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.

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.克隆八个差异表达的PCR片段，并用作北印迹的探针。所有克隆都可以检测与PCR产物中观察到的特异性相对应的特定转录本。结果表明，CDNA的PCR模式可靠地判断其特异性，并且该方法也可以有效地检测和克隆差异表达的基因，即使它们的表达方式在低丰富的类别中。确定了CDNA两端的核苷酸序列。他们被用来搜索GenBank数据库以获取序列同源性。八个中的两个克隆分别鉴定为酿酒酵母p68基因（RNA解旋酶基因）和果蝇Melanogaster bithoraxoi区域。使用抗血清对无组织Calli的总蛋白质对无组织的Calli表达的蛋白质免疫提取。在免疫疗法之前，将免疫提取蛋白与总蛋白质的2-D页谱的比较使我们能够检测7种特有的针对体细胞胚胎的蛋白质。结果表明，免疫学方法是在低丰度下浓缩胚胎发生特异性蛋白的有效方法。