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

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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.