Differential Gene Expression In Somatic Embryogenesis On Dimocarpus Longan Lour

Longan somatic embryogenesis occurred through the following stages- embryogenic callus, globular embryoid, heart-shaped embryoid, torpedo-shaped embryoid, cotyledonary embryoid and matured cotyledonary embryoid, which was similar to that of zygotic embryo. Synchronized embryogenic cultures at different developmental stages were obtained by controlling 2,4-D concentration and developmental time. High-quality DNA of longan embryogenic cultures were extracted by SDS ,CTAB and LiCl methods. DNA extracted by LiCl was better with less contamination and high-quality than that by the other 2 methods. Furthermore, it was cheaper and took less time, which was suitable for the DNA extraction of in vitro materials. High-quality RNA of longan embryogenic cultures for mRNA differential display were also extracted by the Sangon RNA extraction kit.The mRNA differential display system for longan embryogenic cultures was first established, and 30 specific expression cDNA fragments were obtained. Among them, 5 cDNAs(longan1, longan2, longan3, longan4, Iongan4') were proved to be specific expression cDNAs by northern blot and reverse northern blot. Longanl expressed from the embryogenic callus stage to the globular embryoid stages. Longan2 expressed in the whole process of longan somatic embryogenesis, which was probably a housekeeping gene. Longan3 expressed from the globular embryoid stage to the torpedo-shapedembryoid stage. Longan4, longan4' expressed only at the cotyledonary embryoid stage.Sequence analysis showed that an open reading frame(ORF) without the termination code existed at the 3' end of longanl and at the 5' end of longanl and longanl, which suggested that they should be the part of ORF at their 3' or 5' extension end, respectively. The whole length cDNA of these three genes could obtain by RACE or screening cDNA library. Blast results in Genbank showed that there were no homologous genes to longan2, longan3, longan4, longan4' However longanl was highly homology to apx gene(92%) from tomato. It was predicted that longanl was an apx gene in longan somatic embryoids.Proteins in longan somatic embryogenesis changed greatly as the mRNAs did. 13 specific proteins were obtained with horizontal two-dimensional electrophoresis. The PIs of these specific proteins were decreased as somatic embryogenesis occurred and developed, which suggested that the PIs of the specific proteins should be negative relative to the developmental satages of somatic embryoids.The obtained results of the mRNA differential display and two-dimensional electrophoresis of the proteins showed that the cDNAs of embryogenic calli and globular embryoids were more than those of cotyledonary embryoids and matured-cotyledonary embryoids, While the proteins were in turn. It was predicted that some genes existed in transcripting mRNAs or exiting in mRNPs, which would be translated into proteins which were important to somatic embryogenesis at the later stages.