JP4610044B2 - Promoter gene and expression vector - Google Patents

Description

【００６３】
次に、得られた一本鎖cDNAの反応液に、表２に示す試薬を順に加16℃で2.5時間インキュベートすることで第２鎖cDNAを合成した。インキュベート後、反応液を氷中で冷却した。
【００６４】
【表２】

【００６５】
合成した二本鎖cDNAに2μlの2.5U/μl Cloned Pfu DNA polymerase（Stratagene社製）を加え、72℃で30分間インキュベートすることで、合成した二本鎖cDNAの両末端を平滑化した。次いで、フェノール・クロロホルム抽出及びエタノール沈殿を行った後、得られた沈殿を9.0μlのEco RIアダプター溶液に溶解した。得られた溶液9.0μlのうち1.0μlを1.0％アルカリアガロースゲル電気泳動に供試することによって、合成した二本鎖cDNAのサイズを確認した。
次に、残った溶液（8.0μl）に溶解した二本鎖cDNAに、表３に示す組成の溶液を加えて、８℃で一晩インキュベートすることにより、二本鎖cDNAの両末端にEco RIアダプターを付加した。
【００６６】
【表３】

【００６７】
アダプター付加後、反応液を70℃で30分間熱処理し、T4 DNAリガーゼを失活させた。
次いで、ベクターとのライゲーションを行うために、表４に示す組成の溶液を調製し、37℃で30分間インキュベートすることで二本鎖cDNAのEco RI末端のリン酸化を行った。
【００６８】
【表４】

【００６９】
Eco RI末端のリン酸化後、反応液を70℃で30分間熱処理し、T4ポリヌクレオチドキナーゼを失活させた。
二本鎖cDNAがベクターにセンス方向に組み込まれるように、上記二本鎖cDNAをXho Iで消化し、５'末端にEco RIサイト、３'末端にXho Iサイトが生じるように、表５に示す反応液組成で37℃で90分間反応させることにより、３'末端にXho Iサイトを生じさせた。
【００７０】
【表５】

【００７１】
反応後、反応液を室温にまで冷却し、５μlの10×STE緩衝液を加えた。
上記溶液を、1×STE緩衝液で平衡化したSephacryl S-500カラム（Stratagene社製）に供して、未付加のEco RIアダプターとXho I消化産物を除去した。次いで、カラムの溶出画分にフェノールクロロホルム抽出、エタノール沈殿を行い、得られた沈殿を５μlの滅菌水に溶解した。得られたcDNA溶液の濃度は吸光度測定法により定量した。
【００７２】
上記のようにして得られた、５'末端にEco RIサイト、３'末端にXho Iサイトを有するcDNA（100ng）を、クローニングベクターであるZAP Express Vector arms（Stratagene社製）に２UのT4 DNA リガーゼを用いて挿入した。反応は12℃で14時間行った。このライゲーション反応液５μlのうち２μlを、Giga Pack III Gold Packaging Extract（Stratagene社製）を用いて、in vitro パッケージングに供した。
【００７３】
in vitroパッケージングされた組換えバクテリオファージを含むパッケージング溶液30μlに、10mM MgCl₂でOD.600 = 0.5に調整したE. coli XL1-blue MRF'株（Stratagene社製）を600μl加え、37℃で15分間培養した。これを48℃に保温した6.5mlのNZYトップアガロース（0.5％塩化ナトリウム、0.2％硫酸マグネシウム７水和物、0.5％酵母エキス、1％NZアミン及び0.7％アガロースを含み、pH7.5に調整されたもの。）に加え、90mm×130mmの角型シャーレを用いたNZYプレート（0.5％塩化ナトリウム、0.2％硫酸マグネシウム７水和物、0.5％酵母エキス、1％NZアミン及び1.5％アガーを含み、pH7.5に調整されたもの。）上に、１枚当たり約５万個のプラークが形成されるように、計15枚のプレートに播き、合計75万個のプラークからなるcDNAライブラリーを作製した。
【００７４】
（３）シイタケのゲノムDNAライブラリーの調製
液体培養により生育させた菌糸をガラスフィルターで回収し、ペーパータオルで菌糸の水分を可能な限り搾り取った。回収した菌糸約１gを乳鉢に入れ、液体窒素を適量加えて磨砕した。磨砕した菌糸を50mlポリプロピレンチューブに移し、20mlのTESS緩衝液（0.73M シュクロース、10mM トリス緩衝液（pH 8.0）、１mM EDTA（pH8.0）及び１％SDSを含む。）を加えて、65℃で１時間インキュベートした。これに終濃度が１Mとなるように５M NaClを加えて、8,000×gで20分間遠心し、上清を回収した。回収した上清に等量のフェノール・クレゾール試薬（まず、100gのフェノールに、4.7mlのm-クレゾールを加えて50℃で溶解させる。次に、これに８-キノリノールを0.05％となるように加えて、さらに等量の１M NaClで平衡化させた試薬）を加え、1,300×gで５分間遠心して、上清（水層）を回収した。さらに、クロロホルム処理、エタノール沈殿を行って、１mlのTE緩衝液に溶解した。その溶液をRNase Aおよびproteinase Kで処理し、フェノール・クロロホルム処理とエタノール沈殿を行って、適当量のTE緩衝液に溶解し、ゲノムDNA試料とした。
【００７５】
得られたゲノムDNA試料を制限酵素Sau 3AIによって消化し、フェノール・クロロホルム処理後、エタノール沈殿を行って適当量のTE緩衝液に溶解した。得られたDNA断片を用い、Lambda EMBL3/Bam HI Vector Kit（Stratagene社製）によりゲノムDNAライブラリーを作製した。
【００７６】
（４）縮重プライマーの作製
既知の糸状菌類のchs遺伝子において保存性の高いアミノ酸配列［A. R. bowen,et. al.: Proc.Natl.Acad.Sci.USA., 89: 519-523 (1992)］をもとに縮重センスプライマー及び縮重アンチセンスプライマーを合成した。縮重センスプライマーとしては、U1（5'-CTGAAGCTTACNATGTAYAAYGARGAY-3'：配列番号３）を、縮重アンチセンスプライマーとしては、L1（5'-GTTCTCGAGYTTRTAYTCRAARTTYTG-3'：配列番号４）を合成した。ここで、YはT又はCを示し、RはG又はAを示し、NはA、C、G又はTを示す。なお、これら縮重センスプライマー及び縮重アンチセンスプライマーは、日本製粉（株）中央研究所カスタムサービスに委託して合成した。
【００７７】
（５）縮重PCR
上記（３）において得られたゲノムDNA試料を鋳型に、上記（４）において調製した縮重センスプライマー及び縮重アンチセンスプライマーを用いて、縮重PCRを行った。縮重PCRの反応液の組成は表６の通りである。
【００７８】
【表６】

【００７９】
縮重PCR反応は、96℃で30秒間の熱変性、続いて50℃で30秒間のアニーリング、続いて72℃で１分間の伸長反応の条件を１サイクルとして、30サイクル行った。反応終了後、反応液を新しいマイクロチューブに移し、そのうち5μlを1.0％アガロースゲルによる電気泳動に供し、増幅断片の確認を行った。次いで、残りの反応液にフェノール・クロロホルム処理とエタノール沈殿を行い、ペレット化したPCR産物を20μlのTE緩衝液に溶解した。
【００８０】
得られたPCR産物を１％低融点アガロースゲル電気泳動に供し、約 0.9 kbp（既知の糸状菌類のアミノ酸配列等から予測される大きさ）の断片を切り出し、QIAEX II（QIAGEN社製）を用いてゲルからDNAを抽出し、所望のPCR産物を回収した。次いで、回収したPCR産物をTA Cloning Kit（Invitrogen社製）でpCR2.1ベクターにサブクローニングした後、蛍光自動DNAシーケンサー（Parkin Elmer社製、ABI PRISM 310型）により塩基配列を解析した。その結果、得られたPCR産物におけるイントロンを除く領域を配列番号９に示す。また、この配列番号９から考えられるアミノ酸配列を配列番号１０に示す。
【００８１】
そして、この配列番号１０に示すアミノ酸配列と公知のchsとの相同性を調べた結果を図１及び図２に示す。図１から判るように、配列番号１０に示すアミノ酸配列は、ノイロスポラ・クラッサ（Neurospora crassa）におけるchsと高い相同性を示している。また、図２から判るように、配列番号６に示すアミノ酸配列は、スエヒロダケ（Schizophyllu commune）におけるchsと高い相同性を示していた。そこで、プロモーター及びターミネーター領域を含むchs遺伝子の全長配列をクローニングするために、回収したPCR産物を、シイタケ菌糸cDNAライブラリーおよびシイタケゲノムDNAライブラリーをスクリーニングするためのプローブとして用いた。
【００８２】
（６）cDNAライブラリーからのchs遺伝子の単離
chs遺伝子のスクリーニングにおいて、プラークの形成およびライブラリーの増幅はZAP Express cDNA synthesis Kit（Stratagene社製）のマニュアルに基づいて行った。すなわち、上記（２）において調製したcDNAライブラリーから合計50万個のプラークをスクリーニングするため、１×10⁷pfu/mlのファージ溶液５μlを10本用意し、それぞれに10mM MgSO₄でOD600=0.5に調整した大腸菌XL1-Blue MRF'株600μlを加えて、37℃で15分間インキュベートした。次いで、6.5mlのNZYトップアガーを加えて、90mm×130mmの角型NZYプレートに重層し、37℃で６〜８時間インキュベートし、プラークを形成させた。プラークが形成したプレートは４℃で保存した。
【００８３】
予め大きさを合わせて切断したHybond N+ナイロンメンブレン（90mm×130mm、Amersham Life Science社製、以下、単に「メンブレン」という。）をプレートに重ね、２分間放置し、ファージDNAをメンブレンに転写した。次いで、メンブレンをアルカリ変性溶液（0.25 M NaOH及び1.5 M NaClを含む。）で20分間処理し、ファージDNAをアルカリ変性させた。変性後、メンブレンを中和液（0.5M Tris-HCl (pH8.0)、1.5M NaCl）に浸して、室温で５分間振盪し、続いて、リンス溶液（0.2M Tris-HCl (pH7.5)、２×SSC）中で30秒間メンブレンを洗浄後、ペーパータオルにメンブレンを挟んで、80℃で２時間加温することで、ファージDNAをメンブレンに固定した。
【００８４】
次いで、プラークハイブリダイゼーション及びシグナルの検出をECL direct nucleic acid labelling and detection system（Amersham Life Science社製）を用いて行った。すなわち、DNAを固定したメンブレンをハイブリバック（コスモバイオ社製）に入れ、キット付属のハイブリダイゼーション緩衝液を適量加えて、密閉し、42℃で１時間プレハイブリダイゼーションを行った。次いで、溶液を除去し、上記（５）において調製したプローブをペルオキシダーゼ標識したもの50ngを含む５mlのハイブリダイゼーション緩衝液を加え、42℃で４時間ハイブリダイゼーションを行った。ハイブリダイゼーション完了後、メンブレンを0.4％ SDS、36％Ureaを含む0.5×SSCを用い、42℃で20分間の洗浄を２回行った。さらに、２×SSCを用い、室温で５分間の洗浄を２回行った。
【００８５】
このメンブレンを１mlのECL detection reagentで１分間処理し、サランラップで包み、フィルムカセットに入れ、生じたシグナルをHyperfilm-ECL（Amersham Life Science 社製）に感光させた。フィルムを現像したところ、16個の陽性シグナルが得られた。得られたシグナルに対応するプラークをピペットで吸い取り、それを１mlのSM緩衝液（100mM NaCl、100mM MgSO₄、50mM Tris-HCl (pH7.5)及び0.01％ゼラチンを含む。）に懸濁し、20μlのクロロホルムを加えて撹拌した。このファージ懸濁液を適当に希釈して、プレーティングし、その後、上記と同様の手法を複数回繰り返して精製を行い、陽性プラークを得た。
【００８６】
スクリーニング後、ファージミド形成は Zap Express cDNA synthesis kit（Stratagene社製）のマニュアルに基づいて行った。すなわち、１mlのSM緩衝液に懸濁した陽性組換えファージ250μlに、10mM MgSO₄でOD600=1.0に調整した大腸菌XL1-Blue MRF'株200μlと、ExAassist ヘルパーファージを加えて、37℃で15分間インキュベートした。そこへ、３mlのNZY 培地を加えて、さらに37℃で2.5時間インキュベートした。インキュベート後、70℃で20分間加温し、1000×gで15分間遠心して、上清を回収し、ファージミド溶液を得た。次に、ファージミドを大腸菌に組み込むため、ファージミド溶液100μlもしくは10μlに、10mM MgSO₄でOD600=1.0に調整した大腸菌XLOLR株（Stratagene社製）200μlを加えて、37℃で15分間インキュベートした。培養液200μlを50μg/mlのカナマイシンを含むLB固体培地に播き、37℃で一晩インキュベートし、生じたコロニーからファージミドを調製した。
【００８７】
ファージミドの調製はRPM Kit（BIO 101社製）を用いて行った。すなわち、上記で得られたコロニーを50μg/mlのカナマイシンを含む３mlのLB液体培地に接種し、37℃で一晩インキュベートした。培養液をマイクロチューブに取り、10,000×gで１分間遠心して、集菌した。回収した菌体にPre-Lysis緩衝液50μlを加えて、激しく撹拌し、菌体を懸濁した。さらに、100μlのアルカリLysis緩衝液を加えて、穏やかに撹拌し、菌体を完全に溶解させた。次に、100μlの中和溶液を加えて激しく撹拌し、10,000×gで２分間遠心して、上清を回収した。キット付属のSPIN FILTERによく混ぜたGlass Milkを250μl加えて、そこへ回収した上清を加えてよく撹拌し、マイクロチューブにSPIN FILTERをのせた。10,000×gで１分間遠心し、さらにWash緩衝液350μlを加えて同様に遠心した後、新しいマイクロチューブにSPIN FILTERをのせ、TEを50μl加えて10,000×gで30秒間遠心し、Glass Milkからファージミドを溶出させた。エタノール沈殿後、得られた沈殿を20μlのTE緩衝液に溶解し、塩基配列の決定に供した。
【００８８】
（７）ゲノムDNAライブラリーからのchs遺伝子の単離
上記（３）において作製したゲノムDNAライブラリーから、上記（６）とほぼ同様の手順でchs遺伝子を単離した。プラークの形成及びライブラリーの増幅は、大腸菌XL1-blue MRA株を使用して、Lambda EMBL3 / Bam HI vector kit（Stratagene社製）のマニュアルに基づいて行い、プラークハイブリダイゼーション及びシグナルの検出は、cDNAライブラリーからのchs遺伝子の単離と同じ手順で行った。そして、得られたシグナル付近のプラークを掻き取り、それをSM緩衝液に懸濁した。このファージ懸濁液を適度に希釈して、プレーティングし、上記と同様のスクリーニングを行い、ゲノムにおけるchs遺伝子を含む組換え体ファージを得た。クローン化したファージをそれぞれプレーティングし、37℃で６〜８時間インキュベートしてプラークを形成させた。プラークが形成したプレートに10mlのSM緩衝液を重層し、４℃で12時間以上振盪培養して、ファージをSM緩衝液中に遊離させた。ファージを含むSM緩衝液を15mlのプロピレンチューブに回収し、クロロホルムを終濃度５％となるように加えて、室温に15分間放置し、それを1,500×gで10分間遠心し、上清を回収した。次いで、Wizard Lambda Preps DNA Purification System（Promega社製）を用いて、回収した上清からファージDNAを精製し、塩基配列の決定に供した。
【００８９】
（８）塩基配列の決定
上記（６）および（７）において得られた陽性クローンの塩基配列を、ABI PRISM Dye Terminator Cycle Seqeuencing Ready Reaction Kit, FS（Perkin Elmer社製）を用いてそれぞれ決定した。PCR反応はそのマニュアルに基づいて行い、得られたPCR産物はABI PRISM 310 Genetic Analyzer（Perkin Elmer社製）によって解析した。いずれの陽性クローンに対しても５’末端及び３’末端の塩基配列を決定した。決定した配列は、配列番号１及び２に示す。配列番号１は、chs遺伝子の開始コドンを含む上流約2.3kbpの塩基配列であり、配列番号２はchs遺伝子の終止コドンを含む下流約0.9 kbp の塩基配列である。
【００９０】
〔実施例２〕
発現用組換えベクターの構築
ここでは、chs遺伝子のプロモーター領域を含む発現用組換えベクターを構築した。なおchs遺伝子のターミネーター領域を含む発現用組換えベクターを構築する際も、以下に示す手法を同様に使用することができる。
（１）chs遺伝子のプロモーター領域の単離
単離したchs遺伝子の開始コドンより上流域約 2.2 kbpの塩基配列（配列番号１）より、各種プロモーターに見出される特定塩基配列（TATA box、CAAT box など）が存在していることが判る。そこで、この開始コドンより上流約 2.2 kbpをプロモーター領域（配列番号１における第１塩基から第２２２９塩基に挟まれる領域）とみなし、ゲノムchs遺伝子を鋳型としてPCRを行い、chs遺伝子のプロモーター領域を大量に調製した。
【００９１】
このPCRに際して、センスプライマーとして、翻訳開始点から-2256〜-2227bpの位置に存在する配列CproU（5'-ATAAGAATAATCAGGCTTGTCGTCGAACCG-3'：配列番号５）を有するものを用い、アンチセンスプライマーとして、翻訳開始点から-57〜-28bpの位置に存在する配列CproL（5'-CCTTAGTTGTAGATGGAAATGGTGGGGTGG-3'：配列番号６）を有するものを用いた。なお、これらCproU及びCproLは、日本製粉（株）中央研究所カスタムサービスに委託して合成した。このときのPCRの反応液組成は表７の通りである。
【００９２】
【表７】

【００９３】
このとき、PCR反応は、96℃で30秒間の熱変性、続いて60℃で30秒間のアニーリング、続いて72℃で２分間の伸長反応の条件を１サイクルとして、30サイクル行った。反応終了後、反応液を新しいマイクロチューブに移し、そのうち５μlを１％アガロースゲルによる電気泳動に供し、増幅断片の確認を行った。次いで、残りの反応液にフェノール・クロロホルム処理とエタノール沈殿を行い、ペレット化したPCR産物を20μlのTE緩衝液に溶解した。
【００９４】
得られたPCR産物を１％低融点アガロースゲル電気泳動に供し、約2.2 kbpの断片を切り出した。その断片をQIAEX II（QIAGEN社製）を用いてゲルから抽出し、chs遺伝子のプロモーター領域を単離、精製した。
【００９５】
（２）組換えベクター（pLCHS）の構築
ここでは、組換えベクターの一構築例として、図１に示すように、上記（１）において単離したプロモーター領域と特開平6-319547で開示した pLG-hphプラスミドベクターとを利用し、シイタケを宿主とする組換えベクターpLCHS-hph を構築した。最初に、pLG-hphプラスミドベクターからgpd遺伝子のプロモーター領域を除き、代わりにchs遺伝子のプロモーター領域を組み込んだベクターを構築するため、pLG-hphプラスミドベクターを鋳型として、gpd遺伝子のプロモーター領域を除くようにPCRを行った。得られたDNA断片を１％Sea Plaque Agarose（FMC）を用いて、４℃、50Vの条件で電気泳動した。泳動後、目的のバンドをゲルから切り出し、QIAEX II Gel Extraction Kits（QIAGEN）によってDNAを精製した。エタノール沈澱後、得られたDNAペレットをDNA Blunting Kit（TaKaRa）を用いて平滑末端にし、さらにアルカリフォスファターゼ(Calf intestine)によって脱リン酸化し、フェノール・クロロホルム処理、エタノール沈澱を行って、pLG-hph (-Pgpd)プラスミドベクター（pLG-hphプラスミドベクターからgpd遺伝子のプロモーターを除いた断片）を得た。さらにその断片を平滑末端にし、さらに脱リン酸化を行って、Perfectly Blunt Cloning Kits（Novagen）を用いて、上記（１）で得られたchs遺伝子のプロモーターと連結させ、pLCHS-hphプラスミドベクターを得た。
【００９６】
（３）組換え発現ベクター（pChG-bar）の構築
組換え発現ベクターpChG-barは、pLG-hphプラスミドベクターのhph遺伝子をビアラフォス耐性遺伝子（bar遺伝子）に入れ替えたpLG-barプラスミドベクターと上記（２）で得られたpLCHS-hphプラスミドベクターとを結合させて構築したものである。
【００９７】
はじめに、図３に示すように、pLG-barプラスミドベクターを構築した。すなわち、先ず、pLG-hphプラスミドベクターからhph遺伝子を除去し、代わりにbar遺伝子を組み込んだベクターを構築するためにpLG-hphプラスミドベクターを鋳型として、hph遺伝子を除くようにPCRを行い、pLGプラスミドベクター断片（pLG-hphプラスミドベクターからhph遺伝子を除いた断片）を得た。得られたpLGプラスミドベクター断片は、１％Sea Plaque Agarose（FMC）を用いて、４℃、50Vの条件で電気泳動した。泳動後、目的のバンドをゲルから切り出し、QIAEX II Gel Extraction Kits（QIAGEN）によってDNAを精製した。エタノール沈澱後、得られたDNAペレットをDNA Blunting Kit（TaKaRa）を用いて平滑末端にし、フェノール・クロロホルム処理、エタノール沈澱を行った。一方、bar遺伝子は、pLC-barプラスミドベクター［Yanai, K. et. al., Biosci. Biotech. Biochem., 60, 472-475 (1996)］から単離した。すなわち、pLC-barプラスミドベクターを BamHI で消化し、得られたDNA断片を１％Sea Plaque Agarose（FMC）を用いて、４℃、50Vの条件で電気泳動した。泳動後、目的のバンドをゲルから切り出し、QIAEX II Gel Extraction Kits（QIAGEN）によってDNAを精製した。エタノール沈澱後、得られたDNAペレット Blunting Kit（TaKaRa）を用いて平滑末端にし、フェノール・クロロホルム処理、エタノール沈澱を行って、bar遺伝子断片を得た。そして、このbar遺伝子断片を、上記pLGプラスミドベクター断片と連結させ、pLG-barプラスミドベクターを得た。
【００９８】
次に、シイタケ発現ユニットPchs-bar-Tgpd 断片を得るために、pLG-barプラスミドベクターを鋳型として、pLG-barプラスミドベクターにおけるgpd遺伝子のプロモーター（Pgpd）、bar遺伝子（bar）及びgpd遺伝子のターミネーター（Tgpd）を含む領域をPCRにより増幅した。得られたDNA断片を１％Sea Plaque Agarose（FMC）を用いて、４℃、50Vの条件で電気泳動した。泳動後、目的のバンドをゲルから切り出し、QIAEX II Gel Extraction Kits（QIAGEN）によってPgpd−bar−Tgpdを精製した。エタノール沈澱後、得られたDNAペレットをDNA Blunting Kit（TaKaRa）を用いて平滑末端にした。
【００９９】
一方、pLCHS-hphプラスミドベクターをEhe Iで消化し、末端平滑化、脱リン酸化を行い、フェノール・クロロホルム処理、エタノール沈澱を行って、線状のpLCHS-hphプラスミドベクターを得た。これとPgpd-bar-Tgpd 断片とをPerfectly Blunt Cloning Kits（Novagen）を用いて連結させ、図３に示すようなpChG-bar プラスミドベクターを得た。こうして作出したプラスミドのうち、方向的に正しく挿入されたものを選抜して、大量に調製した。
【０１００】
〔実施例３〕
REMI法によるシイタケの形質転換
（１）プロトプラストの調製
シイタケ菌株の二核菌糸を、0.25×MYPG寒天培地（0.25％麦芽エキス、0.1％酵母エキス、0.1％ペプトン、0.5％グルコース及び1.5％寒天を含む。）で、25℃２週間培養した。生育した菌糸をかきとり、50 ml の0.25×MYPG液体培地で１週間培養した。得られた菌糸はガラスフィルターで集菌し、50mlの0.25×MYPG液体培地に懸濁してポリトロンホモジナイザーで裁断し、100μmのナイロンメッシュで濾過した濾過菌糸をさらに0.25×MYPG液体培地中25℃で、５日間培養した。培養菌糸は100μmのナイロンメッシュで集菌後、クエン酸緩衝液（0.6 Mマンニトールを含む50mM クエン酸緩衝液を含有し、pH5.6に調整されたもの。）で２回洗浄し、菌糸１g当たり10mlの酵素溶液（2.5重量%セルラーゼ（ヤクルト社製）、0.1重量%キチナーゼ（シグマ社製）を含むクエン酸緩衝液を含有する）に菌糸を懸濁し、28℃で３〜４時間インキュベートした。酵素処理した菌糸を40μmのナイロンメッシュで濾過し、濾液を1,500×gで10分間遠心分離して、プロトプラストを沈殿させた。上清を捨て、プロトプラストをSTC 緩衝液（10mM塩化カルシウム、1.2Mソルビトールを含む10mM Tris-HClを含有し、pH7.5に調整されたもの。）で洗浄後、再び１ml のSTC 緩衝液にプロトプラストを懸濁し、顕微鏡でプロトプラストの数を計測した。最終的には、STC緩衝液100μlに0.5〜1.0×10⁷protoplastsとなるように調整して、形質転換用のプロトプラストとした。
【０１０１】
（２）pLCHS-hph及びpChG-barプラスミドベクターの導入
形質転換は、pLCHS-hphプラスミドベクター及びpChG-barプラスミドベクターを１箇所で切断する制限酵素Hind IIIを用いたRestriction Enzyme Mediasted Integration（REMI）法によって行った（特開平11-155568号公報を参照）。すなわち、2.5μgのpLCHS-hphあるいはpChG-barプラスミドベクターと50ユニットのHind IIIとを含む150μlのSTC緩衝液に、上記のプロトプラスト懸濁液100μlを穏やかに加え、氷中で20分間インキュベートした。これに 62.5μlのPEG溶液（10mM塩化カルシウム、60% PEG4000を含む10mMTris-HClを含有し、pH7.5に調整されたもの）を加え、氷中で20分間インキュベートした。さらに3.125mlのPEG溶液を加えて、室温で20分間インキュベートした。次に、10mlのSTC緩衝液を加えて溶液全体を十分に懸濁し、1,500×gで10分間遠心し、プロトプラストを沈殿させた。回収したプロトプラストを４mlのMS（２%麦芽エキス、0.6Mスクロースを含有する）液体培地に懸濁し、25 ℃で3-4日間静置培養した。
【０１０２】
（３）ハイグロマイシン B 耐性菌糸の選抜
プロトプラストから菌糸を再生し、再生した菌糸を1,500×gで10分間遠心することで回収した。回収した菌糸を１mlのMS液体培地に再懸濁し、5μg/mlハイグロマイシンBを含む最少寒天培地（2％グルコース、0.2％酒石酸アンモニウム、0.05％硫酸マグネシウム、0.1％リン酸二水素カリウム、0.112％炭酸ナトリウム、0.132％フマル酸、10ppm硫化鉄、8.8ppm硫化亜鉛、7.2ppm塩化マンガン及び1.5％寒天からなり、pH4.5に調整されたもの）にまき、 25 ℃で５日間培養した。次に、一旦溶解し、50℃程度に冷却させた0.25×MYPG寒天培地に、20μg/mlハイグロマイシンBを加え、それを最少寒天培地上で生育した菌糸上に重層した。25℃で約５日間培養後、増殖してきた菌糸を分離し、新しい20μg/mlハイグロマイシンBを含むMYPG寒天培地に植え替えた。さらに１週間程度培養し、増殖してきた菌糸を新しい20μg/mlハイグロマイシンBを含むMYPG寒天培地に植え替えて培養し、ハイグロマイシン耐性を獲得した菌糸を選抜した。
【０１０３】
（４）ビアラフォス耐性菌糸の選抜
pChG-barプラスミドベクター導入株に関しては、上記（３）で選抜したハイグロマイシンB耐性菌糸を、さらに2μg/mlビアラフォス及び20μg/mlハイグロマイシンBを含むMYPG寒天培地にまき、25℃で約１週間培養後、増殖してきた菌糸を分離し、新しい2μg/mlビアラフォス及び20μg/mlハイグロマイシンBを含むMYPG寒天培地に植え替え、両薬剤耐性株を選抜した。
（５）結果
結果を表８に示す。なお、数値は３連の実験の平均値である。
【０１０４】
【表８】

【０１０５】
表８から判るように、pLG-hphプラスミドベクターによりハイグロマイシン耐性を獲得したシイタケ形質転換体が作出され、本発明の発現用組換えベクターが、シイタケを宿主とする異種遺伝子発現用ベクターとして有効であることを確認した。
【０１０６】
【発明の効果】
以上、詳細に説明したように、本発明によれば、シイタケキチン合成酵素遺伝子の転写開始を指令するプロモーター及び/又は該ターミネーターを含む組換えベクター、該組換えベクターを含む形質転換体、該形質転換体を用いるポリペプチドの製造方法が提供される。
【０１０７】
【配列表】

【０１０８】
【配列表フリーテキスト】
【配列番号３】
プライマー。第１２塩基のｎは、ａ、ｔ、ｃ又はｇである。
【配列番号４〜８】
プライマー。
【図面の簡単な説明】
【図１】配列番号１０のアミノ酸配列とノイロスポラ・クラッサにおけるchsアミノ酸配列との相同性を示す図である
【図２】配列番号１０のアミノ酸配列とスエヒロダケにおけるchsアミノ酸配列との相同性を示す図である
【図３】 pChG-barプラスミドベクターの構築を表わす模式図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a promoter that directs transcription initiation in the chitin synthase gene of basidiomycete shiitake, a terminator that directs transcription termination of the chitin synthase gene, the promoter and / or a recombinant expression vector comprising the terminator, the promoter and The present invention relates to a recombinant vector containing the terminator, a recombinant vector for expression or a transformant containing the recombinant vector, and a method for producing a polypeptide using the transformant.
[0002]
[Prior art]
Recent advances in recombinant DNA technology have led to the development of host vector systems for heterologous gene expression using various cells such as E. coli, Bacillus subtilis, actinomycetes, yeast, filamentous fungi, animal cells and plant cells as hosts. Yes. For example, various useful substances such as insulin, growth hormone, and interferon have been produced so far using E. coli host vector systems. Moreover, in plant breeding, gene transfer from heterologous organisms beyond species that was not possible with the traditional classical mating method is Agrobacterium tumefaciens (Agrobacterium tumefaciensA transgenic plant having the desired properties has been produced by using a host vector system such as Ti plasmid.
[0003]
Of the host vector systems described above, the E. coli host vector system is the most widely used to date. However, when Escherichia coli is used as a host to produce a polypeptide derived from a higher animal such as a human, no sugar chain is added to the produced polypeptide or the polypeptide chain is folded into its original higher-order structure (folding). In many cases, the original physiological activity is not exhibited due to the absence of such activity. Furthermore, Escherichia coli produces various toxic substances in addition to the polypeptide in the production process of the target polypeptide, and thus has a problem that a multi-step purification process is required.
[0004]
In order to solve these problems, host vector systems using eukaryotic cells such as yeast cells and animal cells have been developed as hosts having a glycosylation function and an appropriate folding function. However, when yeast cells are used to produce polypeptides derived from higher animals such as humans, high mannose-type sugar chains that are different from those derived from humans may be added, or the production of the desired polypeptide There are problems such as low. On the other hand, even when animal cells are used, there are many problems such as extremely low yield of the target product and the necessity of an expensive medium. From such a viewpoint, there has been a demand for a host vector system that satisfies the requirements such that sugar chains equivalent to those of higher animal cells can be added, can be cultured at low cost, and safety is high. By the way, basidiomycetes are generally more closely related to animals than yeast [TL Smith: Proc. Natl. Acad. Sci. USA, 86: 7063 (1989)], and therefore have an appropriate glycosylation function and an appropriate folding function. It is thought to have. In particular, shiitake (Lentinula edodes) Has been used for food for many years and is excellent in safety. Moreover, one of the nutritional characteristics of shiitake mushrooms is a high content of ergosterol, which is a vitamin D precursor, and especially in dried shiitake mushrooms. On the other hand, shiitake mushroom contains an anticancer polysaccharide lentinan, and in recent years, the existence of eritadenine having a cholesterol lowering action has been revealed. This suggests that shiitake is promising not only as a food but also as a raw material for medicine. Therefore, the establishment of genetic engineering techniques in shiitake mushrooms is very important in considering shiitake breeding.
[0005]
To date, Miranda D. et al. Have developed mushroom transformation by electroporation [Miranda D. van de Rhee, et.al. Mol. Gen. Genet., 250, 252-258, (1996)], Ming Peng et al. Transformed oyster mushrooms by electroporation [Peng, M., et.al., Curr. Genet., 22, 53-59, (1992)] and Yanai, K. et al. Transformed oyster mushrooms by the polyethylene glycol method [Yanai, K. et. Al., Biosci. Biotech. Biochem., 60, 472-475 (1996)]. Noёl et al., Transformation of Fumotake mushrooms by the polyethylene glycol method [Noёl, T and Labarere, J., Curr. Genet., 25: 432-437 (1994), Noёl, T. et al., Theor. Appl. Genet., 90: 1019-1027 (1995)].
[0006]
However, an effective host vector system for shiitake mushrooms has not been established so far. In Shiitake, an expression vector having a marker gene and expressing a foreign gene in the same vector has not been developed. In general, in order to construct an expression vector that yields a higher expression level, it is necessary to use a promoter with high transcription efficiency to mRNA. Up to now, we have made extensive studies, and an effective method for transforming shiitake mushrooms [Sato, T. et. Al., Biosci. Biotech. Biochem., 62, 2346-2350 (1998), Sato et al. No. 155568 (Japanese Patent Application No. 9-331611)] and expression vectors (pLG and pLT) have been developed (Hirano et al., Japanese Patent Application Laid-Open No. 2000-69975 (Japanese Patent Application No. 10-247470), and Sato et al. (Japanese Patent Application No. 11-342347).
[0007]
On the other hand, chitin is a mycelium component of shiitake mushroom, and therefore a chitin synthase gene (hereinafter referred to as “chs gene”), which is a gene that synthesizes chitin, is considered to be expressed in the growth stage of shiitake mushroom. Therefore, the promoter region of the shiitake chs gene is effective as a promoter for expressing a foreign gene and is considered to be useful for a vector.
However, there is no known recombinant vector containing a shiitake-derived chs gene promoter so far.
[0008]
[Problems to be solved by the invention]
The present invention relates to a promoter that commands the initiation of transcription of the chs gene of basidiomycete shiitake, a terminator that commands the termination of transcription of the chs gene, a recombinant vector for expression containing the promoter and / or terminator, the promoter and / or the terminator And a recombinant vector for expression or a transformant containing the recombinant vector, and a method for producing a polypeptide using the transformant.
[0009]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventors have conducted extensive research, and as a result, isolated the chs gene from cDNA and genomic DNA library prepared from shiitake mycelia, and further isolated the promoter region and terminator region. Successfully completed the present invention.
[0010]
That is, the DNA according to the present invention is a DNA that can function as a promoter shown in the following (a) or (b).
(A) DNA comprising a sequence from the first base to the 2229th base in the base sequence represented by SEQ ID NO: 1.
(B) a DNA having a promoter activity, comprising a base sequence in which at least one base has been deleted, substituted or added in the sequence from the first base to the 2229th base in the base sequence represented by SEQ ID NO: 1;
[0011]
The present invention may also be DNA that hybridizes with the DNA of claim 1 under stringent conditions and has promoter activity.
Furthermore, the present invention is a recombinant vector for expression containing the DNA of claim 1 or 2.
Furthermore, the present invention is a recombinant expression vector in which any DNA having terminator activity is incorporated into the recombinant expression vector according to claim 3.
[0012]
On the other hand, the DNA according to the present invention is a DNA that can function as a terminator, as shown in (c) or (d) below.
(C) DNA comprising a sequence from the 20th base to the 920th base in the base sequence represented by SEQ ID NO: 2.
(D) DNA having a terminator activity, including a base sequence in which at least one base has been deleted, substituted, or added in the sequence from the 20th base to the 920th base in the base sequence represented by SEQ ID NO: 2.
[0013]
The present invention may also be a DNA that hybridizes with the DNA of claim 5 under stringent conditions and has terminator activity.
Furthermore, the present invention is a recombinant vector for expression containing the DNA of claim 5 or 6.
Furthermore, the present invention is a recombinant expression vector in which any DNA having promoter activity is incorporated into the recombinant expression vector according to claim 7.
[0014]
Furthermore, a recombinant vector for expression containing the DNA according to claim 1 or 2 and the DNA according to claim 5 or 6.
Furthermore, the present invention is a recombinant vector in which a gene encoding an arbitrary polypeptide is incorporated in the recombinant vector for expression according to claim 3, 4, 7, or 8.
Furthermore, the present invention is a transformant comprising the expression recombinant vector according to claim 3, 4, 7, or 8.
[0015]
Furthermore, the present invention is a transformant comprising the recombinant vector according to claim 10.
Furthermore, the present invention is a method for producing the polypeptide, comprising culturing or cultivating the transformant according to claim 12, and collecting the polypeptide from the obtained culture or culture.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
1. Isolation of promoter and terminator region of shiitake chs gene
The promoter of the present invention is isolated from the upstream region of the 5 'end of the chs gene, and the terminator is isolated from the downstream region of the 3' end of the chs gene.
The promoter and terminator of the present invention are (1) preparation of shiitake mRNA and cDNA library, (2) preparation of shiitake genome and genomic DNA library, (3) isolation of chs gene, (4) chs gene It can be obtained by determining the nucleotide sequence to be included and (5) isolating the promoter and terminator regions. Hereinafter, each process is demonstrated in order.
[0017]
(1) Preparation of mRNA and cDNA library
The source of mRNA is Shiitake (Lentinula edodes) Umbrellas, fungi, mycelia, fungi, legs, hyphae, etc., or whole fruit bodies. Alternatively, spore or primary hyphae or secondary hyphae can be used after culturing in a solid medium such as 0.25 × MYPG medium, SMY medium, glucose / peptone medium, etc., then inoculating the mycelium into a liquid medium and growing cells.
[0018]
Preparation of mRNA can be performed by a commonly performed technique. For example, mycelia obtained from a liquid medium are collected by filtration and then frozen with liquid nitrogen. Next, after the frozen mycelium is ground, ISOGEN (manufactured by Nippon Gene) or the like is added to extract the nucleic acid. Extracted nucleic acid is treated with chloroform or phenol reagent to obtain total RNA, and then affinity column method using oligo-dT-cellulose or poly-U-sepharose with Sepharose 2B as carrier, or Straight A's mRNA Isolation System MRNA (poly (A)) using a kit such as Novagen⁺RNA) can be prepared.
[0019]
Using the mRNA thus obtained as a template, a commercially available kit (for example, ZAP Express)^TMOligo dT using cDNA Synthesis Kit (Stratagene)₂₀And a single-stranded cDNA is synthesized by reverse transcriptase, and then a double-stranded cDNA is synthesized from the single-stranded cDNA. Next, an appropriate adapter such as an Eco RI adapter is added to the obtained double-stranded cDNA, and then incorporated into an appropriate cloning vector such as Uni-ZAP XR Vector (Stratagene) or λgt10 (Amersham). Make a replacement vector.
[0020]
When a plasmid vector is used as a cloning vector, the resulting recombinant vector is transformed into E. coli. Here, the transformation of E. coli was performed by Hanahan's method [Hanahan, D., J. Mol. Biol. 166: 557-580 (1983)], that is, competent prepared in the presence of calcium chloride, magnesium chloride or rubidium chloride. This can be done by adding a recombinant vector to the cells. The plasmid vector used here must contain a drug resistance gene such as tetracycline or ampicillin for selection of colonies on the plate. Next, after transforming E. coli with a plasmid vector containing double-stranded cDNA, a cDNA library can be obtained by selecting transformants using tetracycline resistance and ampicillin resistance as indices.
[0021]
On the other hand, when a phage vector is used as a cloning vector, the resulting recombinant vector is packaged in vitro using a kit such as Giga Pack III Gold Packaging Extract (manufactured by Stratagene). Infect. This is cultured using an agar medium such as LB Agar, and a cDNA library can be obtained by forming plaques.
[0022]
(2) Preparation of Shiitake genomic DNA and genomic DNA library
The source of genomic DNA may be part of the fruiting body such as shiitake umbrella, fungi, mycelium, fungus pattern, leg hook, mycelium, or the whole fruiting body, as in (1) above. In addition, after spore or primary hyphae or secondary hyphae is cultured in a solid medium such as 0.25 × MYPG medium, SMY medium, glucose / peptone medium, etc., the mycelium is inoculated into a liquid medium, and grown cells can be used.
[0023]
Genomic DNA can be prepared by a conventional method. That is, for example, first, after harvesting the cells of shiitake in liquid culture by filtration or the like, the cells are frozen with liquid nitrogen and the frozen cells are ground using a mortar. Next, a buffer for DNA extraction is added to the ground cells, and chloroform is further added and vigorously stirred. Thereafter, after removing chloroform, ethanol is gradually added to the aqueous layer portion, and when the DNA is precipitated, the genomic DNA is wound up and dissolved in TE buffer to obtain a genomic DNA solution. Alternatively, genomic DNA can be obtained from the ground bacterial cells using a commercially available kit (for example, ISOPLANT (manufactured by Nippon Gene)).
[0024]
A genomic DNA library can be prepared by a commonly used technique. That is, for example, first, genomic DNA is digested with a restriction enzyme such as the restriction enzyme Sau3AI, treated with phenol / chloroform, and then the DNA fragment is recovered by ethanol precipitation. Then, using a commercially available kit (for example, Lambda EMBL3 / Bam HI Vector Kit (Stratagene)), the fragment is ligated to, for example, λ EMBL3-Bam HI arm using T4 DNA ligase, and the obtained phage DNA Is infected with E. coli. Thereafter, this is cultured using an agar medium such as LB Agar, and a plaque is formed, whereby a genomic DNA library can be obtained.
[0025]
(3) Isolation of the chs gene
The chs gene can be screened from the cDNA library prepared in the above (1) or the genomic DNA library prepared in the above (2) by a usual method (for example, PCR method, plaque hybridization method, etc.). That is, the amino acid sequence is highly conserved in the chs gene of known filamentous fungi [AR bowen, et. Al .: Proc. Natl. Acad. Sci. USA., 89: 519-523 (1992)]. Design and synthesize heavy sense and degenerate antisense primers. These are used to perform degenerate polymerase chain reaction (degenerate PCR). Next, a method of screening from a shiitake cDNA library using the obtained fragment as a probe can be mentioned.
[0026]
The template DNA used for degenerate PCR includes the genomic DNA prepared in (2) above. In addition, U1 (5′-CTGAAGCTTACNATGTAYAAYGARGAY-3 ′: SEQ ID NO: 3) is used as a degenerate sense primer used in this degenerate PCR, and L1 (5′-GTTCTCGAGYTTRTAYTCRAARTTYTG-3 ′) is used as a degenerate antisense primer. SEQ ID NO: 4) can be used. Here, Y represents T or C, R represents G or A, and N represents A or C or G or T or another base. However, in the present invention, primers used for degenerate PCR are not limited to these primers, and any sequence can be used as long as it is designed based on amino acid sequences highly conserved in chs genes such as filamentous fungi. You may have.
[0027]
The base sequence of the amplified DNA fragment obtained by the degenerate PCR is determined using a known method, and it is confirmed that the homology with known chs genes in other basidiomycetes other than shiitake is high. Thereafter, the amplified DNA fragment is directly labeled with an enzyme such as peroxidase or alkaline phosphatase, or³²P,³⁵After radiolabeling with a radioisotope such as S, it is used as a probe and hybridized with a nitrocellulose or nylon membrane filter to which the genomic DNA library obtained in (2) above is denatured and immobilized. A phage clone can be identified from the obtained positive signal, and the shiitake chs gene can be cloned.
[0028]
Thereafter, the amplified DNA fragment is directly labeled with an enzyme such as peroxidase or alkaline phosphatase, or³²P,³⁵After radiolabeling with a radioisotope such as S, it is used as a probe and hybridized with a nitrocellulose or nylon membrane filter to which the genomic DNA library obtained in (2) above is denatured and immobilized. A phage clone can be identified from the obtained positive signal, and the shiitake chs gene can be cloned.
[0029]
(4) Determination of base sequence
The phage clone obtained by the above screening (3) is subcloned into an appropriate commercially available plasmid vector such as pBlueScript SK (+) (Stratagene) or pCR2.1 (Invitrogen), for example, cycle sequencing. Analyze nucleotide sequences such as methods. The base sequence is determined using an automatic base sequencer (for example, ABI PRISM 310 Genetic Analyzer, manufactured by Perkin Elmer). As a result, a base sequence of about 2.3 kbp including the start codon of the chs gene (SEQ ID NO: 1) and a base sequence of about 0.9 kbp including the stop codon of the chs gene (SEQ ID NO: 2) are determined.
[0030]
(5) Isolation of promoter region and terminator region of chs gene
Isolation of the promoter region and terminator region of the chs gene is based on the sense primer and the region of about 2.2 kbp upstream of the chs gene containing the promoter region based on the base sequence determined in (4) above. An antisense primer is synthesized, and a sense primer and an antisense primer are synthesized so as to sandwich a region of about 0.9 kbp downstream of the chs gene containing the terminator region. Next, using these synthesized primers, the chs gene promoter was obtained by PCR using the genomic chs gene cloned from the genomic DNA library prepared in (2) above or the genomic DNA prepared in (2) above as a template. Amplify the region and terminator region. Thereby, the promoter region and terminator region of the chs gene can be isolated.
[0031]
Here, when isolating the promoter region, for example, CproU (5′-ATAAGAATAATCAGGCTTGTCGTCGAACCG-3 ′: SEQ ID NO: 5) is used as the sense primer, and for example, CproL (5′-CCTTAGTTGTAGATGGAAATGGTGGGGTGG- 3 ′: SEQ ID NO: 6) can be used. According to PCR using these CproU and CproL, it is possible to amplify a region between the first base and the 2229th base in SEQ ID NO: 1. When the terminator region is isolated, for example, CterU (5′-AACGCGTTGATTCTGATA-3 ′: SEQ ID NO: 7) is used as the sense primer, and for example, CterL (5′-TTGTGGATGTCGGAGTAG-3) is used as the antisense primer. ': SEQ ID NO: 8) can be used. According to PCR using these CterU and CterL, the region between the 20th base and the 920th base in SEQ ID NO: 2 can be amplified.
[0032]
Examples of the base sequence of the DNA having the promoter activity of the present invention from the first base to the 2229th base in SEQ ID NO: 1 and the base sequence of the DNA having the terminator activity of the present invention from the 20th base to the 920th base in SEQ ID NO: 2 However, as long as it has promoter activity in the former case and terminator activity in the latter case, mutations such as deletion, substitution, addition, etc. of at least one base may occur in the base sequence, Mutations may be introduced. Here, the deletion, substitution and addition include not only 1 to 10 short deletions, substitutions and additions, but also long deletions, substitutions and additions of 10 to 50 bases, and further 50 to 100 bases.
[0033]
In order to introduce a mutation into DNA, a known method such as the Kunkel method or the Gapped duplex method, or a method based thereon can be employed. For example, using a mutagenesis kit (for example, Mutant-K or Mutant-G (TaKaRa)) using site-directed mutagenesis, or using a LA PCR in vitro Mutagenesis series kit from TaKaRa Mutations can be introduced.
[0034]
  Further, DNAs that hybridize with the above promoter region or terminator region under stringent conditions and have promoter activity or terminator activity are also included in the DNA of the present invention..
[0035]
The DNA of the present invention has at least 90% or more, preferably 95% or more, more preferably 98% or more homology with the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, and promoter activity or It also includes DNA having terminator activity. The numerical value of the homology is obtained, for example, by executing a command of the Maxim matching method using DNASIS (Hitachi Software Engineering) which is sequence analysis software. The parameters at that time are default settings (initial settings).
[0036]
Whether the DNA having a base sequence other than SEQ ID NO: 1 has promoter activity or the DNA having a base sequence other than SEQ ID NO: 2 has terminator activity, for example, upstream of a gene serving as a marker or It can be confirmed by constructing an expression vector having the DNA of interest downstream and testing for expression of the marker gene.
[0037]
Once the base sequence of the DNA of the present invention is determined, the subsequent chemical synthesis, PCR using cDNA or genomic DNA containing the DNA of the present invention as a template, or a DNA fragment having the base sequence as a probe By hybridizing, the DNA of the present invention can be obtained.
[0038]
2. Construction of recombinant vector for expression of the present invention
The recombinant vector for expression of the present invention can be constructed by linking the promoter region and / or terminator region of the chs gene obtained in 1. above to an appropriate plasmid vector (for example, pUC19 vector).
[0039]
As an example, a recombinant vector for expression pLCHS1 can be constructed by linking the promoter region and terminator region of the chs gene to the pUC19 vector. Further, the expression recombinant vector pLCHS can be constructed by ligating the promoter region of the chs gene and the gpd terminator region of the pLG vector disclosed in JP-A-6-319547 to an appropriate plasmid vector. Furthermore, by combining the pLCHS vector and the pLG vector, a foreign gene expression vector pChG vector containing a marker gene for selection originally possessed by the pLG vector can be constructed.
[0040]
It is desirable to use a recombinant expression vector in combination with a marker gene that is effective in actually confirming the introduction of a foreign gene. Therefore, the recombinant vector for expression of the present invention includes, for example, a hygromycin B phosphotransferase (hph) gene [Griz and Davis, Gene, 25, which imparts resistance to the antibiotic hygromycin B downstream of the promoter region of the chs gene. : 179-188 (1983)], phosphinothricin acetyltransferase (bar) gene [Murakami et al., Mol. Gen. Genet., 205: 42-50 (1986)] that confers resistance to the herbicide bialaphos Can be connected. The recombinant vector for expression thus produced is introduced into shiitake mushrooms by various gene introduction methods such as PEG method, electroporation method, REMI method (Restriction Enzyme-Mediated Integration method, see JP-A-11-155568). A transformant can be obtained using resistance to the drug as an index.
[0041]
3. Production of polypeptides using the recombinant vector for expression of the present invention
Using the recombinant vector for expression of the present invention, the desired polypeptide can be obtained by genetic engineering in shiitake mushrooms.
[0042]
2. A recombinant vector can be prepared by linking (inserting) a gene encoding a useful polypeptide such as insulin, growth hormone, tyrosinase, laccase, peroxidase, etc. into the recombinant vector for expression of it can. As a method for inserting a gene encoding the desired polypeptide into the vector of the present invention, the purified DNA is cleaved with an appropriate restriction enzyme and inserted into the restriction enzyme site or the multiple cloning site of the vector of the present invention. And the like. A transformant can be obtained by introducing the obtained recombinant vector into shiitake mushroom by a method such as PEG method, electroporation method or REMI method.
[0043]
Subsequently, the target transformant can be obtained by culturing or cultivating the obtained transformant and collecting it from the culture or the cultivated product. When the polypeptide of interest is produced in shiitake mycelium, the mycelium is cultured.
In the present invention, the method for culturing the mycelium of the transformant is carried out according to the usual method used for cultivation of shiitake mushrooms.
[0044]
As a medium for cultivating a transformant obtained using shiitake as a host, it contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by shiitake.
As long as the medium can be efficiently used, either a natural medium or a synthetic medium may be used.
As the carbon source, carbohydrates such as glucose, fructose, sucrose, starch, maltose and dextrin are used.
[0045]
Nitrogen sources include inorganic salts such as ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium phosphate, or ammonium salts of organic acids or other nitrogen-containing compounds, as well as peptone, meat extract, corn steep liquor, casamino acid, NZ amine Etc. are used.
[0046]
Examples of inorganic substances that can be used include monopotassium phosphate, dipotassium phosphate, calcium chloride, magnesium sulfate, sodium carbonate, ferrous sulfate, zinc sulfate, manganese chloride, and calcium carbonate.
[0047]
The mycelium is preferably cultured for several days to two months at 25 ° C. under aerobic to slightly aerobic conditions such as shaking culture in a liquid medium, aeration and agitation culture, or stationary culture in a solid medium. Passaging can be performed by inoculating a new medium with a part of the grown mycelium. During the culture, antibiotics such as hygromycin and bialaphos may be added to the medium as necessary.
[0048]
After the culture, if the desired polypeptide is produced in the mycelium, the mycelium is crushed.
On the other hand, when the target polypeptide is secreted outside the mycelium, the culture solution is used as it is, or the mycelium is removed by centrifugation or the like to obtain a supernatant. By using general biochemical methods used for polypeptide isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, the culture The target polypeptide can be isolated and purified from the inside.
[0049]
When the target polypeptide is produced in shiitake fruiting bodies, the fruiting bodies can be launched using shiitake mushroom strains. Examples of the method for starting shiitake fruiting bodies include bacterial bed culture.
[0050]
In order to carry out fungal bed culture, first, inoculum is prepared. Here, the “inoculum” is used as a seed in shiitake cultivation, and refers to a microbial cell or culture cultured purely under appropriate conditions. Examples of such inoculum include liquid inoculum prepared in a liquid medium, liquid inoculum obtained by fragmenting mycelia, those prepared on an agar medium, and sawdust inoculum.
[0051]
Preparation of the inoculum can be performed using a known method, and the method is not particularly limited, but here, an example using the sawdust seed culture method is shown as an example. The medium is prepared by mixing sawdust and rice bran in a ratio of 10: 1 to a moisture content of 63%. This is filled with 500 g of polypropylene incubator (800 ml), covered and autoclaved at 121 ° C. for 40 minutes. After allowing to cool to room temperature, Shiitake strain is inoculated and cultured. The culture conditions are not particularly limited because those skilled in the art can appropriately set the conditions. Such culture can be performed, for example, by culturing for about 50 days at 20 ° C. and a relative humidity of 65%.
[0052]
Next, bacterial bed culture is performed using the inoculum obtained as described above. Here, the “fungal bed” refers to a medium prepared by inoculating a seed medium for the purpose of cultivation of shiitake mushrooms, or one in which the bacteria spread. Here, the period from inoculation of the inoculum to the maturation of the fungus bed is referred to as “culture period”, and the period from the emergence of fruiting bodies to harvest is referred to as “occurrence period”. This bacterial bed culture can be appropriately performed by those skilled in the art, and the method is not particularly limited, but examples thereof include the following methods.
[0053]
The moisture content of the medium made of sawdust: Hokuken Bidel (10: 1) is adjusted to 63%, 2.5 kg of the medium is packed into a polypropylene bag with a filter, and formed into a rectangular parallelepiped (density 0.7 g / cm). A hole with a diameter of 2 cm is opened in the center of the medium until it reaches the bottom, and this is sterilized in an autoclave at 121 ° C. for 40 minutes and allowed to cool overnight at room temperature. About 17 g of the above sawdust seeds are inoculated into this culture medium, and the bag is quickly closed with a heat sealer. This is cultured in the dark at 20 ° C. and 65% relative humidity. This culture is carried out until the mycelium spreads over the entire fungus bed and is fully ripe, but the culture period is usually 102 to 116 days.
[0054]
Finally, fruit bodies are generated from the fungus bed obtained by the above-mentioned fungus bed culture. Since this operation can be appropriately performed by those skilled in the art, the method is not particularly limited, and examples thereof include the following methods.
[0055]
Open the bag that has been cultivated until it is fully matured, take out the microbial bed, generate fruit bodies under conditions of temperature 16 ° C., humidity 85%, illuminance 300 lux (every 12 hours), and harvest. This generation period is not particularly limited because it can be appropriately set by those skilled in the art. After completion of the development, after performing a water immersion treatment (about 20 hours), a second generation is performed by performing culture for generation again. Furthermore, after harvesting, the water treatment is performed again, and the fruiting body can be generated as in the second time.
[0056]
Thereafter, when the target polypeptide is produced in the microbial body or fruit body, the microbial body or fruit body is crushed. On the other hand, when the target polypeptide is secreted outside the cells, the liquid culture solution is used as it is, or the cells are removed by centrifugation or the like to obtain a supernatant. By using general biochemical methods used for polypeptide isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, the culture The target polypeptide can be isolated and purified from the inside.
[0057]
【Example】
The present invention will be described more specifically with reference to the following examples. However, these examples are for illustrative purposes and do not limit the technical scope of the present invention.
[Example 1]
Isolation of the promoter and terminator regions of the chs gene
(1) Preparation of shiitake mycelium
Hokuken Sangyo Co., Ltd. Shiitake strain Hokuken 57 dinuclear mycelium is inoculated into 0.25 x MYPG agar medium (containing 0.25% malt extract, 0.1% yeast extract, 0.1% peptone, 0.5% glucose and 1.5% agar). And cultured at 25 ° C. for about 2 weeks. The grown mycelium was scraped from the agar medium and inoculated into a 200 ml Erlenmeyer flask containing 100 ml of 0.25 × MYPG liquid medium. After inoculation, the cells were cultured with shaking at 25 ° C. for about 2 to 4 weeks to grow mycelia.
[0058]
(2) Preparation of shiitake mycelium cDNA library
The mycelium was recovered by filtering the liquid culture solution obtained in (1) above with a glass filter. Water was sufficiently removed with a paper towel, and hyphae having a wet weight of about 1 g was ground to a powder using a mortar and pestle in the presence of liquid nitrogen. Leave for 2 minutes to remove liquid nitrogen, transfer the ground mycelium to a polypropylene centrifuge tube, add 10 ml ISOGEN (Nippon Gene), stir vigorously at room temperature for 10 minutes, and then add 2 ml of chloroform. In addition, it was stirred vigorously for 1 minute at room temperature. This is a pulverized liquid.
[0059]
The ground liquid was centrifuged at 10,000 × g and 4 ° C. for 5 minutes, and the aqueous layer was recovered. In order to remove proteins and DNA in the aqueous layer, 5 ml of ISOGEN and 1 ml of chloroform were added again and stirred vigorously. Then, after centrifuging at 10,000 × g and 4 ° C. for 5 minutes, the aqueous layer was collected again and subjected to phenol / chloroform treatment and isopropanol precipitation to obtain a precipitate.
[0060]
This precipitate is dissolved in 1 ml of DEPC-treated water (diethyl pyrocarbonate dissolved to 0.1% in sterilized water and autoclaved), then 250 μl of 10M lithium chloride solution is added and cooled at -80 ° C for 1 hour. did. Then, it was centrifuged at 12,000 × g and 4 ° C. for 15 minutes. After removing the supernatant, the pellet was washed with 70% ethanol and dissolved in 200 μl of DEPC-treated water. Centrifugation was performed at 10,000 × g and 4 ° C. for 5 minutes, and the supernatant was collected, followed by ethanol precipitation to obtain total RNA. The obtained total RNA was dissolved in 200 μl of DEPC-treated water, and it was confirmed that the total RNA was prepared in good quality by an absorbance measurement method and formaldehyde-denatured agarose gel electrophoresis.
[0061]
Then, from the total RNA, Straight A's^TMPoly (A) using mRNA Isolation System (Novagen)⁺RNA was purified. The amount of RNA obtained was 20 μg when measured with a UV spectrometer. Poly (A) obtained⁺ZAP Express using RNA as a template^TMDouble-stranded cDNA was synthesized using cDNA Synthesis Kit (Stratagene). Specifically, first, reverse transcriptase (MMLV-RTase) 3.5 μl (20 U / μl) was added to a solution having the composition shown in Table 1 and incubated at 37 ° C. for 1 hour to synthesize single-stranded cDNA. After incubation, the reaction was cooled in ice.
[0062]
[Table 1]

[0063]
Next, the reagents shown in Table 2 were sequentially added to the obtained single-stranded cDNA reaction solution and incubated at 16 ° C. for 2.5 hours to synthesize second-strand cDNA. After incubation, the reaction was cooled in ice.
[0064]
[Table 2]

[0065]
2 μl of 2.5 U / μl Cloned Pfu DNA polymerase (Stratagene) was added to the synthesized double-stranded cDNA and incubated at 72 ° C. for 30 minutes to smooth both ends of the synthesized double-stranded cDNA. Subsequently, phenol / chloroform extraction and ethanol precipitation were performed, and the obtained precipitate was dissolved in 9.0 μl of Eco RI adapter solution. The size of the synthesized double-stranded cDNA was confirmed by subjecting 1.0 μl of 9.0 μl of the obtained solution to 1.0% alkaline agarose gel electrophoresis.
Next, a solution having the composition shown in Table 3 is added to the double-stranded cDNA dissolved in the remaining solution (8.0 μl), and incubated at 8 ° C. overnight, whereby Eco RI is added to both ends of the double-stranded cDNA. An adapter was added.
[0066]
[Table 3]

[0067]
After adding the adapter, heat the reaction solution at 70 ° C for 30 minutes.Four DNA ligase was inactivated.
Next, in order to perform ligation with the vector, a solution having the composition shown in Table 4 was prepared and incubated at 37 ° C. for 30 minutes to phosphorylate the Eco RI end of the double-stranded cDNA.
[0068]
[Table 4]

[0069]
After phosphorylation of Eco RI terminal, the reaction solution was heat treated at 70 ° C. for 30 minutes to inactivate T4 polynucleotide kinase.
The double-stranded cDNA is digested with Xho I so that the double-stranded cDNA is incorporated into the vector in the sense direction, and an Eco RI site is generated at the 5 ′ end and an Xho I site is generated at the 3 ′ end. By reacting for 90 minutes at 37 ° C. with the reaction solution composition shown, an Xho I site was generated at the 3 ′ end.
[0070]
[Table 5]

[0071]
After the reaction, the reaction solution was cooled to room temperature, and 5 μl of 10 × STE buffer was added.
The solution was applied to a Sephacryl S-500 column (manufactured by Stratagene) equilibrated with 1 × STE buffer to remove unadded Eco RI adapter and Xho I digestion product. Subsequently, phenol chloroform extraction and ethanol precipitation were performed on the elution fraction of the column, and the obtained precipitate was dissolved in 5 μl of sterilized water. The concentration of the obtained cDNA solution was quantified by an absorbance measurement method.
[0072]
The cDNA (100 ng) having the Eco RI site at the 5 ′ end and the Xho I site at the 3 ′ end and 2U T4 DNA obtained as described above in the ZAP Express Vector arms (Stratagene) was used. Insertion was performed using ligase. The reaction was carried out at 12 ° C. for 14 hours. Of 5 μl of this ligation reaction solution, 2 μl was subjected to in vitro packaging using Giga Pack III Gold Packaging Extract (Stratagene).
[0073]
10 mM MgCl in 30 μl of packaging solution containing recombinant bacteriophage packaged in vitro₂600 μl of E. coli XL1-blue MRF ′ strain (manufactured by Stratagene) adjusted to OD.600 = 0.5 was added and cultured at 37 ° C. for 15 minutes. 6.5 ml of NZY top agarose (0.5% sodium chloride, 0.2% magnesium sulfate heptahydrate, 0.5% yeast extract, 1% NZ amine and 0.7% agarose, adjusted to pH 7.5, kept at 48 ° C. NZY plate (including 0.5% sodium chloride, 0.2% magnesium sulfate heptahydrate, 0.5% yeast extract, 1% NZ amine, and 1.5% agar) Prepare a cDNA library consisting of a total of 750,000 plaques by plating on a total of 15 plates so that approximately 50,000 plaques are formed on each plate. did.
[0074]
(3) Preparation of Shiitake genomic DNA library
The mycelium grown by liquid culture was collected with a glass filter, and the moisture of the mycelium was squeezed out as much as possible with a paper towel. About 1 g of the collected mycelium was put in a mortar, and an appropriate amount of liquid nitrogen was added and ground. Transfer the ground mycelium to a 50 ml polypropylene tube and add 20 ml of TESS buffer (containing 0.73 M sucrose, 10 mM Tris buffer (pH 8.0), 1 mM EDTA (pH 8.0) and 1% SDS). Incubated for 1 hour at 65 ° C. To this, 5M NaCl was added so that the final concentration was 1M, and the mixture was centrifuged at 8,000 × g for 20 minutes, and the supernatant was collected. To the collected supernatant, add an equal amount of phenol / cresol reagent (first add 4.7 ml of m-cresol to 100 g of phenol and dissolve at 50 ° C. Next, add 8-quinolinol to 0.05%. In addition, a reagent equilibrated with an equal volume of 1M NaCl) was added, and the mixture was centrifuged at 1,300 × g for 5 minutes to recover the supernatant (aqueous layer). Furthermore, chloroform treatment and ethanol precipitation were performed and dissolved in 1 ml of TE buffer. The solution was treated with RNase A and proteinase K, treated with phenol / chloroform and ethanol precipitated, dissolved in an appropriate amount of TE buffer, and used as a genomic DNA sample.
[0075]
The obtained genomic DNA sample was digested with the restriction enzyme Sau 3AI, treated with phenol / chloroform, ethanol precipitated, and dissolved in an appropriate amount of TE buffer. Using the obtained DNA fragment, a genomic DNA library was prepared by Lambda EMBL3 / Bam HI Vector Kit (Stratagene).
[0076]
(4) Preparation of degenerate primers
Degenerate sense based on amino acid sequence [AR bowen, et. Al .: Proc. Natl. Acad. Sci. USA., 89: 519-523 (1992)] highly conserved in known chs genes of filamentous fungi Primers and degenerate antisense primers were synthesized. U1 (5′-CTGAAGCTTACNATGTAYAAYGARGAY-3 ′: SEQ ID NO: 3) was synthesized as a degenerate sense primer, and L1 (5′-GTTCTCGAGYTTRTAYTCRAARTTYTG-3 ′: SEQ ID NO: 4) was synthesized as a degenerate antisense primer. Here, Y represents T or C, R represents G or A, and N represents A, C, G, or T. In addition, these degenerate sense primers and degenerate antisense primers were synthesized by consigning to Nippon Flour Mills Central Research Laboratory Custom Service.
[0077]
(5) Degenerate PCR
Using the genomic DNA sample obtained in (3) above as a template, degenerate PCR was performed using the degenerate sense primer and degenerate antisense primer prepared in (4) above. The composition of the degenerate PCR reaction solution is shown in Table 6.
[0078]
[Table 6]

[0079]
The degenerate PCR reaction was performed for 30 cycles, with the heat denaturation at 96 ° C. for 30 seconds, followed by annealing at 50 ° C. for 30 seconds, followed by extension reaction at 72 ° C. for 1 minute. After completion of the reaction, the reaction solution was transferred to a new microtube, 5 μl of which was subjected to electrophoresis on a 1.0% agarose gel, and the amplified fragment was confirmed. Subsequently, the remaining reaction solution was treated with phenol / chloroform and ethanol precipitated, and the pelleted PCR product was dissolved in 20 μl of TE buffer.
[0080]
The obtained PCR product was subjected to 1% low-melting point agarose gel electrophoresis, and a fragment of about 0.9 kbp (size predicted from the amino acid sequence of a known filamentous fungus) was excised and used with QIAEX II (manufactured by QIAGEN) DNA was extracted from the gel and the desired PCR product was recovered. Subsequently, the recovered PCR product was subcloned into a pCR2.1 vector using a TA Cloning Kit (Invitrogen), and then the nucleotide sequence was analyzed with a fluorescent automatic DNA sequencer (Parkin Elmer, ABI PRISM 310 type). As a result, a region excluding introns in the obtained PCR product is shown in SEQ ID NO: 9. Further, the amino acid sequence considered from SEQ ID NO: 9 is shown in SEQ ID NO: 10.
[0081]
And the result of having investigated the homology of this amino acid sequence shown to sequence number 10 and well-known chs is shown in FIG.1 and FIG.2. As can be seen from FIG. 1, the amino acid sequence shown in SEQ ID NO: 10 shows high homology with chs in Neurospora crassa. As can be seen from FIG. 2, the amino acid sequence shown in SEQ ID NO: 6 showed high homology with chs in Schizophyllu commune. Therefore, in order to clone the full-length sequence of the chs gene including the promoter and terminator regions, the collected PCR products were used as probes for screening the shiitake mycelium cDNA library and shiitake genomic DNA library.
[0082]
(6) Isolation of chs gene from cDNA library
In the chs gene screening, plaque formation and library amplification were performed based on the manual of ZAP Express cDNA synthesis Kit (Stratagene). That is, in order to screen a total of 500,000 plaques from the cDNA library prepared in (2) above, 1 × 10⁷Prepare 10 5 μl pfu / ml phage solutions, each containing 10 mM MgSO_FourThen, 600 μl of E. coli XL1-Blue MRF ′ strain adjusted to OD600 = 0.5 was added and incubated at 37 ° C. for 15 minutes. Next, 6.5 ml of NZY top agar was added and overlaid on a 90 mm × 130 mm square NZY plate and incubated at 37 ° C. for 6-8 hours to form plaques. Plaque-formed plates were stored at 4 ° C.
[0083]
Hybond N + nylon membrane (90 mm × 130 mm, manufactured by Amersham Life Sciences, hereinafter simply referred to as “membrane”), which had been cut to a size in advance, was placed on the plate and allowed to stand for 2 minutes to transfer the phage DNA to the membrane. Next, the membrane was treated with an alkaline denaturing solution (containing 0.25 M NaOH and 1.5 M NaCl) for 20 minutes to denature the phage DNA. After denaturation, the membrane was immersed in a neutralization solution (0.5 M Tris-HCl (pH 8.0), 1.5 M NaCl), shaken at room temperature for 5 minutes, and then rinsed (0.2 M Tris-HCl (pH 7.5). ) After washing the membrane in 2 × SSC) for 30 seconds, the membrane was sandwiched between paper towels and heated at 80 ° C. for 2 hours to immobilize the phage DNA on the membrane.
[0084]
Subsequently, plaque hybridization and signal detection were performed using an ECL direct nucleic acid labeling and detection system (Amersham Life Science). That is, the membrane on which the DNA was immobilized was placed in a hybrid bag (manufactured by Cosmo Bio), an appropriate amount of a hybridization buffer attached to the kit was added, sealed, and prehybridized at 42 ° C. for 1 hour. Next, the solution was removed, 5 ml of hybridization buffer containing 50 ng of the peroxidase-labeled probe prepared in (5) above was added, and hybridization was performed at 42 ° C. for 4 hours. After completion of hybridization, the membrane was washed twice at 42 ° C. for 20 minutes using 0.5 × SSC containing 0.4% SDS and 36% Urea. Further, using 2 × SSC, washing was performed twice at room temperature for 5 minutes.
[0085]
This membrane was treated with 1 ml of ECL detection reagent for 1 minute, wrapped in Saran wrap, placed in a film cassette, and the resulting signal was exposed to Hyperfilm-ECL (Amersham Life Science). When the film was developed, 16 positive signals were obtained. Against the signal obtainedMeetPipette off the plaque to be treated and add it to 1 ml of SM buffer (100 mM NaCl, 100 mM MgSO_Four, 50 mM Tris-HCl (pH 7.5) and 0.01% gelatin. And 20 μl of chloroform was added and stirred. This phage suspension was appropriately diluted and plated, and then purified by repeating the same procedure as described above several times to obtain positive plaques.
[0086]
After screening, phagemid formation was performed based on the manual of Zap Express cDNA synthesis kit (Stratagene). That is, 250 μl of positive recombinant phage suspended in 1 ml of SM buffer was added to 10 mM MgSO._Four200 μl of E. coli XL1-Blue MRF ′ strain adjusted to OD600 = 1.0 with ExAassist helper phage was added and incubated at 37 ° C. for 15 minutes. Thereto, 3 ml of NZY medium was added and further incubated at 37 ° C. for 2.5 hours. After incubation, the mixture was heated at 70 ° C. for 20 minutes, centrifuged at 1000 × g for 15 minutes, and the supernatant was collected to obtain a phagemid solution. Next, to incorporate the phagemid into E. coli, add 10 mM MgSO to 100 μl or 10 μl of the phagemid solution._Four200 μl of E. coli XLOLR strain (Stratagene) adjusted to OD600 = 1.0 was added and incubated at 37 ° C. for 15 minutes. 200 μl of the culture solution was seeded on an LB solid medium containing 50 μg / ml kanamycin and incubated overnight at 37 ° C., and a phagemid was prepared from the resulting colonies.
[0087]
Phagemid was prepared using RPM Kit (manufactured by BIO 101). That is, the colonies obtained above were inoculated into 3 ml of LB liquid medium containing 50 μg / ml kanamycin and incubated overnight at 37 ° C. The culture solution was taken in a microtube and centrifuged at 10,000 × g for 1 minute to collect the cells. To the collected cells, 50 μl of Pre-Lysis buffer was added and vigorously stirred to suspend the cells. Furthermore, 100 μl of alkaline Lysis buffer was added and gently stirred to completely dissolve the cells. Next, 100 μl of neutralization solution was added and stirred vigorously, and centrifuged at 10,000 × g for 2 minutes to recover the supernatant. 250 μl of Glass Milk mixed well with the SPIN FILTER included in the kit was added, and the collected supernatant was added and stirred well, and the SPIN FILTER was placed on the microtube. Centrifuge at 10,000 × g for 1 minute, add 350 μl of Wash buffer, and centrifuge in the same manner. Place SPIN FILTER in a new microtube, add 50 μl of TE, centrifuge at 10,000 × g for 30 seconds, and add phagemid from Glass Milk. Was eluted. After ethanol precipitation, the obtained precipitate was dissolved in 20 μl of TE buffer and used for determination of the base sequence.
[0088]
(7) Isolation of chs gene from genomic DNA library
From the genomic DNA library prepared in (3) above, the chs gene was isolated in substantially the same manner as in (6) above. Plaque formation and library amplification are performed using E. coli XL1-blue MRA strain based on the manual of Lambda EMBL3 / Bam HI vector kit (Stratagene). Plaque hybridization and signal detection are performed using cDNA. The procedure was the same as that for isolating the chs gene from the library. The plaque near the obtained signal was scraped off and suspended in SM buffer. This phage suspension was appropriately diluted, plated, and screened in the same manner as described above to obtain a recombinant phage containing the chs gene in the genome. Each cloned phage was plated and incubated at 37 ° C. for 6-8 hours to form plaques. The plaque-formed plate was overlaid with 10 ml of SM buffer, and cultured with shaking at 4 ° C. for 12 hours or more to release phages in SM buffer. Collect SM buffer containing phage into a 15 ml propylene tube, add chloroform to a final concentration of 5%, leave at room temperature for 15 minutes, centrifuge at 1500 xg for 10 minutes, and collect supernatant did. Next, phage DNA was purified from the collected supernatant using Wizard Lambda Preps DNA Purification System (Promega) and subjected to determination of the base sequence.
[0089]
(8) Determination of base sequence
The base sequences of the positive clones obtained in the above (6) and (7) were determined using ABI PRISM Dye Terminator Cycle Seqeuencing Ready Reaction Kit, FS (Perkin Elmer). PCR reaction was performed based on the manual, and the obtained PCR product was analyzed by ABI PRISM 310 Genetic Analyzer (Perkin Elmer). The base sequences of the 5 'end and 3' end were determined for any positive clone. The determined sequences are shown in SEQ ID NOs: 1 and 2. SEQ ID NO: 1 is a base sequence of about 2.3 kbp upstream including the start codon of the chs gene, and SEQ ID NO: 2 is a base sequence of about 0.9 kbp downstream including the stop codon of the chs gene.
[0090]
[Example 2]
Construction of recombinant vector for expression
Here, an expression recombinant vector containing the chs gene promoter region was constructed. In addition, when constructing a recombinant vector for expression containing the terminator region of the chs gene, the following method can be used in the same manner.
(1) Isolation of the chs gene promoter region
From the base sequence (SEQ ID NO: 1) of about 2.2 kbp upstream from the start codon of the isolated chs gene, it can be seen that specific base sequences (TATA box, CAAT box, etc.) found in various promoters exist. Therefore, about 2.2 kbp upstream from this initiation codon is regarded as a promoter region (region sandwiched between the 1st base and 2229th base in SEQ ID NO: 1), PCR is performed using the genomic chs gene as a template, and a large amount of the promoter region of the chs gene is obtained. Prepared.
[0091]
In this PCR, a sense primer having the sequence CproU (5′-ATAAGAATAATCAGGCTTGTCGTCGAACCG-3 ′: SEQ ID NO: 5) present at a position of −2256 to −2227 bp from the translation start point is used, and the translation start is performed as the antisense primer. The one having the sequence CproL (5′-CCTTAGTTGTAGATGGAAATGGTGGGGTGG-3 ′: SEQ ID NO: 6) present at a position of −57 to −28 bp from the point was used. These CproU and CproL were synthesized by consigning to a custom service of Nippon Flour Mills Co., Ltd. The PCR reaction solution composition at this time is as shown in Table 7.
[0092]
[Table 7]

[0093]
At this time, the PCR reaction was carried out for 30 cycles, with the heat denaturation at 96 ° C. for 30 seconds, followed by annealing at 60 ° C. for 30 seconds, followed by extension reaction at 72 ° C. for 2 minutes as one cycle. After completion of the reaction, the reaction solution was transferred to a new microtube, 5 μl of which was subjected to electrophoresis on a 1% agarose gel, and the amplified fragment was confirmed. Subsequently, the remaining reaction solution was treated with phenol / chloroform and ethanol precipitated, and the pelleted PCR product was dissolved in 20 μl of TE buffer.
[0094]
The obtained PCR product was subjected to 1% low melting point agarose gel electrophoresis, and a fragment of about 2.2 kbp was excised. The fragment was extracted from the gel using QIAEX II (manufactured by QIAGEN), and the promoter region of the chs gene was isolated and purified.
[0095]
(2) Construction of recombinant vector (pLCHS)
Here, as an example of construction of a recombinant vector, as shown in FIG. 1, the promoter region isolated in the above (1) and the pLG-hph plasmid vector disclosed in JP-A-6-319547 are used. A recombinant vector pLCHS-hph was constructed as a host. First, in order to construct a vector in which the promoter region of the gds gene is removed from the pLG-hph plasmid vector and the promoter region of the chs gene is incorporated instead, the promoter region of the gpd gene should be removed using the pLG-hph plasmid vector as a template. PCR was performed. The obtained DNA fragment was electrophoresed using 1% Sea Plaque Agarose (FMC) at 4 ° C. and 50 V. After electrophoresis, the target band was excised from the gel, and the DNA was purified by QIAEX II Gel Extraction Kits (QIAGEN). After ethanol precipitation, the resulting DNA pellet was blunted using a DNA Blunting Kit (TaKaRa), dephosphorylated with alkaline phosphatase (Calf intestine), treated with phenol / chloroform, and ethanol precipitated to give pLG-hph A (-Pgpd) plasmid vector (a fragment obtained by removing the gpd gene promoter from the pLG-hph plasmid vector) was obtained. The fragment was further blunt ended, further dephosphorylated, and ligated with the promoter of the chs gene obtained in (1) above using Perfectly Blunt Cloning Kits (Novagen) to obtain a pLCHS-hph plasmid vector. It was.
[0096]
(3) Construction of recombinant expression vector (pChG-bar)
The recombinant expression vector pChG-bar combines the pLG-bar plasmid vector obtained by replacing the hph gene of the pLG-hph plasmid vector with the bialaphos resistance gene (bar gene) and the pLCHS-hph plasmid vector obtained in (2) above. It was built by letting.
[0097]
First, as shown in FIG. 3, a pLG-bar plasmid vector was constructed. Specifically, first, the hph gene was removed from the pLG-hph plasmid vector, and PCR was performed using the pLG-hph plasmid vector as a template to remove the hph gene in order to construct a vector in which the bar gene was incorporated instead. A vector fragment (a fragment obtained by removing the hph gene from the pLG-hph plasmid vector) was obtained. The obtained pLG plasmid vector fragment was electrophoresed using 1% Sea Plaque Agarose (FMC) at 4 ° C. and 50 V. After electrophoresis, the target band was excised from the gel, and the DNA was purified by QIAEX II Gel Extraction Kits (QIAGEN). After ethanol precipitation, the resulting DNA pellet was blunt-ended using DNA Blunting Kit (TaKaRa), treated with phenol / chloroform, and ethanol precipitated. On the other hand, the bar gene was isolated from a pLC-bar plasmid vector [Yanai, K. et. Al., Biosci. Biotech. Biochem., 60, 472-475 (1996)]. That is, the pLC-bar plasmid vector was digested with BamHI, and the resulting DNA fragment was electrophoresed using 1% Sea Plaque Agarose (FMC) at 4 ° C. and 50 V. After electrophoresis, the target band was excised from the gel, and the DNA was purified by QIAEX II Gel Extraction Kits (QIAGEN). After ethanol precipitation, the obtained DNA pellet was blunted using a Blunting Kit (TaKaRa), treated with phenol / chloroform and ethanol precipitated to obtain a bar gene fragment. Then, this bar gene fragment was ligated with the pLG plasmid vector fragment to obtain a pLG-bar plasmid vector.
[0098]
Next, in order to obtain the Shiitake expression unit Pchs-bar-Tgpd fragment, the pLG-bar plasmid vector was used as a template, the promoter (Pgpd), the bar gene (bar) and the terminator of the gpd gene in the pLG-bar plasmid vector. The region containing (Tgpd) was amplified by PCR. The obtained DNA fragment was electrophoresed using 1% Sea Plaque Agarose (FMC) at 4 ° C. and 50 V. After electrophoresis, the target band was cut out from the gel, and Pgpd-bar-Tgpd was purified by QIAEX II Gel Extraction Kits (QIAGEN). After ethanol precipitation, the obtained DNA pellet was blunt-ended using a DNA Blunting Kit (TaKaRa).
[0099]
On the other hand, the pLCHS-hph plasmid vector was digested with Ehe I, subjected to end blunting, dephosphorylation, phenol / chloroform treatment, and ethanol precipitation to obtain a linear pLCHS-hph plasmid vector. This and the Pgpd-bar-Tgpd fragment were ligated using Perfectly Blunt Cloning Kits (Novagen) to obtain a pChG-bar plasmid vector as shown in FIG. Among the plasmids produced in this way, those that were correctly inserted were selected and prepared in large quantities.
[0100]
Example 3
Transformation of shiitake mushroom by REMI method
(1) Preparation of protoplast
The binuclear mycelium of Shiitake strain was cultured in 0.25 × MYPG agar medium (containing 0.25% malt extract, 0.1% yeast extract, 0.1% peptone, 0.5% glucose and 1.5% agar) at 25 ° C. for 2 weeks. The grown mycelium was scraped off and cultured in 50 ml of 0.25 × MYPG liquid medium for 1 week. The obtained mycelium was collected with a glass filter, suspended in 50 ml of 0.25 × MYPG liquid medium, cut with a polytron homogenizer, and filtered through a 100 μm nylon mesh and further filtered at 25 ° C. in a 0.25 × MYPG liquid medium at 25 ° C. Cultured for 5 days. Cultured mycelia are collected with a 100 μm nylon mesh, washed twice with citrate buffer (containing 50 mM citrate buffer containing 0.6 M mannitol, adjusted to pH 5.6), and per 1 g of mycelia The mycelium was suspended in 10 ml of enzyme solution (containing a citrate buffer containing 2.5% by weight cellulase (manufactured by Yakult) and 0.1% by weight chitinase (manufactured by Sigma)) and incubated at 28 ° C. for 3 to 4 hours. The enzyme-treated mycelium was filtered through a 40 μm nylon mesh, and the filtrate was centrifuged at 1,500 × g for 10 minutes to precipitate protoplasts. Discard the supernatant, wash the protoplasts with STC buffer (containing 10 mM Tris-HCl containing 10 mM calcium chloride and 1.2 M sorbitol, adjusted to pH 7.5), and then again into protoplasts in 1 ml of STC buffer. Was suspended, and the number of protoplasts was counted with a microscope. Finally, 0.5-1.0 x 10 in 100 μl of STC buffer⁷Protoplasts for transformation were prepared by adjusting to protoplasts.
[0101]
(2) Introduction of pLCHS-hph and pChG-bar plasmid vectors
Transformation was performed by the Restriction Enzyme Mediasted Integration (REMI) method using restriction enzyme Hind III that cuts the pLCHS-hph plasmid vector and the pChG-bar plasmid vector at one place (see JP-A-11-155568). . Specifically, 100 μl of the above protoplast suspension was gently added to 150 μl of STC buffer containing 2.5 μg of pLCHS-hph or pChG-bar plasmid vector and 50 units of Hind III, and incubated for 20 minutes in ice. To this was added 62.5 μl of PEG solution (containing 10 mM calcium chloride, 10 mM Tris-HCl containing 60% PEG4000, adjusted to pH 7.5) and incubated in ice for 20 minutes. An additional 3.125 ml of PEG solution was added and incubated at room temperature for 20 minutes. Next, 10 ml of STC buffer was added to fully suspend the whole solution, and centrifuged at 1,500 × g for 10 minutes to precipitate protoplasts. The collected protoplasts were suspended in 4 ml of MS (containing 2% malt extract and 0.6M sucrose) and cultured at 25 ° C. for 3-4 days.
[0102]
(3) Selection of hygromycin B resistant hyphae
The mycelium was regenerated from the protoplasts, and the regenerated mycelia were collected by centrifugation at 1,500 × g for 10 minutes. The collected mycelium is resuspended in 1 ml of MS liquid medium and a minimum agar medium containing 2 μg / ml hygromycin B (2% glucose, 0.2% ammonium tartrate, 0.05% magnesium sulfate, 0.1% potassium dihydrogen phosphate, 0.112% Sodium carbonate, 0.132% fumaric acid, 10 ppm iron sulfide, 8.8 ppm zinc sulfide, 7.2 ppm manganese chloride, and 1.5% agar adjusted to pH 4.5) and cultured at 25 ° C. for 5 days. Next, 20 μg / ml hygromycin B was added to a 0.25 × MYPG agar medium once dissolved and cooled to about 50 ° C., and this was layered on the mycelium grown on the minimum agar medium. After culturing at 25 ° C. for about 5 days, the growing mycelium was isolated and replanted on a MYPG agar medium containing fresh 20 μg / ml hygromycin B. Further, the mycelium that had been cultured for about one week and proliferated was transplanted to a new MYPG agar medium containing 20 μg / ml hygromycin B and cultured, and hyphae that had acquired hygromycin resistance were selected.
[0103]
(4) Selection of bialaphos resistant hyphae
For the pChG-bar plasmid vector-introduced strain, the hygromycin B resistant mycelium selected in (3) above is further spread on a MYPG agar medium containing 2 μg / ml bialaphos and 20 μg / ml hygromycin B for about 1 week at 25 ° C. After cultivation, the mycelium that had proliferated was isolated and replanted on a MYPG agar medium containing 2 μg / ml bialaphos and 20 μg / ml hygromycin B, and both drug-resistant strains were selected.
(5) Results
The results are shown in Table 8. The numerical value is an average value of three experiments.
[0104]
[Table 8]

[0105]
As can be seen from Table 8, a shiitake transformant that has acquired hygromycin resistance was produced by the pLG-hph plasmid vector, and the recombinant vector for expression of the present invention is effective as a heterologous gene expression vector using shiitake as a host. I confirmed that there was.
[0106]
【The invention's effect】
As described above in detail, according to the present invention, a promoter instructing transcription initiation of a shiitake chitin synthase gene and / or a recombinant vector containing the terminator, a transformant containing the recombinant vector, A method for producing a polypeptide using the transformant is provided.
[0107]
[Sequence Listing]

[0108]
[Sequence Listing Free Text]
[SEQ ID NO: 3]
Primer. N of the 12th base is a, t, c or g.
[SEQ ID NOs: 4-8]
Primer.
[Brief description of the drawings]
FIG. 1 is a view showing the homology between the amino acid sequence of SEQ ID NO: 10 and the chs amino acid sequence in Neurospora crassa.
FIG. 2 is a view showing the homology between the amino acid sequence of SEQ ID NO: 10 and the chs amino acid sequence in Shirohirotake.
FIG. 3 is a schematic diagram showing the construction of a pChG-bar plasmid vector.

Claims (11)

A DNA comprising a sequence from the first base to the 2229th base in the base sequence represented by SEQ ID NO: 1, and capable of functioning as a promoter.   A recombinant expression vector containing the DNA according to claim 1.   An expression recombinant vector in which any DNA having terminator activity is incorporated into the expression recombinant vector according to claim 2. A DNA comprising a sequence from the 20th base to the 920th base in the base sequence represented by SEQ ID NO: 2, which can function as a terminator.   A recombinant vector for expression containing the DNA according to claim 4.   An expression recombinant vector in which any DNA having promoter activity is incorporated into the expression recombinant vector according to claim 5.   A recombinant vector for expression containing the DNA according to claim 1 and the DNA according to claim 4.   A recombinant vector in which a gene encoding an arbitrary polypeptide is incorporated into the recombinant vector for expression according to claim 2, 3, 5 or 6.   A transformant comprising the expression recombinant vector according to claim 2, 3, 5 or 6.   A transformant comprising the recombinant vector according to claim 8.   A method for producing the polypeptide, comprising culturing or cultivating the transformant according to claim 10, and collecting the polypeptide from the obtained culture or culture.

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