Studies on the structure and expression of genes that governs leaf senescence

叶片衰老调控基因的结构与表达研究

基本信息

批准号：
03404002
负责人：
WATANABE Akira
金额：
$ 19.58万
依托单位：
The University of Tokyo (1993-1994)Nagoya University (1991-1992)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1994
项目状态：
已结题

项目摘要

We were successful in isolating several cDNA clones for senescence-related genes by newly developed procedures. One of the procedures is that cDNA sequences are selected by proteins expressed in vitro if they have ability to be imported by isolated intact chloroplasts. By this procedure a sequence was found to code for a new protein which has similarity to a stress protein expressed in E.coli cells when they are infected by a phage. Another one is a modified direct display RT-PCR.Three independent cDNA clones were isolated, one of which codes for protein which has homology to branched-chain alpha-ketoacid dehydrogenase. This enzyme is one of the three subunits of pyruvate dehydrogenase complex known in mammals. Another clone had a sequence homologous to the second subunit of this complex. These observations suggest that senescing leaf cells become deficient in supply of energy from photosynthesis and are preparing to utilized branched chain amino acids such as Leu, Val, and Ile release … More d from degraded protein for a energy source by burning their carbon skeleton in TCA cycle. The last clone showed similarity in amino acid sequence to glucosidases known in plants. Expression of the gene in senescing leaf cells may suggest that the encoded enzyme converts various glucosides to glucose which can also used to generate energy.Expression of several known protease genes were also examined by northern hybridization with cDNA probes. Genes for proteasome subunits were not found to be specifically expressed in senescing cells, but a nuclear gene for the regulatory subunit of chloroplast Clp protease and a gene for cathepsin-like protease know to localize in vacuoles are expressed at high levels in those cells.Two out of the three genes for cytosolic glutamine synthetase had been shown to be expressed in the later stage of leaf senescence. By examining the effects of inhibitors of the enzyme and of feeding amino acids, one on the two was found to be regulated by the level of Glu/Gln in the cell. Less

我们成功地通过新开发的程序将几个cDNA克隆隔离为传感相关基因。其中之一是，如果蛋白质有能力通过分离的完整叶绿体进口，则在体外表达的cDNA序列选择。通过此过程，发现一个序列可以编码一种新蛋白质，该蛋白与在大肠杆菌细胞中被噬菌体感染时具有相似之处。另一个是修改的直接显示RT-PCR。分离了三个独立的cDNA克隆，其中一种代码为蛋白质，它与分支链α-酮酸脱氢酶具有同源性。该酶是丙酮酸脱氢酶复合物的三个亚基之一。另一个克隆具有与该复合物的第二个亚基同源的序列。这些观察结果表明，感应叶片细胞从光合作用中不足以供应，并准备利用分支链氨基酸，例如Leu，Val和Ile释放……从降解的蛋白质中，通过在TCA循环中燃烧其碳骨架，从降解的蛋白质来源。最后一个克隆在氨基酸序列中与植物中已知的葡萄糖酶相似。基因在感应叶细胞中的表达可能表明，编码的酶将各种糖苷转化为葡萄糖，也可以用于产生能量。还通过北部杂交与cDNA问题来表达几种已知蛋白酶基因。未发现用于蛋白酶的基因在传感细胞中特异性表达，但是叶绿体CLP蛋白酶的调节亚基的核基因和类似组织蛋白酶的蛋白酶的基因在真空中含量很高，在这些细胞中很高表达。通过检查酶抑制剂和摄食氨基酸的抑制剂的影响，发现两种抑制剂受细胞中的GLU/GLN水平调节。较少的