成果報告書詳細
管理番号20120000000143
タイトル*平成23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2012/8/3
報告書年度2011 - 2011
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
和文要約和文要約等以下本編抜粋:
1. 研究開発の内容及び成果等
1) 有用形質を持つP. ellipsoidea突然変異体のゲノム解析
1)-1 Resequencing
これまでに分離してきたP. ellipsoidea突然変異体に現れた有用形質がどのような遺伝子の変異によってもたらされたのかを知るために、突然変異株のゲノム解析を行っている。本年度は、脂質高蓄積、低クロロフィル含有などの形質を持つ12の変異株からゲノムDNAを抽出し、次世代シーケンサーを用いてゲノム塩基配列を解読した。
1)-2 変異遺伝子の機能推定
得られた突然変異体ゲノム塩基配列と野生株のゲノム塩基配列との比較解析を、現在行っている。次年度は同定された突然変異部位の遺伝子について、その機能をin silico解析によって推定する。
英文要約Title; R&D on Molecular Breeding of Microalgae for Improvement Oil Productivity
FY2011-2013 Annual report

In our previous studies, we have isolated several mutants of the green alga, Pseudochoricystis ellipsoidea, which have improved traits for biofuel production (e.g. low antenna-size mutant). The whole genome sequences of the 12 different mutants were determined. Based on the acquired genomic sequences of them, mutated genes were identified, and we are currently annotating the mutated genes to predict their functions.

We also have successfully transformed P. ellipsoidea cells with a particle bombardment method. The transformation efficiency, however, was low, and further improvement of the method was required. In this study, relevant operating parameters of the particle bombardment method were optimized using the G418 gene as a selectable marker. G418 resistant colonies appeared on agar plates were examined by colony PCR to evaluate the transformation efficiency. By optimization of particle bombardment conditions, we successfully improved the transformation efficiency by 6-fold.

TALEN is a new artificial endonuclease, consisting of a transcription activator-like (TAL) effector domain originally found in the plant pathogen, Xanthomonas spp, and the FokI nuclease. In addition, the TALEN element contains a nuclear localization sequence (NLS) which allows the translocation of TALEN from the cytoplasm into the nucleus, where TALEN recognizes a specific DNA sequence and cleaves DNA in the vicinity of the binding site. The binding specificity of the TAL domain could be modified by changing specific amino acids in the TAL effector. In this study, we screened putative NLS sequences in the genome of P. ellipsoidea, and found that some of the NLS sequences in P. ellipsoidea were very similar to the NLS sequence found in the original TALEN element. Orotidine 5’-phosphate decarboxylase (OMP decarboxylase) is an enzyme involved in the pyrimidine biosynthesis. The genes for OMP decarboxylase have been used as a selectable marker in the recombinant DNA technology; and therefore we chose the gene as a target for the TALEN-based mutagenesis. Based on the OMP decarboxylase gene sequence in P. ellipsoidea, we designed a custom-made TALEN for the inactivation of the OMP decarboxylase gene in P. ellipsoidea. We are in the final step, i. e. the introduction of a P. ellipsoidea promoter upstream of the TALEN element to construct plasmids for the site-specific mutagenesis of the OMP decarboxylase gene.

Endogenous microRNAs (miRNAs) are potent gene silencer at the translational level in eukaryotes. Artificial miRNAs (amiRNAs) have been constructed based on the miRNA sequences in plants and animals. To develop an amiRNA methodology applicable to P. ellipsoidea, we have to first identify miRNAs in P. ellipsoidea. Towards the goal, we purified mature miRNA. The sequences of the miRNA will soon be determined.

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