JP2004081022A - Pure culture of aphanothece sacrum - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for isolation and pure culture of Aphanothece sacrum using a culture medium prepared according to an individual ordor for isolation and pure culture of freshwater algae growing in the special environment. <P>SOLUTION: The method for the pure culture of the Aphanothece sacrum is characterized by culturing algal bodies of the Aphanothece sacrum in a culture medium for the culturing the freshwater algae containing at least Ca, Cu, Fe, Mg, Mn and Zn as metal elements in the following weight ratios of Ca 1, Cu 0.00025-0.00049, Fe 0.017-0.068, Mg 0.160-0.313, Mn 0.060 and Zn 0.0012 and further sulfate ions at 5.8-11.7 mg/L concentration, nitrate ions at 2.8-56.6 mg/L concentration and phosphate ions at 1.4-28.6 mg/L concentration. <P>COPYRIGHT: (C)2004,JPO

Description

【００１８】
【表２】

【００１９】
上記の結果から、４種の培地の中ではＡＳ−ＩＩ培地がスイゼンジノリ細胞の生育にもっとも適しており、硫酸イオンがスイゼンジノリ細胞の生育に影響していること、並びに硝酸イオン、リン酸イオン量が多いほど夾雑するバクテリアの生育が著しいことが示唆された。また、ＡＳ−ＩＶ培地でも生育が認められることから、ＡＳ−ＩＶ培地での結果を加味してＡＳ−ＩＩ培地のＣａ、Ｃｕ、Ｆｅ、Ｍｇ、Ｍｎ　及びＺｎの量比をさらに調整した。
【００２０】
硫酸マグネシウム、塩化マグネシウム、硫酸銅及び酒石酸鉄の量を半量とした培地（ＡＳ−１／２ＭＦ）、硫酸マグネシウム、塩化マグネシウム及び硫酸銅の量を半量とし、さらに酒石酸鉄の量を１／４とした培地（ＡＳ−１／２Ｍ１／４Ｆ）を調製した（下記表３参照）。これらの培地１００ｍＬを３００ｍＬ用三角フラスコにスイゼンジノリ藻体（約５ｍｍ角の小塊：湿重量約０．５　ｇ）を入れ光照射（約２５０　ｌｕｘ）下、２５±２℃でゆっくりと振盪（５０ｒｐｍ）させながら８日間インキュベートして藻体の生育を比較した。藻体の生育状態を表４に示す。
【００２１】
【表３】

【００２２】
【表４】

【００２３】
上記結果から、ＡＳ−ＩＩ培地の硫酸マグネシウム、塩化マグネシウム及び硫酸銅の量を１／２としてさらに酒石酸鉄の量を１／４としたＡＳ−１／２Ｍ１／４Ｆ培地がスイゼンジノリ藻体の生育に適していることが示唆された。
【００２４】
スイゼンジノリ藻体の生育が観察され、真核藻類の著しい生育が見られなかったＡＳ−ＩＩ　培地、ＡＳ−１／２Ｍ１／４Ｆ培地と自然状態のスイゼンジノリにおけるＣａの含有量を１　としたときの　Ｃａ、Ｃｕ、Ｆｅ、Ｍｇ、Ｍｎ、Ｚｎの重量比は表５のとおりであり、実質的に等しい比率であった。
【００２５】
【表５】

【００２６】
スイゼンジノリ藻体の生育率の良かったＡＳ−１／２Ｍ１／４Ｆ培地をＡＳＴ培地と命名し、Ａ．　ｓａｃｒｕｍ純粋培養のための培地とした。
実施例２：Ａ．　ｓａｃｒｕｍ　の単離
原料のスイゼンジノリ（Ａｐｈａｎｏｔｈｅｃｅ　ｓａｃｒｕｍ　）は福岡県甘木市の遠藤金川堂のものを用いる。この藻体（約１．５ｇ）を無菌水でよく洗浄後、無菌水１００ｍｌと共に、Ａｃｅ　ｈｏｍｏｇｅｎｉｚｅｒ　ＡＭ−９（Ｎｉｈｏｎｓｅｉｋｉ　Ｋａｉｓｈａ）で細かくすりつぶし、Ｇ１ガラスろ過器（細孔の大きさ：１００〜１２０μｍ）に通した。ヘマサイトメーターに一部をとり、顕微鏡で観察してスイゼンジノリの細胞が１つずつ分かれていることを確認すると共に細胞数を測定し、この菌数をもとに殺菌済みＡＳＴ液体培地５ｍｌ入った試験管に１個ずつ細胞が植菌できるように希釈、植菌し、２５℃、５，０００ｌｕｘ蛍光灯下で約１ヶ月間培養した。２１００本のサンプルを作成したうち、約７割の試験管にスイゼンジノリと思われる細胞が認められた。
【００２７】
これら増殖の認められた試験管のサンプルを用い、ポリペプトン（０．１％）と酵母抽出物（０．１％）を含む有機物入りＡＳＴ培地に増殖の認められた試験管から０．１ｍｌずつ植菌した。もし、従属栄養細菌がコンタミネーションしていたら、２〜３日でこれらの試験管は白濁する。約１０日たっても白濁しないサンプルの試験管を第１次純粋スイゼンジノリと考え、同様な操作を最低２回行なったのち、残った試験管（１本）から株分けを２回行なった。最終的に４本の試験管を用い、従属栄養細菌の混在のテストを行ない、白濁しないサンプルを選抜した。
【００２８】
以上の処理を行った藻体約　４〜５ｇ　に　ＡＳＴ　培地を　３０ｍｌ　加え、ホモジェナイザーで細かくすりつぶした。これを　Ｇ２ガラスろ過器（細孔の大きさ：４０〜５０μｍ）に通した後に容量　５０ｍｌ　のスクリューキャップのチューブに移し、遠心機（Ｃｅｎｔｒｉｆｕｇｅ　０５Ｐ−２１、Ｈｉｔａｃｈｉ）で遠心分離して沈殿を回収した。この沈殿を容量　２ｍｌのマイクロチューブに移し、ソニケーター（Ｓｏｎｉｃａｔｏｒ^ＴＭ　Ｗ−２２０、Ｈｅａｔ　Ｓｙｓｔｅｍｓ−　Ｕｌｔｒａｓｏｎｉｃｓ）を用いて超音波処理し、卓上遠心機（ＭＴＸ−１５０、ローター：ＴＭＡ−Ｓ２６、ＴＯＭＹ）で遠心分離して細胞のみの沈殿を回収した。沈殿を　ＡＳＴ　培地にとかし、ヨードキサノールの濃度で４０〜４５％　の　ＯｐｔｉＰｒｅｐ^ＴＭ　（Ａｘｉｓ−Ｓｈｉｅｌｄ）の密度勾配に重層し、超遠心機（７０Ｐ−７２、ローター：ＳＲＰ２８ＳＡ−１６５、Ｈｉｔａｃｈｉ）で　２５，０００ｒｐｍ、２時間超遠心した。中心付近に見られた細胞層をマイクロチューブに回収し、等量〜９倍量の　ＡＳＴ　培地を加え、卓上遠心機で遠心分離して細胞のみの沈殿を回収した。沈殿を適当量の　ＡＳＴ　培地にとかし、ヘマサイトメーターに一部をとり、顕微鏡で観察してスイゼンジノリの細胞が１つずつ分かれていることを確認すると共に細胞数を測定し、この菌数をもとに　０．６％　のアガロースを含むＡＳＴ培地のプレートに１６，０００個の細胞をまき、２５〜２７℃、２，０００〜３，０００ｌｕｘの蛍光灯下で約１ヶ月間培養した。
【００２９】
形成されたコロニーの内、付近にコンタミネーションのコロニーが観察されなかったコロニーを選択し、その中から　ＬＩＶＥ／ＤＥＡＤ　ＢａｃＬｉｇｈｔ（Ｍｏｌｅｃｕｌａｒ　Ｐｒｏｂｅ）で染色して蛍光顕微鏡で観察したときに細胞間にコンタミネーションのシグナルが見られなかったクローンを選抜した。
【００３０】
このクローン化された藻体を先の有機物入りＡＳＴ培地の他、ＡＳＴ　培地に牛肉抽出物（０．３％）とペプトン（０．５％）を加えた培地（Ｂ−ＩＩＩ　ｍｅｄｉｕｍ）、ＡＳＴ　培地に酢酸ナトリウム（０．０５％）、グルコース（０．０５％）、トリプトン（０．０５％）、酵母抽出物（０．０３％）を加えた培地（Ｂ−Ｖ　ｍｅｄｉｕｍ）、ＡＳＴ　培地に酵母抽出物（０．１％）とトリプトン（０．２％）を加えた培地（ＹＴ　ｍｅｄｉｕｍ）を用いて２５〜２７℃、６０ｒｐｍ、２，０００〜３，０００ｌｕｘの蛍光灯下で１週間培養し、コンタミネーションが増殖してこないことを確認した。
実施例３：Ａ．　ｓａｃｒｕｍ　の単離
原料のスイゼンジノリ（Ａｐｈａｎｏｔｈｅｃｅ　ｓａｃｒｕｍ　）は福岡県甘木市の遠藤金川堂のものを用いる。この藻体約　４〜５ｇ　に　ＡＳＴ　培地を　３０ｍｌ　加え、ホモジェナイザーで細かくすりつぶした。これを　Ｇ２ガラスろ過器（細孔の大きさ：４０〜５０μｍ）に通した後に容量　５０ｍｌ　のスクリューキャップのチューブに移し、遠心機（Ｃｅｎｔｒｉｆｕｇｅ　０５Ｐ−２１、Ｈｉｔａｃｈｉ）で遠心分離して沈殿を回収した。この沈殿を容量　２ｍｌのマイクロチューブに移し、ソニケーター（Ｓｏｎｉｃａｔｏｒ^ＴＭ　Ｗ−２２０、Ｈｅａｔ　Ｓｙｓｔｅｍｓ　−　Ｕｌｔｒａｓｏｎｉｃｓ）を用いて超音波処理し、卓上遠心機（ＭＴＸ−１５０、ローター：ＴＭＡ−Ｓ２６、ＴＯＭＹ）で遠心分離して細胞のみの沈殿を回収した。沈殿を　ＡＳＴ　培地にとかし、ヨードキサノールの濃度で４０〜４５％　の　ＯｐｔｉＰｒｅｐ^ＴＭ　（Ａｘｉｓ−Ｓｈｉｅｌｄ）の密度勾配に重層し、超遠心機（７０Ｐ−７２、ローター：ＳＲＰ２８ＳＡ−１６５、Ｈｉｔａｃｈｉ）で　２５，０００ｒｐｍ、２時間超遠心した。中心付近に見られた細胞層をマイクロチューブに回収し、等量〜９倍量の　ＡＳＴ　培地を加え、卓上遠心機で遠心分離して細胞のみの沈殿を回収した。沈殿を適当量の　ＡＳＴ　培地にとかし、ヘマサイトメーターに一部をとり、顕微鏡で観察してスイゼンジノリの細胞が１つずつ分かれていることを確認すると共に細胞数を測定し、この菌数をもとに　０．６％　のアガロースを含むＡＳＴ培地のプレートに１６，０００個の細胞をまき、２５〜２７℃、２，０００〜３，０００ｌｕｘの蛍光灯下で約１ヶ月間培養した。
【００３１】
形成されたコロニーの内、付近にコンタミネーションのコロニーが観察されなかったコロニーを選択し、その中から　ＬＩＶＥ／ＤＥＡＤ　ＢａｃＬｉｇｈｔ（Ｍｏｌｅｃｕｌａｒ　Ｐｒｏｂｅ）で染色して蛍光顕微鏡で観察したときに細胞間にコンタミネーションのシグナルが見られなかったクローンを選抜した。
【００３２】
このクローン化された藻体を先の有機物入りＡＳＴ培地の他、ＡＳＴ　培地に牛肉抽出物　（０．３％）とペプトン（０．５％）を加えた培地（Ｂ−ＩＩＩ　ｍｅｄｉｕｍ）、ＡＳＴ　培地に酢酸ナトリウム（０．０５％）、グルコース（０．０５％）、トリプトン（０．０５％）、酵母抽出物（０．０３％）を加えた培地（Ｂ−Ｖ　ｍｅｄｉｕｍ）、ＡＳＴ　培地に酵母抽出物（０．１％）とトリプトン（０．２％）を加えた培地（ＹＴ　ｍｅｄｉｕｍ）を用いて２５〜２７℃、６０ｒｐｍ、２，０００〜３，０００ｌｕｘの蛍光灯下で１週間培養し、コンタミネーションが増殖してこないことを確認した。
実施例４：自然状態のスイゼンジノリのフェレドキシン遺伝子の部分塩基配列の決定
自然状態のスイゼンジノリを滅菌水で十分水洗いしたものから　ＩＳＯＰＬＡＮＴ^ＴＭ（日本ジーン）を用い、以下のようにして　ＤＮＡ　を抽出した。
【００３３】
藻体約１ｇ　に対してキットの　Ｓｏｌｕｔｉｏｎ　Ｉ　を３倍量加えて撹拌後、キットの　Ｓｏｌｕｔｉｏｎ　ＩＩ　を藻体の１．５倍量加えて５０℃で１５〜１２０分反応させた。キットの　Ｓｏｌｕｔｉｏｎ　ＩＩＩ　を藻体の１．５倍量加えて氷上に１５分置き、遠心機（Ｃｅｎｔｒｉｆｕｇｅ　０５Ｐ−２１、Ｈｉｔａｃｈｉ）を用いて　３，０００ｒｐｍ　で　１０分間遠心分離した後、水層を別のチューブに移した。２倍量のエタノールを加えて遠心機（ＳＣＲ　２０ＢＡ、ローター：ＲＰＲ　２０−２、Ｈｉｔａｃｈｉ）で　７，０００ｒｐｍ　で　１０分間遠心分離して沈殿を回収した。沈殿を　ＴＥ　バッファー　０．５ｍｌ　に溶かし、フェノール・クロロフォルム（１：１）を等量加えてよく撹拌した。１２，０００ｒｐｍ　で　５分間遠心分離した後、水層を別のマイクロチューブに移した。水層とフェノール・クロロフォルム層の間にゲルに由来する中間層が見られなくなるまでフェノール・クロロフォルム処理を繰り返した。水層の１０分の１量の酢酸ナトリウムと、２倍量の　１００％エタノールを加えて混合した後、−２０℃で１０分間放置した。卓上遠心機（ＭＴＸ−１５０、ローター：ＴＭＡ−Ｓ２６、ＴＯＭＹ）で　４℃、１５，０００ｒｐｍ　で　１０分間遠心分離した後、上清を廃棄した。１００μｌ　の滅菌水を加えよく撹拌し、５０℃　で１０分加温して、これを以下の　ＰＣＲ　によるスイゼンジノリのフェレドキシン遺伝子の増幅反応の鋳型　ＤＮＡ　溶液とした。
【００３４】
スイゼンジノリのフェレドキシンタンパク質として既に報告されている２種類のアミノ酸配列を元に次の配列のプライマーを設計して、以下の配列を持つＤＮＡを合成した。

この配列をプライマーに、スイゼンジノリから調製した　ＤＮＡ　を鋳型に用いて　ＰＣＲ　によるスイゼンジノリのフェレドキシン　Ｉ　及びフェレドキシン　ＩＩ　遺伝子の増幅行った。反応液の組成および反応条件は下記のとおりである。
Ａ：
反応液組成
鋳型　ＤＮＡ　　　　　　　　　　　　　　　　　　　　１μｇ
ＴａＫａＲａ　Ｔａｑ　　　　　　（ＴａＫａＲａ）　　　　　　　　０．２５μｌ
１０ｘ　ＰＣＲ　バッファー　　（ＴａＫａＲａ）　　　　　　　　５μｌ
ｄＮＴＰ　Ｍｉｘ　　　　　　　（ＴａＫａＲａ）　　　　　　　　４μｌ
プライマー　１＊　　　　　（０．０１ｍＭ）　　　　　　　　１μｌ
プライマー　２＊＊　　　　　（０．０１ｍＭ）　　　　　　　　１μｌ
滅菌水　　　　　　　　　　　　　ｕｐ　ｔｏ　５０μｌ
＊　ＡｓＦｄ−ＩＮ　　：ＧＡＣＴＣＣＣＧＡＴＧＧＡＧＡＣＡＡＴＧ
または
ＡｓＦｄ−ＩＩＮ　：ＧＡＧＧＴＡＧＣＡＧＡＴＧＡＴＣＡＧＡＣＣ
＊＊ＡｓＦｄ−ＩＣ　　：ＴＣＴＣＣＧＧＴＡＧＧＡＴＡＡＧＣＣＡＣ
または
ＡｓＦｄ−ＩＩＣ　：ＣＴＣＣＴＧＴＡＧＧＡＴＡＡＧＣＡＡＣＧＣ

この反応により増幅された　ＤＮＡ　断片を　ＳＵＰＲＥＣＴＭ−０２（Ｔａｋａｒａ）を用いて濃縮し、１ｘ　ＴＡＥ　−　２％　アガロースゲルを用いた電気泳動で、目的とする約　０．３ｋｂ　のＤＮＡ　断片をプライマーなどと分離した後、目的の位置のゲルを切り出して細かく刻んで　ＳＵＰＲＥＣＴＭ−０１（Ｔａｋａｒａ）に移して、卓上遠心機で遠心して増幅された　ＤＮＡ　断片を回収した。
【００３５】
回収した断片を「ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ」（Ｉｎｖｉｔｒｏｇｅｎ）を用い、下記の反応液の組成および反応条件で　ｐＣＲ２．１　ベクターと連結させた。
Ｂ：
反応液組成
ＰＣＲ　ｐｒｏｄμｃｔ　　　　　　　　　　　　　　　　　１μｌ
ｐＣＲ２．１ＴＭ　ｖｅｃｔｏｒ　　　　　　　　（２５ｎｇ／μｌ）　　　２μｌ
１０ｘ　Ｌｉｇａｔｉｏｎ　バッファー　　　　（Ｉｎｖｉｔｒｏｇｅｎ）　　１μｌ
Ｔ４　ＤＮＡ　Ｌｉｇａｓｅ　　　　　　　　　（Ｉｎｖｉｔｒｏｇｅｎ）　　１μｌ
滅菌水　　　　　　　　　　　　　　　　ｕｐ　ｔｏ　１０μｌ
反応条件
１６℃ｘ　　一晩
大腸菌　ＩＮＶαＦ’（Ｉｎｖｉｔｒｏｇｅｎ）５０μｌ　に、ＩＮＶαＦ’　に付属していた　０．５　Ｍβ−メルカプトエタノール　２μｌ　と連結反応液　２μｌ　を加えて氷上に３０分置いた。この大腸菌液を４２℃で３０秒処理して形質転換させた後、氷上に２分置いた。ＩＮＶαＦ’　に付属していた　ＳＯＣ　培地を　２００μｌ　加えて３７℃、１７０ｒｐｍ　で１時間振盪培養した。５０μｇ　／μｌ　のアンピシリンを含む　ＬＢ　寒天培地上に　４０ｍｇ／ｍｌ　の　Ｘ−Ｇａｌ　ＤＭＦＡ溶液を４０μｌ　塗布した後に１００μｌ　の大腸菌液をまき、３７℃で一晩培養した。
【００３６】
プレート上に形成されたいくつかの白いコロニーを拾い、Ｌｙｓｉｓ　バッファー（２０ｍＭ　Ｔｒｉｓ−ＨＣｌ　ｐＨ　８．０、５０ｍＭ　ＫＣｌ、０．１％　Ｔｗｅｅｎ　２０）１０μｌ　に溶かした。これを９５℃で５分処理したもの　５μｌ　を鋳型に、ベクター上の配列をプライマーにしてＰＣＲ　を行った。反応液の組成および反応条件は下記のとおりである。
Ｃ：
反応液組成
鋳型　ＤＮＡ　　　　　　　　　　　　　　　　５μｌ
ＴａＫａＲａ　Ｔａｑ　　　　　　（ＴａＫａＲａ）　　　　　０．２５μｌ
１０ｘ　ＰＣＲ　バッファー　　（ＴａＫａＲａ）　　　　　５μｌ
ｄＮＴＰ　Ｍｉｘ　　　　　　　（ＴａＫａＲａ）　　　　　４μｌ
ｐｒｉｍｅｒ　ｓｅｔＡ＊　　　　　（０．０１ｍＭ　ｅａｃｈ）　　　１μｌ
滅菌水　　　　　　　　　　　　ｕｐ　ｔｏ　５０μｌ

ＰＣＲ　産物を電気泳動し、目的とするサイズの　ＤＮＡ　断片が含まれた　ＰＣＲ　産物を鋳型に　Ｄｙｅ　Ｐｒｉｍｅｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｃｏｒｅ　Ｋｉｔ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ）を用いてシーケンシング反応を行った。反応液の組成および反応条件は下記のとおりである。
Ｄ：
反応液組成
鋳型　ＤＮＡ　　　　　　　（１０ｎｇ／μｌ）　　　　　　　１μｌ
５ｘ　Ｓｅｑ　バッファー　　　　　　　　　　　　　　　１μｌ
ｄ／ｄｄＮＴＰ　　　　　　　　　　　　　　　　　　　　１μｌ
Ｄｙｅ　ｐｒｉｍｅｒ　　　　　　（０．４μＭ）　　　　　　　　１μｌ
Ｄｉｌｕｔｅｄ　Ｅｎｚｙｍｅ＊　　　　　　　　　　　　　　　　１μｌ
＊Ｄｉｌｕｔｅｄ　ＥｎｚｙｍｅＡｍｐｌｉＴａｑ　ＤＮＡ　ｐｏｌｙｍｅｒａｓｅ，　ＦＳ　　１μｌ
５ｘ　Ｓｅｑ　バッファー　　　　　　　　　　　　　　　１μｌ
滅菌水　　　　　　　　　　　　　　　　　　　　５μｌ

この反応産物を　ＤＮＡ　シーケンサー（ＤＳＱ−１、Ｓｈｉｍａｚｕ）にかけて塩基配列を決定し、この部分塩基配列（配列表の配列番号１及び２参照）から推定される部分アミノ酸配列が、既報のフェレドキシンタンパク質の部分アミノ酸配列と一致したことを確認した（図１及び図２参照）。
実施例５：単離した藻体の同定
実施例２または３でクローン化された藻体がスイゼンジノリの本体である　Ａ．　ｓａｃｒｕｍ　かどうかを同定する。藻体の外観は、細胞の長径が約６μｍの桿形である点、細胞はゲルに埋まった状態にある点など、自然状態のスイゼンジノリの外観と一致する。また、以下の方法で単離した藻体からスイゼンジノリのフェレドキシン遺伝子の部分塩基配列が検出されるかどうかを確認した。
【００３７】
実施例２または３で単離した藻体に対して　ＩＳＯＰＬＡＮＴ　ＩＩ　^ＴＭ（日本ジーン）を用いて以下のように　ＤＮＡ　を抽出した。
【００３８】
藻体約１ｇ　に対してキットの　Ｓｏｌｕｔｉｏｎ　Ｉ　を３倍量加えて撹拌後、キットの　Ｓｏｌｕｔｉｏｎ　ＩＩ　を藻体の１．５倍量加えて５０℃で１５〜２０分反応させた。キットの　Ｓｏｌｕｔｉｏｎ　ＩＩＩ−Ａ　を藻体の１倍量、キットの　Ｓｏｌｕｔｉｏｎ　ＩＩＩ−Ｂ　を藻体の１．２倍量加えて氷上に１５分置き、遠心機（Ｃｅｎｔｒｉｆｕｇｅ　０５Ｐ−２１、Ｈｉｔａｃｈｉ）を用いて　３，０００ｒｐｍ　で　１０分間遠心分離した後、水層を別のチューブに移した。２倍量のエタノールを加えて遠心機（ＳＣＲ　２０ＢＡ、ローター：ＲＰＲ　２０−２、Ｈｉｔａｃｈｉ）で　７，０００ｒｐｍ　で　１０分間遠心分離して沈殿を回収した。沈殿を　ＴＥ　バッファー　０．５ｍｌ　に溶かし、フェノール・クロロフォルム（１：１）を等量加えてよく撹拌した。１２，０００ｒｐｍ　で　５分間遠心分離した後、水層を別のマイクロチューブに移した。水層とフェノール・クロロフォルム層の間にゲルに由来するとみられる中間層が見られなくなるまでフェノール・クロロフォルム処理を繰り返した。水層に１０分の１量の酢酸ナトリウムと、２〜２．５倍量の　１００％　エタノールを加えて混合した後、−２０℃で１０分間放置した。卓上遠心機（ＭＴＸ−１５０、ローター：ＴＭＡ−Ｓ２６、ＴＯＭＹ）で　４℃、１５，０００ｒｐｍ　で　１０分間遠心分離した後、上清を廃棄した。上清を十分除いた後、１００μｌ　の滅菌水を加えよく撹拌し、５０℃　で１０分加温して以下の　ＰＣＲ　によるフェレドキシン遺伝子の増幅反応の鋳型　ＤＮＡ　溶液とした。
【００３９】
実施例４に記載のプライマーを用いて、単離した藻体の　ＤＮＡ　からフェレドキシン遺伝子の部分塩基配列を増幅した。反応液の組成および反応条件は実施例４のＡとおりである。
【００４０】
この反応により増幅された　ＤＮＡ　断片を「ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ」（Ｉｎｖｉｔｒｏｇｅｎ）を用い、実施例４のＢの反応液の組成および反応条件で　ｐＣＲ２．１　ベクターと連結させた。大腸菌　ＩＮＶαＦ’（Ｉｎｖｉｔｒｏｇｅｎ）５０μｌ　に、ＩＮＶαＦ’　に付属していた　０．５　Ｍβ−メルカプトエタノール　２μｌ　と連結反応液　２μｌ　を加えて氷上に３０分置いた。この大腸菌液を４２℃で３０秒処理した後、氷上に２分置いた。ＩＮＶαＦ’　に付属していた　ＳＯＣ　培地を　２００μｌ　加えて３７℃、１７０ｒｐｍ　で１時間振盪培養した。５０μｇ　／μｌ　のアンピシリンを含む　ＬＢ　寒天培地上に　４０ｍｇ／ｍｌ　の　Ｘ−Ｇａｌ　ＤＭＦＡ溶液を４０μｌ　塗布した後に５０μｌ　の大腸菌液をまき、３７℃で一晩培養した。
【００４１】
プレート上に形成されたいくつかの白いコロニーを拾い、Ｌｙｓｉｓ　バッファー（２０ｍＭ　Ｔｒｉｓ−ＨＣｌ　ｐＨ　８．０、５０ｍＭ　ＫＣｌ、０．１％　Ｔｗｅｅｎ　２０）１０μｌ　に溶かした。これを９５℃で５分処理したもの　５μｌ　を鋳型に、ｐＣＲ２．１　ベクター上の配列をプライマーにして　ＰＣＲ　を行った。反応液の組成および反応条件は実施例４の　Ｃ　のとおりである。ＰＣＲ　産物を電気泳動して目的とするサイズの　ＤＮＡ　断片が含まれた　ＰＣＲ　産物を選択し、これを鋳型に　Ｄｙｅ　Ｐｒｉｍｅｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｃｏｒｅ　Ｋｉｔ（ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍｓ）を用いてシーケンシング反応を行った。反応液の組成および反応条件は実施例４の　Ｄ　の通りである。この反応産物を　ＤＮＡ　シーケンサー（ＤＳＱ−１、Ｓｈｉｍａｚｕ）にかけて塩基配列を決定し、この部分塩基配列から推定される部分アミノ酸配列が、既報のフェレドキシンタンパク質の部分アミノ酸配列と一致したことを確認した（図３参照）。
【００４２】
今回クローン化された藻体は自然状態のスイゼンジノリが持つ塩基配列を持ち、外観が自然状態のスイゼンジノリに似ていることから、スイゼンジノリの本体である　Ａ．　ｓａｃｒｕｍ　であると同定し、これをＡ．　ｓａｃｒｕｍ　ＫｕＸ　３と命名した。この藻体は、経済産業省産業技術総合研究所生命工学工業技術研究所に、ブダペスト条約のもとで国際寄託されている（受託番号ＦＥＲＭ　ＢＰ−７３１５）。
実施例６：Ａ．　ｓａｃｒｕｍ　ＫｕＸ　３　の１６Ｓ　ｒＲＮＡ　遺伝子の部分塩基配列の解析
対象とする藻体が実施例２または３でクローン化された藻体　Ａ．　ｓａｃｒｕｍ　　ＫｕＸ　３　かどうかを同定するための指標として　１６Ｓ　ｒＲＮＡ　遺伝子の部分塩基配列を解析した。
【００４３】
実施例５で抽出した単離した藻体の　ＤＮＡ　を以下の　ＰＣＲ　による１６Ｓ　ｒＲＮＡ　遺伝子の増幅反応の鋳型　ＤＮＡ　溶液とした。
【００４４】
１６Ｓ　ｒＲＮＡ　遺伝子を増幅するためのユニバーサルプライマーを用いて、単離した藻体の　ＤＮＡから　１６Ｓ　ｒＲＮＡ　遺伝子の部分塩基配列を増幅した。反応液の組成および反応条件は下記のとおりである。
反応液組成
鋳型　ＤＮＡ　　　　　　　　　　　　５μｌまたは１μｇ
ＴａＫａＲａ　Ｔａｑ　　　　　　　　（ＴａＫａＲａ）　　　　　　０．２５μｌ
１０ｘ　ＰＣＲ　バッファー　　　　（ＴａＫａＲａ）　　　　　　５μｌ
ｄＮＴＰ　Ｍｉｘ　　　　　　　　　（ＴａＫａＲａ）　　　　　　４μｌ
プライマー　１６Ｓ−２７ｆ＊　　　　（０．０１ｍＭ）　　　　　　１μｌ
プライマー　１６Ｓ−１５２５ｒ＊＊　　（０．０１ｍＭ）　　　　　　１μｌ
滅菌水　　　　　　　　　　　　　ｕｐ　ｔｏ　５０μｌ
＊　１６Ｓ−２７ｆ　　　：　ＡＧＡＧＴＴＴＧＡＴＣＭＴＧＧＣＴＣＡＧ
＊＊１６Ｓ−１５２５ｒ　：　ＡＡＧＧＡＧＧＴＧＷＴＣＣＡＲＣＣ

この反応により増幅された　ＤＮＡ　断片を「ＴＡ　Ｃｌｏｎｉｎｇ　Ｋｉｔ」（Ｉｎｖｉｔｒｏｇｅｎ）を用い、実施例４のＢの反応液の組成および反応条件で　ｐＣＲ２．１　ベクターと連結させた。大腸菌　ＩＮＶαＦ’（Ｉｎｖｉｔｒｏｇｅｎ）５０μｌ　に、ＩＮＶαＦ’　に付属していた　０．５　Ｍβ−メルカプトエタノール　２μｌ　と連結反応液　２μｌ　を加えて氷上に３０分置いた。この大腸菌液を４２℃で３０秒処理した後、氷上に２分置いた。ＩＮＶαＦ’　に付属していた　ＳＯＣ　培地を　２００μｌ　加えて３７℃、１７０ｒｐｍ　で１時間振盪培養した。５０μｇ　／μｌ　のアンピシリンを含む　ＬＢ　寒天培地上に　４０ｍｇ／ｍｌ　の　Ｘ−Ｇａｌ　ＤＭＦＡ溶液を４０μｌ　塗布した後に５０μｌ　の大腸菌液をまき、３７℃で一晩培養した。
【００４５】
プレート上に形成されたいくつかの白いコロニーを拾い、Ｌｙｓｉｓ　バッファー（２０ｍＭ　Ｔｒｉｓ−ＨＣｌ　ｐＨ　８．０、５０ｍＭ　ＫＣｌ、０．１％　Ｔｗｅｅｎ　２０）１０μｌ　に溶かした。これを９５℃で５分処理したもの　５μｌ　を鋳型に、ｐＣＲ２．１　ベクター上の配列をプライマーにして　ＰＣＲ　を行った。反応液の組成および反応条件は実施例４のＣのとおりである。ＰＣＲ　産物を電気泳動して目的とするサイズの　ＤＮＡ　断片が含まれた　ＰＣＲ　産物を選択し、これを鋳型に　Ｄｙｅ　Ｐｒｉｍｅｒ　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｃｏｒｅ　Ｋｉｔ（ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍｓ）を用いてシーケンシング反応を行った。反応液の組成および反応条件は実施例４のＤのとおりである。この反応産物を　ＤＮＡ　シーケンサー（ＤＳＱ−１、Ｓｈｉｍａｚｕ）にかけて塩基配列を決定し、この部分塩基配列（配列表の配列番号３参照）がラン藻の　１６Ｓ　ｒＲＮＡ　遺伝子の部分塩基配列に相同性があることを確認した（図４参照）。
実施例７：Ａ．　ｓａｃｒｕｍ　の純粋培養
クローン化されたＡ．　ｓａｃｒｕｍ　の純粋培養条件を確立するため、ＡＳＴ　培地の特徴である硝酸塩及びリン酸塩の塩濃度の有効範囲を検討した。
【００４６】
硝酸カリウムとリン酸水素二カリウムを除いた　ＡＳＴ　培地を１００ｍｌ　の三角フラスコに　３０ｍｌ　ずつ分注した。これに、終濃度がそれぞれ　０．１ｘＮＰ、０．５ｘＮＰ、１ｘＮＰ、５ｘＮＰ、１０ｘＮＰ、５０ｘＮＰ（硝酸カリウム：　９．２１６ｍｇ／Ｌ、リン酸水素二カリウム：　５．２４８ｍｇ／Ｌ　を１ｘ　ＮＰ　とする）となるようにＮＰ　の溶液を　３０ｍｌ　ずつ混合した後、オートクレーブ滅菌した。十分冷めたところでオートクレーブ滅菌した　１００ｍｌ　の三角フラスコに　２０ｍｌ　ずつ分注した。これに今回単離した細胞塊　０．０２ｇ　を加えて　８００ｌｕｘ　の連続明条件、２５℃、６０ｒｐｍ　の培養条件で振盪培養を行い１週間ごとの細胞塊の湿重量を量った。
【００４７】
２週間培養した結果　０．５ｘＮＰ〜１０ｘＮＰ（硝酸イオン濃度　２．８ｍｇ／Ｌ〜５６．５ｍｇ／Ｌ　リン酸イオン濃度１．４ｍｇ／Ｌ〜２８．６ｍｇ／Ｌ）において湿重量の増加が見られた（下記表６参照）。
【００４８】
【表６】

【００４９】
【配列表】

【図面の簡単な説明】
【図１】自然状態のスイゼンジノリのフェレドキシン　Ｉ　遺伝子の部分塩基配列。
【図２】自然状態のスイゼンジノリのフェレドキシン　ＩＩ　遺伝子の部分塩基配列。
【図３】自然状態のスイゼンジノリと単離した細胞（ＫｕＸ　３）のフェレドキシン遺伝子の部分塩基配列の比較。
【図４】単離した細胞（ＫｕＸ　３）の１６Ｓ　ｒＲＮＡ　遺伝子の部分塩基配列とその相同性検索の結果。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a pure cultivation technique of edible freshwater cyanobacterium, Suiseninori.
[0002]
[Prior art]
When isolating and purifying freshwater algae from nature, the nutritional requirements of the target algae and other detailed growth conditions (eg, water temperature, light, etc.) must be considered. However, in practice, it is difficult to examine the nutritional requirements of the target alga and other detailed growth conditions before isolation, and the target alga could not be specifically isolated and purified. Many of the algae that have been isolated and purified to date are based on existing media (eg, BG-11, MA, MDM, CT, etc.) devised for culturing freshwater algae and algae that have grown under culture conditions. It is obtained by aggressive selection, and if there is a target algae, the algae is not always obtained with an existing medium and culture conditions.
[0003]
For example, the edible freshwater cyanobacterium Suizenjinori is a special cyanobacterium that can grow only in a limited environment such as the spring water of the Aso Mountain Water System, and is threatened with extinction due to deterioration of water quality. To date, many researchers have attempted isolation and pure culture on existing media for freshwater algae (eg, BG-11, MA, MDM, CT, etc.) and culture conditions, but none have been successful. .
[0004]
Aphaenothece sacrum is a freshwater unicellular cyanobacterium (cyanobacterium), which is known as a species unique to Japan. A. Sacrum forms irregularly spread strata, unique to this species, and can be as long as 10 cm or more in large ones. In Japan, this foliate has been used for food since ancient times. Sacrum is a valuable freshwater algae that has been designated as a natural monument. However, this freshwater algae can only grow in the clear spring waters of the Aso Mountains, and has become extinct due to the deterioration of its water quality, and is currently only cultivated in Amagi City, Fukuoka Prefecture. Efforts have been made to breed this valuable freshwater algae in a steady effort, but they have not escaped the threat of extinction and are questioned if aquaculture can continue in the future.
[0005]
A. Sacrum is present in W.C. F. R. It was first introduced to the world as Phylderma sacrum in Illustration des Algus du Japan (1872) by ｉｎｇSurningar. However, it was later transferred to the genus Aphanothece by Kiichi Okada (1953). A. A series of studies on ferredoxins extracted from Sacrum algal bodies (Wada et al., 1974, Hase et al., 1975, 1976, 1978) have been described in a phylogenetic study of cyanobacteria. Sacrum was shown to be in a special position (Wilmotte 1994). However, A. The fact that sacrum can only grow in a limited area of Japan and has not yet been successfully cultured in pure culture has hindered subsequent genetic and physiological studies.
[0006]
A. Attempts to purify sacrum in pure culture have been made by many researchers to date, but none of them has been successful. For example, the present inventors isolated a single-celled cyanobacterium from Suiseninori using a BG-11 medium that has been widely used for cultivation in cyanobacteria, and filed a patent application as KuX (Japanese Patent Application No. Hei 8-191605). However, as a result of the subsequent analysis, this cyanobacterium was found to have A. It turned out that it was not sacrum. The reason is as follows. sacrum is a cyanobacterium having a special auxotrophy, and does not grow on freshwater algae cultures developed to date, such as BG-11, MDM, MA, CT, etc. The growth of other algae is fast. Sacrum is hindered, and as a result, It seems that sacrum could not be isolated.
[0007]
[Problems to be solved by the invention]
An object of the present invention is to isolate and purify a water-liquorice maize using a custom-made medium for isolating and purifying a freshwater algae growing in a special environment.
[0008]
[Means for Solving the Problems]
The present inventors have proposed A.I. Focusing on the metal elements contained in the {sacrum algal body, a basal medium was constructed based on at least the weight ratio of Ca, Cu, Fe, Mg, Mn} and Zn per ash. As a result of adjusting the quantitative ratio of each metal ion and other components in consideration of the influence on the growth of sacrum cells, this time, A. A medium (AST) suitable for the growth of sacrum was found, and this was used to produce A. sacrum. Sacrum cells were successfully cultured in pure culture and isolated.
[0009]
Thus, according to the present invention, a watermelon alga body is used as a metal element with Ca, Cu, Fe, Mg, Mn and Zn in the following weight ratios:
Ca 1
Cu 0.00025-0.00049
Fe 0.017 to 0.068
Mg 0.160 ~ 0.313
Mn 0.060
Zn 0.0012
And a sulfate ion at a concentration of {5.8 mg / L to 11.7 mg / L, a nitrate ion at a concentration of {2.8 mg / L to 56.5 mg / L}, and a phosphate ion at {1.4 mg / L. The present invention provides a method for purely cultivating Aphanothece sacrum, which comprises culturing in a freshwater algal culture medium containing a concentration of の 28.6 mg / L.
[0010]
Hereinafter, the cyanobacterium A.1 showing the special auxotrophy provided by the present invention. The isolation and pure culture of sacrum will be described in more detail.
[0011]
BEST MODE FOR CARRYING OUT THE INVENTION
The present inventors have focused on the metal elements contained in the L. albicans, based on the weight ratio of at least Ca, Cu, Fe, Mg, Mn and Zn of the metal elements contained in the L. alba bodies based on the ash content. A basal medium was constructed and The amount ratio of each metal ion and other components was adjusted while considering the effect on the growth of sacrum cells. A medium suitable for growing sacrum (hereinafter referred to as AST medium) was prepared.
[0012]
Using this AST medium, we succeeded in isolating and purifying pure unicellular cyanobacterial cells from algal cells cultured in Amagi City, Fukuoka Prefecture. This unicellular cyanobacterium is A. cerevisiae. The ferredoxin gene was isolated using an oligonucleotide (PCR primer) for gene amplification designed based on the amino acid sequence of each of the two types of ferredoxins identified to confirm that the ferredoxin was a sacrum. The cyanobacteria isolated and purely cultured by determining Sacrum was identified.
[0013]
A. The procedure of pure culture and identification of {sacrum} will be described more specifically below by way of Examples.
[0014]
【Example】
Example 1: Preparation of basal medium
The amount of metal element per ash contained in dried alga bodies of Suiseninori has already been reported (Shokukenkenhoku Technical Report 37: 163-173, 1980). Based on this report, for each metal element of Ca, Cu, Mg, Mn and Zn, the content of Ca was assumed to be 1 (approximately 20 mg / L: equal to the Ca concentration of the sweet-water-cultured Koganegawa of Amagi tree). The ratio was set to be equal to the ash content ratio (see Table 5 below), and Fe was set to a value close to the ratio to Ca at a concentration at which precipitation did not occur in consideration of solubility. In addition, metal salts commonly used in algae culture medium are added, and the ratio of Ca to Na is not considered important for Na and K in consideration of being actively taken up and excreted in cells. NO₃ ⁻, CO₃ ^2--, PO₄ ^3-The basic composition of the medium was constructed as metal ions to add as a metal salt. Further, trace elements such as B, Co, Ni, Se, and Mo, which are required for algae culture, were added in an amount included in other algae culture media to prepare a basic culture medium (AS-I culture medium).
[0015]
A plastic petri dish containing a water-absorbing algae body (approximately 2 to 4 cm long) cultivated in Amagi City, Fukuoka Prefecture, as a small lump (wet weight 0.31 g) of about 5 mm square and containing 5 mL of a basic medium AS-I medium (Falcon), and the growth of algal cells was observed at 25 ± 2 ° C. under light irradiation (about 250 lux). One month later, cells were present in the alga, but the wet weight of the alga was almost unchanged (0.38 kg). Therefore, the influence on the growth of the water-swallower cells was examined by changing the ratio of the components constituting the AS-I medium without changing the ratio of Ca, Cu, Fe, Mg, Mn and Zn.
[0016]
Medium (AS-II medium) in which half of sulfated salt added as Mg is chloride (AS-II medium), medium in which half of magnesium sulfate is replaced with magnesium chloride and the amount of nitrate, phosphate, carbonate, etc. is doubled (AS-III medium) and a medium (AS-IV medium) in which the amounts of Mg, Ca, Mn, Fe, Zn, Cu and Co were reduced to half were prepared (see Table 1 below). 100 mL of these mediums are placed in a 300 mL Erlenmeyer flask, into which a daffodil alga body (a small lump of about 5 mm square: wet weight of about 0.5 g) is put and slowly shaken at 25 ± 2 ° C. (50 rpm) under light irradiation (about 250 lux) After incubation for 8 days, the growth of algal cells was compared. Table 2 shows the growth state of the alga bodies.
[0017]
[Table 1]

[0018]
[Table 2]

[0019]
From the above results, among the four types of media, the AS-II medium was most suitable for the growth of N. japonica cells, that the sulfate ions affected the growth of N. nastoma cells, and that the amounts of nitrate ions and phosphate ions were higher. It was suggested that the larger the number, the more contaminant bacteria grew. In addition, since growth was observed in the AS-IV medium, the ratio of Ca, Cu, Fe, Mg, Mn and Zn in the AS-II medium was further adjusted in consideration of the results in the AS-IV medium.
[0020]
A medium (AS-1 / 2MF) in which the amounts of magnesium sulfate, magnesium chloride, copper sulfate and iron tartrate are reduced to half, the amounts of magnesium sulfate, magnesium chloride and copper sulfate are reduced to half, and the amount of iron tartrate is reduced to 1/4. A prepared medium (AS-1 / 2M1 / 4F) was prepared (see Table 3 below). 100 mL of these mediums are put into a 300 mL Erlenmeyer flask, into which a daffodil alga body (about 5 mm square small mass: wet weight of about 0.5 kg) is put and slowly shaken at 25 ± 2 ° C (50 rpm) under light irradiation (about 250 lux). ) And incubated for 8 days to compare the growth of algae. Table 4 shows the algal growth state.
[0021]
[Table 3]

[0022]
[Table 4]

[0023]
From the above results, the AS-1 / 2M1 / 4F medium, in which the amounts of magnesium sulfate, magnesium chloride and copper sulfate in the AS-II medium were reduced to 1/2 and the amount of iron tartrate was reduced to 1/4, was used for the growth of the water-absorbing alga body. Suggested to be suitable.
[0024]
Assuming that the Ca content in the AS-II medium, the AS-1 / 2M1 / 4F medium, and the natural state of the watermelon were 1%, the Ca content was 1%. , Cu, Fe, Mg, Mn, and Zn by weight are as shown in Table 5, and were substantially equal.
[0025]
[Table 5]

[0026]
The AS-1 / 2M1 / 4F medium, which had a good growth rate of the water-wort algae, was named AST medium.培 地 sacrum was used as a medium for pure culture.
Example 2: A. Isolation of {sacrum}
As the raw material, Aphanothece {sacrum} is from Endo Kanagawado of Amagi City, Fukuoka Prefecture. This algal body (about 1.5 g) is thoroughly washed with sterile water, and then finely ground with 100 ml of sterile water using ACE homogenizer AM-9 (Nihonseiiki Kaisha), and a G1 glass filter (pore size: 100 to 120 μm). Passed. A portion of the cell was taken on a hemacytometer and observed under a microscope to confirm that the cells of P. perfringens were separated one by one and the number of cells was measured. Based on the number of cells, 5 ml of a sterilized AST liquid medium was placed. Each test tube was diluted and inoculated so that cells could be inoculated one by one, and cultured at 25 ° C. under a 5,000 lux fluorescent lamp for about one month. Of the 2,100 samples prepared, approximately 70% of the test tubes contained cells that seemed to be watermelon.
[0027]
Using a sample of the test tube in which the growth was observed, 0.1 ml each was inoculated from the test tube in which the growth was observed in an AST medium containing an organic substance containing polypeptone (0.1%) and yeast extract (0.1%). Bacteria. If heterotrophic bacteria are contaminated, these tubes become cloudy in 2-3 days. A test tube of a sample that did not become turbid even after about 10 days was regarded as a primary pure water bud, and the same operation was performed at least twice, and the remaining test tubes (one tube) were separated twice. Finally, the test for heterotrophic bacteria was performed using four test tubes, and a sample that did not become cloudy was selected.
[0028]
{30 ml} of {AST} medium was added to about {4-5 g} of the algae treated as described above, and the mixture was finely ground with a homogenizer. This was passed through a {G2 glass filter (pore size: 40 to 50 μm), transferred to a screw cap tube with a volume of {50 ml}, and centrifuged with a centrifuge (Centrifuge 05P-21, Hitachi) to collect a precipitate. . The precipitate was transferred to a microtube having a volume of 2 ml, and sonicator (Sonicator) was used.^TMUltrasonic treatment was performed using W-220, Heat Systems- Ultrasonics, and the mixture was centrifuged with a tabletop centrifuge (MTX-150, rotor: TMA-S26, TOMY) to collect a precipitate of only cells. The precipitate is dissolved in {AST} medium and the concentration of iodoxanol is 40-45% of {OptiPrep}.^TMIt was overlaid on a density gradient of (Axis-Shield) and ultracentrifuged at 25,000 rpm for 2 hours in an ultracentrifuge (70P-72, rotor: SRP28SA-165, Hitachi). The cell layer observed near the center was collected in a microtube, an equal volume to 9-fold amount of {AST} medium was added, and the mixture was centrifuged with a tabletop centrifuge to collect a cell-only precipitate. Dissolve the precipitate in an appropriate amount of {AST} medium, take a part of it in a hemacytometer, observe under a microscope to confirm that the cells of Suiseninori are separated one by one, and count the number of cells. Then, 16,000 cells were seeded on a plate of AST medium containing {0.6%} agarose, and cultured at 25 to 27 ° C under a fluorescent lamp of 2,000 to 3,000 lux for about one month.
[0029]
Among the formed colonies, those colonies in which no contamination colonies were observed were selected, stained with \ Live / Dead \ BacLight (Molecular \ Probe), and contaminated between cells when observed with a fluorescence microscope. A clone in which no signal was observed was selected.
[0030]
This cloned algal body is used in addition to the above-mentioned AST medium containing organic matter, a medium (B-III medium) in which beef extract (0.3%) and peptone (0.5%) are added to an AST medium, and an AST medium. Medium (BV medium) containing sodium acetate (0.05%), glucose (0.05%), tryptone (0.05%) and yeast extract (0.03%) in yeast, yeast in AST medium The cells were cultured for 1 week in a medium (YT medium) containing an extract (0.1%) and tryptone (0.2%) under a fluorescent lamp of 2,000 to 3,000 lux at 25 to 27 ° C. and 60 rpm. It was confirmed that the contamination did not grow.
Example 3: A. Isolation of {sacrum}
As the raw material, Aphanothece {sacrum} is used from Endo Kanagawado of Amagi City, Fukuoka Prefecture. {30 ml} of {AST} medium was added to about {4-5 g} of the algal cells, and the mixture was ground with a homogenizer. This was passed through a G2 glass filter (pore size: 40 to 50 μm), transferred to a screw cap tube with a volume of {50 ml}, and centrifuged with a centrifuge (Centrifuge 05P-21, Hitachi) to collect a precipitate. . The precipitate was transferred to a microtube having a volume of 2 ml, and sonicator (Sonicator) was used.^TMUltrasonic treatment was performed using W-220, Heat Systems-Ultrasonics, and the mixture was centrifuged with a tabletop centrifuge (MTX-150, rotor: TMA-S26, TOMY) to collect a precipitate of only cells. The precipitate is dissolved in {AST} medium, and the concentration of iodoxanol is 40 to 45% of {OptiPrep}.^TMIt was overlaid on a density gradient of (Axis-Shield) and ultracentrifuged at 25,000 rpm for 2 hours in an ultracentrifuge (70P-72, rotor: SRP28SA-165, Hitachi). The cell layer observed near the center was collected in a microtube, an equal volume to 9-fold amount of {AST} medium was added, and the mixture was centrifuged with a tabletop centrifuge to collect a cell-only precipitate. Dissolve the precipitate in an appropriate amount of {AST} medium, take a part of it in a hemacytometer, observe under a microscope to confirm that the cells of Suiseninori are separated one by one, and count the number of cells. Then, 16,000 cells were seeded on a plate of AST medium containing {0.6%} agarose, and cultured at 25 to 27 ° C under a fluorescent lamp of 2,000 to 3,000 lux for about one month.
[0031]
Among the formed colonies, those colonies in which no contamination colonies were observed were selected, stained with \ Live / Dead \ BacLight (Molecular \ Probe), and contaminated between cells when observed with a fluorescence microscope. A clone in which no signal was observed was selected.
[0032]
This cloned algal body is used in addition to the above-mentioned AST medium containing organic substances, a medium (B-III medium) in which beef extract (0.3%) and peptone (0.5%) are added to an AST medium, and an AST medium. Medium (BV medium) containing sodium acetate (0.05%), glucose (0.05%), tryptone (0.05%) and yeast extract (0.03%) in yeast, yeast in AST medium The cells were cultured for 1 week in a medium (YT medium) containing an extract (0.1%) and tryptone (0.2%) under a fluorescent lamp of 2,000 to 3,000 lux at 25 to 27 ° C. and 60 rpm. It was confirmed that the contamination did not grow.
Example 4: Determination of the partial nucleotide sequence of the ferredoxin gene of the natural waterweed L.
From a natural water lily, which has been thoroughly washed with sterilized water.^TM(Nippon Gene) was used to extract {DNA} as follows.
[0033]
To about 1 g of algal cells, 3 times the amount of the solution {I} of the kit was added and stirred, and then 1.5 times the amount of the solution {II} of the kit was added to the algal cells, followed by reaction at 50 ° C for 15 to 120 minutes. After adding 1.5 times the algal cells of the kit {Solution III}, the mixture was placed on ice for 15 minutes, and centrifuged at {3,000 rpm} for {10 minutes} using a centrifuge (Centrifuge 05P-21, Hitachi). Transferred to tube. A 2-fold amount of ethanol was added, and the mixture was centrifuged at {7,000 rpm} for 10 minutes in a centrifuge (SCR-20BA, rotor: RPR-20-2, Hitachi) to collect a precipitate. The precipitate was dissolved in {TE} buffer {0.5 ml}, an equal amount of phenol / chloroform (1: 1) was added, and the mixture was stirred well. After centrifugation at 12,000 rpm for 5 minutes, the aqueous layer was transferred to another microtube. The phenol / chloroform treatment was repeated until no intermediate layer derived from the gel was observed between the aqueous layer and the phenol / chloroform layer. After adding and mixing 1/10 amount of sodium acetate and 2 times amount of 100% ethanol of the aqueous layer, the mixture was allowed to stand at -20 ° C for 10 minutes. After centrifugation at {4 ° C., 15,000 rpm} for 10 minutes using a tabletop centrifuge (MTX-150, rotor: TMA-S26, TOMY), the supernatant was discarded. 100 μl of sterile water was added, and the mixture was stirred well and heated at 50 ° C. for 10 minutes to obtain a template {DNA} solution for the following amplification reaction of the ferredoxin gene of Magnaporthe oryzae by PCR.
[0034]
A primer having the following sequence was designed based on the two amino acid sequences already reported as ferredoxin proteins of L. fern, and a DNA having the following sequence was synthesized.

Using this sequence as a primer and {DNA} prepared from L. indica as a template, the ferredoxin {I} and ferredoxin {II} genes of L. indica were amplified by {PCR}. The composition of the reaction solution and the reaction conditions are as follows.
A:
Reaction liquid composition
Template DNA 1μg
TaKaRa Taq (TaKaRa) 0.25 μl
10x PCR buffer (TaKaRa) 5μl
dNTP Mix (TaKaRa) 4 μl
Primer 1 * (0.01 mM) 1 μl
Primer {2 **} (0.01 mM) 1 μl
Sterile water up to 50μl
* {AsFd-IN}: GACTCCCCGATGGAGACAATG
Or
AsFd-IIN: GAGGTAGCAGATGATCAGAACC
** AsFd-IC: TCTCCGGGTAGGATAAGCCAC
Or
AsFd-IIC: CTCCTGTAGGATAAGCAACGC

The {DNA} fragment amplified by this reaction was concentrated using {SUPRECTM-02 (Takara), and the desired approximately {0.3 kb} DNA fragment was separated from primers and the like by electrophoresis using 1x {TAE}-{2%} agarose gel. After that, the gel at the desired position was cut out, cut into small pieces, transferred to {SUPRECTM-01 (Takara), and centrifuged with a tabletop centrifuge to recover the amplified {DNA} fragment.
[0035]
The recovered fragment was ligated to the {pCR2.1} vector using “TA Cloning Kit” (Invitrogen) under the following reaction solution composition and reaction conditions.
B:
Reaction liquid composition
PCR prodμct 1 μl
pCR2.1TM vector (25 ng / μl) 2 μl
10 x ligation buffer 1 μl (Invitrogen)
T4 DNA Ligase (Invitrogen) 1 μl
Sterilized water up to 10 μl
Reaction conditions
16 ° C x overnight
Escherichia coli {INVαF ”(Invitrogen) (50 μl) was added with {0.5} Mβ-mercaptoethanol {2 μl} and ligation reaction solution (2 μl) attached to INVαF ′, and the mixture was placed on ice for 30 minutes. This Escherichia coli solution was treated at 42 ° C. for 30 seconds to transform, and then placed on ice for 2 minutes. {SOC} medium attached to INVαF ′ was added to the medium, and 200 μl of the medium was added thereto, followed by shaking culture at 37 ° C. and 170 rpm for 1 hour. 40 μl of a 40 mg / ml X-Gal DMFA solution was applied to {LB} agar medium containing 50 μg / μl of ampicillin, then 100 μl of Escherichia coli was sowed and cultured at 37 ° C. overnight.
[0036]
Some white colonies formed on the plate were picked and dissolved in 10 μl of Lysis buffer (20 mM Tris-HCl pH 8.0, 50 mM KCl, 0.1% Tween 20). This was treated at 95 ° C. for 5 minutes, and PCR was performed using {5 μl} as a template and the sequence on the vector as a primer. The composition of the reaction solution and the reaction conditions are as follows.
C:
Reaction liquid composition
Template DNA 5μl
TaKaRa Taq (TaKaRa) 0.25 μl
10x PCR buffer (TaKaRa) 5μl
dNTP Mix (TaKaRa) 4 μl
Primer setA * (0.01 mM each) 1 μl
Sterile water up to 50μl

The PCR product was subjected to electrophoresis, and a sequencing reaction was carried out using a Dye Primer Cycle Sequencing Core Kit (Applied Biosystems) using a {PCR} product containing a {DNA} fragment of the desired size as a template. The composition of the reaction solution and the reaction conditions are as follows.
D:
Reaction liquid composition
Template {DNA} (10 ng / μl) 1 μl
5x Seq buffer 1µl
d / ddNTP 1μl
Dye primer (0.4 μM) 1 μl
Diluted Enzyme * 1 μl
* Diluted EnzymeAmpliTaq DNA polymerase, FS 1 μl
5x Seq buffer 1µl
Sterile water 5μl

This reaction product was subjected to {DNA} sequencer (DSQ-1, Shimazu) to determine the nucleotide sequence, and the partial amino acid sequence deduced from this partial nucleotide sequence (see SEQ ID NOs: 1 and 2 in the sequence listing) was converted to the previously reported part of the ferredoxin protein. It was confirmed that the sequence matched the amino acid sequence (see FIGS. 1 and 2).
Example 5: Identification of isolated algae
The algae cloned in Example 2 or 3 is the main body of the water lily. Identify {sacrum}. The appearance of the algal body is consistent with the appearance of the natural water-swallow, such as that the major axis of the cells is rod-shaped with a diameter of about 6 μm, and that the cells are buried in a gel. In addition, it was confirmed whether or not the partial base sequence of the ferredoxin gene of water-crested swallowtail was detected from the alga bodies isolated by the following method.
[0037]
For the algal cells isolated in Example 2 or 3, {ISOPLANT II}^TM{DNA} was extracted using (Nippon Gene) as follows.
[0038]
To about 1 g of algal cells, 3 times the amount of the solution {I} of the kit was added and stirred. After that, 1.5 times the amount of the solution {II} of the kit was added to the algal cells, and the mixture was reacted at 50 ° C for 15 to 20 minutes. Add {Solution {III-A}} of the kit to 1-fold amount of alga body and 1.2-fold amount of {Solution III-B} of the kit to alga body, place on ice for 15 minutes, and use a centrifuge (Centrifuge 05P-21, Hitachi). After centrifugation at 3,000 rpm for 10 minutes, the aqueous layer was transferred to another tube. A 2-fold amount of ethanol was added, and the mixture was centrifuged at {7,000 rpm} for 10 minutes in a centrifuge (SCR-20BA, rotor: RPR-20-2, Hitachi) to collect a precipitate. The precipitate was dissolved in {TE} buffer {0.5 ml}, an equal amount of phenol / chloroform (1: 1) was added, and the mixture was stirred well. After centrifugation at 12,000 rpm for 5 minutes, the aqueous layer was transferred to another microtube. The phenol / chloroform treatment was repeated until no intermediate layer, which is considered to be derived from gel, was found between the aqueous layer and the phenol / chloroform layer. The aqueous layer was mixed with 1/10 amount of sodium acetate and 2-2.5 times the amount of {100%} ethanol, and allowed to stand at -20 ° C for 10 minutes. After centrifugation at {4 ° C., 15,000 rpm} for 10 minutes using a tabletop centrifuge (MTX-150, rotor: TMA-S26, TOMY), the supernatant was discarded. After sufficiently removing the supernatant, 100 μl of sterilized water was added, the mixture was stirred well, and heated at 50 ° C. for 10 minutes to obtain a template {DNA} solution for the ferredoxin gene amplification reaction by the following {PCR}.
[0039]
Using the primers described in Example 4, the partial base sequence of the ferredoxin gene was amplified from {DNA} of the isolated algal cells. The composition of the reaction solution and the reaction conditions are the same as A in Example 4.
[0040]
The {DNA} fragment amplified by this reaction was ligated to the {pCR2.1} vector using "TA Cloning Kit" (Invitrogen) under the composition and reaction conditions of the reaction solution of Example B. Escherichia coli {INVαF ”(Invitrogen) (50 μl) was added with {0.5} Mβ-mercaptoethanol {2 μl} and ligation reaction solution (2 μl) attached to INVαF ′, and the mixture was placed on ice for 30 minutes. This E. coli solution was treated at 42 ° C. for 30 seconds and then placed on ice for 2 minutes. {SOC} medium attached to INVαF ′ was added to the medium, and 200 μl of the medium was added thereto, followed by shaking culture at 37 ° C. and 170 rpm for 1 hour. 40 μl of a 40 mg / ml X-Gal DMFA solution was applied to an LB agar medium containing 50 μg / μl of ampicillin, and then 50 μl of Escherichia coli was sowed and cultured at 37 ° C. overnight.
[0041]
Some white colonies formed on the plate were picked and dissolved in 10 μl of Lysis buffer (20 mM Tris-HCl pH 8.0, 50 mM KCl, 0.1% Tween 20). This was treated at 95 ° C for 5 minutes, and {PCR} was performed using {5 μl} as a template and the sequence on the pCR2.1 vector as a primer. The composition of the reaction solution and the reaction conditions are as shown in {C} of Example 4. The PCR product was electrophoresed to select a PCR product containing a DNA fragment of a desired size, and a sequencing reaction was performed using the product as a template with Dye Primer Cycle Sequencing Core Kit (Applied Biosystems). The composition of the reaction solution and the reaction conditions are as shown in {D} of Example 4. The reaction product was subjected to {DNA} sequencer (DSQ-1, Shimazu) to determine the nucleotide sequence, and it was confirmed that the partial amino acid sequence deduced from this partial nucleotide sequence matched the partial amino acid sequence of the previously reported ferredoxin protein (FIG. 3).
[0042]
The algae cloned this time has the base sequence of natural water-lily, and its appearance is similar to that of natural water-light, so it is the main body of water-powered wild-flower. {Sacrum}, which was identified as A. Sacrum KuX 3. This alga body has been deposited internationally under the Budapest Treaty with the National Institute of Advanced Industrial Science and Technology, the Ministry of Economy, Trade and Industry, under the Budapest Treaty (Accession No. FERM @ BP-7315).
Example 6: A. Analysis of partial nucleotide sequence of 16S rRNA gene of {sacrum} KuX {3}
The algal cells of interest were cloned in Example 2 or 3 {A. The partial base sequence of the {16S} rRNA} gene was analyzed as an indicator for identifying whether it was {sacrum} KuX {3}.
[0043]
The {DNA} of the isolated algae extracted in Example 5 was used as a template {DNA} solution for the amplification reaction of 16S {rRNA} gene by the following {PCR}.
[0044]
The partial base sequence of the {16S} rRNA} gene was amplified from the isolated algal DNA using universal primers for amplifying the 16S {rRNA} gene. The composition of the reaction solution and the reaction conditions are as follows.
Reaction liquid composition
Template DNA 5μl or 1μg
TaKaRa Taq (TaKaRa) 0.25 μl
10x PCR buffer (TaKaRa) 5μl
dNTP Mix (TaKaRa) 4 μl
Primer {16S-27f *} (0.01 mM) 1 μl
Primer {16S-1525r **} (0.01 mM) {1 μl
Sterile water up to 50μl
* {16S-27f}: {AGAGTTTGATCMTGGCTCAG}
** 16S-1525r: AAGGAGGTGWTCCARCC

The {DNA} fragment amplified by this reaction was ligated to the {pCR2.1} vector using "TA Cloning Kit" (Invitrogen) under the composition and reaction conditions of the reaction solution of Example B. Escherichia coli {INVαF ”(Invitrogen) (50 μl) was added with {0.5} Mβ-mercaptoethanol {2 μl} and ligation reaction solution (2 μl) attached to INVαF ′, and the mixture was placed on ice for 30 minutes. This E. coli solution was treated at 42 ° C. for 30 seconds and then placed on ice for 2 minutes. {SOC} medium attached to INVαF ′ was added to the medium, and 200 μl of the medium was added thereto, followed by shaking culture at 37 ° C. and 170 rpm for 1 hour. 40 μl of a 40 mg / ml X-Gal DMFA solution was applied to an LB agar medium containing 50 μg / μl of ampicillin, and then 50 μl of Escherichia coli was sowed and cultured at 37 ° C. overnight.
[0045]
Some white colonies formed on the plate were picked and dissolved in 10 μl of Lysis buffer (20 mM Tris-HCl pH 8.0, 50 mM KCl, 0.1% Tween 20). This was treated at 95 ° C for 5 minutes, and {PCR} was performed using {5 μl} as a template and the sequence on the pCR2.1 vector as a primer. The composition of the reaction solution and the reaction conditions are as in C of Example 4. The PCR product was electrophoresed to select a PCR product containing a DNA fragment of a desired size, and a sequencing reaction was performed using the product as a template with Dye Primer Cycle Sequencing Core Kit (Applied Biosystems). The composition of the reaction solution and the reaction conditions are as shown in D of Example 4. This reaction product was subjected to {DNA} sequencer (DSQ-1, Shimazu) to determine the nucleotide sequence, and this partial nucleotide sequence (see SEQ ID NO: 3 in the sequence listing) was homologous to the partial nucleotide sequence of cyanobacterial {16S} rRNA} gene. Was confirmed (see FIG. 4).
Example 7: A. Pure culture of {sacrum}
Cloned A. In order to establish the conditions for pure culture of {sacrum}, the effective range of the salt concentration of nitrate and phosphate, which is a characteristic of the AST medium, was examined.
[0046]
The {AST} medium from which potassium nitrate and dipotassium hydrogen phosphate had been removed was dispensed {30 ml} into a 100 ml Erlenmeyer flask. The final concentrations are respectively {0.1 × NP, 0.5 × NP, 1 × NP, 5 × NP, 10 × NP, 50 × NP (potassium nitrate: {9.216 mg / L, dipotassium hydrogen phosphate: {5.248 mg / L}, 1 × {NP}). NP} solution was mixed in {30 ml} each, and then sterilized in an autoclave. When cooled sufficiently, the mixture was dispensed in {20 ml} portions into a {100 ml} Erlenmeyer flask which had been sterilized by autoclaving. The cell mass {0.02 g} isolated this time was added thereto, followed by shaking culture under {800 lux} continuous light conditions, 25 ° C. and 60 rpm culture conditions, and the wet weight of the cell mass was measured every week.
[0047]
As a result of culturing for 2 weeks, an increase in wet weight was observed at 0.5 × NP to 10 × NP (nitrate concentration 硝酸 2.8 mg / LＬ56.5 mg / L phosphate concentration 1.4 mg / L ／ 28.6 mg / L). (See Table 6 below).
[0048]
[Table 6]

[0049]
[Sequence list]

[Brief description of the drawings]
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a partial nucleotide sequence of a ferredoxin {I} gene of a wild water lily, Lynchus vulgaris.
FIG. 2 is a partial nucleotide sequence of a ferredoxin {II} gene of a natural watermelon, L. indica.
FIG. 3. Comparison of partial nucleotide sequences of ferredoxin gene between natural water-liquorice and isolated cells (KuX # 3).
FIG. 4 shows the partial nucleotide sequence of the 16S rRNA gene of the isolated cell (KuX 3) and the result of homology search thereof.

Claims (4)

The water-absorptive alga body is prepared by using at least Ca, Cu, Fe, Mg, Mn and Zn as metal elements in a weight ratio Ca 1 shown below.
Cu 0.00025-0.00049
Fe 0.017-0.068
Mg 0.160-0.313
Mn 0.060
Zn 0.0012
And a sulfate ion at a concentration of 5.8 mg / L to 11.7 mg / L, a nitrate ion at a concentration of 2.8 mg / L to 56.5 mg / L and a phosphate ion at 1.4 mg / L. A pure culture method of Aphanothece sacrum, characterized by culturing in a freshwater algal culture medium containing a concentration of 濃度 28.6 mg / L. Pure culture of Aphanothece @ sacrum. The partial base sequence of the ferredoxin gene of Aphanothece @ sacrum shown in SEQ ID NOs: 1 and 2 in the sequence listing. A partial nucleotide sequence of the 16S rRNA gene of Aphanothe sacrum shown in SEQ ID NO: 3 in the sequence listing.

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