Conferring freezing tolerance to plants by accumulation of trehalose

通过海藻糖的积累赋予植物耐冻性

• 批准号: 15580109
• 负责人: HONJOH Ken-ichi
• 金额: $ 2.37万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15580109/
• 关键词: freezing tolerance; trehalose; trehalase; trehalose 6-phosphate synthase; trehalose 6-phosphate phosphatase; transgenic plants

We tried to isolate a cDNA clone corresponding to a gene encoding trehalase from Nicotiana tabacum and express the coding region of the clone in E.coli. As the expressed protein formed an inclusion body, it did not show its enzymatic activity. However, it was used as an antigen for construction of polyclonal antibodies. Expression of trehalase in wild type tobacco plants was investigated by using the antibiodies. The result showed that the level of expresson of the enzyme was very low. Twenty-two transgenic tobacco plants, carrying the gene encoding trehalase in antisense direction, were also constructed. However, the accumulation of trehalose was not confirmed in the transgenic plants.Next, we tried to isolate cDNA clones corresponding to genes encoding trehalose 6-phosphate synthase (tps) and trehalose 6-phosphate phosphatase (tpp) from N.tabacum For obtaining the PCR products, primers were designed based on the conserved regions of two enzymes from other higher plants. The sizes of amplified PCR fragments corresponding to tps and tpp were about 650 bp and 450 bp, respectively. The nucleotide sequences of the amplified fragments were confirmed. By using the fragments as probes, a cDNA library was screened. After screening of the library for tpp gene, one positive clone was obtained. The cDNA clone was 1854-bp size and coded for 384 amino acids. The result of homology search showed that the deduced amino acid sequence of the cDNA clone was quite similar to those of TPPs form other higher plants. On the other hand, no cDNA clone corresponding to tps gene was obtained. By combination of 5'RACE and 3'RACE methods, nucleotide sequence of the partial fragment of cDNA for tps was determined. The size was 2177 bp and the fragment codes for 676 amino acids.

我们试图从烟草中分离出与编码海藻糖酶基因相对应的cDNA克隆，并在大肠杆菌中表达该克隆的编码区。由于表达的蛋白形成包涵体，因此不显示其酶活性。然而，它被用作构建多克隆抗体的抗原。使用抗体研究了野生型烟草植物中海藻糖酶的表达。结果表明该酶的表达水平很低。还构建了二十二株转基因烟草植物，其携带反义方向编码海藻糖酶的基因。然而，在转基因植物中并未证实海藻糖的积累。 接下来，我们尝试从烟草中分离出编码海藻糖6-磷酸合酶（tps）和海藻糖6-磷酸磷酸酶（tpp）的基因对应的cDNA克隆，为了获得PCR产物，根据来自其他高等植物的两种酶的保守区域设计了引物。对应于tps和tpp的扩增PCR片段的大小分别约为650 bp和450 bp。确认了扩增片段的核苷酸序列。通过使用片段作为探针，筛选cDNA文库。对该文库进行tpp基因筛选，获得1个阳性克隆。 cDNA 克隆大小为 1854 bp，编码 384 个氨基酸。同源性搜索结果显示，推导的cDNA克隆的氨基酸序列与其他高等植物TPPs的氨基酸序列非常相似。另一方面，没有获得对应于tps基因的cDNA克隆。通过结合5'RACE和3'RACE方法，确定了tps cDNA部分片段的核苷酸序列。该片段大小为 2177 bp，编码 676 个氨基酸。