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Identification of high molecular weight proteins in rice endosperm and analysis of C_4-photosynthetic genes.

水稻胚乳中高分子量蛋白的鉴定及C_4-光合基因分析

基本信息

批准号：
09556001
负责人：
SASAHARA Takeo
金额：
$ 7.62万
依托单位：
Yamagata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
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

Total andosperm proteins of japonica and indica cultivars, and their F_2 progeny were analyzed by two-dinmensional polyacrylamide gel electro-phoresis (2D-PAGE). Nine different high molecular weight (HMW) proteins were detected in the F_2 progeny fits with a ratio of 1 : 1 : 1 : 1 due to double fertilization. The dominantgenes appeared to regulate expression of the high molecular weight protein sub- units, and dose effect of dominant genes was detected.The proteins which were extracted from endosperm of the rice seed were separated by 2D-PAGE, and were electroblotted onto a PVDF membrane. The partial amino acid sequences of the proteins were determined by automatic Edman degradation. The N-terninal sequences and the internal amino acid sequences were deter-mined. Structural homology searches resulted in identification of seven HMW proteins (enzymes), namely, starch debranching enzyme precursor (96 kD), pyruvate orthophosphate dikinase (94kD), malate dehydrogenase (45 kD), phosphoglycerate mutase (69 kD), enolase (57 kD), D-xylulose reductase (54 kD), and UDP-glucose pyrophosphorylase (51kD). In addition, three of the proteins appeared to be related to proteins in the heat shock family, i.e., chloroplast atroma heat shock related protein (HSP) (74 kD), Heat shock 70 kD protein (71 kD), and chloroplastchaperonin (67 kD). The three proteins (60, 43 and 42 kD) were found to have no homology with those of known plant and animal proteins. .These results may suggest that sucrose transferred from leaves to endosperm are consumed for sturch synthesis and glycolysis. Expression of the gene for pyruvate orthophosphate dikinase that is the key enzyme for C_4-dicarboxylic acid cycle was strong in the early stage of grain filling, The result might show it is concerned with carbon and amino acidmetabolysms.

用双向聚丙烯酰胺凝胶电泳（2D-PAGE）分析了粳稻、籼稻及其F_2代的胚乳总蛋白。在双受精F_2后代中检测到9种不同的高分子量蛋白，其比例为1：1：1：1。从水稻种子胚乳中提取蛋白质，经2D-PAGE电泳分离后，电印迹到PVDF膜上，电泳结果表明，显性基因对水稻胚乳中高分子量蛋白质亚基的表达具有明显的调控作用，并存在剂量效应。通过自动Edman降解测定蛋白质的部分氨基酸序列。测定了N端序列和内部氨基酸序列。结构同源性检索鉴定出7种HMW蛋白（酶），即淀粉脱支酶前体（96 kD）、丙酮酸正磷酸二激酶（94 kD）、苹果酸脱氢酶（45 kD）、磷酸甘油酸脱氢酶（69 kD）、烯醇化酶（57 kD）、D-木酮糖还原酶（54 kD）和UDP-葡萄糖焦磷酸化酶（51 kD）。此外，其中三种蛋白质似乎与热休克家族中的蛋白质相关，即，叶绿体萎缩热休克相关蛋白（HSP）（74 kD）、热休克70 kD蛋白（71 kD）和叶绿体伴侣蛋白（67 kD）。这三个蛋白质（60、43和42 kD）与已知的植物和动物蛋白质没有同源性。这些结果可能表明，从叶片转移到胚乳中的蔗糖主要用于淀粉合成和糖酵解。C_4-二羧酸循环的关键酶丙酮酸正磷酸二激酶基因在灌浆前期表达较强，这可能与碳代谢和氨基酸代谢有关。