エンドウ原形質膜におけるATPアーゼとホスファチジルイノシトールリン資質リン酸化酵素の共精製

The plasma membrance ATPase was partially purified by a linear glycerol density gradient centrifugation of the detergent-solubilized plasma membrance poteins and subsequent separation by a size-exclusion column chromatogrphy. A purified ATPase preparation is shown to contain a 97.6kDa protein that was cross-reacted with an antibody raised against mung bean H+-ATPase. The preparation also exhibited the phosphorylation of exogenous phosphatidylionsitol(PI) when supplized with [γ-32P]ATP. These results indicate that one form plasma membrance ATPase is co-purified with PI kinase.エンドウの上胚軸組織から分離した原形質膜画分におけるATPアーゼとホスファチジルイノシトールリン脂質リン酸化酵素との相互作用を解析する目的で、双方の原形質膜画分からの可溶化とそれらの部分精製を試みた。原形質膜のTritonX－100可溶化画分をグリセロール連続密度勾配遠心分画に供し、得られた活性画分をさらに分子ふるいカラムクロマトグラフィーによって分離した。この結果、ＡＴＰアーゼとホスファチジルイイノシトールリン脂質リン酸化酵素は共精製され、非変性条件下では双方の活性を分けることができなかった

共生真菌アクレモニウムエンドファイトの形質転換

Conditions have been developed for transforming protoplasts of the Acremonium endophyte by PEG 4000 and electroporation. Transformation by PEG exhibited a higher number of transformants than by electroporation. lntegration of iaaM gene into the genome was examined by PCR and Southern blot hybridization analysis. PCR product showed that transformants banded at around 1.7 kb corresponding to the size of iaaM gene. Hybridization of the digests of genomic DNA with iaaM gene as DNA probe showed that the number of hybridized band signals was different between transformant and non-transformant. These results might indicate that PEG is an effective method for the transformation of Acremonium endophyte and that there are repeated copies of the iaaM homologous sequences in the genome of Acremonium.アクレモニウムエンドファイトの形質転換の条件を検討した。アクレモニウムエンドファイトからプロトプラストを調製し、PEG4000とエレクトロポレーションを用いて形質転換を試みた。その結果、PEGで形質転換すると、エレクトロポレーションによる場合よりも多くの形質転換体が得られた。PCR解析によってiaaM遺伝子のゲノムへの導入を確認したところ、形質転換体のPCR産物はiaaM遺伝子のサイズに相当する約1.7kbのバンドを持っていた。一方、iaaM遺伝子をプローブとしたサザンブロット解析においては、形質転換体と非形質転換体の間にはハイブリダイズした断片数に違いがあることが明らかとなった。以上の結果は、アクレモニウムエンドファイトの形質転換にはPEG法が有効であること、アクレモニウムエンドファイトのゲノム中にはiaaM遺伝子様配列の反復コピーが存在することを示唆している

Development of bone-bonding hafnium metal and titanium-hafnium alloys by chemical surface modification

Hafnium (Hf) has attracted considerable attention as a component of biomedical titanium (Ti) alloys with low Young\u27s moduli and/or shape‐memory functionalities, because its cytotoxicity is as low as that of Ti. The drawback of metals is that their bone‐bonding ability is generally low. It is known that apatite formation in the body is a prerequisite for bone‐bonding. Although several chemical treatments have been proposed for preparing Ti for bone‐bonding, there have been no similar investigations for Hf. In the present study, NaOH‐ and heat‐treatments were applied to pure Hf and Ti‐Hf alloys and their bone‐bonding ability was assessed in vitro with the use of simulated body fluid (SBF). After NaOH‐ and heat‐treatments, anatase formed on alloys with low Hf content (20–40% (atom%) Hf); mixtures of sodium titanate and hafnium titanate formed on alloys with similar Ti and Hf content (60% Hf); and hafnium oxide formed on alloys with high Hf content (80% Hf and pure Hf). Precipitates of apatite were observed on all the metals in SBF, except for the alloy with 60% Hf. We speculated that the hafnium titanate formed on this alloy had a low apatite‐forming ability owing to its high negative surface charge, which inhibited P adsorption. The apatite‐forming abilities of the Ti‐Hf alloys strongly depended on their Hf content. The present results indicate that Hf‐based materials have good potential for bone‐bonding

A Volatile Substance, β-Caryophyllene, from Talaromyces wortmannii Promotes Growth and Tolerance to Diseases on Several Plants

A plant growth-promoting fungus, Talaromyces wortmannii strain FS2 was isolated from an agricultural field at Okayama Pref. FS2 enhanced seed germination, root elongation and leaf growth of Brassica rapa var perviridis (Komatsuna). Such plant growth-promoting effect was observed in the same sealed chamber where FS2 was cultured on PDA medium separated from seedlings, suggesting effective volatile compound(s). GC‒MS analysis showed that FS2 emitted at least seven terpenoids, of which a volatile was identified as β‒caryophyllene. β‒caryophyllene alone promoted the growth of cucumber, Nicotiana benthamiana and barley. Furthermore β‒caryophyllene increased the yield of cucumber fruits. Interestingly, we found that β‒caryophyllene conditioned these plants to be resistant to respective diseases caused by Colletotrichum orbiculare, Botrytis cinerea or Blumeria graminis f. sp hordei. The findings indicate that β‒caryophyllene has desirable dual features and therefore, it is available to cultivation of many crops.岡山県総社市の圃場から分離した植物生育促進菌Talaromyces wortmannii FS2が生産するβ-caryophylleneは，コマツナ（アブラナ科）のみならず，キュウリ（ウリ科），タバコ（ナス科）およびオオムギ（イネ科）など広汎な植物に対して，生育促進作用および耐病性増進作用を示したことから，有用な農業資材として利用可能であるものと考察した

病原菌シグナルによるエンドウ原形質膜におけるホスファチジルイノシトールリン脂質のリン酸化とリゾリン脂質生成の制御

Effects of elicitor and suppressor from a pea pathogen, Mycosphaerella pinodes, on Pl etabolism in pea plasma membrane were examined in vitro. The elicitor induced rapid phosphorylation of phosphatidylinositols as well as production of lysophospholipid in plasma membranes, but these responses were severely inhibited by the suppressor. These results indicate that a membrane-associated phospholipase A is regulated coordinately by fungal signals, together with Pl metabolism, and that it may participate in signal transduction pathways leading to defense responses. To evaluate a possible rote of phospholipase A activation in induction of a pea defense response, the effect of free fatty acid on induction of a phytoalexin accumulation was also examined. When pea leaves were treated with linoleic- or linolenic acid, most commonly released in plant cells by phospholipase A, the accumulation of pisatin was induced even in the absence of the elicitor. It is, therefore, conceivable that free fatty acid(s) released from plasma membrane is also implicated in the early stage of elicitor-signal transduction in pea.エンドウの上胚軸組織により分離した原形質膜画分を褐紋病菌の生産するエリシターで処理すると、ホスファチジルイノシトールリン脂質の急速なリン酸化とリゾリン脂質の生成が誘導されたが、同菌より調製したサプレッサーの共存下では双方とも著しく阻害された。本結果は、ポリホスホイノシチド代謝系と同調的に作動するホリパーゼA活性化が存在すること、さらに、原形質膜における病原菌シグナルの受容・応答には複数の資質代謝系が介在する可能性を示唆している。一方、ホスホリパーゼAの活性化の役割を調べる目的で、本酵素によって原形質膜から生成されると考えられる脂肪酸(リノール酸ならびにリノレン酸)をエンドウ葉に処理したところ、エリシターの非存在下においてもファイトアレキシンであるピサチンの生成が誘導されることが示された。以上から、ポリホスホイノシチド代謝系と同調的に働くホスホリパーゼAがエリシターシグナルの初期伝達に深く関連しているものと考えられた

Structure and expression of 12-oxophytodienoate reductase (OPR) subgroup I gene in pea and oxidoreductase activity of their recombinant proteins

Recently, we observed that expression of a pea gene (S64) encoding an oxophytodienoic acid reductase (OPR) was induced by a suppressor of pea defense responses, secreted by the pea pathogen Mycosphaerella pinodes. Because it is known that OPRs are usually encoded by families of homologous genes, we screened for genomic and cDNA clones encoding members of this putative OPR family in pea. We isolated five members of the OPR gene family from a pea genomic DNA library, and amplified six cDNA clones, including S64, by RT-PCR (reverse transcriptase-PCR). Sequencing analysis revealed that S64 corresponds to PsOPR2, and the amino acid sequences of the predicted products of the six OPR-like genes shared more than 80% identity with each other. Based on their sequence similarity, all these OPR-like genes code for OPRs of subgroup I, i.e., enzymes which are not required for jasmonic acid biosynthesis. However, the genes varied in their exon/intron organization and in their promoter sequences. To investigate the expression of each individual OPR-like gene, RT-PCR was performed using gene-specific primers. The results indicated that the OPR-like gene most strongly induced by the inoculation of pea plants with a compatible pathogen and by treatment with the suppressor from M. pinodes was PsOPR2. Furthermore, the ability of the six recombinant OPR-like proteins to reduce a model substrate, 2-cyclohexen-1-one (2-CyHE), was investigated. The results indicated that PsOPR1, 4 and 6 display robust activity, and PsOPR2 has a most remarkable ability to reduce 2-CyHE, whereas PsOPR3 has little and PsOPR5 does not reduce this compound. Thus, the six OPR-like proteins can be classified into four types. Interestingly, the gene structures, expression profiles, and enzymatic activities used to classify each member of the pea OPR-like gene family are clearly correlated, indicating that each member of this OPR-like family has a distinct function.</p

Isolation and Identification of a Plant Growth-Promoting Fungus from an Agricultural Field in Okayama Prefecture

A plant growth-promoting fungus was isolated from an agricultural field in Okayama Prefecture, Japan. The strain FS2, which enhanced seed germination, root elongation and leaf growth of Brassica rapa var. perviridis, was identified as Talaromyces wortmannii based on ITS1 sequence and its morphology.本研究では，実際の生産圃場から植物生育促進菌（PGPF）の探索を試み，コマツナの生育を促進するFS2株を分離した．FS2株の形態観察並びにのITS1領域の系統樹解析から本菌をTalaromyces wortmanniiと同定した

エンドウのエリシター誘導性遺伝子発現におけるAAAGモチーフとPsDof1タンパク質の関与

Recently, we, isolated cDNA clone, PsDof1, from clicitor-treated pea cDNA library. The putative gene product, a PsDof1, encodes DNA binding protein that specifically binds the DNA fragment containing AAAG core sequence. In this paper we report that GST-PsDof1 fusion protein specifically binds to the promoter region containing AAAG core sequence(s) of PsCHS1, one of the elicitor-inducible genes encoding chalcone synthase (CHS). Furthermore the addition of DNA fragment containing AAAG motif to the 35S minimal promoter provided the elicitor-responsibility in transient transfection assay using pea protoplasts. These results suggest that PsDof1 might be involved in defense responses by acitivating the transcription by a binding to AAAG core sequence in the promoter of the defense-related genes in pea.エリシターを処理したエンドウ上胚軸由来のRNAから作成されたcDNAライブラリーからエリシター処理により発現が増高する遺伝子候補のcDNAとしてPsDof1が単離された．大腸菌で生産されたGST-PsDof１融合タンパク質はAAAG配列をコアとするDNAに結合することが明らかにされている．本論文ではGST-PsDof１がエリシター応答性遺伝子の一つ，PsCHS1のプロモーター上のAAAGまたはCTTT配列を有する断片に特異的に結合することを明らかにした．更にAAAG配列のエリシター応答性シスエレメントとしての機能を解析するため，AAAG配列を４回繰り返したユニットをCaMV35Ｓの最小プロモーターとCATレポーター遺伝子に連結したキメラ遺伝子を構築し，エンドウプロトプラストにエレクトロポレーション法により導入した．CAT活性を指にプロモーター活性を調べたところ，AAAG配列を有するプロモーターは，エリシター処理により活性化されることが明らかとなった．これらの結果はPsDof１がエリシター応答性防御遺伝子のプロモーター上のAAAG配列に結合し，転写を活性化させる可能性を示唆している

エンドウの推定ZnフィンガーDNA結合性タンパク質のcDNAクローニング

We constructed cDNA library from pea epicotyls treated with fungal elicitor for 5hrs, and performed differential screening using the individual 32P-cDNA probe derived from poly(A)+ RNA prepared from elicitor- and water-treated epicotyls. As a result of the screening, we have isolated from elicitor- and water-treated epicotyls. As a results of the screening, we have isolated about 90 cDNA clones as candidates for elicitor-inducible genes, and their nucleotide sequences have been partially determined. One of these clones, E31 was a pea homolog of the putative zinc-finger proteins, Ljpzf in Lotus japonicus and Gmpz in soybean. E31 possesses 1,726bp insert, and encodes an open reading frame corresponding to position 82 amino acids from N-terminus to the C-terminal end in Ljpzf. The protein product of E31 was designated as Pspzf. Pspzf also possesses nuclear localization signal(NLS), HKRK, and Cys3His2Cys3(RIngH2) motif at the same position to LjpZF and putative C-terminal end of the deduced amino acid sequences, respectively. Since zinc-finger motif is one of the well-known DNA-binding domains, Ljpzf and Pspzf might be able to bins to a particular DNA sequence and regulate transcriptional activity in plants.エリシターを処理したエンドウの上胚軸からcDNAライブラリーを作成し、水処理上胚軸をコントロールに differential screening を行い、エリシター応答性cDNAの候補として90個のクローンを単離した。そららのクローンの1つE31は1,716bpのインサートを有し、ミヤコグサ、ダイズでcDNAがクローニングされているLjpzf、Gmpzfのエンドウにおけるホモローグをコードしていると考えられ、その推定翻訳産物をPspzfと命名した。E31は、Ljpzfの推定アミノ酸配列の82アミノ酸目からカルボキシル末端側をコードしていると考えられた。LjpzfやPspzfの推定アミノ酸配列中には核移行シグナルHKRKが存在していたこと、カルボキシル末端側にはCys3His2Cys3もモチーフとするRing H2 fingerドメインが存在していたことより、LjpzfやPspzfは、核内に局在するDNA結合性のZn fingerタンパク質の1種として遺伝子発現の制御に機能している可能性が示唆された