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Cloning of the genes that were related to control regeneration-pathways in rice calli.

与控制水稻愈伤组织再生途径相关的基因的克隆。

基本信息

批准号：
06660005
负责人：
YOSHIDA Kaoru t
金额：
$ 1.34万
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 (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06660005/
关键词：
Differential display Cloning RT-PCR Regeneration Somatic embryo Adventitious shoot Rice Protein Differential screening 分子生物学

项目摘要

To detect gene expressions during regenration, we applied two novel methods to rice culture system, in which we could separate morphogenic pathways, embryogenesis and organogenesis.1.Simplified differential displayReverse-transcribed cDNAs from mRNA populations isolated from organogenetic calli, embryogenic calli, and unorganized calli were used as templates for PCR with a short arbitrary RAPD primer. Eight differentially expressed PCR fragments were cloned and used as probes for Northern blots. All clones could detect specific transcripts corresponding to the specificity observed in PCR products. The results revealed that PCR patterns of cDNAs are reliable to judge their specificity and that the method is also effective to detect and clone differential expressed genes even if their expressions are in the low abundant class. Nucleotide sequences of both ends of cDNAs were detemined. They were used to search the Genbank database for sequence homology. Two clones out of eight were identified as Saccharomyces cerevisiae p68 gene (RNA helicase gene) and Drosophila melanogaster bithoraxoid region, respectively.2.ImmunosubractionTo enrich embryogenesis-specific proteins, we used the affinity chromatography. The proteins that were expressed in unorganized calli were immunosubtracted from total proteins of embryogenic calli using the antiserum against total proteins of unorganized calli. Comparison of 2-D PAGE profile of immunosubtracted proteins with that of total proteins before immunosubtraction allowed us to detect 7 proteins specific to somatic embryogenesis. The result indicated that the method of immunosubtraction is an effective method to concentrate embryogenesis-specific proteins at low abundance.

为了检测水稻再生过程中基因的表达，我们采用了两种新的方法，在水稻培养系统中，我们可以分开形态发生途径，胚胎发生和器官发生。1.简化的差异显示从器官发生愈伤组织，胚性愈伤组织和无组织愈伤组织中分离的mRNA群体的反转录cDNA作为模板，用一个短的随机RAPD引物进行PCR。克隆了8个差异表达的PCR片段并用作北方印迹的探针。所有克隆都能检测到与PCR产物中观察到的特异性相对应的特异性转录物。结果表明，cDNA的PCR图谱可以可靠地判断其特异性，该方法也可以有效地检测和克隆差异表达基因，即使它们的表达是在低丰度类。测定cDNA两端的核苷酸序列。将它们用于搜索Genbank数据库中的序列同源性。其中2个克隆分别鉴定为酿酒酵母p68基因（RNA解旋酶基因）和果蝇双胸区。2.免疫印迹采用亲和层析法富集胚胎发生特异性蛋白。用无组织愈伤组织总蛋白的抗血清从胚性愈伤组织总蛋白中免疫扣除在无组织愈伤组织中表达的蛋白。比较免疫消减蛋白与免疫消减前总蛋白的2-D PAGE图谱，我们检测到7个体细胞胚胎发生特异性蛋白。结果表明，免疫消减法是一种有效的富集低丰度胚胎发生特异蛋白的方法。