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Immunohistochemical analysis on control of regeneration-pathways in rice calli

水稻愈伤组织再生途径控制的免疫组织化学分析

基本信息

批准号：
03454036
负责人：
YOSHIDA Kaoru
金额：
$ 3.58万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

To isolate proteins and gene that control two regeneration-pathways (embryogenesis and organogenesis) in rice calli.1. Four proteins (a, b, c, d) specific to embryogenic calli and two proteins (e, f) specific to regeneration (common in both embryogenic and organogenic calli) were detected by 2 - D PAGE analysis. Partial amino acid sequences of protein a were determined.2. After affinity chromatography of ConA, ten glycoproteins specific to embryogenic calli were detected.3. To isolate rare proteins that control regeneration, immuno-subtraction method was applied. The protein expressed in unorganized calli were subtracted from total proteins of embryogenic or organogenic calli by affinity chromatography using the antiserum against extracts of unorganized calli. Six proteins specific to embryogenic calli, two proteins specific to organogenic calli and a protein specific to regeneration were newly detected.4. To isolate and clone rare genes that are differentially expressed, a novel method of differential screening of random amplified cDNAs with RAPD primers was applied. A number of differentially expressed transcripts in rice calli could be detected and cloned by this method. Northern blot analysis also revealed that the detected differentially expressed transcripts included rare genes and that a gene specific to embryogenic calli was exclusively expressed in zygotic embryos.

分离控制水稻胚状体发生和器官发生两条再生途径的蛋白和基因. 2 - D PAGE分析检测到4种胚性愈伤组织特异性蛋白（a，B，c，d）和2种再生特异性蛋白（e，f）。测定了a蛋白的部分氨基酸序列.经ConA亲和层析后，检测到10种胚性愈伤组织特异性糖蛋白.为了分离控制再生的稀有蛋白，应用免疫消减方法。通过亲和层析，使用针对无组织愈伤组织提取物的抗血清，从胚性或器官发生愈伤组织的总蛋白中减去无组织愈伤组织中表达的蛋白。新检测到6个胚性愈伤组织特异性蛋白、2个器官性愈伤组织特异性蛋白和1个再生特异性蛋白.为了分离和克隆差异表达的稀有基因，采用RAPD引物对随机扩增的cDNA进行差异筛选。利用该方法可以检测并克隆水稻愈伤组织中的一系列差异表达转录本。北方印迹分析还表明，检测到的差异表达的转录本包括罕见的基因，并特异性的胚性愈伤组织的基因只在合子胚中表达。