The Expression Analysis Of Related Genes In Panax Ginseng Hairy Roots At The Early Stage Of Embryogenesis

Panax ginseng(Araliaceae, Panax), a perennial herb, is a valuable medicinal herb whose main active secondary metabolite named ginsenosides ha potent effects on regulating central nervous system, building immunity, relieving fatigue, antioxidation, anti-tumor, anti-diabetes and so on acording to reasearch. P. ginseng has considerable economic value and broad application in production of durgs, cosmetics and health care products. However because of rare resource of wild P. ginseng, long period time of cultivation and rigorous cultivation condition, plants of P. ginseng with high quality are in short supply and expensive.This study is based on the expression of several realted genes in P. ginseng hairy roots at the early stage of embryogenesis, aiming to provide a strong background for the system building of embryogenesis of P. ginseng hairy roots. P. ginseng hairy roots which are developed from roots infected by Agrobacterium rhizogenes show many advantages in embryogenesis of P. ginseng, therefore it has been widely used in rapid propagation and species-preservation of P. ginseng. In this study, we developed callus and embryonic callus of P. ginseng hairy roots, and analyzed the expression of LBD29, MAPK4, MAPK6, WRKY3, WRKY6 and WRKY27 in P. ginseng at the early stage of embryogenesis using Semi-quantity RT-PCR and Real-time quantitative PCR. The reasults are showed as below:1. Four different kinds of tissue materials in this study were develped and collected through subculture of P. ginseng hairy roots, induction and multiplication of callus and embryonic callus.2. According to a large variety of related research of plants genes involved in embryogenesis, LBD29, MAPK4, MAPK6, WRKY3, WRKY6 and WRKY27 were designated as objects for reasearch. Based on the sequence of these genes in other species, using the tools of BLAST, we designed primers and successfully cloned and sequenced these genes in P. ginseng, providing sequence data for further study of them.3. Semi-quantity RT-PCR analyzed the expression of related genes in P. ginseng at different stage of embryogenesis. Using calibrated 5S rRNA in P. ginseng as a standard, we analyzed the expression of LBD29, MAPK4, MAPK6, WRKY3, WRKY6 and WRKY27 in P. ginseng at different stage of embryogenesis. The results showed that all of the six displayed variation of expression level at different stage of embryogenesis, indicating their relevance to embryogenesis of P. ginseng hairy roots at early stage.4. Real-time quantitative PCR analyzed the expression of the six genes in P. ginseng at different stage of embryogenesis. The results in Real-time quantitative PCR were consistent with that in Semi-quantity RT-PCR. The change of expression level of LBD29, MAPK4, MAPK6, WRKY3, WRKY6 and WRKY27 at different stages of embryogenesis indicated their important role in embryogenesis of P. ginseng at early stage.5. The CDS of WRKY6 in P. ginseng was cloned and sequenced. The length of WRKY6 cloned from P. ginseng was 1041 bp, and it was identified as the CDS(Gen Bank: KT277547.1) of WRKY6 in P. ginseng, as the blast of WRKY6 s in P. ginseng and Panax quinquefolius showed the similarity of 99.61% in nucleotide sequence and of 97.63% in amino acid sequence.