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Gateway technology is a universal cloning approach that enables rapid cloning of DNA fragments into mul- tiple Gateway-compatible destination vectors using λ phage site-specific recombination , eliminating the requirement to work with restriction enzymes and ligase. But a problem using this system for making entry clone is the expensive- ness and long time to buy the enzyme. To solve this problem , we created the TA cloning entry vector that contained a T-tail in each 3 ' -end through modification of pDONR207. The TA cloning approach can construct entry clones simply , economically and rapidly. Using Gateway T vectors prepared by this improved method , prokaryotic expres- sion vector and eukaryotic expression vector for SOS2 gene were constructed. Through the methods of prokaryotic ex- pression and transient gene expression in Arabidopsis protoplasts , it proved that the SOS2 gene expressed well in both

Chitosan (CHN) specially induced the activities of 39 kD and 42 kD protein kinases in ginseng cells, which could be suppressed by an inhibitor of mitogen-activated protein kinase (MAPK) pathway, PD98059. The immunoprecipitation (IP) using MAPK antibody or kinase assay in vitro also showed that CHN-induced 42 kD and 39 kD protein kinases belonged to the MAPK family. PD98059 suppressed CHN-induced transcriptions of ginseng squalene synthase and ginseng squalene epoxidase genes (gss and gse), CHN-induced accumulation of beta-Amyrin synthase (beta-AS) and synthesis of saponin. These results showed that CHN-induced activities of MAPKs were necessary for the CHN-induced saponin synthesis. EGTA and LaCl3 suppressed CHN-induced 39 kD and 42 kD MAPK activities. Ruthenium red (RR) could suppress CHN-induced 39 kD activity. All of them suppressed CHN-induced saponin synthesis. These results indicated that CHN-induced increment of cytosolic calcium was necessary for CHN-induced saponin synthesis. PD98059 also suppressed CHN-induced oxidative burst (including the increment of activity of plasma membrane NADPH oxidase and production of H2O2), but diphenylene iodonium (DPI), dimethylthiourea (DMTU) and 2,5-dihydroxycinnamic acid methyl ester (DHC) could not suppress CHN-induced MAPK activities, which indicated that MAPK was possibly function upstream of CHN-induced oxidative burst.