciliates Nassula against the cyanobacteria Oscillatoria and the Amoeba Vampyrellida against the green alga Scenedesmus.
そこで、分子育種技術を用いて、微細藻類そのものに捕食生物に対する耐性を付与する生物学的手法が有効な手法として考えられる。しかし、現在まで微細藻類及び動物プランクトンの捕食−被食関係の分子メカニズムに関する研究は殆どなされておらず知見に乏しい。それゆえ、生物学的手法による抑制した例は藍藻における一報のみに留まっている (非特許文献１：Simkovsky, R., Daniels, E. F., Tang, K., Huynh, S. C., Golden, S. S., Brahamsha, B. (2012). Impairment of O-antigen production confers resistance to grazing in a model amoeba-cyanobacterium predator-prey system. Proc Natl Acad Sci USA, 109(41), 16678-16683)。非特許文献１によれば、藍藻のランダム変異体ライブラリーからアメーバによる捕食が認められなかった株を獲得したとある。また、この株の詳細な解析の結果、細胞表面のO抗原の欠損が捕食作用からの回避に寄与することを明らかにしている。しかし、この機構は原核生物に特有のものであり、これまで真核微細藻類において生物学的手法を用いて、捕食生物の抑制法を確立した報告はない。 Therefore, a biological method for imparting resistance to predatory organisms to the microalgae itself by using molecular breeding technology is considered as an effective method. However, until now, little research has been conducted on the predator-prey relationship molecular mechanism of microalgae and zooplankton, and there is little knowledge. Therefore, there is only one example of suppression by biological methods in blue-green algae (Non-Patent Document 1: Simkovsky, R., Daniels, EF, Tang, K., Huynh, SC, Golden, SS, Brahamsha, B. (2012). Impairment of O-antigen production confers resistance to grazing in a model amoeba-cyanobacterium predator-prey system. Proc Natl Acad Sci USA, 109 (41), 16678-16683). According to Non-Patent Document 1, a strain in which predation by amoeba was not observed was obtained from a random mutant library of cyanobacteria. In addition, detailed analysis of this strain has revealed that deficiency of O antigen on the cell surface contributes to avoidance from predation. However, this mechanism is peculiar to prokaryotes, and there have been no reports of establishing a method for suppressing predators using biological methods in eukaryotic microalgae.
一方、植物においては、植物細胞が元来有している有毒物質を産生させることで捕食生物に対する防御能（化学的防御）を向上させる試みが知られている。例えば、非特許文献２では、モデル高等植物Arabidopsis thalianaにおいてクロロフィラーゼ（Chlorophyllase; CLH）を高発現させた形質転換植物を作製し、当該形質転換植物を与えた捕食生物の生存率が低下することを見出している。クロロフィラーゼ（EC: 3.1.1.14）はクロロフィルを加水分解し、フィチル基とクロロフィリドを生成する反応を触媒する酵素である。 On the other hand, in plants, attempts are known to improve the defense ability (chemical defense) against predators by producing toxic substances originally possessed by plant cells. For example, in Non-Patent Document 2, a transformed plant in which a high expression of chlorophyllase (CLH) is produced in the model higher plant Arabidopsis thaliana is produced, and the survival rate of the predator to which the transformed plant is given is reduced. I'm finding out. Chlorophyllase (EC: 3.1.1.14) is an enzyme that catalyzes the reaction that hydrolyzes chlorophyll to produce phytyl groups and chlorophyllides.
しかしながら、微細藻類において、遺伝子操作技術や遺伝子情報を利用した分子育種によって捕食生物に対する耐性を付与するといった技術は知られていない。そこで、本発明は、このような実情に鑑み、捕食生物に対する耐性・防御能に優れた形質転換微細藻類及び当該形質転換微細藻類の培養方法を提供することを目的とする。 However, in microalgae, there is no known technology for imparting resistance to predators by genetic engineering technology or molecular breeding using genetic information. Therefore, in view of such circumstances, it is an object of the present invention to provide a transformed microalgae having excellent resistance and defense ability against predatory organisms and a method for culturing the transformed microalgae.
上述した目的を達成するため本発明者が鋭意検討した結果、クロロフィラーゼ遺伝子を発現可能に導入した形質転換微細藻類は、クロロフィラーゼ活性により捕食生物に対して高度に毒性を発揮し、その結果、捕食生物によるコンタミネーションを抑制できることを見出し、本発明を完成するに至った。 As a result of diligent studies by the present inventor to achieve the above-mentioned object, the transformed microalgae into which the chlorophyllase gene has been introduced in an expressible manner are highly toxic to predators due to the chlorophyllase activity, and as a result, We have found that contamination by predatory organisms can be suppressed, and have completed the present invention.
本発明は以下を包含する。
（１）微細藻類に対して、クロロフィルを基質としてクロロフィリドを生成するクロロフィラーゼをコードするクロロフィラーゼ遺伝子を発現可能に導入してなる形質転換微細藻類。
（２）上記クロロフィラーゼ遺伝子は、以下（ａ）又は（ｂ）のタンパク質をコードすることを特徴とする（１）記載の形質転換微細藻類。
（ａ）配列番号２のアミノ酸配列からなるタンパク質
（ｂ）配列番号２のアミノ酸配列に対して８０％以上の同一性を有するアミノ酸配列からなり、且つ、クロロフィラーゼ活性を有するタンパク質
（３）上記クロロフィラーゼ遺伝子を導入する微細藻類は、Naviculaceae（フナガタケイソウ科）に属する珪藻類であることを特徴とする（１）記載の形質転換微細藻類。
（４）上記Naviculaceae（フナガタケイソウ科）に属する珪藻類は、Fistulifera属、Mayamaea属、Navicula属、及びSeminavis属からなる群から選ばれる1つの属に属する珪藻類であることを特徴とする（３）記載の形質転換微細藻類。
（５）上記クロロフィラーゼ遺伝子を導入する微細藻類は、Fistulifera solarisであることを特徴とする（１）記載の形質転換微細藻類。
（６）上記（１）〜（５）いずれかに記載の形質転換微細藻類を培地にて培養する、形質転換微細藻類の培養方法。
（７）屋外における培養であることを特徴とする（６）記載の形質転換微細藻類の培養方法。
（８）培養した形質転換微細藻類から生産目的物質を回収することを特徴とする（６）記載の形質転換微細藻類の培養方法。
（９）上記生産目的物質は、微細藻類が産生するオイル成分であることを特徴とする（８）記載の形質転換微細藻類の培養方法。
The present invention includes the following.
(1) Transformed microalgae obtained by introducing a chlorophyllase gene encoding a chlorophyllase that produces chlorophyllide using chlorophyll as a substrate into microalgae in an expressible manner.
(2) The transformed microalga according to (1), wherein the chlorophyllase gene encodes the protein of (a) or (b) below.
(A) Protein consisting of the amino acid sequence of SEQ ID NO: 2 (b) Protein consisting of an amino acid sequence having 80% or more identity with respect to the amino acid sequence of SEQ ID NO: 2 and having chlorophyllase activity (3) The above chloro The transformed microalgae according to (1), wherein the microalgae into which the filler gene is introduced are diatoms belonging to Naviculaceae (Funagatakeisou family).
(4) The diatoms belonging to the above Naviculaceae (Funagatakeisou family) are characterized by being diatoms belonging to one genus selected from the group consisting of the genus Fistulifera, the genus Mayamaea, the genus Navicula, and the genus Seminavis (). 3) The transformed microalgae described.
(5) The transformed microalgae according to (1), wherein the microalgae into which the chlorophyllase gene is introduced is Fistulifera solaris.
(6) A method for culturing transformed microalgae, wherein the transformed microalgae according to any one of (1) to (5) above is cultured in a medium.
(7) The method for culturing transformed microalgae according to (6), which comprises culturing outdoors.
(8) The method for culturing transformed microalgae according to (6), which comprises recovering a production target substance from the cultured transformed microalgae.
(9) The method for culturing transformed microalgae according to (8), wherein the production target substance is an oil component produced by microalgae.
本発明に係る形質転換微細藻類は、クロロフィラーゼ遺伝子を発現可能に有するもののクロロフィラーゼに起因する毒性が影響せず生育することができる。その一方、本発明に係る形質転換微細藻類は、クロロフィラーゼ遺伝子を発現可能に有することで、捕食した生物を死滅させることができる。このように、本発明に係る形質転換微細藻類は、捕食生物に対する優れた防御能を有している。 Although the transformed microalgae according to the present invention can express the chlorophyllase gene, they can grow without being affected by the toxicity caused by the chlorophyllase. On the other hand, the transformed microalgae according to the present invention can kill predatory organisms by having the chlorophyllase gene expressible. As described above, the transformed microalgae according to the present invention have an excellent protective ability against predators.
本発明に係る形質転換微細藻類の培養方法では、上述のように、形質転換微細藻類が捕食生物に対する優れた防御能を有するため、培養系における当該捕食生物のコンタミネーションを防止することができる。これにより、本発明に係る形質転換微細藻類の培養方法を適用することによって、形質転換微細藻類が産生する生産目的物質を低コストに製造することができる。 In the method for culturing transformed microalgae according to the present invention, as described above, the transformed microalgae have an excellent protective ability against predators, so that contamination of the predators in the culture system can be prevented. Thereby, by applying the method for culturing the transformed microalgae according to the present invention, the production target substance produced by the transformed microalgae can be produced at low cost.
以下、本発明に係る形質転換微細藻類及び形質転換微細藻類の培養方法を、図面を参照して詳細に説明する。 Hereinafter, the method for culturing the transformed microalgae and the transformed microalgae according to the present invention will be described in detail with reference to the drawings.
本発明に係る形質転換微細藻類は、宿主である微細藻類に対してクロロフィラーゼ遺伝子を発現可能に導入したものである。ここで、クロロフィラーゼ遺伝子とは、特に限定されず、クロロフィラーゼ(Chlorophyllase; CLH、EC: 3. 1. 1. 14)をコードする遺伝子を広く意味する。クロロフィラーゼは、クロロフィルを加水分解し、フィチル基とクロロフィリドを生成する反応を触媒する酵素を意味する。クロロフィラーゼに関しては、参考文献１（Jeffrey, S. W., Hallegraeff, G. M. (1987). Chlorophyllase distribution in ten classes of phytoplankton: a problem for chlorophyll analysis. Mar Ecol Prog Ser, 293-304）に記載されるように、微細藻類を含む多くの植物細胞においてその活性が認められている。本発明に係る形質転換微細藻類を作製する際に、当該参考文献１に開示されたクロロフィラーゼ活性が認められた植物からクロロフィラーゼ遺伝子を定法に従って同定・単離し、得られたクロロフィラーゼ遺伝子を使用することができる。また、クロロフィラーゼ遺伝子としては、特開２００１−８６９９０号公報に開示されたシロザ由来のクロロフィラーゼ遺伝子及びシロイヌナズナ由来のクロロフィラーゼ遺伝子等を使用することができる。 The transformed microalgae according to the present invention are those in which the chlorophyllase gene is expressably introduced into the host microalgae. Here, the chlorophyllase gene is not particularly limited, and broadly means a gene encoding chlorophyllase (CLH, EC: 3.1. 1. 14). Chlorophyllide means an enzyme that catalyzes the reaction that hydrolyzes chlorophyll to produce phytyl groups and chlorophyllides. Regarding chlorophyllase, as described in Reference 1 (Jeffrey, SW, Hallegraeff, GM (1987). Chlorophyllase distribution in ten classes of phytoplankton: a problem for chlorophyll analysis. Mar Ecol Prog Ser, 293-304). Its activity has been observed in many plant cells including microalgae. When producing the transformed microalgae according to the present invention, the chlorophyllase gene was identified and isolated from the plant in which the chlorophyllase activity was recognized disclosed in Reference 1 according to a conventional method, and the obtained chlorophyllase gene was used. can do. Further, as the chlorophyllase gene, a chlorophyllase gene derived from white goosefoot, a chlorophyllase gene derived from Arabidopsis thaliana, etc. disclosed in JP-A-2001-86990 can be used.
一例として、シロイヌナズナ由来のクロロフィラーゼ遺伝子を使用することができる。シロイヌナズナ由来のクロロフィラーゼ遺伝子は、配列番号２のアミノ酸配列からなるタンパク質をコードしている。シロイヌナズナ由来のクロロフィラーゼ遺伝子におけるコーディング領域の塩基配列を配列番号１に示す。 As an example, a chlorophyllase gene derived from Arabidopsis can be used. The Arabidopsis thaliana-derived chlorophyllase gene encodes a protein consisting of the amino acid sequence of SEQ ID NO: 2. The nucleotide sequence of the coding region in the Arabidopsis thaliana-derived chlorophyllase gene is shown in SEQ ID NO: 1.
ただし、クロロフィラーゼ遺伝子としては、配列番号１及び２で特定されるものに限定されず、例えば、配列番号２のアミノ酸配列に対して８０％以上の同一性、好ましくは８５％以上の同一性、より好ましくは９０％以上の同一性、更に好ましくは９５％以上の同一性を有するアミノ酸配列からなり、且つ、クロロフィラーゼ活性を有するタンパク質をコードするものを使用することもできる。ここで、アミノ酸配列間の同一性の値は、BLASTアルゴリズムを実装したBLASTNやBLASTXプログラムにより算出することができる（デフォルトの設定）。なお、同一性の値は、一対のアミノ酸配列をペアワイズ・アライメント分析した際に完全に一致するアミノ酸残基を算出し、比較した全アミノ酸残基中の上記アミノ酸残基数の割合として算出される。 However, the chlorophyllase gene is not limited to those specified by SEQ ID NOs: 1 and 2, and for example, 80% or more identity, preferably 85% or more identity with respect to the amino acid sequence of SEQ ID NO: 2. It is also possible to use a protein having an amino acid sequence having 90% or more identity, more preferably 95% or more identity, and encoding a protein having chlorophyllase activity. Here, the value of identity between amino acid sequences can be calculated by a BLASTN or BLASTX program that implements the BLAST algorithm (default setting). The value of identity is calculated as the ratio of the number of amino acid residues to the total amino acid residues compared by calculating the amino acid residues that completely match when a pair of amino acid sequences are pairwise aligned. ..
また、クロロフィラーゼ遺伝子は、例えば、配列番号２のアミノ酸配列に対して、１又は数個のアミノ酸が置換、欠失、挿入又は付加されたアミノ酸配列を有し、クロロフィラーゼ活性を有するタンパク質をコードするものでも良い。ここで、数個とは、例えば、２〜７０個、好ましくは２〜５０個、より好ましくは２〜３０個、最も好ましくは２〜１５個である。 Further, the chlorophyllase gene encodes a protein having chlorophyllase activity, for example, having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added to the amino acid sequence of SEQ ID NO: 2. It may be something to do. Here, the number is, for example, 2 to 70, preferably 2 to 50, more preferably 2 to 30, and most preferably 2 to 15.
さらにまた、クロロフィラーゼ遺伝子は、例えば、配列番号１の塩基配列からなるDNAの相補鎖の全部又は一部に対して、ストリンジェントな条件下でハイブリダイズし、かつクロロフィラーゼ活性を有するタンパク質をコードするものでもよい。ここでいう「ストリンジェントな条件」とはいわゆる特異的なハイブリッドが形成され、非特異的なハイブリッドが形成されない条件を意味し、例えばMolecular Cloning: A Laboratory Manual(Third Edition)を参照して適宜決定することができる。具体的には、サザンハイブリダイゼーションの際の温度や溶液に含まれる塩濃度、及びサザンハイブリダイゼーションの洗浄工程の際の温度や溶液に含まれる塩濃度によりストリンジェンシーを設定することができる。より詳細には、ストリンジェントな条件としては、例えば、ナトリウム濃度が25〜500mM、好ましくは25〜300mMであり、温度が42〜68℃、好ましくは42〜65℃である。より具体的には、5×SSC(83mM NaCl、83mMクエン酸ナトリウム)、温度42℃である。 Furthermore, the chlorophyllase gene encodes a protein that hybridizes under stringent conditions to all or part of the complementary strand of DNA consisting of the nucleotide sequence of SEQ ID NO: 1, and has chlorophyllase activity. It may be something to do. The "stringent condition" here means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed, and is appropriately determined by referring to, for example, Molecular Cloning: A Laboratory Manual (Third Edition). can do. Specifically, the stringency can be set by the temperature at the time of Southern hybridization and the salt concentration contained in the solution, and the temperature at the time of the washing step of Southern hybridization and the salt concentration contained in the solution. More specifically, stringent conditions include, for example, a sodium concentration of 25-500 mM, preferably 25-300 mM, and a temperature of 42-68 ° C, preferably 42-65 ° C. More specifically, it has 5 × SSC (83 mM NaCl, 83 mM sodium citrate) and a temperature of 42 ° C.
上述したように、配列番号１と異なる塩基配列からなる遺伝子、又は配列番号２とは異なるアミノ酸配列をコードする遺伝子が、クロロフィラーゼ遺伝子として機能するか否かは、当該遺伝子を適当なプロモーターとターミネーター等の間に組み込んだ発現ベクターを作製し、この発現ベクターを用いて例えば大腸菌等の宿主を形質転換し、発現するタンパク質のクロロフィラーゼ活性を測定すればよい。クロロフィラーゼ活性とは、クロロフィルを加水分解し、フィチル基とクロロフィリドを生成する反応を触媒する活性である。したがって、クロロフィラーゼ活性は、例えば、基質であるクロロフィルの減少量及び/又は生成物であるクロロフィリドの増加量に基づいて評価することができる。 As described above, whether or not a gene having a base sequence different from SEQ ID NO: 1 or a gene encoding an amino acid sequence different from SEQ ID NO: 2 functions as a chlorophyllase gene depends on the appropriate promoter and terminator. An expression vector incorporated between the above and the like may be prepared, and the expression vector may be used to transform a host such as Escherichia coli and measure the chlorophyllase activity of the expressed protein. The chlorophyllase activity is an activity that catalyzes a reaction that hydrolyzes chlorophyll to produce a phytyl group and chlorophyllide. Therefore, chlorophyllase activity can be evaluated, for example, on the basis of a decrease in the substrate chlorophyll and / or an increase in the product chlorophyllide.
ここで、微細藻類とは、単細胞性の光合成生物群であって藻類に分類される生物を意味する。微細藻類には、緑藻類、黄色鞭毛藻類、珪藻類、渦鞭毛藻類、単細胞真核藻類、淡水性単細胞緑藻類（クロレラ）及びシアノバクテリア類に分類されるものが含まれる。本発明に係る形質転換微細藻類は、これら如何なる分類の微細藻類を宿主として使用することができる。 Here, the microalgae means organisms that are unicellular photosynthetic organisms and are classified as algae. Microalgae include those classified into green algae, yellow algae, diatoms, whirlpool algae, unicellular eukaryotic algae, freshwater unicellular green algae (chlorella) and cyanobacteria. The transformed microalgae according to the present invention can use these microalgae of any classification as a host.
一例として、本発明に係る形質転換微細藻類の宿主として使用できる珪藻類とは、単に珪藻とも呼称され、珪藻綱（Bacillariophyceae）に属するものを挙げることができる。中でも、オイル蓄積能を有する珪藻類とすることが好ましい。珪藻類としては、中心目（Centrales）に属する藻類及び羽状目（Pennales）に属する藻類が挙げられる。中心目（Centrales）に属する藻類としては、シクロテラ属（Cyclotella）、ミニディスクス属（Minidiscus）、タラシオシラ属（Thalassiosira）及びキートケロス属（Chaetoceros）に属する藻類等が挙げられ、羽状目（Pennales）に属する藻類としては、マヤマエア属（Mayamaea）、ディメレグラムマ属（Dimeregramma）、シリンドロテカ属（Cylindrotheca）、ナビキュラ属（Navicula）、及びフィストゥリフェラ属（Fistulifera）に属する藻類等が挙げられる。 As an example, the diatoms that can be used as a host for the transformed microalgae according to the present invention are also simply referred to as diatoms and may belong to the class Bacillariophyceae. Above all, it is preferable to use diatoms having an oil storage ability. Examples of diatoms include algae belonging to the order Centrales and algae belonging to the order Pennales. Examples of algae belonging to the genus Centrales include algae belonging to the genus Cyclotella, the genus Minidiscus, the genus Thalassiosira, and the genus Chaetoceros. Examples of the algae belonging to the genus Mayamaea include algae belonging to the genus Mayamaea, Dimeregramma, Cylindrotheca, Navicula, and Fistulifera.
より具体的に、オイル蓄積能を有する珪藻類としては、好ましくはFistulifera属又はMayamaea属に属する珪藻類である。 More specifically, the diatoms having an oil storage ability are preferably diatoms belonging to the genus Fistulifera or the genus Mayamaea.
オイル蓄積能を有するFistulifera属に属する珪藻類として、特にWO2010/116611に開示されているFistulifera sp. JPCC DA0580株（Navicula sp. JPCC DA0580株から改名、本明細書ではFistulifera solaris JPCC DA0580株とも称する）を例示することができる。なお、Fistulifera solaris JPCC DA0580株は、WO2010/116611に記載されているように、FERM BP-11201（FERM P-21788より移管）として独立行政法人 製品評価技術基盤機構 特許生物寄託センター (IPOD, NITE) (〒292-0818千葉県木更津市かずさ鎌足2-5-8) に国際寄託されている。 As diatoms belonging to the genus Fistulifera having oil storage ability, especially the Fistulifera sp. JPCC DA0580 strain disclosed in WO2010 / 116611 (renamed from Navicula sp. JPCC DA0580 strain, also referred to as Fistulifera solaris JPCC DA0580 strain in this specification). Can be exemplified. As described in WO2010 / 116611, the Fistulifera solaris JPCC DA0580 strain was designated as FERM BP-11201 (transferred from FERM P-21788) by the National Institute of Technology and Evaluation Patent Organism Depositary (IPOD, NITE). It has been internationally deposited at (2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture 292-0818).
また、オイル蓄積能を有するMayamaea属に属する珪藻類として、Mayamaea sp. JPCC CTDA0820株を例示することができる。Mayamaea sp. JPCC CTDA0820株は、たとえ10℃といった低温環境下においてオイル蓄積能を示すといった特徴を有する珪藻類である。Mayamaea sp. JPCC CTDA0820株は、電源開発株式会社にて分譲可能に保存されている。 Moreover, as a diatom belonging to the genus Mayamaea having an oil storage ability, the Mayamaea sp. JPCC CTDA0820 strain can be exemplified. The Mayamaea sp. JPCC CTDA0820 strain is a diatom that has the characteristic of exhibiting oil storage ability even in a low temperature environment such as 10 ° C. The Mayamaea sp. JPCC CTDA0820 strain is stored for sale by Electric Power Development Co., Ltd.
さらに、オイル蓄積能を有する珪藻類としては、上述したFistulifera属又はMayamaea属に属する珪藻類以外にも例えば、Navicula属やSeminavis属に属する珪藻類を挙げることができる。すなわち、オイル蓄積能を有する珪藻類としてはFistulifera属、Mayamaea属、Navicula属、及びSeminavis属などを含むNaviculaceae（フナガタケイソウ科）に属する珪藻類を使用することができる。 Further, examples of the diatoms having an oil storage ability include diatoms belonging to the genus Navicula and the genus Seminavis, in addition to the diatoms belonging to the genus Fistulifera or the genus Mayamaea described above. That is, as the diatoms having an oil storage ability, diatoms belonging to Naviculaceae (Funagatakeisou family) including the genus Fistulifera, the genus Mayamaea, the genus Navicula, and the genus Seminavis can be used.
ここで、オイル蓄積能を有するとは、特に限定されないが、例えば炭素数１６〜２４の脂肪族炭化水素を蓄積する能力を有すると言い換えることができる。但し、オイル蓄積能を有する珪藻類とは、炭素数１５以下の脂肪族炭化水素や、炭素数２５以上の脂肪族炭化水素を生成及び蓄積するものであっても良い。 Here, the ability to store oil is not particularly limited, but can be paraphrased as having the ability to store aliphatic hydrocarbons having 16 to 24 carbon atoms, for example. However, the diatoms having an oil storage ability may be those that produce and accumulate aliphatic hydrocarbons having 15 or less carbon atoms and aliphatic hydrocarbons having 25 or more carbon atoms.
また、本発明に係る形質転換微細藻類は、糖、タンパク質、オイル或いは化合物等を産生する能力を有する微細藻類や、単細胞真核藻類に分類されるユーグレナ藻や濃緑色の単細胞微細藻類であるスピルリナ等のそれ自体が食品用途や化粧品用途を有する微細藻類を宿主として作製しても良い。宿主として使用できる微細藻類としては、アスタキサンチンを産生するHaematococcus pluvialis、抗酸化物質を産生するChlorella vulgaris、β-カロテン等のカロテノイドを産生するDunaliella salina、その抽出物が化粧品等に利用されるAphanizomenon flos-aquae、その粉体等が食品サプリメントに使用されるArthrospina platensis、ドコサヘキサエン酸を産生するCrypthecodinium cohnii、油脂生産能を有するNeochloris oleoabundans、油脂生産能を有するNannochloropsis oculata及び炭素鎖３０以上の油脂を産生するBotryococcus braunii等を挙げることができる。 The transformed microalgae according to the present invention include microalgae capable of producing sugars, proteins, oils, compounds, etc., Euglenidae classified as unicellular eukaryotic algae, and spirulina, which is a dark green unicellular microalgae. Etc. may be produced as a host of microalgae that themselves have food or cosmetic uses. Microalgae that can be used as hosts include Haematococcus pluvialis, which produces astaxanthin, Chlorella vulgaris, which produces antioxidants, Dunaliella salina, which produces carotenoids such as β-carotene, and Aphanizomenon flos-, whose extract is used in cosmetics. aquae, Arthrospina platensis whose powder is used in food supplements, Crypthecodinium cohnii that produces docosahexaenoic acid, Neochloris oleoabundans that has the ability to produce fats and oils, Nannochloropsis oculata that has the ability to produce fats and oils, and Botryococcus that produces fats and oils with 30 or more carbon chains. Braunii and the like can be mentioned.
上述した微細藻類を宿主としてクロロフィラーゼ遺伝子を発現可能に導入するには、従来公知の形質転換方法を適宜使用することができる。例えば、微細藻類内でクロロフィラーゼ遺伝子を発現させる場合、藻類用の発現ベクター、例えば、pChlamyシリーズのベクター（Invitrogen社）、pOptシリーズのベクター(Chlamydomonas resource center)などを使用することができる。なお、クロロフィラーゼ遺伝子は、宿主となる微細藻類において機能するプロモーターの制御下に配置される。プロモーターとしては、例えば、HSP70A/RbcS2、beta-tubulin、PSAD、CaMV35S、NIT1、Cyc6、SQD2などの過剰発現用プロモーターを挙げることができる。なお、発現ベクターには、3UTR、ターミネーター及び選択マーカー等が含まれていることが好ましい。 In order to introduce the chlorophyllase gene expressively using the above-mentioned microalgae as a host, a conventionally known transformation method can be appropriately used. For example, when the chlorophyllase gene is expressed in microalgae, an expression vector for algae, for example, a pChlamy series vector (Invitrogen), a pOpt series vector (Chlamydomonas resource center), or the like can be used. The chlorophyllase gene is placed under the control of a promoter that functions in the host microalgae. Examples of the promoter include promoters for overexpression such as HSP70A / RbcS2, beta-tubulin, PSAD, CaMV35S, NIT1, Cyc6, and SQD2. The expression vector preferably contains 3UTR, a terminator, a selectable marker, and the like.
微細藻類細胞への発現ベクターの導入は、公知の方法によって行うことができ、例えば、エレクトロポレーション法、ボンバードメント法、グラスビーズ法及びアグロバクテリウム法などにより行うことができる。 The expression vector can be introduced into microalgae cells by a known method, for example, an electroporation method, a bombardment method, a glass bead method, an Agrobacterium method, or the like.
また、得られた形質転換微細藻類は、通常の培地にて培養することができる。培地としては、宿主とした微細藻類に応じた組成の培地を準備することができる。例えば、微細藻類が淡水藻類である場合、蒸留水にTris、Glycylglycine、HEPES、TAPS、Bicine及びMES等の緩衝剤を適量加え、さらに各種栄養塩を適量加え、所望の値にpHを調整することで培地を作製することができる。また、微細藻類が、海水藻類である場合、蒸留水に緩衝材及び栄養塩類（NaCl、MgSO4・7H2O、KCl及びCaCl2・2H2O）を適量加え、各種栄養塩を適量加え、所望の値にpHを調整することで培地を作製することができる。 In addition, the obtained transformed microalgae can be cultured in a normal medium. As the medium, a medium having a composition corresponding to the microalgae used as a host can be prepared. For example, when the microalgae are freshwater algae, add an appropriate amount of buffers such as Tris, Glycylglycine, HEPES, TAPS, Bicine and MES to distilled water, and then add an appropriate amount of various nutrients to adjust the pH to the desired value. Can be prepared with. Moreover, microalgae, when a sea algae, distilled water buffer material and nutrients (NaCl, MgSO 4 · 7H 2 O, KCl , and CaCl 2 · 2H 2 O) was added an appropriate amount was added an appropriate amount of various nutrients, A medium can be prepared by adjusting the pH to a desired value.
特に、本発明に係る形質転換微細藻類は、発現可能に導入されたクロロフィラーゼ遺伝子を備えている。クロロフィラーゼ遺伝子の発現に起因して本発明に係る形質転換微細藻類の生育、増殖及び物質生産に対する悪影響はなく、捕食生物に対して高度に毒性を発揮する。捕食生物としては、特に限定されず、微細藻類を特異的又は非特異的に捕食する生物全般を意味する。一例としては、微細藻類を捕食するアメーバ等の原生生物が挙げられる。アメーバ以外にも捕食生物としては、例えば、無色鞭毛虫類や繊毛虫類、ツボカビ類、さらに渦鞭毛藻や黄金色藻などにみられる藻類捕食性の微細藻類などを挙げることができる。 In particular, the transformed microalgae according to the present invention include an expressively introduced chlorophyllase gene. The expression of the chlorophyllase gene does not adversely affect the growth, growth and substance production of the transformed microalgae according to the present invention, and is highly toxic to predators. The predator is not particularly limited, and means all organisms that specifically or non-specifically prey on microalgae. One example is protists such as amoeba, which prey on microalgae. Examples of predatory organisms other than amoeba include colorless flagellates, ciliates, algae, and algae-predatory microalgae found in dinophyceae and golden algae.
以上のように、本発明に係る形質転換微細藻類は、捕食生物に対する毒性を有するため、培養系における捕食生物のコンタミネーションによる被害を防止することができる。例えば、本発明に係る形質転換微細藻類を利用して物質生産等を行う場合、実験室レベルと比較して大規模な培養系を適用することが好ましい。また、大規模な培養系は、通常、屋外に設置される場合が多い。このように、大規模培養、特に屋外大規模培養においては、周囲の環境に曝されることから捕食生物のコンタミネーションが大きな問題となる。コンタミネーションによる問題としては、培養対象である微細藻類の増殖不良や死滅、生産目的物質の生産量低下或いは低品質化等をあげることができる。 As described above, since the transformed microalgae according to the present invention are toxic to predators, it is possible to prevent damage caused by contamination of predators in the culture system. For example, when a substance is produced using the transformed microalgae according to the present invention, it is preferable to apply a large-scale culture system as compared with the laboratory level. In addition, large-scale culture systems are usually installed outdoors in many cases. As described above, in large-scale culture, particularly outdoor large-scale culture, contamination of predators becomes a big problem because it is exposed to the surrounding environment. Problems due to contamination include poor growth and death of the microalgae to be cultured, a decrease in the production amount of the production target substance, or a decrease in quality.
本発明に係る形質転換微細藻類を利用することで、これら捕食生物のコンタミネーションの問題を回避し、形質転換微細藻類の増殖を維持し、生産目的物質の生産量及び/又は品質を維持することができる。したがって、本発明に係る形質転換微細藻類を利用することによって、生産目的物質（形質転換微細藻類自体を含む）を低コストに生産することが可能となる。 By utilizing the transformed microalgae according to the present invention, the problem of contamination of these predators is avoided, the growth of the transformed microalgae is maintained, and the production amount and / or quality of the production target substance is maintained. Can be done. Therefore, by utilizing the transformed microalgae according to the present invention, it is possible to produce a production target substance (including the transformed microalgae itself) at low cost.
一例として屋外の大量培養系としては、特に限定されないが、レースウェイ型培養装置、カラム型培養装置及びパネル型培養装置を挙げることができる。これら培養装置には、未滅菌f/2培地を100〜200Lの規模で使用し、パドルやスターラーで培地を攪拌しながら形質転換微細藻類を培養することができる。このとき、これら培養装置では、温度調節しながら培養しても良いし、また、CO2吹き込みによりpH制御しながら培養してもよい。さらに、大規模な屋外培養系としては、攪拌機として浮遊式水耕機を備える円形ポンド型培養装置（例えば、直径5m、培養内容積10,000L）を適用することができる。この円形ポンド型培養装置は、例えば水深0.5mの培養槽とし、f/2培地及び人工海水を培養液としてCO2吹き込みによりpHを7.7〜8.5の範囲にコントロールしながら培養することができる。また、円形ポンド型培養装置では、水中ポンプにより培養液を輸送することができ、また40W浮遊式攪拌装置により培養液を攪拌しながら形質転換微細藻類の培養を継続することができる。 As an example, the outdoor mass culture system is not particularly limited, and examples thereof include a raceway type culture device, a column type culture device, and a panel type culture device. Unsterilized f / 2 medium can be used in these culture devices on a scale of 100 to 200 L, and transformed microalgae can be cultured while stirring the medium with a paddle or stirrer. At this time, in these culturing devices, culturing may be performed while controlling the temperature, or culturing may be performed while controlling the pH by blowing CO 2 . Further, as a large-scale outdoor culture system, a circular pound type culture device (for example, a diameter of 5 m and a culture internal volume of 10,000 L) equipped with a floating hydroponic machine as a stirrer can be applied. This circular pound type culture device can be used in a culture tank having a depth of 0.5 m, for example, and can be cultured using f / 2 medium and artificial seawater as a culture solution while controlling the pH in the range of 7.7 to 8.5 by injecting CO 2 . Further, in the circular pond type culture device, the culture solution can be transported by a submersible pump, and the cultured microalgae can be continued while stirring the culture solution by a 40 W floating stirrer.
以下、実施例により本発明を更に詳細に説明するが、本発明の技術的範囲は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to the following Examples.
1. 実験方法
1-1. 使用した試薬及び機器
試薬の調製及び実験操作にはMilli-Q Integral 5 バイオタイプシステム (メルク株式会社製)を用いて処理した蒸留水、または超純水を用いた。色素抽出及び高速液体クロマトグラフィー(High performance liquid chromatography; HPLC)分析に用いた試薬は全て和光純薬工業株式会社のHPLCグレードとし、その他の試薬は全て試薬特級品又はそれに準ずるものを使用した。F. solaris細胞数の計数にはバクテリアカウンター (サンリード硝子株式会社製)、アメーバ細胞数の計数にはフックスローゼンタール (サンリード硝子株式会社製)を用いた。F. solarisの前培養にはロータリーシェーカーNR-20 (タイテック株式会社製)を用いて行った。遠心分離には微量高速冷却遠心機MX-305 (株式会社トミー精工社製)を用いた。PCR反応及びシーケンシングに用いたプライマーはライフテクノロジーズジャパンに合成を委託し、サーマルサイクラーはVerti 96-well サーマルサイクラー (Applied Biosystems社製)を使用した。シーケンス解析の際の、シーケンサーには3730x DNA Analyzer (Applied Biosystems社製)を使用し、シーケンス反応にはBigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific社製)を用いた。このシーケンス解析はタカラバイオ株式会社に委託し行った。HPLC分析の際の分析システムにはWaters e2695 Separation Module及び2489 UV/Vis Detector (日本ウォーターズ株式会社製)を用いた。
1. Experimental method
1-1. Reagents and equipment used Distilled water treated with the Milli-Q Integral 5 biotype system (manufactured by Merck Group, Inc.) or ultrapure water was used for the preparation and experimental operation of the reagents. All reagents used for dye extraction and high performance liquid chromatography (HPLC) analysis were HPLC grade of Wako Pure Chemical Industries, Ltd., and all other reagents used were special reagent products or equivalent. F. A bacterial counter (manufactured by Sunlead Glass Co., Ltd.) was used to count the number of solaris cells, and Hooks Rozental (manufactured by Sunlead Glass Co., Ltd.) was used to count the number of amoeba cells. F. solaris was precultured using a rotary shaker NR-20 (manufactured by TIETECH Co., Ltd.). A micro high-speed cooling centrifuge MX-305 (manufactured by Tomy Seiko Co., Ltd.) was used for centrifugation. The primers used for the PCR reaction and sequencing were synthesized by Life Technologies Japan, and the thermal cycler used was a Verti 96-well thermal cycler (manufactured by Applied Biosystems). For sequence analysis, a 3730x DNA Analyzer (manufactured by Applied Biosystems) was used for the sequencer, and a BigDye Terminator v3.1 Cycle Sequencing Kit (manufactured by Thermo Fisher Scientific) was used for the sequence reaction. This sequence analysis was outsourced to Takara Bio Inc. A Waters e2695 Separation Module and a 2489 UV / Vis Detector (manufactured by Nippon Waters Co., Ltd.) were used as the analysis system for HPLC analysis.
1-2. 使用藻体及び培養方法
海洋珪藻 Fistulifera solaris JPCC DA0580株 (以下、F. solaris)の培養にはhalf strength of Guillard’s f solution (以下、f/2培地) (75mg NaNO3、5mg NaH2PO4・H2O、0.5μg vitamin B12、0.5μg biotin、100μg thiamine HCl、30mg Na2SiO3・9H2O、4.36mg Na2EDTA、3.15mg FeCl3・6H2O、10μg CoCl4・5H2O、22μg ZnSO4・7H2O、180μg MnCl2・4H2O、9.8μg CuSO4・5H2O、6.3μg Na2MoO4・2H2O)を用いた。なお、塩濃度の調整にはマリンアートSF-1 (株式会社富田製薬社製)を37.0g/l用いて行った。藻体の前培養として、100 ml三角フラスコ内にて、アンピシリン50μg/ml含有f/2培地50 mlを用いて14日間振盪培養 (回転数120rpm、温度25℃、光量子束密度50μmol/m2/、Light/Dark=24h/0h)した。なお、形質転換体の維持には500μg/ml G418 (ロシュ・ダイアグノスティックス株式会社製)含有f/2培地を用い、継代は1週間ごとに行った。本培養として、扁平フラスコ内にてf/2培地500mlを用いて7日間培養(温度25℃、流量0.8l/min/l、CO2濃度2%、光量子束密度130μmol/m2/、Light/Dark=24h/0h)した。
1-2. Algae used and culture method For culturing the marine diatom Fistulifera solaris JPCC DA0580 strain (hereinafter, F. solaris), half strength of Guillard's f solution (hereinafter, f / 2 medium) (75 mg NaNO 3 , 5 mg NaH 2) PO 4 · H 2 O, 0.5μg vitamin B 12, 0.5μg biotin, 100μg thiamine HCl, 30mg Na 2 SiO 3 · 9H 2 O, 4.36mg Na 2 EDTA, 3.15mg FeCl 3 · 6H 2 O, 10μg CoCl 4 · 5H 2 O, was used 22μg ZnSO 4 · 7H 2 O, 180μg MnCl 2 · 4H 2 O, 9.8μg CuSO 4 · 5H 2 O, the 6.3μg Na 2 MoO 4 · 2H 2 O). The salt concentration was adjusted using 37.0 g / l of Marine Art SF-1 (manufactured by Tomita Pharmaceutical Co., Ltd.). As a preculture of algae, shake culture for 14 days in a 100 ml Erlenmeyer flask using 50 ml of f / 2 medium containing 50 μg / ml of ampicillin (rotation speed 120 rpm, temperature 25 ° C, photon bundle density 50 μmol / m 2 / , Light / Dark = 24h / 0h). To maintain the transformant, an f / 2 medium containing 500 μg / ml G418 (manufactured by Roche Diagnostics Co., Ltd.) was used, and passage was performed weekly. As the main culture, culture in a flat flask using 500 ml of f / 2 medium for 7 days (temperature 25 ° C, flow rate 0.8 l / min / l, CO 2 concentration 2%, photon bundle density 130 μmol / m 2 /, Light / Dark = 24h / 0h).
1-3. アメーバの単離及び培養方法
本研究に用いたアメーバは電源開発株式会社若松研究所 (Kitakyushu、Fukuoka、Japan)より単離した。2016年6月及び9月のF. solaris屋外培養試験において、F. solarisの顕著な死滅が認められた培養槽 (d=5m、V=10kL)の培養液を採取し、顕微鏡観察を行った。その結果、アメーバ運動を行う、形態的に異なる生物を確認し、以下の操作により、培養を行った。アメーバの培養は25cm2ベントキャップフラスコ (イワキ株式会社製)内にて行った。f/2培地10mlに対し、アメーバ細胞及びF. solaris細胞約5×106cellsを加え、室温、暗所下にて静置培養した。F. solaris細胞の添加は週に1度の頻度で行った。
1-3. Isolation and culture method of amoeba The amoeba used in this study was isolated from Wakamatsu Research Institute, Electric Power Development Co., Ltd. (Kitakyushu, Fukuoka, Japan). In the F. solaris outdoor culture test in June and September 2016, the culture solution of the culture tank (d = 5 m, V = 10 kL) in which remarkable death of F. solaris was observed was collected and observed under a microscope. .. As a result, morphologically different organisms that perform amoeboid movement were confirmed, and cultured by the following operations. The amoeba was cultured in a 25 cm 2 vent cap flask (manufactured by Iwaki & Co., Ltd.). About 5 × 10 6 cells of amoeba cells and F. solaris cells were added to 10 ml of f / 2 medium, and the cells were statically cultured at room temperature in a dark place. F. solaris cells were added once a week.
1-4. 顕微鏡観察
35mmφフィルターボトムディッシュ (松浪硝子工業株式会社製)に、f/2培地4ml、F. solaris細胞1×106cells、及びアメーバ細胞1×103cellsを加え、室温、暗所下にて静置した。アメーバ細胞の様子を倒立型蛍光顕微鏡OLYMPUS IX-70 (OLYMPUS株式会社製)を用いて観察した。観察したアメーバ細胞の面積をimageJを用いて算出した。
1-4. Microscopic observation
To a 35 mmφ filter bottom dish (manufactured by Matsunami Glass Industry Co., Ltd.), add 4 ml of f / 2 medium, 1 × 10 6 cells of F. solaris cells, and 1 × 10 3 cells of amoeba cells, and leave them at room temperature in a dark place. did. The state of amoeba cells was observed using an inverted fluorescence microscope OLYMPUS IX-70 (manufactured by OLYMPUS Corporation). The observed area of amoeba cells was calculated using imageJ.
形質転換体におけるGFP蛍光の確認は、正立型蛍光顕微鏡OLYMPUS BX-51 (OLYMPUS株式会社製)を用いて行った。クロロフィル自家蛍光及びGFP蛍光の観察にはそれぞれ、U-MWIB3 (Ex:460-495nm、Em:510nm)、U-MNIGA3 (Ex:540-550nm、Em:575-625nm)をフィルターユニットとして用いた。また、光源にはファイバ光源システムU-HGLGPS (OLYMPUS株式会社製)水銀ランプ(強度100%)を用い、露光時間はそれぞれ、1/10秒及び1秒とした。 Confirmation of GFP fluorescence in the transformant was performed using an upright fluorescence microscope OLYMPUS BX-51 (manufactured by OLYMPUS Corporation). U-MWIB3 (Ex: 460-495nm, Em: 510nm) and U-MNIGA3 (Ex: 540-550nm, Em: 575-625nm) were used as filter units for observing chlorophyll autofluorescence and GFP fluorescence, respectively. A fiber light source system U-HGLGPS (manufactured by OLYMPUS Corporation) mercury lamp (intensity 100%) was used as the light source, and the exposure times were 1/10 second and 1 second, respectively.
1-5. 18S rDNAシーケンス解析
アメーバ及びF. solarisそれぞれ約1×106cells及び1×107cellsからNucleoSpin Tissue XS (タカラバイオ株式会社)を用いてゲノムDNAを抽出した。得られたゲノムDNAをテンプレートとし、PrimeStar Max (タカラバイオ株式会社) によりPCR増幅 ((98℃で1min)×1サイクル、(98℃で10sec、55℃で10sec、72℃で10 sec)×30サイクル、(72℃で3min)×1サイクル)を行った。プライマーには2種類のプライマーセット1500F-1500R及びMoonA-MoonBを使用した(表１)。
1-5. 18S rDNA sequence analysis Genomic DNA was extracted from amoeba and F. solaris from about 1 × 10 6 cells and 1 × 10 7 cells, respectively, using Nucleo Spin Tissue XS (Takara Bio Inc.). Using the obtained genomic DNA as a template, PCR amplification by PrimeStar Max (Takara Bio Inc.) ((98 ° C for 1 min) x 1 cycle, (98 ° C for 10 sec, 55 ° C for 10 sec, 72 ° C for 10 sec) x 30 A cycle (3 min at 72 ° C. x 1 cycle) was performed. Two types of primer sets 1500F-1500R and MoonA-MoonB were used as primers (Table 1).
1%アガロースゲル電気泳動にてバンドを確認後、QIAquick PCR Purification Kit (Qiagen社製)を用いてPCR 産物を精製した。その後、pCR-Blunt II-TOPO vector (Thermo Fisher Scientific)にサブクローニングし、このプラスミドをOne Shot TOP10 Chemically Competent E. coliに対し、ヒートショック法により導入した。カナマイシン (50μg/ml)含有LB寒天培地に播種し培養(37℃、12時間)後、得られたコロニーをカナマイシン (50μg/ml)含有LB液体培地5mlで培養 (12時間、37℃、120rpm)し、QIAprep Spin Miniprep Kit (Qiagen)を用いてプラスミドを抽出した。制限酵素EcoRI(England Biolabs社製)にて処理後 (37℃、15分)、電気泳動にてインサートの確認ができたものについてシーケンス解析を行った。解析に使用したプライマーはユニバーサルプライマーであるM13-P5、M13-21及びそれぞれのサンプルに対し独自に設計したプライマーを用いた。得られた塩基配列をクエリとし、National Center for Biotechnology Information (NCBI)のGenBankをデータベースとしてBlastn相同性検索を行った。 After confirming the band by 1% agarose gel electrophoresis, the PCR product was purified using the QIAquick PCR Purification Kit (manufactured by Qiagen). Then, it was subcloned into pCR-Blunt II-TOPO vector (Thermo Fisher Scientific), and this plasmid was introduced into One Shot TOP10 Chemically Competent E. coli by the heat shock method. After seeding and culturing on LB agar medium containing kanamycin (50 μg / ml) (37 ° C, 12 hours), the obtained colonies were cultured in 5 ml of LB liquid medium containing kanamycin (50 μg / ml) (12 hours, 37 ° C, 120 rpm). Then, the plasmid was extracted using the QIAprep Spin Miniprep Kit (Qiagen). After treatment with the restriction enzyme EcoRI (manufactured by England Biolabs) (37 ° C, 15 minutes), sequence analysis was performed on those whose inserts could be confirmed by electrophoresis. The primers used for the analysis were the universal primers M13-P5 and M13-21 and the primers originally designed for each sample. Using the obtained nucleotide sequence as a query, Blastn homology search was performed using GenBank of the National Center for Biotechnology Information (NCBI) as a database.
1-6. In silico解析
高等植物Arabidopsis thaliana由来CLH1 (NCBI Gene ID: 838554)及びCLH2 (834408)の配列をクエリとし、F. solaris、モデル珪藻Phaeodactylum tricornutum及びThalassiosira pseudonanaの全遺伝子配列に対してBlastn相同性検索を行った。また、モチーフ配列解析ツールMEMEのMASTを用いてセリンリパーゼモチーフPS00120 [LIV]-{KG}-[LIVFY]-[LIVMST]-G-[HYWV]-S-{YAG}-G-[GSTAC]を有する遺伝子を抽出した。
1-6. In silico analysis Using the sequences of CLH1 (NCBI Gene ID: 838554) and CLH2 (834408) derived from the higher plants Arabidopsis thaliana as queries, Blastn for the entire gene sequences of F. solaris, model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. A homology search was performed. In addition, using the motif sequence analysis tool MEME MAST, the serine lipase motif PS00120 [LIV]-{KG}-[LIVFY]-[LIVMST] -G- [HYWV] -S- {YAG} -G- [GSTAC] The gene to have was extracted.
1-7. 色素分析
本培養を行った藻体をセルカウントし、5×108cellsとなるよう遠心回収 (8,000g、25℃、5分)し、直ちに液体窒素にて凍結後、色素の抽出まで-80℃で保存した。色素抽出はJeffreyら及びHuらの方法に従い、アセトンを用いて行った (Jeffrey, S. W., Hallegraeff, G. M. (1987). Chlorophyllase distribution in ten classes of phytoplankton: a problem for chlorophyll analysis. Mar Ecol Prog Ser, 293-304、Hu, X., Tanaka, A., Tanaka, R. (2013). Simple extraction methods that prevent the artifactual conversion of chlorophyll to chlorophyllide during pigment isolation from leaf samples. Plant Methods, 9(1), 19)。藻体に対し-30℃の90%アセトン1mlを添加し、氷水中にて10分間超音波破砕 (W-170ST、本多電子株式会社製)を行った。遠心分離 (13,000g、4℃、5min)後のアセトン層を回収し、残渣に対して4ml、及び5mlの90%アセトンを用いて同様に抽出を行った。これら10mlのアセトン層をフィルター濾過 (DISMIC 13-JP、ADVANTEC社製)し、CLH不活性化条件色素サンプルとした。また、CLH活性化条件として50%アセトン1mlを添加し、室温で30分インキュベーションした。その後4mlの100%アセトンを用いて反応を停止させ、先と同様に超音波処理後、上清を回収した。残渣に対し、5mlの90%アセトンを用いて抽出後、回収したアセトン層をフィルター濾過し、CLH活性化条件色素サンプルとした。
1-7. Dye analysis The algae cells that had undergone the main culture were cell-counted, centrifuged (8,000 g, 25 ° C, 5 minutes) to obtain 5 × 10 8 cells, immediately frozen in liquid nitrogen, and then the dye was used. Stored at -80 ° C until extraction. Dye extraction was performed with acetone according to the method of Jeffrey et al. And Hu et al. (Jeffrey, SW, Hallegraeff, GM (1987). Chlorophyllase distribution in ten classes of phytoplankton: a problem for chlorophyll analysis. Mar Ecol Prog Ser, 293. -304, Hu, X., Tanaka, A., Tanaka, R. (2013). Simple extraction methods that prevent the artifactual conversion of chlorophyll to chlorophyllide during pigment isolation from leaf samples. Plant Methods, 9 (1), 19) .. 1 ml of 90% acetone at -30 ° C was added to the algae, and ultrasonic crushing (W-170ST, manufactured by Honda Electronics Corporation) was performed in ice water for 10 minutes. The acetone layer after centrifugation (13,000 g, 4 ° C., 5 min) was recovered, and the residue was similarly extracted with 4 ml and 5 ml of 90% acetone. These 10 ml acetone layers were filtered through a filter (DISMIC 13-JP, manufactured by ADVANTEC) to prepare a CLH inactivating condition dye sample. In addition, 1 ml of 50% acetone was added as a CLH activation condition, and the mixture was incubated at room temperature for 30 minutes. Then, the reaction was stopped with 4 ml of 100% acetone, sonicated in the same manner as before, and the supernatant was recovered. The residue was extracted with 5 ml of 90% acetone, and the recovered acetone layer was filtered to prepare a CLH activation condition dye sample.
Zapataらに倣い、抽出した色素サンプルのHPLC分析を行った (Zapata, M., Rodriguez, F., Garrido, J. L. (2000). Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase C8 column and pyridine-containing mobile phases. Mar Ecol Prog Ser, 29-45)。固定相にはWaters Symmetry C8 Column (150×4.6nm、3.5μm、100Å、日本Waters株式会社製)を用い、移動相にはメタノール:アセトニトリル:0.25Mピリジン液=2:1:1(v:v:v)、及びメタノール:アセトニトリル:アセトン=1:3:1(v:v:v)を用いた。色素サンプルは100%メタノールを用いて2倍希釈し、10μlを分析に供した。664nmにおける吸光度をモニタリングし、クロマトグラムを作成した。標準試料には100%メタノールで調製したクロロフィルa (和光純薬工業株式会社)、及びクロロフィリドa (DHI Lab Products、HOERSHOLM社製)を用いた。クロロフィルa標準試料の濃度はマイクロプレートリーダーSH-9000 (コロナ電気株式会社製)にて663nm及び645nmにおける吸光度を測定し、Mackenneyの式 (Chlorophyll a(μg/ml)=12.72×A663-2.69×A645)に基づいて算出した。 HPLC analysis of the extracted dye sample was performed following Zapata et al. (Zapata, M., Rodriguez, F., Garrido, JL (2000). Separation of chlorophylls and carotenoids from marine phytoplankton: a new HPLC method using a reversed phase. C8 column and pyridine-containing mobile phases. Mar Ecol Prog Ser, 29-45). Waters Symmetry C8 Column (150 x 4.6 nm, 3.5 μm, 100 Å, manufactured by Japan Waters Corp.) is used for the stationary phase, and methanol: acetonitrile: 0.25 M pyridine solution = 2: 1: 1 (v: v) for the mobile phase. : V) and methanol: acetonitrile: acetone = 1: 3: 1 (v: v: v) were used. Dye samples were diluted 2-fold with 100% methanol and 10 μl was subjected to analysis. The absorbance at 664 nm was monitored and a chromatogram was prepared. Chlorophyll a (Wako Pure Chemical Industries, Ltd.) and chlorophyllide a (DHI Lab Products, manufactured by HOERSHOLM) prepared with 100% methanol were used as standard samples. The concentration of the chlorophyll a standard sample was measured by measuring the absorbance at 663 nm and 645 nm with a microplate reader SH-9000 (manufactured by Corona Electric Co., Ltd.), and the Mackenney formula (Chlorophyll a (μg / ml) = 12.72 × A 663 -2.69 × Calculated based on A 645 ).
1-8. プラスミドの構築
pSP-GFP/GAPDHプラスミド (Maeda, Y., Tateishi, T., Niwa, Y., Muto, M., Yoshino, T., Kisailus, D., Tanaka, T. (2016). Peptide-mediated microalgae harvesting method for efficient biofuel production. Biotechnol Biofuels, 9(1), 10)はenhanced Green Fluorescence Protein (eGFP)遺伝子の上流に当該株由来グリセルアルデヒド 3-リン酸デヒドロゲナーゼ(GAPDH)遺伝子のプロモーター配列とEcoRVサイトを、下流にP. tricornutum由来のフコキサンチンクロロフィルa/c結合タンパク質A(fcpA)遺伝子のターミネーター配列を有している。このプラスミドのEcoRVサイトに人工合成したA. thaliana由来CLH遺伝子(972bp)を導入し、pSP-CLH-GFP/GAPDHプラスミドを構築した(図１)。
1-8. Construction of plasmid
pSP-GFP / GAPDH plasmid (Maeda, Y., Tateishi, T., Niwa, Y., Muto, M., Yoshino, T., Kisailus, D., Tanaka, T. (2016). Peptide-mediated microalgae harvesting The method for efficient biofuel production. Biotechnol Biofuels, 9 (1), 10) has the promoter sequence and EcoRV site of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene derived from the strain upstream of the enhanced Green Fluorescence Protein (eGFP) gene. It has a terminator sequence of the fucoxanthin chlorophyll a / c binding protein A (fcpA) gene derived from P. tricornutum downstream. The artificially synthesized A. thaliana-derived CLH gene (972 bp) was introduced into the EcoRV site of this plasmid to construct the pSP-CLH-GFP / GAPDH plasmid (Fig. 1).
1-9. パーティクルガン法によるF. solarisの形質転換
50%グリセロール中に懸濁された60mg/mlタングステン粒子 (直径0.6μm)懸濁液 (高純度化学研究所製) 50μlに対し、5μg分のpSP-CLH-GFP/GAPDH、2.5M CaCl2 50μl及び0.1Mスペルミジン 20μlを添加し、粒子上へのプラスミド固定化処理を行った。培養3日目 (対数増殖期中期)の藻体を遠心回収(8,500g、25℃、10分間)し、5×107cellsをf/2寒天培地 (直径3.6cmシャーレ)上に塗布し、風乾させた。Biolistic PDS-1000/He Particle Delivery System (Bio-Rad Laboratories社製)内にシャーレをセットし、打ち出し圧力1,100psiにてプラスミド導入を行った。その後、復活培養 (25℃、24時間、50μmol/m2/s)を行い、f/2液体培地を加え、セルスクレーパーで細胞を回収した。回収した藻体を500μg/ml G418含有f/2寒天培地 (直径9.0cmシャーレ)に塗布し、21日間培養を行った (25℃、140μmol/m2/s)。形成した薬剤耐性コロニーを爪楊枝を用いて、500μg/ml G418含有f/2培地入り96穴プレートに移し、培養 (25℃、140μmol/m2/s、7日間)した。
1-9. Transformation of F. solaris by particle gun method
60 mg / ml tungsten particles suspended in 50% glycerol (0.6 μm in diameter) suspension (manufactured by High Purity Chemical Laboratory) 50 μl, 5 μg of pSP-CLH-GFP / GAPDH, 2.5M CaCl 2 50 μl And 20 μl of 0.1 M spermidine were added, and plasmid immobilization treatment was performed on the particles. Algae on the 3rd day of culture (middle logarithmic growth phase) were collected by centrifugation (8,500 g, 25 ° C, 10 minutes), and 5 × 10 7 cells were applied on f / 2 agar medium (3.6 cm diameter petri dish). It was air-dried. A petri dish was set in the Biolistic PDS-1000 / He Particle Delivery System (manufactured by Bio-Rad Laboratories), and the plasmid was introduced at a launch pressure of 1,100 psi. Then, revival culture (25 ° C., 24 hours, 50 μmol / m 2 / s) was performed, f / 2 liquid medium was added, and cells were collected with a cell scraper. The recovered algae were applied to f / 2 agar medium (diameter 9.0 cm petri dish) containing 500 μg / ml G418 and cultured for 21 days (25 ° C, 140 μmol / m 2 / s). The formed drug-resistant colonies were transferred to a 96-well plate containing f / 2 medium containing 500 μg / ml G418 using a toothpick and cultured (25 ° C., 140 μmol / m 2 / s, 7 days).
1-10. 導入遺伝子領域のPCR増幅
液体培地にて培養したクローン及び野生株を遠心回収 (8,500g、25℃、5分間)し、NucleoSpin Tissue XS(タカラバイオ株式会社)を用いてゲノムDNAを抽出した。CLH-GFP遺伝子領域の一部 (1,205bp)を増幅するようにプライマーForward (5’-CGC AAC TAC TTC TAC TCT GAC G-3’(配列番号９))、Reverse (5’-GAC GTT GTG GCT GTT GTA GTT G-3’(配列番号１０))を設計し、Prime STAR Maxを用いてPCR増幅((98℃で1min)×1サイクル、(98℃で10sec、63℃で10sec、72℃で10sec)×30サイクル、(72℃で3min)×1サイクル)を行った。ポジティブコントロールとしてpSP-CLH-GFP/GAPDHプラスミドをテンプレートとし、ネガティブコントロールとして超純水をテンプレートとした。また、ゲノムDNAの抽出の確認のために、1500FRプライマーを用いて増幅を行った。PCR増幅産物の1%アガロースゲル電気泳動を行い、目的のバンドを確認した。
1-10. PCR amplification of transgene region Clone and wild strain cultured in liquid medium are collected by centrifugation (8,500 g, 25 ° C, 5 minutes), and genomic DNA is obtained using NucleoSpin Tissue XS (Takara Bio Co., Ltd.). Extracted. Primers Forward (5'-CGC AAC TAC TTC TAC TCT GAC G-3'(SEQ ID NO: 9)), Reverse (5'-GAC GTT GTG GCT) to amplify part of the CLH-GFP gene region (1,205 bp) GTT GTA GTT G-3'(SEQ ID NO: 10)) was designed and PCR amplified using Prime STAR Max ((98 ° C for 1 min) x 1 cycle, (98 ° C for 10 sec, 63 ° C for 10 sec, 72 ° C). 10 sec) × 30 cycles, (3 min at 72 ° C) × 1 cycle) were performed. The pSP-CLH-GFP / GAPDH plasmid was used as a template as a positive control, and ultrapure water was used as a template as a negative control. In addition, in order to confirm the extraction of genomic DNA, amplification was performed using a 1500FR primer. The target band was confirmed by 1% agarose gel electrophoresis of the PCR amplification product.
1-11. Reverse Transcription (RT)-PCR
凍結保存した野生株及び形質転換体 (5×108cells)を乳鉢・乳棒を用いて破砕した。破砕物に対してPlant RNA Isolation Reagent (Invitrogen社製) を用い、プロトコールに従ってTotal RNAを抽出した。得られたTotal RNAに対しDNaseI (タカラバイオ株式会社)を加え、インキュベーションし (37℃、30分間)、更にRNeasy Mini Kit (Qiagen)を用いて精製した。処理後のTotal RNA 1μgを鋳型とし、PrimeScript II 1st strand cDNA Synthesis Kit (タカラバイオ株式会社)を用いてcDNAを合成した。得られたcDNA 1μgをテンプレートとし、CLH遺伝子に対するプライマーForward (5’-TTG ATC CAG TGG CAG GAA CT-3’(配列番号１１))、Reverse (5’-GGC ATC ACG TTG TTC CAC TT-3’(配列番号１２))により、PCR増幅((98℃で1min)×1サイクル、(98℃で10sec、63℃で5sec、72℃で10sec)×30サイクル、(72℃で3min)×1サイクル)を行った。なお、cDNAの合成を確認するために、GAPDH遺伝子に対するプライマーForward (5’-TTG GAG TCA ATG GCT TTG GC-3’(配列番号１３))、Reverse (5’-TTC ACC ATC ACT ACG TTC GC-3’(配列番号１４))を設計し、同様に増幅した。増幅産物に対し、Agilent DNA 1000 Kit (Agilent Technologies社製)を用いてAgilent 2100 Bioanalyzer (Agilent Technologies)によるキャピラリー電気泳動を行なった。
1-11. Reverse Transcription (RT)-PCR
The cryopreserved wild strains and transformants (5 × 10 8 cells) were crushed using a mortar and pestle. Total RNA was extracted from the crushed material using Plant RNA Isolation Reagent (manufactured by Invitrogen) according to the protocol. DNase I (Takara Bio Inc.) was added to the obtained Total RNA, incubated (37 ° C., 30 minutes), and further purified using RNeasy Mini Kit (Qiagen). Using 1 μg of the treated Total RNA as a template, cDNA was synthesized using the PrimeScript II 1st strand cDNA Synthesis Kit (Takara Bio Inc.). Using 1 μg of the obtained cDNA as a template, primers for the CLH gene Forward (5'-TTG ATC CAG TGG CAG GAA CT-3'(SEQ ID NO: 11)), Reverse (5'-GGC ATC ACG TTG TTC CAC TT-3') (SEQ ID NO: 12)), PCR amplification ((98 ° C for 1 min) x 1 cycle, (98 ° C for 10 sec, 63 ° C for 5 sec, 72 ° C for 10 sec) x 30 cycles, (72 ° C for 3 min) x 1 cycle ) Was performed. In order to confirm the synthesis of cDNA, primers for the GAPDH gene Forward (5'-TTG GAG TCA ATG GCT TTG GC-3'(SEQ ID NO: 13)), Reverse (5'-TTC ACC ATC ACT ACG TTC GC- 3'(SEQ ID NO: 14)) was designed and amplified in the same manner. Capillary electrophoresis of the amplified product with an Agilent 2100 Bioanalyzer (Agilent Technologies) was performed using the Agilent DNA 1000 Kit (manufactured by Agilent Technologies).
1-12. アメーバによるF. solarisの捕食実験
F. solaris野生株及び形質転換体を、f/2培地500mlを用いて本培養 (25℃、130μmol/m2/s、0.8l/min/l、2%CO2)した。7日間後の培養液を、新たなf/2培地500mlに対し、初期濃度1×106cells/mlとなるように加えた。更に、アメーバの培養液を、先のフラスコ内にそれぞれ1×103cells/mlとなるように加えた。25℃、100μmol/m2/s、0.8l/min/l、2%CO2の条件で7日間培養を行い、24時間ごとに培養液を1ml回収した。F. solaris及びアメーバの細胞数を計数し、それぞれの増殖曲線を作成した。また、光量子束密度500μmol/m2/sとして同様に検討を行った。
1-12. F. solaris predation experiment by amoeba
F. solaris wild strains and transformants were main-cultured (25 ° C., 130 μmol / m 2 / s, 0.8 l / min / l, 2 % CO 2 ) using 500 ml of f / 2 medium. After 7 days, the culture medium was added to 500 ml of fresh f / 2 medium at an initial concentration of 1 × 10 6 cells / ml. Further, the amoeba culture solution was added to each of the above flasks at 1 × 10 3 cells / ml. The cells were cultured at 25 ° C., 100 μmol / m 2 / s, 0.8 l / min / l, and 2 % CO 2 for 7 days, and 1 ml of the culture solution was collected every 24 hours. The number of cells of F. solaris and amoeba was counted, and the growth curves of each were created. The same study was conducted with a photon flux density of 500 μmol / m 2 / s.
2. 結果及び考察
2-1. F. solaris捕食性アメーバのキャラクタリゼーション
2-1-1. 形態観察
F. solaris培養槽より単離された2種のアメーバ(A、B)に対し、顕微鏡観察を行い、形態的特徴を獲得した。いずれのアメーバも仮足(pseudopod)と呼ばれる細胞質の一時的な突出を用いた運動(=アメーバ運動)をすることが確認された。それぞれの有する仮足は形態的に異なっており、アメーバAは丸みを帯びた葉状仮足を進行方向に対し突出させていることが確認された。一方でアメーバBでは、dactylopodiumと見られる仮足を進行方向に限らず複数突出させていることが確認された。また、いずれのアメーバにおいても細胞内にF. solaris細胞を取り込んでいることが確認された。クロロフィル蛍光を確認した結果、アメーバ細胞内のF. solaris細胞内では色素体の形状を確認できなかったことから、細胞内構造が維持されていないことが示唆された。培養2週間後の細胞を確認した結果、アメーバBでは顕著な形態的変化は認められなかったものの、アメーバAは多くの細胞がシスト形成していることが確認された。これはF. solaris細胞が枯渇したために休止状態となったためであると考えられる。
2. Results and considerations
2-1. F. solaris Predatory amoeba characterization
2-1-1. Morphological observation
Two types of amoebas (A and B) isolated from the F. solaris culture tank were observed under a microscope to acquire morphological characteristics. It was confirmed that all amoeboids perform exercise (= amoeboid movement) using a temporary protrusion of the cytoplasm called pseudopodia. The pseudopodia possessed by each were morphologically different, and it was confirmed that Amoeba A had a rounded foliate pseudopodia protruding in the direction of travel. On the other hand, in Ameba B, it was confirmed that multiple pseudopodia, which are considered to be dacty lopodium, are projected regardless of the direction of travel. In addition, it was confirmed that F. solaris cells were incorporated into the cells of all amoebas. As a result of confirming chlorophyll fluorescence, the shape of the plastid could not be confirmed in the F. solaris cell in the amoeba cell, suggesting that the intracellular structure was not maintained. As a result of confirming the cells 2 weeks after culturing, it was confirmed that many cells of Amoeba A formed cysts, although no remarkable morphological changes were observed in Amoeba B. It is considered that this is because F. solaris cells were depleted and became dormant.
ImageJを用いてアメーバ細胞の面積を測定した。その結果、アメーバAの運動細胞では179.4±59.5μm2(n=30)であり、シスト細胞では62.6±16.5μm2(n=28)であり、シスト形成により細胞が縮小することが示唆された。また、アメーバBは205.0±118.4μm2(n=42)であり、アメーバAと比較してわずかに大きいことが示唆された。その一方で、アメーバAでは100.4-337.1μm2、アメーバBでは76.2-594.9μm2のサイズの細胞が観察され、アメーバBの方が細胞サイズのばらつきが大きいことが示唆された。以上の形態観察より、F. solaris培養槽から単離された2種のアメーバは形態的に大きく異なることが示唆された。 The area of amoebic cells was measured using ImageJ. As a result, it was 179.4 ± 59.5 μm 2 (n = 30) in the motor cells of amoeba A and 62.6 ± 16.5 μm 2 (n = 28) in the cyst cells, suggesting that the cells shrink due to cyst formation. .. The amoeba B was 205.0 ± 118.4 μm 2 (n = 42), suggesting that it was slightly larger than the amoeba A. On the other hand, amoeba A in 100.4-337.1Myuemu 2, the size of the cells of amoebae B in 76.2-594.9Myuemu 2 was observed, towards the amoeba B has been suggested that variations in cell size is large. From the above morphological observations, it was suggested that the two amoebas isolated from the F. solaris culture tank were morphologically significantly different.
2-1-2. 18S rDNA配列に基づく分子系統解析
形態的に異なることが示された2種のアメーバ(A、B)の18S rDNA配列に基づく分子系統解析を行った。プライマーとして1500F及びR並びにMoonA及びBを用いてPCR増幅を行った結果、いずれのプライマーを用いた場合においても、増幅産物が確認された。しかし、MoonA及びBプライマーを用いた場合、アメーバA、B及びF. solarisゲノムのいずれについても、500-2,000bpの非特異的な増幅が確認された。その一方で1500F及びRプライマーを用いた場合、アメーバAでは約1,800bpのシングルバンドが得られ、アメーバBでは3本のバンド(2,000、1,800及び1,200bp)が得られた。また、いずれのバンドもF. solarisゲノム由来のバンド(約1,800 bp)とは長さが異なったことから、F. solaris由来ではないことが考えられた。そのため、1500F及びRプライマーによる増幅産物を対象にシーケンス解析を行った。サブクローニング後のアメーバA由来の2クローンをシーケンス解析した結果、共に1,871bpの一致した配列が獲得された。Blastnによる相同性検索の結果、Euplaesiobystra hypersalinica strain A2 (Accession No.: FJ222604.1)と高い相同性(88%)を示した。E. hypersalinicaに関する報告は少ないものの、当該株は韓国の高塩濃度環境中(20%)から単離された株である (Park, J. S., Simpson, A. G., Brown, S., Cho, B. C. (2009). Ultrastructure and molecular phylogeny of two heterolobosean amoebae, Euplaesiobystra hypersalinica gen. et sp. nov. and Tulamoeba peronaphora gen. et sp. nov., isolated from an extremely hypersaline habitat. Protist, 160(2), 265-283)。
2-1-2. Molecular phylogenetic analysis based on 18S rDNA sequence We performed molecular phylogenetic analysis based on the 18S rDNA sequence of two amoebas (A and B) that were shown to be morphologically different. As a result of PCR amplification using 1500F and R and Moon A and B as primers, an amplification product was confirmed regardless of which primer was used. However, when Moon A and B primers were used, non-specific amplification of 500-2,000 bp was confirmed for all of the amoeba A, B and F. solaris genomes. On the other hand, when 1500F and R primers were used, a single band of about 1,800 bp was obtained with amoeba A, and three bands (2,000, 1,800 and 1,200 bp) were obtained with amoeba B. In addition, since each band was different in length from the band derived from the F. solaris genome (about 1,800 bp), it was considered that it was not derived from F. solaris. Therefore, sequence analysis was performed on the amplification products with 1500F and R primers. As a result of sequence analysis of two clones derived from amoeba A after subcloning, matching sequences of 1,871 bp were obtained. As a result of homology search by Blastn, it showed high homology (88%) with Euplae siobystra hypersalinica strain A2 (Accession No .: FJ222604.1). Although there are few reports on E. hypersalinica, the strain was isolated from a high salinity environment (20%) in South Korea (Park, JS, Simpson, AG, Brown, S., Cho, BC (2009). ). Ultrastructure and molecular phylogeny of two heterolobosean amoebae, Euplaesiobystra hypersalinica gen. Et sp. Nov. And Tulamoeba peronaphora gen. Et sp. Nov., Isolated from an extremely hypersaline habitat.
また、アメーバB由来の4クローンを解析した結果、長さ(1,790、2,094、2,090及び2,092bp)及び配列が異なることが示された。その一方で、いずれの配列もNeoparamoeba branchiphilaと最も高い相同性(99%)を示した。したがって、複数種のアメーバが混在していたためではなく、18S rRNA遺伝子における塩基多型によって様々な配列が得られたと考えられる。18S rRNA遺伝子における塩基多型は真核生物において確認されており、アメーバにおいても報告されている。なお、N. branchiphilaはサーモンのエラの感染症を引き起こすことが広く知られている。以上、18S rDNA配列に基づく分子系統解析の結果、アメーバAはヘテロロボサ生物のEuplaesiobystra属、アメーバBはアメーボゾア生物のNeoparamoeba属に分類されることが示唆された。以降アメーバAをEuplaesiobystra sp.、アメーバBをNeoparamoeba sp.と称する。ヘテロロボサ及びアメーボゾアはそれぞれ真核生物において主要な系統の1つであり、シアノバクテリア培養槽より同時に単離されたことが報告されている。 In addition, as a result of analyzing 4 clones derived from amoeba B, it was shown that the length (1,790, 2,094, 2,090 and 2,092 bp) and the sequence were different. On the other hand, both sequences showed the highest homology (99%) with Neoparamoeba branchiphila. Therefore, it is considered that various sequences were obtained by the base polymorphism in the 18S rRNA gene, not because multiple kinds of amoeba were mixed. Single nucleotide polymorphisms in the 18S rRNA gene have been confirmed in eukaryotes and have also been reported in amoeba. It is widely known that N. branchiphila causes salmon gill infection. As a result of molecular phylogenetic analysis based on the 18S rDNA sequence, it was suggested that amoeba A is classified into the genus Euplaesiobystra of the heterolobosa organism, and amoeba B is classified into the genus Neoparamoeba of the amoebozoa organism. Hereinafter, amoeba A will be referred to as Euplae siobystra sp., And amoeba B will be referred to as Neoparamoeba sp. Heterolobosa and Amoebozoa are each one of the major strains in eukaryotes, and it has been reported that they were simultaneously isolated from cyanobacterial culture tanks.
以上、顕微鏡観察に基づく形態観察、及び18S rDNA配列に基づく分子系統解析により、2種のF. solaris捕食性アメーバは形態的及び系統的に大きく異なり、F. solarisの捕食者はある種には限られないことが示唆された。 As described above, by morphological observation based on microscopic observation and molecular phylogenetic analysis based on 18S rDNA sequence, the two types of F. solaris predatory amoebas differ greatly morphologically and systematically, and some predators of F. solaris are found. It was suggested that it was not limited.
2-2. F. solaris野生株におけるクロロフィラーゼ(CLH)活性評価
CLH活性はこれまで微細藻類を含む多くの植物細胞において見出されている一方で、その有無は種特異的であり、系統からその有無を判断することも難しい。そこでまず初めに、In silico解析及び色素分析により、F. solaris野生株におけるCLH活性の有無を評価した。
2-2. Evaluation of chlorophyllase (CLH) activity in F. solaris wild-type strains
While CLH activity has been found in many plant cells including microalgae, its presence or absence is species-specific, and it is difficult to determine its presence or absence from strains. Therefore, first of all, the presence or absence of CLH activity in the F. solaris wild-type strain was evaluated by in silico analysis and dye analysis.
Blastnを用いて、A. thaliana由来CLH遺伝子の塩基配列と相同性を持つ遺伝子を検索した結果、モデル珪藻P. tricornutum及びT. pseudonanaを含め、3種の珪藻において相同性を有する遺伝子の存在は認められなかった。これはA. thalianaにおいてパラログのCLH1及びCLH2遺伝子の塩基配列の相同性は40%であるように、CLHは配列の相同性が低いためであると考えられる。一方で、セリンリパーゼモチーフPS00120が共通モチーフとして保存されていることが示唆されている。したがってF. solarisの全遺伝子における当該モチーフを検索した結果、34遺伝子においてその存在が認められた(E-value<e-35)。また、これらの内、16遺伝子がCLHと同様のα/β hydrolase superfamilyであった。以上の解析から、F. solaris野生株においてCLH様活性を示すタンパク質をコードする遺伝子が複数存在することが示唆された。 As a result of searching for genes homologous to the nucleotide sequence of the CLH gene derived from A. thaliana using Blastn, the existence of genes homologous in three types of diatoms, including the model diatoms P. tricornutum and T. pseudonana, was found. I was not able to admit. This is considered to be due to the low sequence homology of CLH, as in A. thaliana, the nucleotide sequence homology of the CLH1 and CLH2 genes of the paralog is 40%. On the other hand, it is suggested that the serine lipase motif PS00120 is preserved as a common motif. Therefore, as a result of searching for the motif in all genes of F. solaris, its existence was confirmed in 34 genes (E-value <e -35 ). Of these, 16 genes were α / β hydrolase superfamily similar to CLH. From the above analysis, it was suggested that there are multiple genes encoding proteins showing CLH-like activity in the F. solaris wild-type strain.
F. solarisにおいてCLH様遺伝子の存在が示唆されたため、CLH不活性化条件(90%アセトン)及びCLH活性化条件(50%アセトン)にて色素を抽出し、HPLC分析によってCLH活性を評価した。各サンプルより得られたクロマトグラムと、クロロフィルa及びクロロフィリドa標品より得られたクロマトグラムとを比較した結果、CLH不活性化条件サンプルではクロロフィルa由来のピーク(Rt:33.2min)が確認された (図２A)。この前後に現れた小さなピークは、抽出又は分析過程にて生じたクロロフィルaの酸化物によるものであると考えられる。また、CLH活性化条件サンプルのクロマトグラムにおいては、クロロフィルa由来のピークに加え、クロロフィリドa由来のピーク(Rt:9.3min)が認された(図２B)ことから、クロロフィリドaの存在が認められた。 Since the presence of the CLH-like gene was suggested in F. solaris, the dye was extracted under CLH inactivation conditions (90% acetone) and CLH activation conditions (50% acetone), and CLH activity was evaluated by HPLC analysis. As a result of comparing the chromatogram obtained from each sample with the chromatogram obtained from the chlorophyll a and chlorophyllide a preparations, the peak derived from chlorophyll a (Rt: 33.2 min) was confirmed in the CLH inactivating condition sample. (Fig. 2A). The small peaks appearing before and after this are considered to be due to the oxide of chlorophyll a generated during the extraction or analysis process. In addition, in the chromatogram of the CLH activation condition sample, in addition to the peak derived from chlorophyll a, a peak derived from chlorophyllide a (Rt: 9.3 min) was observed (Fig. 2B), and thus the presence of chlorophyllide a was observed. Admitted.
珪藻において、CLHは高等植物と同様に葉緑体外に存在することが示唆されている。50%アセトン溶液中においては、CLHとクロロフィルaが反応し、クロロフィリドaが生じる。したがって、細胞破砕を行わずに、CLH不活性化条件である90%アセトンによる抽出を行った色素サンプルではクロロフィリドaは検出されず、一方でCLH活性を維持した場合にのみクロロフィリドが検出されたと考えられる。また、植物細胞においてクロロフィルからクロロフィリドを生成する酵素として、CLHに加え、クロロフィルデフィチラーゼ (CLD1)が近年同定されている。しかしながら、この酵素は7%以上のアセトン溶液中にて著しく失活することが示されている。したがって、今回確認されたクロロフィリドaはF. solarisにおけるCLH様タンパク質によって生じたものであると考えられた。以上のことから、F. solaris野生株はCLH活性を有することが示唆された。 In diatoms, CLH has been suggested to be extrachloroplast, similar to higher plants. In a 50% acetone solution, CLH reacts with chlorophyll a to produce chlorophyllide a. Therefore, chlorophyllide a was not detected in the dye sample extracted with 90% acetone, which is the CLH inactivating condition without cell disruption, while chlorophyllide was detected only when CLH activity was maintained. It is thought that it was. In addition to CLH, chlorophyll defitilase (CLD1) has recently been identified as an enzyme that produces chlorophyllide from chlorophyll in plant cells. However, this enzyme has been shown to be significantly inactivated in greater than 7% acetone solution. Therefore, the chlorophyllide a confirmed this time was considered to be generated by the CLH-like protein in F. solaris. From the above, it was suggested that the F. solaris wild strain has CLH activity.
2-3. CLH活性向上形質転換体の作出
F. solaris野生株に対し、高等植物A. thaliana由来CLH遺伝子を発現可能に導入してなる形質転換F. solarisを作出した。F. solaris 1.25×109cellsに対してpSP-CLH-GFP/GAPDHプラスミドを導入したところ、薬剤耐性株を33コロニー獲得した。この時の形質転換効率は2.6colonies/108cellsであり、ポジティブコントロールとしてpSP-GFP/GAPDHプラスミドを導入した場合の2.7colonies/108cells (8colonies/3.0×108cells)とほぼ同等の値であった。これらのコロニーを液体培地にて培養し、1か月以上安定して培養可能であった19クローン(#1-19)について、PCR増幅によりゲノム上への目的遺伝子の導入を確認した。その結果、19クローン中7クローン (#5、#6、#8、#10、#11、#12、#17)において、プラスミドをテンプレートとしたポジティブコントロールと同様の位置に目的のシングルバンド (1,206bp)が確認された(図３)ことから、CLH-GFP遺伝子のゲノム上への導入が示唆された。その他のクローンについて、18S rDNAユニバーサルプライマーを用いた場合に増幅産物 (約1,800bp)が得られたことから、ゲノムDNAは抽出できていたと考えられ、目的遺伝子のバンドが確認されなかったクローンでは、薬剤耐性遺伝子のみが導入されたこと、または培養期間中におけるCLH-GFP遺伝子の欠失が示唆された。更に、これら7クローンについてGFP蛍光観察を行った結果、全てのクローンについてGFP蛍光が認められたことから、全てのクローンにおいてCLH-GFP遺伝子の発現が示唆された。
2-3. Creation of CLH activity-enhancing transformants
A transformed F. solaris was created by introducing the CLH gene derived from the higher plant A. thaliana into a wild strain of F. solaris in an expressible manner. When the pSP-CLH-GFP / GAPDH plasmid was introduced into F. solaris 1.25 × 10 9 cells, 33 drug-resistant strains were acquired. The transformation efficiency at this time was 2.6 colonies / 10 8 cells, which is almost the same value as 2.7 colonies / 10 8 cells ( 8 colonies / 3.0 × 10 8 cells) when the pSP-GFP / GAPDH plasmid was introduced as a positive control. Met. These colonies were cultured in a liquid medium, and the introduction of the target gene into the genome was confirmed by PCR amplification of 19 clones (# 1-19) that could be stably cultured for 1 month or longer. As a result, in 7 out of 19 clones (# 5, # 6, # 8, # 10, # 11, # 12, # 17), the target single band (1,206) was placed at the same position as the positive control using the plasmid as a template. bp) was confirmed (Fig. 3), suggesting the introduction of the CLH-GFP gene into the genome. For the other clones, the amplification product (about 1,800 bp) was obtained when the 18S rDNA universal primer was used. Therefore, it is considered that the genomic DNA could be extracted, and the clone in which the band of the target gene was not confirmed was found. It was suggested that only the drug resistance gene was introduced, or that the CLH-GFP gene was deleted during the culture period. Furthermore, as a result of GFP fluorescence observation of these 7 clones, GFP fluorescence was observed in all clones, suggesting the expression of CLH-GFP gene in all clones.
CLH-GFP遺伝子のゲノム上への導入、及び発現が示唆された7クローンについてCLH活性を算出し、野生株と比較した。CLH活性は、一般的に用いられているクロロフィリドa/(クロロフィルa+クロロフィリドa)のモル比から算出した。その結果、CLH活性は3クローン(#5、#12、#17)において野生株と比較して有意な活性の向上が認められた(図４A)。特に、野生株と比較した場合の活性の向上の最大値はクローン#12における1.54±0.10倍であった。また、野生株及び7クローン全てにおいて、クロロフィルaプールサイズ(クロロフィルa+クロロフィリドa(fg/cell))に有意な差は認められなかった(図４B)ことから、CLH活性の向上は外来遺伝子の導入や発現等によるクロロフィル量の減少によるものではなく、導入したCLHの活性によることが示唆された。なお、微細藻類のクロロフィルプールサイズは種や培養条件によって異なるものの、珪藻P. tricornutumにおけるクロロフィルaプールサイズは50-300fg/cellであることから、今回のF. solarisにおける値は妥当であると考えられる。 CLH activity was calculated for 7 clones suggesting the introduction and expression of the CLH-GFP gene into the genome and compared with the wild-type strain. CLH activity was calculated from the molar ratio of commonly used chlorophyllide a / (chlorophyll a + chlorophyllide a). As a result, CLH activity was significantly improved in 3 clones (# 5, # 12, # 17) as compared with the wild-type strain (Fig. 4A). In particular, the maximum increase in activity compared to the wild strain was 1.54 ± 0.10 times in clone # 12. In addition, no significant difference was observed in chlorophyll a pool size (chlorophyll a + chlorophyllide a (fg / cell)) in the wild-type strain and all 7 clones (Fig. 4B), indicating that the improvement in CLH activity was due to the foreign gene. It was suggested that it was not due to the decrease in the amount of chlorophyll due to introduction or expression, but due to the activity of the introduced CLH. Although the chlorophyll pool size of microalgae varies depending on the species and culture conditions, the chlorophyll a pool size in the diatom P. tricornutum is 50-300 fg / cell, so the value in F. solaris this time is considered to be appropriate. Be done.
CLH活性の向上したクローン#5、#12、#17に対してRT-PCRを行い、CLH遺伝子のmRNAレベルでの発現を確認した。野生株及び上記3クローンからRNAを抽出し、cDNAを合成後、導入したCLH-GFP遺伝子領域(151bp)に対しPCR増幅を行った。その結果、野生株から得られたcDNAをテンプレートとした場合では目的のバンドは得られなかった。同一のテンプレートに対してGAPDH遺伝子(95bp)を標的とした場合では目的の位置にバンドが得られたことから、cDNAは獲得されていると考えられる。また、CLH-GFP発現プラスミド及び形質転換体からcDNAをテンプレートとした場合では、いずれも目的の位置にバンドが確認された。以上より、クローン#5、#12、#17において導入したCLH遺伝子の発現が示唆され、これによってCLH活性が向上したと考えられた。 RT-PCR was performed on clones # 5, # 12, and # 17 with improved CLH activity to confirm the expression of the CLH gene at the mRNA level. RNA was extracted from the wild-type strain and the above three clones, cDNA was synthesized, and then PCR amplification was performed on the introduced CLH-GFP gene region (151 bp). As a result, the target band could not be obtained when the cDNA obtained from the wild strain was used as a template. When the GAPDH gene (95 bp) was targeted for the same template, a band was obtained at the target position, suggesting that cDNA has been acquired. In addition, when cDNA was used as a template from the CLH-GFP expression plasmid and the transformant, a band was confirmed at the desired position. From the above, the expression of the CLH gene introduced in clones # 5, # 12, and # 17 was suggested, and it was considered that the CLH activity was improved by this.
2-4. CLH活性の向上によるアメーバ捕食能への影響評価
獲得された形質転換体において最もCLH活性の向上が認められたクローン#12を用いて、アメーバによる捕食実験を行い、その影響を評価した。F. solaris野生株及び形質転換体を2種類のアメーバEuplaesiobystra sp.及びNeoparamoeba sp.の存在下で培養し、F. solaris及びアメーバの細胞数を基に生育曲線を作成した。その結果、アメーバ非存在下では野生株及び形質転換体間で顕著な差は見られず、共に培養4日目に定常期に達したことから、外来性CLH遺伝子の導入による生育への影響は認められなかった(図５A、実線がクローン#12、破線が野生株)。また、Euplaesiobystra sp.存在下では、非存在下と比較して顕著なF. solaris細胞数の減少が確認され、野生株及び形質転換体共に同様の傾向を示した(図５B、実線がクローン#12、破線が野生株)。その一方で、Neoparamoeba sp.存在下では、Euplaesiobystra sp.存在下と同様にF. solaris細胞数の減少は認められたものの、4日目以降では野生株と形質転換体の細胞数に有意な差がみられた(図５C、実線がクローン#12、破線が野生株)。特に7日目においては、コントロールと比較して野生株では79.4%細胞濃度が減少した一方で、形質転換体においては62.5%であった。
2-4. Evaluation of the effect of improved CLH activity on ameba predation ability Using clone # 12, which was found to have the most improved CLH activity in the acquired transformants, an amoeba predation experiment was conducted to evaluate the effect. did. F. solaris wild-type strains and transformants were cultured in the presence of two types of amoeba Euplae siobystra sp. And Neoparamoeba sp., And a growth curve was created based on the cell numbers of F. solaris and amoeba. As a result, no significant difference was observed between the wild strain and the transformant in the absence of amoeba, and both reached the stationary phase on the 4th day of culture. Therefore, the effect of the introduction of the exogenous CLH gene on growth was observed. No evidence was observed (Fig. 5A, solid line is clone # 12, dashed line is wild strain). In addition, in the presence of Euplae siobystra sp., A remarkable decrease in the number of F. solaris cells was confirmed as compared with the absence, and both the wild strain and the transformant showed the same tendency (Fig. 5B, solid line is clone #. 12, the dashed line is the wild strain). On the other hand, in the presence of Neoparamoeba sp., Although the number of F. solaris cells decreased as in the presence of Euplae siobystra sp., There was a significant difference in the number of cells between the wild strain and the transformant after the 4th day. (Fig. 5C, solid line is clone # 12, dashed line is wild strain). Especially on day 7, the cell concentration was reduced by 79.4% in the wild strain compared to the control, while it was 62.5% in the transformant.
また、この時のアメーバの生育曲線を作成した結果、Euplaesiobystra sp.では野生株及び形質転換体を与えた場合で生育に顕著な差は認められなかった(図６A、実線がクローン#12の存在下、破線が野生株の存在下)。一方、Neoparamoeba sp.では野生株を与えた場合と比較して、形質転換体を与えた場合、顕著に細胞増殖が抑制されることが認められた(図６B、実線がクローン#12の存在下、破線が野生株の存在下)。したがって、Neoparamoeba sp.の捕食によるF. solaris形質転換体細胞数減少の抑制は、アメーバ細胞数の減少に起因すると考えられた。 In addition, as a result of creating the growth curve of amoeba at this time, no significant difference was observed in the growth of Euplae siobystra sp. When the wild strain and the transformant were given (Fig. 6A, solid line is the presence of clone # 12). Below, the dashed line is under the presence of wild strains). On the other hand, in Neoparamoeba sp., Cell proliferation was significantly suppressed when the transformant was given as compared with the case where the wild strain was given (Fig. 6B, solid line is in the presence of clone # 12). , The dashed line is in the presence of wild strains). Therefore, it was considered that the suppression of the decrease in the number of F. solaris transformant cells by the predation of Neoparamoeba sp. Was due to the decrease in the number of amoeba cells.
上記の通り、CLH活性の向上によってその捕食能への影響が示唆されたNeoparamoeba sp.について、光量子束密度100、500μmol/m2/sにて形質転換体に対する捕食実験を行った。Neoparamoeba sp.非存在下において、F. solarisは、いずれの光量子束密度においても同様の生育を示し、100μmol/m2/s以上での光量子束密度による影響は認められなかった(図７A、円形マーカーはNeoparamoeba sp.非存在下、菱形マーカーはNeoparamoeba sp.存在下、破線が100μmol/m2/s条件、実線が500μmol/m2/s条件)。これは100μmol/m2/sにおいて既に光化学系が還元状態となったためであると考えられる。Neoparamoeba sp.存在下においては、いずれの光量子束密度条件においてもF. solaris細胞濃度の減少が認められたが、500μmol/m2/sにおいては有意な減少の抑制が認められた。また、アメーバの生育についても、強光条件下において顕著に抑制されることが確認されたことから(図７B、破線が100μmol/m2/s条件、実線が500μmol/m2/s条件)、光量依存的なアメーバによる捕食の影響の低減が示唆された。本研究では、クロロフィル類による光毒性(光照射による一重項酸素の発生)に着目していることから、強光条件下においてアメーバ細胞内でより多量の一重項酸素が発生したことが考えられる。 As described above, Neoparamoeba sp., Which was suggested to have an effect on predation ability by improving CLH activity, was subjected to a predation experiment on transformants at a photon flux density of 100 and 500 μmol / m 2 / s. In the absence of Neoparamoeba sp., F. solaris showed similar growth at all photon flux densities, with no effect of photon flux densities above 100 μmol / m 2 / s (Fig. 7A, circular). The marker is in the absence of Neoparamoeba sp., The diamond-shaped marker is in the presence of Neoparamoeba sp., The broken line is 100 μmol / m 2 / s condition, and the solid line is 500 μmol / m 2 / s condition). It is considered that this is because the photochemical system has already been reduced at 100 μmol / m 2 / s. In the presence of Neoparamoeba sp., A decrease in F. solaris cell concentration was observed under all photon flux density conditions, but a significant decrease was observed at 500 μmol / m 2 / s. In addition, it was confirmed that the growth of amoeba was significantly suppressed under strong light conditions (Fig. 7B, broken line under 100 μmol / m 2 / s condition, solid line under 500 μmol / m 2 / s condition). It was suggested that the effect of predation by light-dependent amoebas was reduced. Since this study focuses on phototoxicity (generation of singlet oxygen by light irradiation) due to chlorophylls, it is considered that a larger amount of singlet oxygen was generated in amoebae cells under strong light conditions.
以上の結果より、CLH活性の向上により、F. solarisの捕食生物であるNeoparamoeba sp.の抑制が可能であること、加えて、その作用には光量が重要な因子となることが示唆された。 From the above results, it was suggested that the improvement of CLH activity can suppress Neoparamoeba sp., Which is a predator of F. solaris, and that the amount of light is an important factor for its action.
Claims (9)
（ａ）配列番号２のアミノ酸配列からなるタンパク質
（ｂ）配列番号２のアミノ酸配列に対して８０％以上の同一性を有するアミノ酸配列からなり、且つ、クロロフィラーゼ活性を有するタンパク質 The transformed microalga according to claim 1, wherein the chlorophyllase gene encodes the protein according to (a) or (b) below.
(A) Protein consisting of the amino acid sequence of SEQ ID NO: 2 (b) Protein consisting of an amino acid sequence having 80% or more identity with respect to the amino acid sequence of SEQ ID NO: 2 and having chlorophyllase activity
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| JP2001086990A (en) * | 1999-09-20 | 2001-04-03 | Kagome Co Ltd | Dna encoding chlorophyllase and plant transformed by the same |
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| US20170267984A1 (en) * | 2016-03-18 | 2017-09-21 | Exxonmobil Research And Engineering Company | Chlorophyllase overproduction to enhance photosynthetic efficiency |
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| JP2001086990A (en) * | 1999-09-20 | 2001-04-03 | Kagome Co Ltd | Dna encoding chlorophyllase and plant transformed by the same |
| JP2016034925A (en) * | 2014-08-04 | 2016-03-17 | 国立大学法人東京農工大学 | Novel diatom protein and use thereof |
| US20170267984A1 (en) * | 2016-03-18 | 2017-09-21 | Exxonmobil Research And Engineering Company | Chlorophyllase overproduction to enhance photosynthetic efficiency |
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