Conferring freezing tolerance to plants by accumulation of trehalose

我们试图从烟草中分离海藻糖酶编码基因的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。利用该表达载体研究了海藻糖酶在野生型烟草中的表达。结果表明，该酶在大肠杆菌中的表达水平很低。还构建了22株反义转海藻糖酶基因烟草植株。其次，我们尝试从普通烟草中分离海藻糖6-磷酸合成酶（tps）和海藻糖6-磷酸酶（tpp）基因的cDNA克隆。扩增出的tps和tpp片段大小分别约为650 bp和450 bp。对扩增片段的核苷酸序列进行了确认。以该片段为探针，筛选cDNA文库。对文库中的tpp基因进行筛选，得到一个阳性克隆。该cDNA克隆大小为1854 bp，编码384个氨基酸。同源性搜索结果表明，该cDNA克隆推导的氨基酸序列与其它高等植物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。采用5 'RACE和3' RACE相结合的方法，测定了tps部分cDNA片段的核苷酸序列。该片段大小为2177 bp，编码676个氨基酸。

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.