成果報告書詳細
管理番号20140000000090
タイトル*平成25年度中間年報 「バイオマスエネルギー技術研究開発 戦略的次世代バイオマスエネルギー利用技術開発事業(次世代技術開発) 油分生産性の優れた微細藻類の育種・改良技術の研究開発」
公開日2014/5/2
報告書年度2013 - 2013
委託先名株式会社デンソー 学校法人中央大学
プロジェクト番号P10010
部署名新エネルギー部
和文要約1. 有用形質を持つP. ellipsoidea 突然変異体のゲノム解析
 本年度は新たに取得した18 系統の変異体(Pseudococcomyxa sp. KJ 株で取得した変異体を含む)についてゲノムシークエンスを行った。低クロロフィル変異体(5P)に再度変異を導入して得られた高油脂生産型の変異体のうち、デンプンの蓄積が少ないという形質が共通していた3 系統(5P-n2, 5P-n18,5P-n35)について、ゲノムの変異箇所を調べた。
2. 形質転換効率の改良
 Pseudococcomyxa sp. KJ 株でもP. ellipsoidea 由来の発現プラスミドや導入手法が適用できる事が示された。Pseudococcomyxa sp. KJ 株において、GFP によるレポーターアッセイ系を確立することに成功した。
3. 遺伝子ノックダウン・ノックアウト技術の開発
 相同組換えの効率を高めるためにUMPS 遺伝子破壊プラスミドの再構築を行った。
4. セルフクローニングシステムの構築
 G418 耐性株・ウラシル非要求性形質転換体を4 株選抜した。 
5. 油分生産性改良株の構築
アンテナ・クロロフィル(クロロフィルb)が減少した株を合計19 株取得することができた。これらの株は全てクロロフィルa/b 比が4.0 以上であり、その内5 株はクロロフィルb が検出限界以下であった。
英文要約1. Genome analyses of P. ellipsoidea mutants exhibiting improved properties for biofuel production.
We have isolated three pleiotrophic mutants with low-chlorophyll-, high-triglyceride- and low-starch contents by 1-methyl-2-nitro-1-nitrosoguanidine (NTG) mutagenesis. The genome analyses of these mutants showed that they have allelic mutations in the same gene encoding a dual-specificity tyrosine-(Y)-phosphorylation regulated kinase (DYRK).
2. Development of gene knockout technology in P. ellipsoidea
We constructed a plasmid which may increase the chance of the disruption of the uridine monophosphate synthetase (UMPS) gene through a homologous recombination. The plasmid contains a non-functional UMPS gene, the middle part of which was replaced by a similar size of DNA consisting of the neo gene flanked by a promoter and a terminator. The circular single-stranded DNA of the plasmid was introduced into P. ellipsoidea cells by particle bombardment and transformants were selected on agar plates containing G418 and uracil.
3. Construction of transgenic P. ellipsoidea with improved lipid productivity
In P. ellipsoidea, cellular contents of triglycerides increased when the alga was cultivated under nitrogen deficiency. MYB7 is a transcription factor of P. ellipsoidea which has a typical MYB domain involved in the DNA binding. We have observed that the MYB7 gene is induced under nitrogen deficiency prior to the triglyceride accumulation. When a cDNA of MYB7 placed downstream of a constitutive promoter was introduced into P. ellipsoidea, the triglyceride productivity of the transformant was 8 to 36% higher than that of the wild-type strain. This result suggested that MYB7 positively controls the triglyceride biosynthesis.
4. Development of technology for stable expression of transgenes in Pseudococcomyxa sp. strain KJ
To monitor gene expression from various promoters in Pseudococcomyxa sp. strain KJ, we synthesized codon-optimized eGFP (PeeGFP). Two expression cassettes consisting of PeeGFP flanked either by the s-Tublin1 gene promoter and the-actin gene terminator or that flanked by the RuBisCO small subunit gene promoter and terminator, originated from P. ellipsoidea were constructed. By using particle bombardment method, the cassettes were introduced into Pseudococcomyxa sp. strain KJ, resulting in the isolation of 13 PeeGFP transformants. The expression of GFP in these transformants under variety of culture conditions was readily determined by measuring fluorescence intensity.
5. Creation of mutants of Pseudococcomyxa sp. strain KJ with high oil productivity
In our previous study, the oil productivity of a low-chlorophyll mutant of P. ellipsoidea (strain 5P) was higher than the wild-type strain. The genome analysis of strain 5P revealed that the chlorophllide a oxygenase (CAO) gene of this strain was defective. Thus, we tried to knock out the CAO gene by the transcription activator-like effecter nuclease (TALEN) method.
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