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Gene-technological manipulation of phospholipid composition in Arabidopsis thaliana in relation to freezing tolerance

拟南芥磷脂组成与耐冻性的基因技术操作

基本信息

批准号：
09640763
负责人：
NISHIDA Ikuo
金额：
$ 2.37万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

(1) CTP : phosphoethanolamine cytidylyltransferase (EC2.7.7.14, ECT) is a rate-limiting enzyme in the biosynthesis of phosphatidylethanolamine in plant cells. I have cloned a cDNA for Arabidopsis ECT by PCR, using primers designed from Arabidopsis genome databases (Accession no. g3786005).(2) A cDNA for Brassicanapus CTP : phosphocholine cytidylyltransferase (EC2.7.7.15, CCT) library was introduced into Arabidopsis thaliana by an inplanta transformation method. One of transformants showed changes in the levels of phospholipids. Correlation between the changes in the lipid composition and frost resistance is under investigation.(3) INY102B/YCpGPSS was transformed with the pFL61 (URA3) cDNA library constructed from roots of Brassica napus. From 1 x 10^6 Ura^+ transformants, 100 Etn^+ transformants were isolated, and 16 of them were characterized. They are all identical and a representative clone contained 1,894 bp with an open-reading frame of 1,473 bp. Deduced amino-acid sequence contained 490 amino-acid residues with a calculated molecular mass of 55,153. A homologous Arabidopsis cDNA clone identified by a BLAST search to Arabidopsis EST sequences was also characterized. The Arabidopsis clone contained 1,739 bp with an open-reading frame of 1,449 bp. Deduced amino-acid sequence contained 482 amino-acid residues with a calculated molecular mass of 54,153. Search for homology in GenBank nucleotide sequence database revealed that both clones are homologous to pyridoxal-5'-phosphate-dependent histidine decarboxylases, and a putative pyridoxal phosphate-binding motif for Group II decarboxylases (Sandmeier et al. 1994) was conserved in both proteins. Therefore, our results are the first to suggest that histamine could be synthesized in plant roots.

（1）CTP：磷酸乙醇胺胞苷酰转移酶（EC2.7.7.14，ECT）是植物细胞中磷脂酰乙醇胺生物合成的限速酶。我使用从拟南芥基因组数据库设计的引物（登录号g3786005），通过PCR克隆了拟南芥ECT的cDNA。（2）通过植物内转化方法将甘蓝CTP：磷酸胆碱胞苷酰转移酶（EC2.7.7.15，CCT）文库引入拟南芥。一种转化体显示出磷脂水平的变化。脂质成分变化与抗冻性之间的相关性正在研究中。(3)用从甘蓝型油菜根构建的pFL61(URA3)cDNA文库转化INY102B/YCpGPSS。从 1 x 10^6 Ura^+ 转化子中分离出 100 个 Etn^+ 转化子，并对其中 16 个进行了表征。它们都是相同的，代表性克隆包含 1,894 bp，开放阅读框为 1,473 bp。推导的氨基酸序列包含490个氨基酸残基，计算分子量为55,153。还对通过对拟南芥 EST 序列进行 BLAST 搜索鉴定的同源拟南芥 cDNA 克隆进行了表征。拟南芥克隆包含 1,739 bp，开放阅读框为 1,449 bp。推导的氨基酸序列包含482个氨基酸残基，计算分子量为54,153。在 GenBank 核苷酸序列数据库中搜索同源性表明，这两个克隆与吡哆醛-5'-磷酸依赖性组氨酸脱羧酶同源，并且推定的 II 组脱羧酶的磷酸吡哆醛结合基序（Sandmeier 等，1994）在这两种蛋白质中都是保守的。因此，我们的结果首次表明组胺可以在植物根部合成。