Approaches with Arabidopsis thaliana Mutants to the Regulatory Genes for Photosynthesis

拟南芥突变体光合作用调控基因的研究

• 批准号: 02804056
• 负责人: KOBAYASHI Hirokazu
• 金额: $ 1.09万
• 依托单位: University of Shizuoka (1991)Nagoya University (1990)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02804056/
• 关键词: Photosynthesis Genes; Regulatory Genes; Mutants; Arabidopsis thaliana; RuBisCO; rbcS; cab; PCR

A. thaliana is most suitable for handling in the aspect of molecular generics to thoroughly Investigate mechanisms goveming the transcriptional regulation.1. A Model Experiment for Screening Photosynthetic MutantsA. thaliana grew on the agar medium of Gamborg B5 in supplementation with glucose or sucrose even in the presence of the inhibitor of photosynthetic electron transport, 3-(3, 4-dich ; orophenyQ-1, 1-dimethylurea(DCMU), although it did not grow without sugar sources under that condition. The resulting A. thaliana grown with inhibited level of photosynthetic activity, generated high red fluorescence upon exposure to a long wave UV light.2. Mutagenesis of A thaliana transformed with Reporter and Suicide GonesGenes for P-glucuronidase(GUS)and chloramphenicol acetyltransferase(CAT)as reporter genes, as well as a gene for alcohol dehydrogenase(ADH)as a suicide gene, were tried to introduce into A. thaliana prior to mutagenesis with ethylmethanesuffonate(EMS). This strategy may promi … More se to make the selection of objective mutants easily in combination with screening by high red fluorescence. We have focused on members of genes for small subunit of ribulose 1, 5 -bisphosphate carboxylase/oxygenase(RuBisCO, rbcS-3B)and chlorophyll a/binding proteins(cabl). Polymerase chain reaction(PCR)with thermostable DNA polymerases associated with a 3'-5' proofreading exonuclease activity, was used to quickly obtain approximately 1.5-kilobase pair(kbp)DNA fragments covering promoters and their upstream regions of rbcS-38and cabl, as nucleotide sequences of these genes have been published. A gene for ADH(approximately 2.2 kbp)was similarly amplilied from A. thaliana DNA. The fragments possessing the promoter and regulatory activities of rbcS-3B and cabl were-placed upstream of genes for GUS, CAT, and ADH, and tried to introduce Into A. thaliana(Columbia and Bensheim, adhl. The transgenic A. thaliana is mutagenized with EMS, and mutants which less express rbcS-3B or cabl in green leaves is being screened. Less

拟南芥最适合在分子仿制药方面进行彻底研究转录调控机制。 1．筛选光合突变体的模型实验拟南芥在添加葡萄糖或蔗糖的琼脂培养基上生长。光合电子传递抑制剂 3-(3, 4-dich; orophenyQ-1, 1-二甲基脲(DCMU)虽然在没有糖源的情况下不能生长，但在光合活性水平受到抑制的情况下，在长波紫外光照射下产生高红色荧光。2．用报告基因和自杀 GonesGenes 转化的拟南芥，其中 P-葡萄糖醛酸酶 (GUS) 和氯霉素乙酰转移酶 (CAT) 作为报告基因，以及酒精基因脱氢酶（ADH）作为自杀基因，在用甲基磺酸乙酯（EMS）诱变之前尝试将其引入拟南芥中，该策略可能有助于使目标突变体的选择与高红色荧光筛选相结合。我们重点关注核酮糖 1, 5-二磷酸羧化酶/加氧酶小亚基基因成员（RuBisCO、rbcS-3B）和使用与 3'-5' 校对核酸外切酶活性相关的热稳定 DNA 聚合酶进行叶绿素 a/结合蛋白 (cabl) 快速获得覆盖启动子及其启动子的大约 1.5 千碱基对 (kbp) DNA 片段。 rbcS-38和cabl的上游区域，因为这些基因的核苷酸序列已经公布，ADH基因（大约2.2）。 kbp)同样从拟南芥DNA中扩增出具有rbcS-3B和cabl的启动子和调节活性的片段，并将其置于GUS、CAT和ADH基因的上游，并尝试引入拟南芥(Columbia和Columbia)。 Bensheim，adhl。用 EMS 诱变转基因拟南芥，并正在开发在绿叶中较少表达 rbcS-3B 或 cabl 的突变体。筛选较少。

项目成果

H.Kobayashi,J.Ngernprasirtsiri,and T.Akazawa: "Transcriptional regulation and DNA methylation in plastids during transitional conversion of chloroplasts to chromoplasts" EMBO Journal. 9. 307-313 (1990)

H.Kobayashi、J.Ngernprasirtsiri 和 T.Akazawa：“叶绿体向有色体转变过程中质体中的转录调控和 DNA 甲基化”EMBO 杂志。

T.tsuge,and H.Kobayashi: "Molecular analysis of genes for pathogenicity of Alternaria alternata Japanese pear pathotype,a hostーspecific toxin producer" In Molecular Strategies of Pathogens and Host Plants,edited by S.S.Patil,S.Ouchi,D.Mills,and C.Vance,Sp

T.tsuge 和 H.Kobayashi：“对宿主特异性毒素生产者 Alternaria alternata Japanese pear 致病型致病性基因的分子分析”，《病原体和宿主植物的分子策略》，由 S.S.Patil、S.Ouchi、D 编辑。米尔斯和 C. 万斯，Sp。

H.Kobayashi,A.M.Viale,T.Takabe,T.Akazawa,K.Wada,K.Shinozaki,K.Kobayashi,and M.Sugiura: "Sequence and expression of genes encoding the large and small subunits of ribulose 1,5ーbisphosphate carboxylase/oxygenase from Chromatium vinosum" Gene. 97. 55-62 (199

H. Kobayashi、A. M. Viale、T. Takabe、T. Akazawa、K. Wada、K. Shinozaki、K. Kobayashi 和 M. Sugiura：“编码核酮糖 1,5- 大亚基和小亚基的基因的序列和表达来自 Chromatium v​​inosum 的二磷酸羧化酶/加氧酶”基因。97. 55-62 (199

J.Ngernprasirtsiri,H.Kobayashi,and T.Akazawa: "Expression of photosynthetic genes is distinctly different between chloroplasts and amyloplasts in liquidーcultured cells of Sycamore(Acer pseudoplatanus L.)" Cell Structure and Function. 15. 273-283 (1990)

J.Ngernprasirtsiri、H.Kobayashi 和 T.Akazawa：“Sycamore（Acer pseudoplatanus L.）液体培养细胞中叶绿体和淀粉体的光合基因表达明显不同”，细胞结构和功能。 1990)

J.Ngernprasirtsiri,H.Kobayashi,and T.Akazawa: "DNA methylation is a determinative element of photosynthesis gene expreesion in amyloplasts from liquid-cultured cells of Sycamore(Acer pseudoplatanus L.)" Cell Structure and Function. 15. 285-293 (1990)

J.Ngernprasirtsiri、H.Kobayashi 和 T.Akazawa：“DNA 甲基化是美国梧桐液体培养细胞淀粉体中光合作用基因表达的决定性因素”细胞结构和功能。