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

为了检测再生过程中的基因表达，我们将两种新方法应用于水稻培养系统，分别分离了形态发生途径、胚胎发生途径和器官发生途径。采用从有机愈伤组织、胚性愈伤组织和无组织愈伤组织中分离的mRNA群体中逆转录的cdna作为模板，用短的任意RAPD引物进行PCR。克隆了8个差异表达的PCR片段作为Northern blots的探针。所有克隆均能检测到与PCR产物特异性相对应的特异性转录本。结果表明，cdna的PCR图谱可可靠地判断其特异性，该方法对低丰度表达的差异表达基因的检测和克隆也是有效的。测定cdna两端的核苷酸序列。它们被用于在Genbank数据库中搜索序列同源性。8个克隆中鉴定出2个分别为酿酒酵母p68基因（RNA解旋酶基因）和黑腹果蝇双胸腺区。为了丰富胚胎发生特异性蛋白，我们使用亲和层析。用抗胚性愈伤组织总蛋白的抗血清，从胚性愈伤组织总蛋白中提取无组织愈伤组织中表达的蛋白。将免疫减法蛋白的二维PAGE图谱与免疫减法前的总蛋白的二维PAGE图谱进行比较，使我们能够检测到7种体胚发生特异性蛋白。结果表明，免疫减法是一种浓缩低丰度胚胎发生特异性蛋白的有效方法。

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.